Abstract

Many antibiotics lack activity against Gram-negative bacteria because they cannot permeate the outer membrane or suffer from efflux and, in the case of β-lactams, are degraded by β-lactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin component of 1 is rapidly hydrolyzed by purified ADC-1 β-lactamase to release the oxazolidinone. Conjugate 1 is active against clinical isolates of Acinetobacter baumannii as well as strains producing large amounts of ADC-1 β-lactamase. Overall, the results are consistent with siderophore-mediated active uptake, inherent activity of the delivered dual drug, and in the presence of β-lactamases, intracellular release of the oxazolidinone upon cleavage of the cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result demonstrates that Gram-positive oxazolidinone antibiotics can be made to be effective against Gram-negative bacteria by β-lactamase triggered release.

Targeting Inosine-5′-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis

Ana Trapero^†

, Angela Pacitto^‡, Vinayak Singh^§^#, Mohamad Sabbah^†, Anthony G. Coyne^†

, Valerie Mizrahi^§

, Tom L. Blundell*^‡, David B. Ascher*^‡^∥, and Chris Abell*^†

^† Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

^‡ Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom

^§ MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Rondebosch 7701, Cape Town, South Africa

^∥ Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3052, Australia

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.7b01622

Publication Date (Web): March 16, 2018

*T.L.B. email: tom@cryst.bioc.cam.ac.uk., *D.B.A: phone, +61 3903 54794; e-mail, david.ascher@unimelb.edu.au., *C.A.: phone, +44 (0) 1223 336405; e-mail, ca26@cam.ac.uk.

ACS AuthorChoice - This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

Abstract

Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5′-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible (Mth) IMPDH ΔCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ΔCBS–IMP–inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit.

Introduction

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb), which can be transmitted through the air as droplets. The infection predominantly affects the lungs, but it can spread to other parts of the body, especially in patients with a suppressed immune system.

The World Health Organization (WHO) has estimated that nearly one-third of the world’s population is infected with Mtb, leading to 1.8 million TB deaths in 2015.(1) Although there has been a slow decline in new TB cases and TB-related deaths in recent years, the emergence and spread of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mtb has increased the threat that this disease poses for global public health. According to the WHO, approximately 480,000 cases of MDR-TB emerged in 2015, and the cure rate of those patients was only 50%.(1)

Current TB treatments require combinations of four first-line drugs, isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin, which must be taken for six months or longer.(2) Resistant strains are not susceptible to the standard drugs, and although MDR-TB is treatable using second-line drugs, such treatments have a number severe side effects.(3) Consequently, there is an urgent need for the development of novel and more effective drugs for the treatment of drug resistant TB.

Inosine-5′-monophosphate dehydrogenase (IMPDH, E.C. 1.1.1.205) has received considerable interest in recent years as an important target enzyme for immunosuppressive,(4) anticancer,(5,6) and antiviral drugs.(7) Most recently, IMPDH has emerged as a promising antimicrobial drug target.(8−11)

IMPDH catalyzes the first unique step in the de novo synthesis of guanine nucleotides, the oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP) with the concomitant reduction of the cofactor nicotinamide adenine dinucleotide (NAD⁺) to NADH (Figure 1).(12) XMP is then subsequently converted to guanosine 5′-monophosphate (GMP) by a GMP synthetase.(13)

Figure 1. Purine nucleotide biosynthesis. The commonly occurring guanine nucleotide biosynthetic and salvage reactions are shown, as is the adenine nucleotide biosynthetic pathway. The IMPDH reaction is boxed. NK, nucleoside kinase; HPRT, hypoxanthine phosphoribosyl transferase; XPRT, xanthine phosphoribosyl transferase; GPRT, guanine phosphoribosyl transferase; GMPS, guanosine 5′-monophosphate synthetase; GMPR, guanosine 5′-monophosphate reductase; ADSS, adenylosuccinate synthetase; ADSL, adenylosuccinate lyase.

IMPDH has been deemed essential in every pathogen analyzed to date, including Mtb, Staphylococcus aureus, and Pseudomonas aeruginosa, which are three of the most serious bacterial threats.(14−16) However, this has been somewhat contradictory,(17) in comparing cell versus animal work. IMPDH is an ubiquitous enzyme present in several eukaryotes, bacteria, and protozoa.(18) The IMPDH reaction involves two chemical transformations. The first step of the IMPDH catalyzed reaction involves the attack of catalytic Cys on substrate IMP followed by hydride transfer to NAD⁺, forming the covalent enzyme intermediate E-XMP*. In the second step, E-XMP* is hydrolyzed to XMP.(11) The enzyme exists in two different conformations, an open form that accommodates both the substrate and cofactor during the first step and a closed form where the active site flap moves into the NAD⁺-binding site for the E-XMP* hydrolysis.(19)

In recent years, there has been considerable effort aimed at identifying small molecule inhibitors of IMPDH as potential antitubercular agents. X-ray crystal structures of a truncated form of the Mtb enzyme in complex with some of these compounds have been reported.(20−26)

In antibacterial drug discovery, and especially in TB drug discovery, high-throughput screening (HTS) typically identifies a number of leads that show high potency in vitro, but most did not show any translation to an in vivo effect. It is also inevitable that the HTS libraries represent only a small fraction of possible chemical space and so limit confidence in finding a good starting point for subsequent development. Phenotypic screens can potentially lead to the identification of a molecule that modifies a disease phenotype by acting on a previously undescribed target or by acting simultaneously on more than one target.(27) However, for many of these hits the relevant target or targets has not yet been identified, thus preventing further target-based optimization of the compounds.(28,29)

The previously reported IMPDH inhibitors, such as compounds 7759844 (1), MAD1, P41, VCC234718, and DDD00079282 (Figure 2), were identified by phenotypic screening or target based HTS of compound libraries.(21,23−25)

Figure 2. Structures of previously reported IMPDH inhibitors (α K_i values against IMP). All compounds showed uncompetitive inhibition with respect to IMP.

We have sought to develop IMPDH inhibitors using a fragment-based approach. Fragment-based drug discovery (FBDD) is now established in both industry and academia as an alternative approach to high-throughput screening for the generation of hits or chemical tools for drug targets.(30)

We have previously reported the discovery of several series of novel and potent inhibitors using FBDD to target Mtb. Previously we have reported the fragment elaboration strategies that we have applied which have included fragment growing, merging, and linking. Although fragment linking is conceptually the most appealing strategy for fragment elaboration, in practice, this strategy can be challenging where the choice of the optimal fragment linker can be crucial.(31,32)

In the fragment-based approach, biophysical techniques are usually used to identify small chemical compounds (fragments) that bind with low affinity to the drug target. X-ray crystallography is then usually employed to establish the binding mode of the fragment and to facilitate the design of elaborated fragments. The availability of high-resolution X-ray crystal structures of a truncated form of the IMPDH,(21,24,25) in both the substrate-free and substrate/ligand-bound forms, makes this enzyme attractive for a fragment-based approach.

In this work, the discovery of a new class of potent nM inhibitors of IMPDH using a FBDD approach is reported. A library of 960 fragments was screened against Mth IMPDH ΔCBS using a biochemical assay. The fragment hits from this assay were examined using X-ray crystallography, and an X-ray crystal structure of one of the fragment complexes was solved to a resolution of 1.45 Å. Examination of the X-ray crystal structure suggested a strategy of fragment-linking for optimization of this fragment hit.

Results and Discussion

Fragment Screening

An in-house fragment library composed of 960 fragments was screened using a biochemical assay against Mth IMPDH ΔCBS. Mth IMPDH, which shares 85% sequence identity with Mtb IMPDH and is 100% identical in the active site,(24,25) was chosen for the fragment screening and structural studies because it gave higher protein expression yields and better diffracting crystals than the Mtb orthologue. IMPDH activity was monitored spectrophotometrically by measuring the formation of NADH at 340 nm. The biochemical assay was performed at a fragment concentration of 1 mM, and hits were retested in triplicate. Compound 1 (7759844) previously reported as IMPDH inhibitor was used as a positive control in assays (Table 1).(23)

Table 1. Structures and Activities for Compound 1 (7759844) and the Most Potent Fragment Hits Found in the Screen against Mth IMPDH ΔCBS

% Inhibition at 10 μM.

The screen resulted in 18 hits (1.9% hit rate), where a hit was defined as a compound that gave greater than 50% inhibition at a concentration of 1 mM. A complete list of fragment hits identified is included in Table 1. A number of common scaffolds were observed, in particular phenylimidazole (fragments 2–4), aminopyrazole (fragments 5–6), and naphthol (fragments 7–11), and the remaining compounds contained a substituted phenyl or a heterocyclic five membered ring (fragments 12–19).

The IC₅₀ values of six of the most active fragments were measured and the IC₅₀ and ligand efficiency (LE) values of these fragments are summarized in Table 2. The fragment screen provided an array of hits with IC₅₀ ranging from 325 μM to 674 μM and ligand efficiencies from 0.31 to 0.42. The inhibition constant [K_i] with respect to both substrates IMP and NAD⁺ was determined by assaying various concentrations of each inhibitor with five different concentrations of substrate and a fixed saturating concentration of the cosubstrate. The inhibition data for these fragments are summarized in Table 2. All compounds yielded an uncompetitive inhibition pattern with respect to NAD⁺ with K_i values ranging from 262 to 525 μM. Fragments 14, 16, and 19 yielded a mixed inhibition with respect to IMP with K_i values ranging from 126 to 398 μM, and compounds 1, 2, 11, and 18 yielded an uncompetitive inhibition with K_i values ranging from 361 to 609 μM.

Table 2. IC₅₀, Ligand Efficiency, and Alpha K_i Values of Fragment Hits and Compound 1 (7759844) against Mth IMPDH ΔCBS

Compd	IC₅₀ (μM) (LE)^a	IMP αK_i (μM)	NAD αK_i (μM)
1	0.77 ± 0.06 (0.40)	0.86 ± 0.03 (UC)^b	0.55 ± 0.02 (UC)
2	674 ± 53 (0.36)	609 ± 3 (UC)	512 ± 23 (UC)
11	336 ± 6 (0.39)	361 ± 53 (UC)	310 ± 30 (UC)
14	400 ± 7 (0.42)	238 ± 6 (Mixed)	262 ± 39 (UC)
16	433 ± 60 (0.31)	398 ± 81 (Mixed)	525 ± 64 (UC)
18	512 ± 37 (0.37)	554 ± 70 (UC)	452 ± 26 (UC)
19	325 ± 9 (0.37)	126 ± 34 (Mixed)	355 ± 47 (UC)

Ligand efficiency was calculated using the equation LE = (1.37 × pIC₅₀)/HA, where HA means heavy atom, i.e., a non-hydrogen atom.

UC: Uncompetitive inhibition.

Inhibition constants of compound 2 toward full-length Mtb IMPDH were also determined. Compound 2 inhibited full-length Mtb IMPDH enzyme with a K_i value of 572 ± 14 μM with IMP as the substrate and a K_i value of 534 ± 18 μM with NAD as the substrate, which are similar to the K_i values observed for the Mth IMPDH ΔCBS enzyme.

X-ray Structure of Compounds 1 (7759844) and 2

Compound 1 and the six fragment hits (2, 11, 14, 16, 18, and 19) were selected for structural characterization using X-ray crystallography by soaking into preformed crystals of Mth IMPDH ΔCBS as previously described.(20) After molecular replacement, clear electron density was observed in the 2F₀ – F_c difference map (σ = 3.0) for IMP, and in addition density was observed for one molecule of compound 1 (Figure 3A) and two molecules of compound 2 (Figure 3B), which were partially occupying the NAD⁺ binding site. None of the other fragments (11, 14, 16, 18, and 19) showed any electron density in the X-ray crystal structures. Although the kinetic studies of compounds 1 and 2 suggested that these two compounds are uncompetitive with respect to NAD⁺, the binding mode of compounds 1 and 2 closely resembles that of other previously reported uncompetitive inhibitors of Mtb IMPDH.(21,24,25) It has been proposed that the uncompetitive mode of inhibition of IMPDH inhibitors with respect to NAD⁺ is consistent with their binding preferentially to the covalent IMPDH-XMP* intermediate after NADH has been released.(24,25,33)

Figure 3. X-ray crystal structure of compounds 1 and 2 bound to Mth IMPDH ΔCBS. Ligand interactions are represented as dotted lines; hydrogen bonds are represented in red, polar interactions in orange, ionic interactions in yellow, and aromatic and π interactions in green dotted lines. Protein–ligand interactions were analyzed using Arpeggio. (a) Interactions made by 1 (green) in the X-ray crystal structure of the complex of IMPDH with IMP (orange). (b) Interactions made by 2 (yellow) in the X-ray crystal structure of the complex of IMPDH with IMP. (c) Structural alignment of the IMPDH crystal structures of 1 (green) and 2 (yellow).

The structure of Mth IMPDH ΔCBS with compound 1 showed that the inhibitor binds in the NAD pocket in a near identical manner to our recently described IMPDH inhibitors.(24,25) Compound 1 formed strong π interactions with the hypoxanthine group of IMP, P45′, Y471′, and polar interactions with G409, E442, P45′, and G470′. The electron density revealed two molecules of compound 2 within the NAD binding pocket of IMPDH. One molecule of compound 2 stacked with IMP, forming extensive π interactions with the hypoxanthine group of IMP. This fragment was further stabilized through polar interactions, hydrogen bonds, and π interactions to surrounding residues in the active site pocket, including A269, G318, and E442. The other molecule of compound 2 sits closer to the opening of the active site, making polar interactions with N273 and E442, and π interactions with H270 and Y471′.

A comparison of the structure of Mth IMPDH ΔCBS with compound 1 with the fragment 2 structure shows that the two molecules of 2 mimic the position of the larger inhibitor 1 (Figure 3C).

Fragment Elaboration

Fragment 2 was selected as the starting point for exploration because of the ease of synthetic modification and the availability of a X-ray crystal structure to guide the optimization. For chemical elaboration of 2, fragment linking as well as fragment growing were considered. As the two molecules of fragment 2 are found to bind in adjacent regions of the target protein, the fragment-linking approach was the more attractive option. However, before fragment linking, fragment 2 was further optimized with the aim of improving the binding affinity. The structures and inhibitory activities of these compounds against Mth IMPDH ΔCBS are summarized in Tables 3 and 4. The corresponding data for fragment 2 have also been included for comparative purposes.

Table 3. Structures and Activities for Fragment 2 and Compounds 20–28 against Mth IMPDH ΔCBS

Table 4. Structures and Activities for Fragment 2 and Compounds 29–35 against Mth IMPDH ΔCBS

Ligand efficiency was calculated using the equation LE = (1.37 × pIC₅₀)/HA, where HA means heavy atom, i.e., a non-hydrogen atom.

ND: not determined. IC₅₀ values were determined for compounds that showed >50% inhibition. ^c%Inhibition at 10 μM.

All compounds were evaluated at a concentration of 100 μM with Mth IMPDH ΔCBS.

Fragment Growing

The fragment-growing strategy involves using structure-based drug design to form additional interactions by growing out from the starting fragment. Fragment 2 was modified at the 2-position of the imidazole ring to explore the introduction of various aromatic rings linked by a thioacetamide (20–22) to form π interactions with the hypoxanthine group of IMP (Figure S1). Such modifications gave compounds with improved Mth IMPDH ΔCBS inhibition. The phenyl and benzofuran derivatives (20 and 21) showed 13 and 31% inhibition, respectively. Mth IMPDH ΔCBS inhibition was shown to be sensitive to minor modifications of the phenyl substituent groups; for example, the 4-iodo substituted 22 showed 30% inhibition at 100 μM, whereas the nonsubstituted compound, 20, showed 13% inhibition at the same concentration. The effect of the removal of the 4-bromo group was investigated and compounds 23–25 were synthesized. Removal of the bromo substituent in compounds 20–22 (13–31% inhibition at 100 μM) was tolerated (23–25, 3–46% inhibition at 100 μM). The importance of the aromatic amide linked by a thioacetamide was subsequently examined. Replacing the phenyl with isopropyl (26) resulted in complete loss of activity. Substitution of the thioacetamide by a thioacetic acid also led to a complete loss in activity (compounds 27 and 28).

Fragment Linking and SAR

Examination of the X-ray crystal structure of the previously reported inhibitor 1 when overlaid with fragment 2 revealed that the distance between the 4-position of the phenyl ring of fragment 2 and the 2-position of the imidazole ring represents the closest approach of the molecules (Figure S1). On the basis of this structural information, three different linkers were designed to connect the two copies of the fragment 2 at these positions (compounds 29–31). Initially, a thioacetamide and urea linker moieties were examined. Compounds 29 and 30 showed 20% and 56% of Mth IMPDH ΔCBS inhibition, respectively, at 100 μM (Table 4). Interestingly, compound 30 showed a 12-fold improvement in Mth IMPDH ΔCBS inhibitory activity with an IC₅₀ of 58 μM, compared to the fragment 2.

The lactate linker was then used, but all attempts to link 4-phenylimidazole with 4-(4-bromophenyl)-1H-imidazole were unsuccessful. We therefore decided to synthesize compound 31, which contains 1-methyl-4-phenyl-1H-imidazole and 4-(4-bromophenyl)-1H-imidazole linked with a lactate analogue, as in compound 1. Compound 31 (Table 4) showed markedly improved Mth IMPDH ΔCBS inhibition with a LE of 0.29 and IC₅₀ of 0.52 μM, which is 1300-fold more potent compared to the fragment 2. Although compound 31 binds in the cofactor site, the mechanism of inhibition can vary depending on its relative affinities for the E·IMP and E-XMP* complexes.(8,11) Kinetic evaluation of compound 31 showed the mode of Mth IMPDH ΔCBS inhibition was uncompetitive with respect to both IMP and the NAD⁺ cofactor (see Figure S2, Supporting Information) with a K_i value of 0.30 ± 0.02 μM with IMP as the substrate and a K_i value of 0.20 ± 0.01 with NAD as the substrate.

Inhibition constants of compound 31 toward full-length Mtb IMPDH were also determined. Compound 31 inhibited full-length Mtb IMPDH enzyme with a K_i value of 0.61 ± 0.05 μM with IMP as the substrate and a K_i value of 0.39 ± 0.02 μM with NAD as the substrate. The inhibition constants were consistent with the data using the Mth IMPDH ΔCBS enzyme.

Removal of the bromo substituent in compound 29 to give compound 32 was well tolerated (Table 4). The importance of the imidazole group for the inhibitory activity against Mth IMPDH ΔCBS was confirmed by replacing of the 4-(4-bromophenyl)-1H-imidazole substituent of compound 31 with 4-(4-bromophenyl)oxazole (33) which resulted in a 4-fold loss of activity (Table 4). Replacing the 4-(4-bromophenyl)-1H-imidazole of 31 by a phenyl (34) resulted in complete loss of activity (Table 4). However, the 4-iodophenyl derivative 35 demonstrated slightly improved activity (IC₅₀ = 0.47 μM, LE = 0.34) compared to compounds 31 (IC₅₀ = 0.52 μM, LE = 0.29) and 1 (IC₅₀ = 0.77 μM, LE = 0.40). It is noteworthy that LE of compound 35 was comparable to that of the original fragment hit 2 (LE = 0.36) and other reported IMPDH inhibitors.(34)

The importance of the 4-bromo substituent on the phenyl ring in compound 31 was also explored (compounds 36–39, Table 5). Removal of the bromo substituent (36) resulted in a 5-fold loss in activity, whereas replacing this group with an iodine (39) or a morpholine ring (37) resulted in less than 1.3-fold loss in activity. The electronic nature of the substituent in this position had little effect on inhibitory activity. For example, an electron-donating methoxy (38) retained activity comparable to that of the bromine derivative (31). Among them, imidazoles 31, 37–38, and 39 were found to be potent inhibitors of Mth IMPDH ΔCBS with IC₅₀ values ranging from 520 to 690 nM.

Table 5. Structures and Activities for Compounds 31, 36–39, and (S)-31 against Mth IMPDH ΔCBS

Ligand efficiency was calculated using the equation LE = (1.37 × pIC₅₀)/HA, where HA means heavy atom, i.e., a non-hydrogen atom.

The (S)-isomer of 31 was found to bind preferentially (Table 5), with the racemate 31 having approximately half the Mth IMPDH ΔCBS inhibition of the (S)-isomer 31. This observation accords with the results previously reported for other series of IMPDH inhibitors.(21,35)

X-ray Structure of Compound 31

From crystals soaked with compound 31, the 2F₀ – F_c difference map (σ = 3.0) revealed strong density for the inhibitor. The structure of compound 31 (Figure 4A) showed that it bound in a nearly identical manner to compounds 1 and 2 in the NAD binding pocking (Figure 4B), stacking with IMP, and maintaining the interactions with H270, N273, E442, and P45 and Y471 from the neighboring subunit. Compound 31 made additional interactions in the binding pocket, including polar interactions with D267 and N297.

Figure 4. X-ray crystal structure of compound 31 bound to Mth IMPDH ΔCBS. Protein–ligand interactions were analyzed using Arpeggio. (a) Interactions made by 31 in the X-ray crystal structure of the complex of IMPDH with IMP (orange). Ligand interactions are represented as dotted lines; hydrogen bonds are represented in red, polar interactions in orange, ionic interactions in yellow, and aromatic and π interactions in green dotted lines. (b) Structural alignment of the IMPDH crystal structures of 1 (lilac), 2 (yellow), and 31 (green).

Whole-Cell Activity against Mtb

The whole-cell activity of the most potent analogues 31, 33–39, and (S)-31 in vitro was determined against Mtb H37Rv (see Table S1, Supporting Information). No significant inhibition of bacterial growth was detected for any of the compounds (MIC₉₀ ≥ 50 μM) over the tested concentration range (0–100 μM). There are currently ongoing efforts to explain the lack of efficacy of the potent Mth IMPDH ΔCBS inhibitors described which could be caused by low membrane permeability, poor metabolic stability, and/or drug-efflux mechanisms.

Synthetic Chemistry

Compounds 20–29 and 32 were synthesized from 4-phenyl-1H-imidazole-2-thiol or 4-(4-bromophenyl)-1H-imidazole-2-thiol according to the sequence described in Scheme 1. Thioacetic derivatives 27 and 28 were prepared by treatment of 4-phenyl-1H-imidazole-2-thiol or 4-(4-bromophenyl)-1H-imidazole-2-thiol with 2-chloroacetic acid in the presence of NaOH followed by neutralization with hydrochloric acid.

Scheme 1. Synthesis of Thioacetamide and Thioacetic Derivatives 20–29 and 32^a

^aReagent and conditions: (a) 2-chloroacetic acid, NaOH, EtOH, 80 °C, 69–72%; (b) isopropylamine, HATU, DIPEA, EtOAc, rt, 73%; (c) chloroacetyl chloride, TEA, DCM, rt, 89–99%; (d) NaOH, MeOH, H₂O, 70 °C, 69–96%.

Thioacetamide 26 was synthesized by amide coupling between thioacetic derivative 28 and isopropylamine. Similarly, thioacetamides 20–25, 29, and 32 were prepared by cross-linking 4-phenyl-1H-imidazole-2-thiol or 4-(4-bromophenyl)-1H-imidazole-2-thiol with α-chloroacetamides 40–43, which were obtained by acylation of anilines with various substituents with chloroacetyl chloride.

The synthesis of urea 30 was achieved as shown in Scheme 2 by coupling amine 44 and N-(4-(1H-imidazol-4-yl)phenyl)-1H-imidazole-1-carboxamide, which was obtained from commercially available 4-(1H-imidazol-4-yl)aniline as a crude intermediated, in the presence of N,N-diisopropylethylamine. Amine 44 was synthesized from benzyl ((5-(4-bromophenyl)-1H-imidazol-2-yl)methyl)carbamate,(36) followed by the deprotection of the benzyloxycarbonyl group under acidic condition.

Scheme 2. Synthesis of Urea 30^a

^aReagent and conditions: (a) CDI, THF, rt, quant.; (b) HCl (4 M), dioxane, 100 °C, quant.; (c) DIPEA, DMF, rt, 26%.

Imidazole derivatives 31, 36–38, and 39 were prepared following the synthetic procedure outlined in Scheme 3. 2-Aminoimidazoles 50–54 were synthesized according to a published microwave-assisted protocol.(37) In brief, 2-aminoimidazoles 50–54 were prepared by reaction of the commercially available α-haloketones and N-acetylguanidine, followed by deacetylation (Scheme 3). Acid 59 was synthesized starting with imidazole 55, which was prepared by reaction of 2-bromo-4′-hydroxyacetophenone with formamide as reported previously.(38) The phenol 57 was synthesized by alkylation of imidazole 55, followed by deprotection of the methyl ether with BBr₃. Substituted phenol 57 was converted to the ether 58 upon treatment with methyl 2-bromopropionate in the presence of Cs₂CO₃. Enantiomerically pure phenyl ethers were synthesized by using Mitsunobu reaction conditions with ethyl d-lactate (Scheme 4). After the hydrolysis of the ester group, the resulting carboxylic acid 59 was treated with thionyl chloride to give the acid chloride 60, which was reacted with 2-aminoimidazoles 50–54 to afford imidazole derivatives 31, 36–38, and 39.

Scheme 3. Synthesis of 2-Acylaminoimidazole Derivatives 31, 36–38, and 39^a

^aReagent and conditions: (a) acetylguanidine, CH₃CN, μW, 100 °C, 71–88%; (b) (1) 20% H₂SO₄, MeOH:H₂O (1:1 v/v), μW, 100 °C; (2) aq Na₂CO₃, 79–95%; (c) formamide, 150 °C, 99%; (d) CH₃I, Cs₂CO₃, DMF, rt, 88%; (e) BBr₃ (1 M in DCM), −78 °C to rt, DCM, 96%; (f) Cs₂CO₃, methyl 2-bromopropionate, DMF, 60 °C, 92%; (g) NaOH, THF:H₂O (2:1 v/v), 80 °C, 65%; (h) SOCl₂, 80 °C, quant.; (i) TEA, DCM, 40 °C, 61–72%.

Scheme 4. Synthesis of the S-Enantiomer of 31^a

^aReagent and conditions: (a) (1) Ethyl d-lactate, PPh₃, DEAD, THF, rt; (2) NaOH, THF:H₂O (2:1 v/v), rt, 56%; (b) SOCl₂, 80 °C, quant.; (c) 50, TEA, DCM, 40 °C, 69%.

The syntheses of 2-acylaminooxazole 33 and amides 34–35 were achieved as shown in Scheme 5. 2-Acylaminooxazole derivative 33 was obtained by coupling the acid chloride derivative 60 with 2-aminooxazole derivative 61, which was prepared by reaction of 2,4′-dibromoacetophenone with urea. Similarly, amides 34–35 were prepared by coupling the corresponding anilines with the acid chloride derivative 60.

Scheme 5. Synthesis of 2-Acylaminooxazole Derivative 33 and Amides 34–35^a

^aReagent and conditions: (a) urea, CH₃CN, 80 °C, 78%; (b) TEA, DCM, rt (for 34 and 35) or 40 °C (for 33), 64–76%.

Conclusions

FBDD has emerged as a robust approach to identify small molecules that bind to a wide range of therapeutic targets. Fragment elaboration strategies have resulted in the development of a number of compounds that have progressed into clinical trials. Within the area of TB drug discovery, a number of HTS and phenotypic screens have been performed during the past decade. Although HTS identified a number of leads that show high potency in vitro, the translation to an in vivo effect has proven challenging.

This study illustrates the successful application of a fragment-based approach followed by fragment optimization to obtain nanomolar affinity ligands of IMPDH. A library of 960 fragments were screened against Mth IMPDH ΔCBS, and from the screen the phenylimidazole fragment hit 2 (IC₅₀ = 674 μM) was identified. Kinetic experiments showed that 2 was an uncompetitive inhibitor of Mth IMPDH ΔCBS with respect to NAD⁺ and IMP. Two molecules of the fragment 2 were shown to bind at the NAD binding site of the enzyme. The X-ray crystal structure also revealed that one molecule of fragment 2 makes π interactions with IMP and the other molecule sits closer to the opening of the active site, making polar interactions with N273 and E442 and π interactions with H270 and Y471′. This provides potential for further optimization of fragment 2. To explore better the possibilities given by fragment 2, fragment-linking and fragment-growing strategies were employed, resulting in low micromolar to nanomolar affinity compounds. Among them, compounds 31, 35, and 37– 39 were the most potent IMDPH inhibitors of the series described in this work with IC₅₀ values between 0.47 and 0.69 μM, which represent >1000-fold improvement in Mth IMPDH ΔCBS potency over the initial fragment hit. Compound 31 was shown to bind at the NAD binding site of the enzyme, and the X-ray crystal structure also revealed that it makes π interactions with IMP, maintaining the interactions with H270, N273, E442, and P45 and Y471 from the neighboring subunit. Moreover, compound 31 made additional interactions in the binding pocket, including polar interactions with D267 and N297. A comparison of this structure with the fragment 2 structure shows that the two molecules of 2 mimic the position of the larger inhibitor 31. This is the first example of utilizing the fragment-based approach specifically to identify new potent inhibitors of IMPDH. Further structural optimization to improve the cellular activity of these analogues is ongoing with the aim of developing novel classes of anti-TB agents.

Experimental Section

1. Chemistry

1.1. General Experimental Methods

Solvents were distilled prior to use and dried by standard methods. Unless otherwise stated, ¹H and ¹³C NMR spectra were obtained in CDCl₃, MeOD, or DMSO-d₆ solutions using either a Bruker 400 MHz AVANCE III HD Smart Probe, 400 MHz QNP cryoprobe, or 500 MHz DCH cryoprobe spectrometer. Chemical shifts (δ) are given in ppm relative to the residual solvent peak (CDCl₃: ¹H, δ = 7.26 ppm; ¹³C, δ = 77.16 ppm), and the coupling constants (J) are reported in hertz (Hz). Optical rotations were measured on a PerkinElmer Polarimeter 343 at 589 nm (Na D-line), and specific rotations are reported in 10^–1 deg cm² g^–1. Microwave reactions were performed using a Biotage Initiator system under reaction conditions as indicated for each individual reaction.

Reactions were monitored by TLC and LCMS to determine consumption of starting materials. Flash column chromatography was performed using an Isolera Spektra One/Four purification system and the appropriately sized Biotage SNAP column containing KP-silica gel (50 μm). Solvents are reported as volume/volume eluent mixture where applicable.

High resolution mass spectra (HRMS) were recorded using a Waters LCT Premier Time of Flight (TOF) mass spectrometer or a Micromass Quadrapole-Time of Flight (Q-TOF) spectrometer.

Liquid chromatography mass spectrometry (LCMS) was carried out using an Ultra Performance Liquid Chromatographic system (UPLC) Waters Acquity H-class coupled to a Waters SQ Mass Spectrometer detector. Samples were detected using a Waters Acquity TUV detector at 2 wavelengths (254 and 280 nm). Samples were run using an Acquity UPLC HSS column and a flow rate of 0.8 mL/min. The eluent consisted of 0.1% formic acid in water (A) and acetonitrile (B); gradient, from 95% A to 5% A over a period of 4 or 7 min.

All final compounds had a purity greater than 95% by LCMS analysis.

General Method A: Synthesis of Thioacetamides 20–25, 29, and 32

To a solution of the 2-chloroacetamide derivative (0.17 mmol) in MeOH (8 mL) was added 4-(4-bromophenyl)-1H-imidazole-2-thiol or 4-phenyl-1H-imidazole-2-thiol (0.17 mmol), followed by a solution of NaOH (0.67 mmol) in H₂O (2.5 mL). The reaction mixture was stirred at 70 °C for 3 h. After cooling to rt, the solvents were removed in vacuo and the resulting residue was taken up in 30 mL of EtOAc and washed with H₂O (15 mL). The aqueous layer was extracted with EtOAc (3 × 30 mL) and the combined organic layers were washed with brine and dried over MgSO₄. Filtration and evaporation afforded crude products, which were purified as indicated below.

General Method B: Synthesis of Thioacetic Derivatives 27 and 28

A solution of 4-phenyl-1H-imidazole-2-thiol or 4-(4-bromophenyl)-1H-imidazole-2-thiol (2.80 mmol) and NaOH (5.0 mmol) in EtOH (5 mL) was reflux for 1 h. After cooling to rt, a solution of 2-chloroacetic acid (2.80 mmol) in EtOH (2 mL) was added. The reaction was stirred at reflux for an additional 3 h and then cooled to 0 °C. The reaction mixture was diluted with cold water (5 mL) and acidified with 1 M HCl. The precipitated product was collected by filtration and washed with DCM (2 × 2 mL).

General Method C: Synthesis of Compounds 31, 33, and 36–39

A mixture of acid 59 (0.20 mmol) and SOCl₂ (2 mL) was heated at 80 °C for 2 h. The solvent was removed under reduced pressure to give the acid chloride 60 as a white solid. The resulting solid was immediately dissolved in anhydrous DCM, and the resulting solution was added slowly dropwise at 0 °C to a solution of the corresponding substituted 2-aminoimidazoles (0.20 mmol) and TEA (0.80 mmol) in anhydrous DCM (5 mL). The reaction mixture was stirred at 40 °C for 36 h and then diluted with DCM (20 mL) and washed with saturated aqueous NaHCO₃. The aqueous phase layer was then extracted with DCM (2 × 20 mL), and the combined organic layers were dried over MgSO₄ and filtered, and the solvent was removed under reduced pressure to afford a yellow oil, which was purified by flash chromatography, eluting with the solvent system specified.

General Method D: Synthesis of Amides 34–35

A mixture of acid 59 (0.20 mmol) and SOCl₂ (2 mL) was heated at 80 °C for 2 h. The solvent was removed under reduced pressure to give 60 as a white solid. The resulting solid was immediately dissolved in anhydrous DCM, and the resulting solution was added slowly dropwise at 0 °C to a solution of the corresponding aniline (0.20 mmol) and triethylamine (0.80 mmol) in anhydrous DCM (5 mL). The reaction mixture was stirred at rt for 4 h and then diluted with DCM (20 mL) and washed with saturated aqueous NaHCO₃. The aqueous phase layer was then extracted with DCM (2 × 20 mL), and the combined organic layers were dried over MgSO₄ and filtered, and the solvent was removed under reduced pressure to afford a yellow oil, which was purified by flash chromatography, eluting with the solvent system specified.

General Method E: Synthesis of α-Chloroacetamides 40–43

Et₃N (4.77 mmol) followed by a solution of chloroacetyl chloride (4.77 mmol) in DCM (3 mL) were added to a stirred solution of the corresponding aniline (4.38 mmol) in DCM (5 mL) at rt. The reaction mixture was stirred at rt for 2–4 h. The reaction was then diluted with DCM (20 mL) and washed with saturated aqueous NaHCO₃, 1 M HCl, and brine and dried over anhydrous MgSO₄, and the solvent was removed under reduced pressure. Compound 43 was purified by flash chromatography eluting with the solvent system specified, although other analogues were used in subsequent reactions without further purification.

General Method F: Synthesis of Substituted N-(1H-Imidazol-2-yl)acetamides 45–49

A mixture of the corresponding 2-bromoacetophenone derivative (0.38 mmol) and acetylguanidine (1.13 mmol) in anhydrous acetonitrile (3 mL) was heated at 100 °C using microwave irradiation for 15 min. The solvent was removed, and the residue was taken in H₂O (3 mL) and filtered, and the solid was washed with H₂O (2 mL × 2) and DCM (2 mL). The solid obtained was used in the next step without further purification.

General Method G: Synthesis of Substituted 2-Aminoimidazoles 50–54

To a solution of the corresponding substituted N-(1H-imidazol-2-yl)acetamides (0.31 mmol) in a 1:1 v/v mixture of MeOH and H₂O (2.4 mL) was added concentrated H₂SO₄ (0.6 mL), and the reaction mixture was heated at 100 °C under microwave irradiation for 15–30 min. The reaction mixture was concentrated, and the resulting residue was resuspended in H₂O (5 mL), and a saturated aqueous Na₂CO₃ was added until pH 8. The product was extracted into EtOAc (3 × 40 mL). The combined organic fractions were dried over MgSO₄, and the solvent was removed under reduced pressure. The resulting solid was used in the next reaction without further purification.

2-((4-(4-Bromophenyl)-1H-imidazol-2-yl)thio)-N-phenylacetamide (20)

Compound 40 (29 mg, 0.17 mmol) was reacted with 4-(4-bromophenyl)-1H-imidazole-2-thiol (43 mg, 0.17 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 20 (59 mg, 0.15 mmol, 89% yield) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.62 (br s, 2H), 7.56–7.39 (m, 5H), 7.36–7.17 (m, 2H), 7.13–7.00 (m, 1H), 3.82 (s, 2H) ppm. ¹³C NMR (100 MHz, MeOD) δ 169.6, 141.3, 139.6, 132.8, 129.8, 129.8, 127.5, 125.5, 121.4, 121.2, 121.2, 116.7, 39.6 ppm. LCMS (ESI−) m/z 388.0 [M – H]⁻, retention time 2.20 min (100%). HRMS (ESI+): m/z calculated for C₁₇H₁₅BrN₃OS [M + H]⁺: 388.0119. Found: 388.0121.

N-(Benzofuran-5-yl)-2-((4-(4-bromophenyl)-1H-imidazol-2-yl)thio)acetamide (21)

Compound 41 (36 mg, 0.17 mmol) was reacted with 4-(4-bromophenyl)-1H-imidazole-2-thiol (43 mg, 0.17 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 21 (70 mg, 0.16 mmol, 96% yield) as a white solid.¹H NMR (400 MHz, DMSO-d₆) δ 12.50 (br s, 1H), 10.35 (br s, 1H), 7.94 (dd, J = 10.6, 2.0 Hz, 2H), 7.73–7.64 (m, 2H), 7.52–7.44 (m, 4H), 7.34 (dd, J = 8.8, 2.2 Hz, 1H), 6.91 (dd, J = 2.2, 1.0 Hz, 1H), 3.99 (s, 2H) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ 166.5, 150.8, 146.7, 140.2, 139.9, 134.3, 133.6, 131.4, 127.4, 126.2, 119.0, 116.9, 115.7, 111.6, 111.3, 107.0, 37.9. LCMS (ESI−) m/z 428.0 [M – H]⁻, retention time 2.22 min (100%). HRMS (ESI+): m/z calculated for C₁₉H₁₅BrN₃O₂S [M + H]⁺: 428.0068. Found: 428.0065.

2-((4-(4-Bromophenyl)-1H-imidazol-2-yl)thio)-N-(4-iodophenyl)acetamide (22)

Compound 42 (50 mg, 0.17 mmol) was reacted with 4-(4-bromophenyl)-1H-imidazole-2-thiol (43 mg, 0.17 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 22 (60 mg, 0.11 mmol, 69% yield) as a white solid. ¹H NMR (500 MHz, MeOD) δ 7.63–7.58 (m, 4H), 7.56–7.45 (m, 3H), 7.38–7.31 (m, 2H), 3.81 (s, 2H) ppm.¹³C NMR (125 MHz, MeOD) δ 169.6, 139.6, 138.9, 132.7, 127.5, 127.1, 123.0, 116.6, 111.4, 88.1, 39.7 ppm. LCMS (ESI−) m/z 511.7 [M – H]⁻, retention time 2.30 min (100%). HRMS (ESI+): m/z calculated for C₁₇H₁₄BrIN₃OS [M + H]⁺: 513.9086. Found: 513.9087.

N-Phenyl-2-((4-phenyl-1H-imidazol-2-yl)thio)acetamide (23)

Compound 40 (42 mg, 0.25 mmol) was reacted with 4-phenyl-1H-imidazole-2-thiol (44 mg, 0.25 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 23 (61 mg, 0.20 mmol, 82% yield) as a white solid. ¹H NMR (500 MHz, MeOD) δ 7.71 (br s, 2H), 7.54–7.49 (m, 2H), 7.47 (br s, 1H), 7.35 (t, J = 7.4 Hz, 2H), 7.31–7.20 (m, 3H), 7.11–7.03 (m, 1H), 3.83 (s, 2H) ppm. ¹³C NMR (125 MHz, MeOD) δ 168.3, 142.7, 139.5, 138.5, 138.2, 128.4, 128.3, 126.7, 124.4, 124.1, 123.7, 119.8, 38.3 ppm. LCMS (ESI−) m/z 308.0 [M – H]⁻, retention time 1.82 min (95%). HRMS (ESI+): m/z calculated for C₁₇H₁₆N₃OS [M + H]⁺: 310.1014. Found: 310.1004.

N-(Benzofuran-5-yl)-2-((4-phenyl-1H-imidazol-2-yl)thio)acetamide (24)

Compound 41 (36 mg, 0.17 mmol) was reacted with 4-phenyl-1H-imidazole-2-thiol (30 mg, 0.17 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 24 (42 mg, 0.12 mmol, 70% yield) as a white solid. ¹H NMR (500 MHz, MeOD) δ 7.87 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.46 (br s, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.37–7.29 (m, 3H), 7.23 (t, J = 7.4 Hz, 1H), 6.76 (dd, J = 2.2, 0.9 Hz, 1H), 3.84 (s, 2H) ppm. ¹³C NMR (125 MHz, MeOD) δ 169.6, 153.5, 147.4, 140.9, 134.7, 129.8, 129.1, 128.1, 125.8, 118.9, 114.2, 112.1, 107.7, 39.7 ppm. LCMS (ESI−) m/z 348.0 [M – H]⁻, retention time 1.81 min (100%). HRMS (ESI+): m/z calculated for C₁₉H₁₆N₃O₂S [M + H]⁺: 350.0963. Found: 350.0971.

N-(4-Iodophenyl)-2-((4-phenyl-1H-imidazol-2-yl)thio)acetamide (25)

Compound 42 (74 mg, 0.25 mmol) was reacted with 4-phenyl-1H-imidazole-2-thiol (44 mg, 0.25 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 25 (70 mg, 0.16 mmol, 70% yield) as a white solid. ¹H NMR (500 MHz, MeOD) δ 7.66 (d, J = 7.7 Hz, 2H), 7.61–7.54 (m, 2H), 7.44 (br s, 1H), 7.38–7.31 (m, 4H), 7.23 (t, J = 6.7 Hz, 1H), 3.80 (s, 2H) ppm. ¹³C NMR (125 MHz, MeOD) δ 169.7, 140.8, 139.6, 138.9, 129.8, 128.2, 125.8, 123.0, 88.1, 39.8 ppm. LCMS (ESI−) m/z 433.9 [M – H]⁻, retention time 2.13 min (98%). HRMS (ESI+): m/z calculated for C₁₇H₁₅IN₃OS [M + H]⁺: 435.9981. Found: 435.9989.

N-Isopropyl-2-((4-phenyl-1H-imidazol-2-yl)thio)acetamide (26)

A solution of propan-2-amine (22 μL, 0.25 mmol), 2-((4-phenyl-1H-imidazol-2-yl)thio)acetic acid (60 mg, 0.25 mmol), HATU (194 mg, 0.50 mmol), and DIPEA (90 μL, 0.50 mmol) in EtOAc (10 mL) was stirred for 5 h at rt. The reaction mixture was diluted with EtOAc (40 mL) and washed with saturated aqueous NaHCO₃ (20 mL), water (20 mL), and brine (20 mL). The organic layer was dried over MgSO₄ and filtered. The solvent was removed under reduced pressure to give an oil residue, which was purified by flash chromatography (5–100% v/v EtOAc in petroleum ether) to give the desired product as a white solid (50 mg, 0.18 mmol, 73% yield).¹H NMR (500 MHz, MeOD) δ 7.37 (d, J = 7.8 Hz, 2H), 7.14 (s, 1H), 7.05 (t, J = 7.7 Hz, 2H), 6.93 (t, J = 7.4 Hz, 1H), 3.62 (dt, J = 13.1, 6.4 Hz, 1H), 3.00 (s, 2H), 0.78 (d, J = 6.6 Hz, 6H) ppm.¹³C NMR (125 MHz, MeOD) δ 170.3, 141.1, 140.8, 133.7, 129.7, 128.1, 125.7, 119.7, 43.0, 38.8, 22.4 ppm. LCMS (ESI−) m/z 273.9 [M – H]⁻, retention time 1.52 min (100%). HRMS (ESI+): m/z calculated for C₁₄H₁₈N₃OS [M + H]⁺: 276.1171. Found: 276.1172.

2-((4-(4-Bromophenyl)-1H-imidazol-2-yl)thio)acetic Acid (27)

Following general procedure B, from 4-(4-bromophenyl)-1H-imidazole-2-thiol (250 mg, 0.98 mmol) was obtained 27 (220 mg, 0.70 mmol, 72% yield) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.65–7.56 (m, 2H), 7.57–7.39 (m, 3H), 3.80 (s, 2H) ppm. ¹³C NMR (100 MHz, MeOD) δ 172.9, 141.9, 140.2, 132.9, 132.6, 127.5, 121.8, 118.9, 37.4 ppm. LCMS (ESI−) m/z 311.0 [M – H]⁻, retention time 1.59 min (98%). HRMS (ESI+): m/z calculated for C₁₁H₁₀BrN₂O₂S [M + H]⁺: 312.9646. Found: 312.9654.

2-((4-Phenyl-1H-imidazol-2-yl)thio)acetic Acid (28)

Following general procedure B, from 4-phenyl-1H-imidazole-2-thiol (500 mg, 2.80 mmol) was obtained 28 (450 mg, 1.92 mmol, 69% yield) as a white solid. ¹H NMR (500 MHz, MeOD) δ 7.68 (d, J = 7.2 Hz, 2H), 7.59 (s, 1H), 7.41 (t, J = 7.7 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 3.86 (s, 2H) ppm. ¹³C NMR (125 MHz, MeOD) δ 172.7, 141.7, 139.6, 131.6, 130.0, 129.1, 126.0, 118.9, 37.5 ppm. LCMS (ESI+) m/z 235.1 [M + H]⁺, retention time 1.22 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₁₁N₂O₂S [M + H]⁺: 235.0541. Found: 235.0536.

N-(4-(1H-Imidazol-4-yl)phenyl)-2-((4-(4-bromophenyl)-1H-imidazol-2-yl)thio)acetamide (29)

Compound 43 (40 mg, 0.17 mmol) was reacted with 4-(4-bromophenyl)-1H-imidazole-2-thiol (43 mg, 0.17 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 29 (51 mg, 0.12 mmol, 69% yield) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.72 (d, J = 1.1 Hz, 1H), 7.66 (d, J = 8.5 Hz, 4H), 7.59–7.47 (m, 5H), 7.40 (br s, 1H), 3.86 (s, 2H) ppm. ¹³C NMR (125 MHz, MeOD) δ 169.4, 141.2, 138.4, 137.0, 132.8, 127.5, 126.2, 121.4, 116.7, 111.4, 39.7 ppm. LCMS (ESI−) m/z 451.9 [M – H]⁻, retention time 1.65 min (100%). HRMS (ESI+): m/z calculated for C₂₀H₁₇BrN₅OS [M + H]⁺: 454.0337. Found: 454.0334.

1-(4-(1H-Imidazol-4-yl)phenyl)-3-((5-(4-bromophenyl)-1H-imidazol-2-yl)methyl)urea (30)

To a solution of 4-(1H-imidazol-4-yl)aniline (50 mg, 0.31 mmol) in THF (5 mL) was added 1,1-carbonyldiimidazole (76 mg, 0.47 mmol). The mixture was stirred at rt overnight, and then it was filtered. The filter was washed with THF (2 × 3 mL) and the resulting solid (30 mg, 0.12 mmol) was dissolved in DMF (3 mL), and 44 (42 mg, 0.12 mmol) and N,N-diisopropylethylamine (74 μL, 0.48 mmol) were added. The reaction mixture was stirred at rt for 14 h, and then it was poured into water, extracted with EtOAc, dried over Na₂SO₄, and concentrated. After cooling to 0 °C, a 1:5 v/v mixture of MeOH and DCM (12 mL) was added and the suspended solid was collected by filtration and dried at vacuum to yield 30 (35 mg, 0.08 mmol, 26% yield) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.03 (br s, 2H), 8.64 (br s, 1H), 7.73–7.68 (m, 2H), 7.64–7.59 (m, 2H), 7.57 (d, J = 1.9 Hz, 1H), 7.52–7.47 (m, 2H), 7.47–7.39 (m, 2H), 7.39–7.32 (m, 2H), 6.58 (br s, 1H), 4.33 (d, J = 5.5 Hz, 2H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 155.5, 146.9, 140.6, 138.9, 138.9, 135.9, 134.6, 131.7, 128.8, 126.6, 125.1, 125.0, 119.0, 118.4, 118.2, 113.7, 111.7, 37.6 ppm. LCMS (ESI−) m/z 437.0 [M – H]⁻, retention time 5.07 min (97%). HRMS (ESI+): m/z calculated for C₂₀H₁₈BrN₆O [M + H]⁺: 437.0725. Found: 437.0725.

N-(4-(4-Bromophenyl)-1H-imidazol-2-yl)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanamide (31)

4-(4-Bromophenyl)-1H-imidazol-2-amine 50 (48 mg, 0.20 mmol) was reacted with the acid chloride 60 (53 mg, 0.20 mmol) and Et₃N (112 μL, 0.80 mmol) according to general method C. The crude product was purified by flash chromatography (0–20% v/v MeOH in DCM) to give compound 31 as a white solid (64 mg, 0.14 mmol, 67% yield). ¹H NMR (500 MHz, CDCl₃) δ 10.79 (br s, 1H), 9.49 (br s, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.58–7.41 (m, 5H), 7.18–6.97 (m, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.86 (d, J = 6.8 Hz, 1H), 3.71 (s, 3H), 1.65 (d, J = 6.7 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃) δ 171.5, 155.2, 141.8, 141.2, 138.0, 137.2, 131.7, 129.1, 127.6, 126.3, 120.5, 115.8, 115.6, 115.3, 108.1, 74.6, 33.5, 18.5 ppm. LCMS (ESI+) m/z 465.9 [M + H]⁺, retention time 2.73 min (95%). HRMS (ESI+): m/z calculated for C₂₂H₂₁BrN₅O₂ [M + H]⁺: 466.0879. Found: 466.0874. ((S)-31): 69% yield. [α]_D²⁵ −40.2 (c 1.0, CHCl₃). LCMS (ESI+) m/z 466.0 [M + H]⁺, retention time 2.70 min (95%). HRMS (ESI+): m/z calculated for C₂₂H₂₁BrN₅O₂ [M + H]⁺: 466.0879. Found: 466.0879.

N-(4-(1H-Imidazol-4-yl)phenyl)-2-((4-phenyl-1H-imidazol-2-yl)thio)acetamide (32)

Compound 43 (40 mg, 0.17 mmol) was reacted with 4-(4-bromophenyl)-1H-imidazole-2-thiol (30 mg, 0.17 mmol) according to general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded 32 (41 mg, 0.11 mmol, 65% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.46 (br s, 2H), 10.42 (br s, 1H), 7.76 (br s, 2H), 7.68 (d, J = 8.2 Hz, 4H), 7.56 (d, J = 8.5 Hz, 2H), 7.52 (s, 1H), 7.34 (br s, 2H), 7.19 (br s, 1H), 3.99 (s, 2H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 167.0, 141.7, 139.9, 137.7, 136.2, 134.6, 128.9, 126.7, 125.2, 124.6, 119.7, 115.4, 39.5 ppm. LCMS (ESI+) m/z 376.1 [M + H]⁺, retention time 0.88 min (100%). HRMS (ESI+): m/z calculated for C₂₀H₁₈N₅OS [M + H]⁺: 376.1232. Found: 376.1241.

N-(4-(4-Bromophenyl)oxazol-2-yl)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanamide (33)

4-(4-Bromophenyl)oxazol-2-amine 61 (39 mg, 0.16 mmol) was reacted with the acid chloride 60 (43 mg, 0.16 mmol) and Et₃N (90 μL, 0.64 mmol) according to general method C. The crude product was purified by flash chromatography (0–15% v/v MeOH in DCM) to give compound 33 as a white solid (49 mg, 0.10 mmol, 64% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.13 (br s, 1H), 7.81–7.66 (m, 3H), 7.63–7.50 (m, 4H), 7.47 (s, 1H), 7.13 (d, J = 1.3 Hz, 1H), 7.03–6.94 (m, 2H), 4.90 (q, J = 6.6 Hz, 1H), 3.74 (s, 3H), 1.70 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 155.1, 141.7, 138.0, 131.9, 131.7, 130.3, 129.4, 129.3, 127.8, 127.0, 126.7, 126.4, 122.2, 116.0, 115.4, 75.1, 33.5, 18.4 ppm. LCMS (ESI+) m/z 468.9 [M + H]⁺, retention time 2.86 min (95%). HRMS (ESI+): m/z calculated for C₂₂H₂₀BrN₄O₃ [M + H]⁺: 467.0719. Found: 467.0711.

2-(4-(1-Methyl-1H-imidazol-4-yl)phenoxy)-N-phenylpropanamide (34)

Aniline (28 μL, 0.30 mmol) was reacted with the acid chloride 60 (53 mg, 0.20 mmol) and Et₃N (112 μL, 0.80 mmol) according to general method D. The crude product was purified by flash chromatography (0–20% v/v MeOH in DCM) to give compound 34 as a white solid (48 mg, 0.14 mmol, 74% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.25 (br s, 1H), 7.79–7.69 (m, 2H), 7.60–7.52 (m, 2H), 7.47 (d, J = 1.3 Hz, 1H), 7.35 (dd, J = 8.5, 7.3 Hz, 2H), 7.19–7.06 (m, 2H), 7.05–6.97 (m, 2H), 4.82 (q, J = 6.7 Hz, 1H), 3.73 (s, 3H), 1.69 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 155.6, 141.8, 137.9, 137.0, 129.0, 129.0, 126.3, 124.7, 120.0, 116.0, 115.3, 75.6, 33.5, 18.7 ppm. LCMS (ESI+) m/z 322.2 [M + H]⁺, retention time 1.86 min (97%). HRMS (ESI+): m/z calculated for C₁₉H₂₀N₃O₂ [M + H]⁺: 322.1556. Found: 322.1552.

N-(4-Iodophenyl)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanamide (35)

4-Iodoaniline (65 mg, 0.30 mmol) was reacted with the acid chloride 60 (53 mg, 0.20 mmol) and Et₃N (112 μL, 0.80 mmol) according to the general method D. The crude product was purified by flash chromatography (0–20% v/v MeOH in DCM) to give compound 35 as a pink solid (62 mg, 0.14 mmol, 68% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (br s, 1H), 7.77–7.69 (m, 2H), 7.69–7.57 (m, 2H), 7.47 (d, J = 1.4 Hz, 1H), 7.41–7.31 (m, 2H), 7.11 (d, J = 1.3 Hz, 1H), 7.03–6.91 (m, 2H), 4.80 (q, J = 6.7 Hz, 1H), 3.73 (s, 3H), 1.67 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 155.5, 141.7, 137.9, 136.9, 129.0, 126.3, 126.0, 121.8, 116.0, 115.3, 87.9, 75.6, 33.5, 18.6 ppm. LCMS (ESI+) m/z 448.2 [M + H]⁺, retention time 2.47 min (96%). HRMS (ESI+): m/z calculated for C₁₉H₁₉IN₃O₂ [M + H]⁺: 448.0522. Found: 448.0517.

2-(4-(1-Methyl-1H-imidazol-4-yl)phenoxy)-N-(4-phenyl-1H-imidazol-2-yl)propanamide (36)

4-Phenyl-1H-imidazol-2-amine 51 (33 mg, 0.20 mmol) was reacted with the acid chloride 60 (53 mg, 0.20 mmol) and Et₃N (112 μL, 0.80 mmol) according to general method C. The crude product was purified by flash chromatography (0–15% v/v MeOH in DCM) to give compound 36 as a brown solid (57 mg, 0.15 mmol, 72% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.84 (br s, 1H), 9.51 (br s, 1H), 7.78–7.56 (m, 4H), 7.46 (d, J = 1.2 Hz, 1H), 7.39 (t, J = 7.7 Hz, 2H), 7.22–7.26 (m, 1H), 7.14 (s, 1H), 7.11 (d, J = 1.4 Hz, 1H), 7.02–6.89 (m, 2H), 4.89 (q, J = 6.8 Hz, 1H), 3.73 (s, 3H), 1.68 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃) δ 171.3, 155.2, 141.8, 137.9, 129.1, 128.7, 126.9, 126.3, 124.6, 115.9, 115.3, 107.8, 74.5, 33.5, 18.5 ppm. LCMS (ESI+) m/z 388.1 [M + H]⁺, retention time 4.44 min (100%). HRMS (ESI+): m/z calculated for C₂₂H₂₂N₅O₂ [M + H]⁺: 388.1773. Found: 388.1767.

2-(4-(1-Methyl-1H-imidazol-4-yl)phenoxy)-N-(4-(4-morpholinophenyl)-1H-imidazol-2-yl)propanamide (37)

4-(4-Morpholinophenyl)-1H-imidazol-2-amine 52 (40 mg, 0.16 mmol) was reacted with the acid chloride 60 (43 mg, 0.16 mmol) and Et₃N (90 μL, 0.65 mmol) according to general method C. The crude product was purified by flash chromatography (3–15% v/v MeOH in DCM) to give compound 37 as a brown solid (51 mg, 0.11 mmol, 67% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.74 (br s, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.54 (br s, 2H), 7.47 (d, J = 1.4 Hz, 1H), 7.11 (d, J = 1.4 Hz, 1H), 7.03–6.88 (m, 5H), 4.90 (q, J = 6.7 Hz, 1H), 4.06–3.83 (m, 4H), 3.73 (s, 3H), 3.27–3.01 (m, 4H), 1.69 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃) δ 171.5, 155.3, 150.3, 141.8, 141.0, 137.9, 137.8, 128.9, 126.3, 125.5, 115.8, 115.3, 74.4, 66.9, 49.3, 33.6, 18.7 ppm. LCMS (ESI+) m/z 473.2 [M + H]⁺, retention time 2.22 min (100%). HRMS (ESI+): m/z calculated for C₂₆H₂₉N₆O₃ [M + H]⁺: 473.2301. Found: 473.2297.

N-(4-(4-Methoxyphenyl)-1H-imidazol-2-yl)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanamide (38)

4-(4-Methoxyphenyl)-1H-imidazol-2-amine 53 (30 mg, 0.16 mmol) was reacted with the acid chloride 60 (43 mg, 0.16 mmol) and Et₃N (90 μL, 0.65 mmol) according to general method C. The crude product was purified by flash chromatography (1–20% v/v MeOH in DCM) to give compound 38 as a yellow solid (45 mg, 0.11 mmol, 67% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.73 (br s, 1H), 9.62 (br s, 1H), 7.78–7.65 (m, 2H), 7.57 (br s, 2H), 7.46 (d, J = 1.4 Hz, 1H), 7.10 (d, J = 1.3 Hz, 1H), 7.02 (s, 1H), 6.95 (dd, J = 10.5, 8.8 Hz, 4H), 4.88 (q, J = 6.8 Hz, 1H), 3.84 (s, 3H), 3.73 (s, 3H), 1.68 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃) δ 171.4, 158.7, 155.3, 141.8, 140.8, 137.9, 129.1, 126.3, 125.8, 115.8, 115.3, 114.2, 74.7, 55.3, 33.4, 18.4 ppm. LCMS (ESI+) m/z 418.3 [M + H]⁺, retention time 4.69 min (100%). HRMS (ESI+): m/z calculated for C₂₃H₂₄N₅O₃ [M + H]⁺: 418.1879. Found: 418.1875.

N-(4-(4-Iodophenyl)-1H-imidazol-2-yl)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanamide (39)

4-(4-Iodophenyl)-1H-imidazol-2-amine 54 (46 mg, 0.16 mmol) was reacted with the acid chloride 60 (43 mg, 0.16 mmol) and Et₃N (90 μL, 0.65 mmol) according to general method C. The crude product was purified by flash chromatography (1–20% v/v MeOH in DCM) to give compound 39 as a yellow solid (50 mg, 0.10 mmol, 61% yield). ¹H NMR (500 MHz, CDCl₃) δ 10.75 (br s, 1H), 9.42 (br s, 1H), 7.69 (t, J = 8.3 Hz, 4H), 7.53–7.33 (m, 3H), 7.10 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 4.88 (q, J = 6.7 Hz, 1H), 3.71 (s, 3H), 1.66 (d, J = 6.7 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃) δ 171.5, 155.2, 141.7, 138.0, 137.7, 129.1, 126.5, 126.3, 115.8, 115.3, 108.1, 91.8, 74.5, 33.5, 18.5 ppm. LCMS (ESI+) m/z 514.2 [M + H]⁺, retention time 4.61 min (95%). HRMS (ESI+): m/z calculated for C₂₂H₂₁IN₅O₂ [M + H]⁺: 514.0740. Found: 514.0740.

2-Chloro-N-phenylacetamide (40)

Aniline (400 μL, 4.38 mmol) was reacted with chloroacetyl chloride (380 μL, 4.77 mmol) and Et₃N (670 μL, 4.77 mmol) according to general method E, and used in subsequent reactions without further purification. Compound 40 was obtained (720 mg, 4.24 mmol, 96% yield) as a green solid and was used without further purification. ¹H NMR (400 MHz, CDCl₃) δ 8.27 (br s, 1H), 7.58 (d, J = 7.7 Hz, 2H), 7.39 (t, J = 7.9 Hz, 2H), 7.20 (t, J = 7.4 Hz, 1H), 4.22 (s, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 163.9, 136.8, 129.3, 125.4, 120.2, 43.0 ppm. LCMS (ESI+) m/z 170.1 [M + H]⁺, retention time 1.58 min (100%). HRMS (ESI+): m/z calculated for C₈H₉ClNO [M + H]⁺: 170.0373. Found: 170.0366. NMR data is in accordance with literature values.(39)

N-(Benzofuran-5-yl)-2-chloroacetamide (41)

Benzofuran-5-amine (140 mg, 1.05 mmol) was reacted with chloroacetyl chloride (93 μL, 1.16 mmol) and Et₃N (162 μL, 1.16 mmol) according to general method E, and it was used in subsequent reactions without further purification. Compound 41 was obtained (219 mg, 1.04 mmol, 99% yield) as a green solid and was used without further purification. ¹H NMR (400 MHz, CDCl₃) δ 8.41 (br s, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.64 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 8.8, 0.8 Hz, 1H), 7.32 (dd, J = 8.8, 2.2 Hz, 1H), 6.76 (dd, J = 2.2, 0.9 Hz, 1H), 4.23 (s, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 163.9, 152.4, 146.1, 131.9, 127.9, 117.7, 113.3, 111.6, 106.8, 43.0 ppm. LCMS (ESI+) m/z 210.1 [M + H]⁺, retention time 1.71 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₉ClNO₂ [M + H]⁺: 210.0322. Found: 210.0319.

2-Chloro-N-(4-iodophenyl)acetamide (42)

4-Iodoaniline (500 mg, 2.28 mmol) was reacted with chloroacetyl chloride (200 μL, 2.51 mmol) and Et₃N (350 μL, 2.51 mmol) according to general method E. The crude product was purified by flash chromatography (10–100% v/v EtOAc in petroleum ether) to give compound 42 as a brown solid (600 mg, 2.03 mmol, 89% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.32–8.11 (br s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 4.20 (s, 2H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 163.8, 138.1, 136.4, 121.8, 88.7, 42.9 ppm. LCMS (ESI−) m/z 293.7 [M – H]⁻, retention time 2.38 min (100%). HRMS (ESI−): m/z calculated for C₈H₆ClINO [M – H]⁻: 293.9183. Found: 293.9187. NMR data is in accordance with literature values.(40)

N-(4-(1H-Imidazol-4-yl)phenyl)-2-chloroacetamide (43)

4-(1H-Imidazol-4-yl)aniline (200 mg, 1.25 mmol) was reacted with chloroacetyl chloride (109 μL, 1.38 mmol) and Et₃N (192 μL, 1.38 mmol) according to general method E. The crude product was purified by flash chromatography (3–20% v/v MeOH in DCM) to give compound 43 as a yellow solid (264 mg, 1.12 mmol, 89% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 10.43 (br s, 1H), 9.83 (br s, 1H), 7.98 (br s, 1H), 7.77–7.65 (m, 2H), 7.70–7.47 (m, 3H), 4.27 (s, 2H) ppm. LCMS (ESI+) m/z 236.2 [M + H]⁺, retention time 1.15 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₁₀ClN₃O [M + H]⁺: 236.0591. Found: 236.0590.

(5-(4-Bromophenyl)-1H-imidazol-2-yl)methanamine Hydrochloride (44)

4 M HCl (2 mL) was added slowly to a stirred solution of benzyl ((5-(4-bromophenyl)-1H-imidazol-2-yl)methyl)carbamate(36) (105 mg, 0.26 mmol) in dioxane (1 mL). The mixture was stirred at 100 °C for 4 h, and then the solvents were removed under reduced pressure to give the desired product as a white solid (86 mg, 0.26 mmol, 100% yield). ¹H NMR (500 MHz, MeOD) δ 8.00 (s, 1H), 7.75–7.67 (m, 4H), 4.55 (s, 2H) ppm. ¹³C NMR (125 MHz, MeOD) δ 140.8, 136.0, 133.7, 128.4, 127.6, 124.7, 117.8, 111.4, 34.8 ppm. LCMS (ESI−) m/z 250.1 [M – H]⁻, retention time 2.13 min (100%). HRMS (ESI−): m/z calculated for C₁₀H₉BrN₃ [M + H]⁺: 249.9980. Found: 249.9987.

N-(4-(4-Bromophenyl)-1H-imidazol-2-yl)acetamide (45)

Following general method F, from 2-bromo-1-(4-bromophenyl)ethanone (104 mg, 0.38 mmol) was obtained 45 (88 mg, 0.31 mmol, 84% yield) as a green solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.66 (br s, 1H), 11.20 (br s, 1H), 7.72–7.57 (m, 2H), 7.54–7.42 (m, 2H), 7.30 (d, J = 1.6 Hz, 1H), 2.05 (s, 3H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 169.0, 141.9, 135.5, 134.4, 131.7, 126.4, 119.0, 110.4, 23.3 ppm. LCMS (ESI+) m/z 282.1 [M + H]⁺, retention time 2.13 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₁₁BrN₃O [M + H]⁺: 280.0085. Found: 280.0080. NMR data is in accordance with literature values.(37)

N-(4-Phenyl-1H-imidazol-2-yl)acetamide (46)

Following general method F, from 2-bromo-1-phenylethan-1-one (100 mg, 0.50 mmol) was obtained 46 (79 mg, 0.39 mmol, 78% yield) as a green solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.59 (br s, 1H), 11.24 (br s, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.25 (br s, 1H), 7.16 (t, J = 7.4 Hz, 1H), 2.07 (s, 3H) ppm.¹³C NMR (100 MHz, DMSO-d₆) δ 169.0, 141.7, 136.6, 128.9, 126.3, 124.4, 109.6, 23.3 ppm. LCMS (ESI−) m/z 200.0 [M – H]⁻, retention time 1.44 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₁₂N₃O [M + H]⁺: 202.0980. Found: 202.0977. NMR data is in accordance with literature values.(37)

N-(4-(4-Morpholinophenyl)-1H-imidazol-2-yl)acetamide (47)

Following general method F, from 2-bromo-1-(4-(piperidin-1-yl)phenyl)ethan-1-one (106 mg, 0.38 mmol) was obtained 47 (90 mg, 0.31 mmol, 83% yield) as a brown solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.46 (br s, 1H), 11.17 (br s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.06 (br s, 1H), 6.89 (d, J = 8.5 Hz, 2H), 3.93–3.53 (m, 4H), 3.07 (t, J = 4.8 Hz, 4H), 2.04 (s, 3H) ppm.¹³C NMR (125 MHz, DMSO-d₆) δ 168.8, 149.8, 141.4, 136.8, 125.2, 115.6, 113.5, 107.9, 66.6, 49.0, 23.2 ppm. LCMS (ESI−) m/z 285.1 [M – H]⁻, retention time 1.60 min (95%). HRMS (ESI+): m/z calculated for C₁₅H₁₉N₄O₂ [M + H]⁺: 287.1508. Found: 287.1505.

N-(4-(4-Methoxyphenyl)-1H-imidazol-2-yl)acetamide (48)

Following general method F, from 2-bromo-1-(4-methoxyphenyl)ethan-1-one (150 mg, 0.66 mmol) was obtained 48 (110 mg, 0.47 mmol, 71% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.52 (br s, 1H), 11.20 (br s, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.11 (s, 1H), 6.89 (d, J = 8.4 Hz, 2H), 3.75 (s, 3H), 2.06 (s, 3H) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ 168.5, 157.8, 141.2, 136.1, 127.6, 125.3, 113.9, 107.9, 55.1, 22.9 ppm. LCMS (ESI−) m/z 230.1 [M – H]⁻, retention time 1.52 min (96%). HRMS (ESI+): m/z calculated for C₁₂H₁₄N₃O₂ [M + H]⁺: 232.1086. Found: 232.1082. NMR data is in accordance with literature values.(37)

N-(4-(4-Iodophenyl)-1H-imidazol-2-yl)acetamide (49)

Following general method F, from 2-bromo-1-(4-iodophenyl)ethan-1-one (214 mg, 0.66 mmol) was obtained 49 (190 mg, 0.58 mmol, 88% yield) as a green solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.66 (br s, 1H), 11.21 (br s, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.30 (s, 1H), 2.05 (s, 3H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 168.5, 141.4, 137.1, 135.1, 134.3, 126.2, 109.9, 90.9, 22.8. LCMS (ESI−) m/z 326.0 [M – H]⁻, retention time 2.43 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₁₁IN₃O [M + H]⁺: 327.9947. Found: 327.9945.

4-(4-Bromophenyl)-1H-imidazol-2-amine (50)

Following general method G, from N-(4-(4-bromophenyl)-1H-imidazol-2-yl)acetamide 45 (88 mg, 0.31 mmol) was obtained 50 (70 mg, 0.29 mmol, 95% yield) as a brown solid.¹H NMR (500 MHz, DMSO-d₆) δ 7.52 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 7.04 (s, 1H), 5.40 (br s, 2H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 150.4, 134.0, 131.2, 126.0, 125.4, 117.5, 110.5 ppm. LCMS (ESI+) m/z 240.1 [M + H]⁺, retention time 1.50 min (97%). HRMS (ESI+): m/z calculated for C₉H₉BrN₃ [M + H]⁺: 237.9980. Found: 237.9980. NMR data is in accordance with literature values.(37)

4-Phenyl-1H-imidazol-2-amine (51)

Following general method G, from N-(4-phenyl-1H-imidazol-2-yl)acetamide 46 (70 mg, 0.35 mmol) was obtained 51 (47 mg, 0.29 mmol, 84% yield) as a red solid. ¹H NMR (400 MHz, MeOD) δ 7.62–7.46 (m, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.16 (td, J = 7.5, 1.2 Hz, 1H), 6.91 (s, 1H) ppm.¹³C NMR (125 MHz, MeOD) δ 150.3, 132.9, 128.4, 128.1, 125.6, 123.5, 111.2 ppm. LCMS (ESI+) m/z 160.1 [M + H]⁺, retention time 1.45 min (100%). HRMS (ESI+): m/z calculated for C₉H₁₀BrN₃ [M + H]⁺: 160.0875. Found: 160.0873. NMR data is in accordance with literature values.(37)

4-(4-Morpholinophenyl)-1H-imidazol-2-amine (52)

Following general method G, from N-(4-(4-morpholinophenyl)-1H-imidazol-2-yl)acetamide 47 (85 mg, 0.30 mmol) was obtained 52 (60 mg, 0.24 mmol, 82% yield) as a brown solid. ¹H NMR (400 MHz, MeOD) δ 7.47 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.78 (s, 1H), 3.91–3.64 (m, 4H), 3.19–3.02 (m, 4H) ppm. ¹³C NMR (125 MHz, MeOD) δ 149.9, 149.8, 129.3, 125.0, 124.5, 115.8, 113.0, 66.6, 49.4 ppm. LCMS (ESI+) m/z 245.1 [M + H]⁺, retention time 1.50 min (100%). HRMS (ESI+): m/z calculated for C₁₃H₁₇N₄O [M + H]⁺: 245.1402. Found: 245.1390.

4-(4-Methoxyphenyl)-1H-imidazol-2-amine (53)

Following general method G, from N-(4-(4-methoxyphenyl)-1H-imidazol-2-yl)acetamide 48 (83 mg, 0.36 mmol) was obtained 53 (65 mg, 0.34 mmol, 95% yield) as a brown solid. ¹H NMR (400 MHz, MeOD) δ 7.54–7.39 (m, 2H), 7.04–6.85 (m, 2H), 6.78 (s, 1H), 3.81 (s, 3H) ppm. ¹³C NMR (100 MHz, MeOD) δ 158.3, 150.0, 125.9, 125.0, 124.8, 113.6, 109.8, 54.3 ppm. LCMS (ESI+) m/z 190.2 [M + H]⁺, retention time 1.61 min (100%). HRMS (ESI+): m/z calculated for C₁₀H₁₂N₃O [M + H]⁺: 190.0980. Found: 190.0979. NMR data is in accordance with literature values.(37)

4-(4-Iodophenyl)-1H-imidazol-2-amine (54)

Following general method G, from N-(4-(4-iodophenyl)-1H-imidazol-2-yl)acetamide 49 (150 mg, 0.46 mmol) was obtained 54 (103 mg, 0.36 mmol, 79% yield) as a red solid. ¹H NMR (500 MHz, DMSO-d₆) δ 7.58 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.03 (s, 1H), 5.35 (br s, 2H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 150.8, 137.4, 134.6, 133.4, 126.0, 111.1, 90.2 ppm. LCMS (ESI+) m/z 286.1 [M + H]⁺, retention time 2.09 min (100%). HRMS (ESI+): m/z calculated for C₉H₉IN₃ [M + H]⁺: 285.9841. Found: 285.9846.

4-(1H-Imidazol-4-yl)phenol (55)

2-Bromo-1-(4-hydroxyphenyl)ethan-1-one (1.0 g, 4.67 mmol) was dissolved in formamide (5 mL), and the reaction mixture was heated at 150 °C for 24 h. After cooling to rt, the resulting mixture was diluted with EtOAc (40 mL) and washed with saturated aqueous NaHCO₃ (40 mL). The aqueous layer was extracted with EtOAc (2 × 40 mL), and the combined organic layers were dried over MgSO₄, filtered, and concentrated to give 55 (0.75 g, 4.67 mmol, 99% yield) as a brown oil. ¹H NMR (400 MHz, MeOD) δ 8.17 (br s, 1H), 7.68 (d, J = 1.2 Hz, 1H), 7.64–7.44 (m, 2H), 7.25 (d, J = 1.1 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H) ppm.¹³C NMR (100 MHz, MeOD) δ 156.3, 135.1, 125.8, 124.3, 115.1 ppm. LCMS (ESI+) m/z 161.2 [M + H]⁺, retention time 0.54 min (100%). HRMS (ESI+): m/z calculated for C₉H₉N₂O [M + H]⁺: 161.0715. Found: 161.0720. NMR data is in accordance with literature values.(38)

4-(4-Methoxyphenyl)-1-methyl-1H-imidazole (56)

To a solution of 55 (2.77 g, 17 mmol) in DMF (20 mL) containing cesium carbonate (12.4 g, 38 mmol) at rt was added iodomethane (2.4 mL, 38 mmol). The reaction mixture was stirred at rt for 8 h. The reaction mixture was then cooled to 0 °C and quenched with H₂O (70 mL) and extracted with EtOAc (3 × 60 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over MgSO₄, filtered, and concentrated to give 56 (2.8 g, 15 mmol, 88% yield) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 1.3 Hz, 1H), 7.10 (d, J = 1.4 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 3.85 (s, 3H), 3.73 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 158.6, 142.4, 137.8, 127.2, 126.0, 114.8, 114.0, 55.3, 33.5. LCMS (ESI+) m/z 189.2 [M + H]⁺, retention time 1.38 min (100%). HRMS (ESI+): m/z calculated for C₁₁H₁₃N₂O [M + H]⁺: 189.1028. Found: 189.1031. NMR data is in accordance with literature values.(41)

4-(1-Methyl-1H-imidazol-4-yl)phenol (57)

A solution of 4-(4-methoxyphenyl)-1-methyl-1H-imidazole 56 (1.21 g, 6.42 mmol) in anhydrous DCM (30 mL) at −78 °C was treated with BBr₃ (16 mL, 16.05 mmol, 1 M in DCM). The reaction mixture was stirred at −78 °C for 10 min and 2 h at rt. The reaction mixture was then cooled to −78 °C and quenched with MeOH (4 mL). The solvents were then removed under reduced pressure, and the crude product was redissolved in EtOAc (50 mL) and washed with saturated NaHCO₃ solution. The aqueous phase was extracted with EtOAc (2 × 50 mL), and the combined organic fractions were dried with anhydrous MgSO₄ and filtered off, and the solvent was removed under vacuum to give 57 (1.30 g, 6.17 mmol, 96% yield) as a brown solid, which was used in the next step without further purification. ¹H NMR (400 MHz, MeOD) δ 8.99–8.80 (m, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 3.99 (s, 3H) ppm. ¹³C NMR (100 MHz, MeOD) δ 159.1, 135.1, 134.6, 126.9, 117.3, 117.2, 115.8, 35.0 ppm. LCMS (ESI+) m/z 175.1 [M + H]⁺, retention time 1.09 min (100%). HRMS (ESI+): m/z calculated for C₁₀H₁₁N₂O [M + H]⁺: 175.0871. Found: 175.0870.

Methyl 2-(4-(1-Methyl-1H-imidazol-4-yl)phenoxy)propanoate (58)

Cs₂CO₃ (3.46 g, 10.62 mmol) and methyl 2-bromopropanoate (1.29 g, 6.81 mmol) were added to a solution of phenol derivative 57 (1.12 g, 5.32 mmol) in anhydrous DMF (10 mL), and the mixture was stirred at rt for 1 h and at 60 °C for 5 h. After cooling to rt, the reaction was diluted with water (80 mL) and extracted with EtOAc (2 × 80 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over MgSO₄, filtered, and concentrated. The crude product was purified by flash chromatography (0–20% v/v MeOH in DCM) to give 58 (1.30 g, 4.99 mmol, 92% yield) as an orange oil. ¹H NMR (400 MHz, CDCl₃) δ 7.74–7.61 (m, 2H), 7.45 (d, J = 1.3 Hz, 1H), 7.08 (d, J = 1.4 Hz, 1H), 6.96–6.81 (m, 2H), 4.81 (q, J = 6.8 Hz, 1H), 3.77 (s, 3H), 3.71 (s, 3H), 1.64 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 172.8, 156.5, 142.1, 137.8, 128.1, 126.0, 115.3, 115.1, 72.7, 52.3, 33.5, 18.6 ppm. LCMS (ESI+) m/z 261.3 [M + H]⁺, retention time 1.50 min (100%). HRMS (ESI+): m/z calculated for C₁₄H₁₇N₂O₃ [M + H]⁺: 261.1239. Found: 261.1232.

2-(4-(1-Methyl-1H-imidazol-4-yl)phenoxy)propanoic Acid (59)

Methyl 2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanoate 58 (0.53 g, 2.04 mmol) was dissolved in a 2:1 v/v mixture of THF and H₂O (7.5 mL), and then NaOH (0.16 g, 4.08 mmol) was added. The reaction mixture was heated at 80 °C for 40 min. After the reaction mixture was allowed to cool to rt, the solvents were removed. The mixture was then diluted with H₂O (5 mL) and acidified to pH 7–8 using 1 N HCl. The mixture was cooled, and the resulting precipitate was collected by filtration, washed with water (2 × 3 mL) and petroleum ether (2 × 3 mL), and dried in vacuo to afford acid 59 (0.33 g, 1.33 mmol, 65% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.69–7.55 (m, 3H), 7.46 (d, J = 1.3 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 4.81 (q, J = 6.8 Hz, 1H), 3.66 (s, 3H), 1.49 (d, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.3, 156.1, 140.4, 138.2, 127.8, 125.4, 115.8, 114.8, 71.7, 33.1, 18.4. LCMS (ESI+) m/z 247.2 [M + H]⁺, retention time 1.43 min (100%). HRMS (ESI+): m/z calculated for C₁₃H₁₅N₂O₃ [M + H]⁺: 247.1083. Found: 247.1080.

(S)-2-(4-(1-Methyl-1H-imidazol-4-yl)phenoxy)propanoic Acid ((S)-59)

To a solution of 57 (212 mg, 1.21 mmol) in anhydrous THF (5 mL) was added ethyl d-lactate (215 mg, 1.82 mmol). After the reaction mixture was cooled to 0 °C, PPh₃ (478 mg, 1.82 mmol) and DEAD (286 μL, 1.82 mmol) were added, and the reaction mixture was stirred for 16 h at rt. The reaction mixture was poured into ice–water (40 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous MgSO₄, filtered, and concentrated. The crude product was purified by flash chromatography (0–15% v/v MeOH in DCM) to give ethyl (S)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanoate as an orange solid. LCMS (ESI+) m/z 275.3 [M + H]⁺, retention time 2.06 min (94%). Ethyl (S)-2-(4-(1-methyl-1H-imidazol-4-yl)phenoxy)propanoate (200 mg, 0.73 mmol) was dissolved in a 2:1 v/v mixture of THF and H₂O (7.5 mL), and then NaOH (58 mg, 1.46 mmol) was added. The reaction mixture was stirred at rt for 3 h, and the solvents were removed. The residue was dissolved in EtOAc (40 mL) and then extracted with H₂O (40 mL). The aqueous layer was acidified to pH 7–8 using 1 N HCl. The mixture was cooled, and the resulting precipitate was collected by filtration, washed with water (2 × 3 mL) and petroleum ether (2 × 3 mL), and dried in vacuo to afford acid (S)-59 (100 mg, 0.40 mmol, 56% yield) as a white solid. [α]_D²⁵ −51.6 (c 1.0, CHCl₃). LCMS (ESI+) m/z 247.2 [M + H]⁺, retention time 1.43 min (100%). HRMS (ESI+): m/z calculated for C₁₃H₁₅N₂O₃ [M + H]⁺: 247.1083. Found: 247.1079.

4-(4-Bromophenyl)oxazol-2-amine (61)

A mixture of 2-bromo-1-(4-bromophenyl)ethanone (200 mg, 0.72 mmol) and urea (435 mg, 7.2 mmol) in anhydrous acetonitrile (5 mL) was heated at 80 °C for 18 h. The solvent was removed, and the residue was purified by flash chromatography (0–10% v/v MeOH in DCM) to afford 61 (134 mg, 0.56 mmol, 78% yield) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 7.68 (s, 1H), 7.59–7.42 (m, 4H) ppm. ¹³C NMR (125 MHz, MeOD) δ 163.9, 139.8, 132.7, 132.1, 128.8, 127.9, 122.1 ppm. LCMS (ESI+) m/z 238.8 [M + H]⁺, retention time 1.95 min (100%). HRMS (ESI+): m/z calculated for C₉H₈BrN₂O [M + H]⁺: 238.9820. Found: 238.9816.

2. Enzyme Assay

The activity of Mth IMPDH ΔCBS was determined using a plate reader by monitoring the production of NADH in absorbance at 340 nm and corrected for noncatalyzed chemical reactions in the absence of Mth IMPDH ΔCBS. All the measurements were done in the assay buffer (50 mM Tris HCl pH 8, 1 mM DTT, 1 mM EDTA, and 100 mM KCl) at 37 °C with 20 nM Mth IMPDH ΔCBS, 2.8 mM NAD⁺, and 1 mM IMP in a total of 150 μL volume in a 96 well plate-based format, and data were collected for 32 min. The reaction was initiated by the addition of the substrate, IMP, at a concentration of 1 mM. All reactions were performed in triplicate. Prior to reaction initiation, the compounds were preincubated in a buffer with enzyme for 5 min. The inhibitors were dissolved in DMSO-d₆ and diluted to a final concentration of 1% v/v in experimental reactions.

IC₅₀ values were calculated by plotting the percentage of inhibition against the logarithm of inhibitor concentration, and dose–response curves were fitted using Prism software (GraphPad).

The K_i value for NAD⁺ was determined at a constant saturating IMP concentration (1 mM) and five different concentrations of NAD⁺ (0.35, 0.70, 1.0, 1.4, and 2.8 mM) in the presence of increasing concentrations of inhibitor. The value of K_i for IMP was determined at fixed saturating concentration of NAD⁺ (2.8 mM) and different concentrations of IMP (0.12, 0.18, 0.25, 0.5, and 1 mM) and inhibitor.

The initial velocities at various inhibitor concentrations were determined based on the slope in the linear part of each reaction containing the inhibitor and the uninhibited reaction. To determine the inhibition constant (K_i values), the initial rate data versus substrate concentration at different inhibitor concentrations were fit using Prism software (GraphPad) to equations for uncompetitive or mixed inhibition. For each inhibitor concentration, the reciprocal of enzyme reaction velocity versus the reciprocal of the substrate concentration was plotted in a Lineweaver–Burk plot to determine the pattern of inhibition.

3. Protein Purification, Crystallization, and Data Collection of Mth IMPDH ΔCBS

Mth IMPDH was expressed, purified, and crystallized as previously described.(24,25) Briefly, hexahistidine tagged Mth IMPDH ΔCBS in pHat2 was expressed overnight in BL21 DE3 (NEB) cells at 18 °C by addition of 500 μM IPTG. Cells were lysed in 50 mM Hepes, pH 8.0, 500 mM NaCl, 5% glycerol, 10 mM β-mercaptoethanol, and 20 mM imidazole, and the recombinant protein purified using a Hi-Trap IMAC FF column (GE Healthcare) charged with nickel and an elution gradient of up to 300 mM imidazole. The hexahistidine tag was cleaved by TEV protease, and the purified Mth IMPDH ΔCBS was obtained by negative nickel gravity-flow purification(42) and size exclusion chromatography on a Superdex 200 gel filtration column equilibrated in 20 mM Hepes pH 8.0, 500 mM NaCl, 5% glycerol, and 1 mM TCEP step. The recombinant Mth IMPDH ΔCBS was then concentrated to 12.5 mg/mL for crystallization.

Mth IMPDH ΔCBS protein crystallized in 1 μL + 1 μL hanging drops with 100 mM sodium acetate, pH 5.5, 200 mM calcium chloride, and 8–14% isopropanol. Crystals were soaked overnight in drops of well solution + 5 mM IMP and either 5 mM Fragment 2 or Compound 1 or 1 mM Compound 31. Crystals were cryoprotected by passing through drops containing well solution + 25% glycerol and flash-frozen in liquid nitrogen. Data were collected from the crystals at Diamond Light Source beamline.

4. Structure Solution, Ligand Fitting, and Refinement

Data were processed using XDS(43) and Pointless (CCP4). To solve the structure, molecular replacement was performed with Phenix Phaser(44) using a previously solved IMP-bound Mth IMPDH ΔCBS structure as a probe (PDB IDs: 5J5R; 5K4X; 5K4Z). Refinement was performed using Phenix.refine and manually in Coot.(45) IMP and the inhibitors were sequentially fitted into the density using the LigandFit function of Phenix, and the structures were manually refined further using Coot. Information regarding the crystallographic statistics can be found in Table S2. Protein–ligand interactions were analyzed using Arpeggio(46) and CSM-Lig.(47,48) Compound properties were evaluated using pkCSM.(49) All figures made using Pymol (Schrodinger).

5. Drug Susceptibility Testing against Mtb

An Alamar Blue fluorescence-based broth microdilution assay was used to assess the minimum inhibitory concentration (MIC) of compounds against Mtb H37Rv, as described previously.(25,50) Briefly, Mtb H37Rv was grown in standard Middlebrook 7H9 broth (BD) supplemented with OADC (BD), 0.2% glycerol, and 0.05% Tween-80 to midexponential phase. Compounds dissolved in DMSO (1%) were tested in clear-bottomed, round-well 96-well microtiter plates at eight different concentrations using the standard anti-TB drugs, rifampin and isoniazid, as positive controls. An inoculum of ∼10⁵ bacteria was added to each well, and the plates were incubated at 37 °C for 7 d. On day 7, 10 μL of Alamar Blue (Invitrogen) was added to each well, and plates were further incubated at 37 °C for 24 h. The fluorescence (excitation 544 nm; emission 590 nm) was measured in a FLUOstar OPTIMA plate reader (BMG LABTECH, Offenberg, Germany). Data were normalized to the minimum and maximum inhibition controls to generate a dose response curve (% inhibition) from which the MIC₉₀ was determined.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01622.

¹H, ¹³C, and 2D NMR spectra of all new compounds, supplementary Figures S1–S2, whole-cell activity of compounds against M. tuberculosis H37Rv (Table S1), and X-ray data collection and refinement statistics (Table S2) (PDF)
Molecular formula strings data file for all chemical structures mentioned in the manuscript (CSV)

Fragment-Based Approach to Targeting Inosine-5′-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis

Showing 1/2: jm7b01622_si_002.csv

	A	B	C
1	Compound_ID	SMILES	Mth IMPDH IC50 (uM)
2	1	COC1=CC=C(OC(C)C(NC2=CC=C(I)C=C2)=O)C=C1	0.77 ± 0.06
3	2	BrC1=CC=C(C2=CNC=N2)C=C1	674 ± 53
4	3	C1(C2=CC=CC=C2)=CNC=N1	–
5	4	ClC1=CC=C(C2=CNC=N2)C=C1	–
6	5	BrC1=C(C2=CC=CC=C2)NN=C1N	–
7	6	NC1=C(C2=CC=CC(Br)=C2)C=NN1	–
8	7	OC1=C2C=CC=CC2=CC=C1	–
9	8	OC1=CC=C2C=CC=CC2=C1	–
10	9	O=C(O)C1=C(O)C=C2C(O)=CC=CC2=C1	–
11	10	OC1=CC=C2C=CC(OC)=CC2=C1	–
12	11	OC1=CC2=CC(O)=CC=C2C=C1	336 ± 6
13	12	OC1=CC=C(N2C=CN=C2)C=C1	–
14	13	O=C(O)C1=CC=CC=C1N2C=CC=C2	–
15	14	O=C(C1=CC2=C(C=CS2)S1)O	400 ± 7
16	15	N#CC1=CNC2=C1C=CC=C2	–
17	16	CC1=CC=C(C2=NC(C(O)=O)=CS2)C=C1	433 ± 60
18	17	OCC1=CC=C2C(C=CS2)=C1	–
19	18	CC(NC1=CC(C)=CC=C1O)=O	512 ± 37
20	19	OCC1=C(OC(C)(C)C2)C2=CC=C1	325 ± 9
21	20	O=C(NC1=CC=CC=C1)CSC2=NC(C3=CC=C(Br)C=C3)=CN2	–
22	21	O=C(NC1=CC=C(OC=C2)C2=C1)CSC3=NC(C4=CC=C(Br)C=C4)=CN3	–
23	22	O=C(NC1=CC=C(I)C=C1)CSC2=NC(C3=CC=C(Br)C=C3)=CN2	–
24	23	O=C(NC1=CC=CC=C1)CSC2=NC(C3=CC=CC=C3)=CN2	–
25	24	O=C(NC1=CC=C(OC=C2)C2=C1)CSC3=NC(C4=CC=CC=C4)=CN3	–
26	25	O=C(NC1=CC=C(I)C=C1)CSC2=NC(C3=CC=CC=C3)=CN2	–
27	26	O=C(NC(C)C)CSC1=NC(C2=CC=CC=C2)=CN1	–
28	27	O=C(O)CSC1=NC(C2=CC=C(Br)C=C2)=CN1	–
29	28	O=C(O)CSC1=NC(C2=CC=CC=C2)=CN1	–
30	29	O=C(NC1=CC=C(C2=CNC=N2)C=C1)CSC3=NC(C4=CC=C(Br)C=C4)=CN3	–
31	30	O=C(NCC1=NC(C2=CC=C(Br)C=C2)=CN1)NC3=CC=C(C4=CNC=N4)C=C3	58 ± 8
32	31	CN1C=C(C2=CC=C(OC(C)C(NC3=NC(C4=CC=C(Br)C=C4)=CN3)=O)C=C2)N=C1	0.52 ± 0.004
33	32	O=C(NC1=CC=C(C2=CNC=N2)C=C1)CSC3=NC(C4=CC=CC=C4)=CN3	–
34	33	CN1C=C(C2=CC=C(OC(C)C(NC3=NC(C4=CC=C(Br)C=C4)=CO3)=O)C=C2)N=C1	2.21 ± 0.08
35	34	CN1C=C(C2=CC=C(OC(C)C(NC3=CC=CC=C3)=O)C=C2)N=C1	–
36	35	CN1C=C(C2=CC=C(OC(C)C(NC3=CC=C(I)C=C3)=O)C=C2)N=C1	0.47 ± 0.03
37	36	CN1C=C(C2=CC=C(OC(C)C(NC3=NC(C4=CC=CC=C4)=CN3)=O)C=C2)N=C1	2.76 ± 0.21
38	37	CN1C=C(C2=CC=C(OC(C)C(NC3=NC(C4=CC=C(N5CCOCC5)C=C4)=CN3)=O)C=C2)N=C1	0.52 ± 0.01
39	38	CN1C=C(C2=CC=C(OC(C)C(NC3=NC(C4=CC=C(OC)C=C4)=CN3)=O)C=C2)N=C1	0.64 ± 0.03
40	39	CN1C=C(C2=CC=C(OC(C)C(NC3=NC(C4=CC=C(I)C=C4)=CN3)=O)C=C2)N=C1	0.69 ± 0.06
41	40	O=C(CCl)NC1=CC=CC=C1	–
42	41	O=C(CCl)NC1=CC2=C(OC=C2)C=C1	–
43	42	O=C(CCl)NC1=CC=C(I)C=C1	–
44	43	O=C(CCl)NC1=CC=C(C2=CNC=N2)C=C1	–
45	44	BrC1=CC=C(C2=CN=C(CN)N2)C=C1.Cl	–
46	45	O=C(C)NC1=NC(C2=CC=C(Br)C=C2)=CN1	–
47	46	O=C(C)NC1=NC(C2=CC=CC=C2)=CN1	–
48	47	O=C(C)NC1=NC(C2=CC=C(N3CCOCC3)C=C2)=CN1	–
49	48	O=C(C)NC1=NC(C2=CC=C(OC)C=C2)=CN1	–
50	49	O=C(C)NC1=NC(C2=CC=C(I)C=C2)=CN1	–

#Author Present Address

(V.S.) H3D Drug Discovery and Development Centre, Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.

The authors declare no competing financial interest.

Notes

Additional data related to this publication is available at the University of Cambridge data repository: https://doi.org/10.17863/CAM.20512. Structures have been deposited in the Protein Data Bank (PDB codes 5OU1 for IMPDH:IMP:Compound 1; 5OU2 for IMPDH:IMP:Compound 2 and 5OU3 for IMPDH:IMP:Compound 31 complex structures). Authors will release the atomic coordinates and experimental data upon article publication.

Acknowledgments

This work was supported by the Bill and Melinda Gates Foundation (Hit-TB) and FP7 European Project MM4TB Grant no. 260872. D.B.A was supported by a C. J. Martin Research Fellowship from the National Health and Medical Research Council of Australia (APP1072476) and the Jack Brockhoff Foundation (JBF 4186, 2016). D.B.A. and T.L.B. were also funded by a Newton Fund RCUK-CONFAP Grant awarded by The Medical Research Council and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (MR/M026302/1). V.M. and V.S. were also supported by grants from the HHMI (Senior International Research Scholars grant to V.M.), the South African Medical Research Council, and the National Research Foundation.

Abbreviations Used
TB	tuberculosis
Mycobacterium tuberculosis	Mtb
MDR	multidrug resistant
XDR	extensively drug resistant
IMPDH	inosine-5′-monophosphate dehydrogenase
IMP	inosine 5′-monophosphate
CBS	cystathionine β-synthase
XMP	xanthosine monophosphate
NAD⁺	nicotinamide adenine dinucleotide
Mth	Mycobacterium thermoresistible
MIC	minimal inhibitory concentration.
WHO	World Health Organization

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Chen, Liqiang; Pankiewicz, Krzysztof W.

Current Opinion in Drug Discovery & Development (2007), 10 (4), 403-412CODEN: CODDFF; ISSN:1367-6733. (Thomson Scientific)

A review. IMP dehydrogenase (IMPDH) represents an attractive target for the development of anticancer agents; however, there are no drugs aimed at this target for the treatment of cancer currently available on the market. Tiazofurin, a potent IMPDH inhibitor, reached clin. trials with Orphan Drug status for the treatment of patients in blast crisis of chronic myelogenous leukemia (CML); however, it was considered too toxic for application against other malignancies and no development has been reported for this drug since 2002. Formulations of mycophenolic acid, another potent inhibitor of IMPDH, are currently used for the prevention of rejection following transplantation, and against autoimmune diseases. More recently, numerous studies have demonstrated the potential of mycophenolic acid as an anticancer agent, with a phase I clin. trial in patients with advanced multiple myeloma ongoing. Furthermore, synergy between imantinib and mycophenolic acid in CML treatments has also been reported. Related compds. such as mycophenolic adenine dinucleotides, along with second-generation analogs, are undergoing preclin. evaluation, while another inhibitor of IMPDH, AVN-944, is currently in phase I clin. trials to investigate the treatment of hematol. malignancies. This article reviews recent applications of IMPDH inhibitors as anticancer agents, and highlights the progress that has been made in this field.

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6.

Olah, E.; Kokeny, S.; Papp, J.; Bozsik, A.; Keszei, M. Modulation of cancer pathways by inhibitors of guanylate metabolism. Adv. Enzyme Regul. 2006, 46, 176– 190, DOI: 10.1016/j.advenzreg.2006.01.002

[Crossref], [PubMed], [CAS]

6.

Modulation of cancer pathways by inhibitors of guanylate metabolism

Olah, Edith; Kokeny, Szabolcs; Papp, Janos; Bozsik, Aniko; Keszei, Marton

Advances in Enzyme Regulation (2006), 46 (), 176-190CODEN: AEZRA2; ISSN:0065-2571. (Elsevier Ltd.)

A review on current progress in the modulation of cancer pathways by inhibiting guanylate metab., with emphasis on the mol. impact of targeting IMP dehydrogenase (IMP DH). The genetic aspects of cancer and the pathways controlled by cancer genes are discussed, along with the mol. and cellular mechanisms in the antiproliferative effects of IMP DH inhibitors tiazofurin and ribavirin. The role of IMP DH inhibitors in mol. and biochem. targeting of neoplasia is also examd. Evidence shows that the down-regulation of oncogenes by either tiazofurin or ribavirin in different cancer cell lines similarly induces cell-cycle arrest, differentiation and apoptosis. Apoptosis seemed to have a major role in the action of ribavirin, whereas differentiation appeared to be more important in the anticancer effect of tiazofurin.

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7.

Nair, V.; Shu, Q. Inosine monophosphate dehydrogenase as a probe in antiviral drug discovery. Antiviral Chem. Chemother. 2007, 18, 245– 258, DOI: 10.1177/095632020701800501

[Crossref], [PubMed], [CAS]

7.

Inosine monophosphate dehydrogenase as a probe in antiviral drug discovery

Nair, Vasu; Shu, Qingning

Antiviral Chemistry & Chemotherapy (2007), 18 (5), 245-258CODEN: ACCHEH; ISSN:0956-3202. (International Medical Press, Ltd.)

A review. Inosine monophosphate (IMP) dehydrogenase (IMPDH) is a significant enzyme in the purine nucleotide biosynthetic pathway. IMPDH is viewed as an important biol. target in the quest for drugs in the antiviral therapeutic area. This review article is focused on the chem. and biol. of IMPDH inhibitors and the use of IMPDH inhibition data as a probe in antiviral drug discovery. Examples of both inosine 5' monophosphate and NAD+ site-directed inhibitors are presented. Correlation of antiviral activities with IMPDH inhibition is discussed.

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8.

Shu, Q.; Nair, V. Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery. Med. Res. Rev. 2008, 28, 219– 232, DOI: 10.1002/med.20104

[Crossref], [PubMed], [CAS]

8.

Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery

Shu, Qingning; Nair, Vasu

Medicinal Research Reviews (2008), 28 (2), 219-232CODEN: MRREDD; ISSN:0198-6325. (John Wiley & Sons, Inc.)

A review. Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme of de novo purine nucleotide biosynthesis and is viewed as an important target in the quest for discovery of drugs in the antiviral, antibacterial and anticancer therapeutic areas. This review focuses on the medicinal chem., drug discovery and chem. biol. of IMPDH. Examples of IMP and cofactor site-directed inhibitors, allosteric inhibitors and isoform-selective inhibitors are presented. Comparison of IMPDHs from different organisms is also made to facilitate the design of species-selective IMPDH inhibitors for drug discovery. Special emphasis in the review is placed on IMPDH from Mycobacterium tuberculosis.

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9.

Shah, C. P.; Kharkar, P. S. Inosine 5′-monophosphate dehydrogenase inhibitors as antimicrobial agents: recent progress and future perspectives. Future Med. Chem. 2015, 7, 1415– 1429, DOI: 10.4155/fmc.15.72

[Crossref], [PubMed], [CAS]

9.

Inosine 5'-monophosphate dehydrogenase inhibitors as antimicrobial agents: recent progress and future perspectives

Shah, Chetan P.; Kharkar, Prashant S.

Future Medicinal Chemistry (2015), 7 (11), 1415-1429CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)

A review. IMP dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an important target for cancer, bacterial, parasitic and viral infections and autoimmune disorders. Several classes of IMPDH inhibitors are known in the literature. The current review succinctly summarizes the progress made in the design and development of IMPDH inhibitors as antimicrobial agents in last five years or so. The focus is on the inhibitor and enzyme structural features responsible for imparting selectivity for the microbial over the host enzyme. Future perspectives clearly outline the inhibitor design opportunities available in this area to address the present challenges of drug resistance and re-emergence of newer and deadly strains of microbes, posing a serious threat to public.

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10.

Hedstrom, L.; Liechti, G.; Goldberg, J. B.; Gollapalli, D. R. The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors. Curr. Med. Chem. 2011, 18, 1909– 1918, DOI: 10.2174/092986711795590129

[Crossref], [PubMed], [CAS]

10.

The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors

Hedstrom, L.; Liechti, G.; Goldberg, J. B.; Gollapalli, D. R.

Current Medicinal Chemistry (2011), 18 (13), 1909-1918CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)

A review. IMP dehydrogenase (IMPDH) catalyzes the first committed step of GMP biosynthesis, and thus regulates the guanine nucleotide pool, which in turn governs proliferation. Human IMPDHs are validated targets for immunosuppressive, antiviral and anticancer drugs, but as yet microbial IMPDHs have not been exploited in antimicrobial chemotherapy. Selective inhibitors of IMPDH from Cryptosporidium parvum have been discovered that display anti-parasitic activity in cell culture models of infection. X-ray crystal structure and mutagenesis expts. identified the structural features that det. inhibitor susceptibility. These features are found in IMPDHs from a wide variety of pathogenic bacteria, including select agents and multiply drug resistant strains. A second generation inhibitor displays antibacterial activity against Helicobacter pylori, demonstrating the antibiotic potential of IMPDH inhibitors.

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11.

Hedstrom, L. IMP dehydrogenase: structure, mechanism, and inhibition. Chem. Rev. 2009, 109, 2903– 2928, DOI: 10.1021/cr900021w

[ACS Full Text ], [CAS]

11.

IMP Dehydrogenase: Structure, Mechanism, and Inhibition

Hedstrom, Lizbeth

Chemical Reviews (Washington, DC, United States) (2009), 109 (7), 2903-2928CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)

A review. The discovery that IMP dehydrogenase (IMPDH) is amplified in tumors and rapidly proliferating tissues provided the foundation for design of drugs targeting this enzyme. Though yet much success in the cancer arena has not been achieved, IMPDH inhibitors are now widely used in immunosuppressive and antiviral chemotherapy, and IMPDH may also be a target for antimicrobial drugs. Clin. relevance aside, IMPDH is a fascinating enzyme. It traverses several conformations while catalyzing two different chem. transformations, utilizing unusual chem. strategies to promote each reaction. Monovalent cations such as potassium activate IMPDH, possibly by acting as a mol. lubricant to facilitate these conformational changes. The biol. of IMPDH also displays some surprising twists. IMPDH binds nucleic acids and is assocd. with polyribosomes, though the physiol. role of this interaction also has not yet been elucidated. Perhaps most intriguing is the discovery that mutations in IMPDH are assocd. with hereditary retinal disease. These mutations cluster to a subdomain that is not required for enzymic activity, and the function of this subdomain is currently under debate. The paper reviews recent work on the biochem. of IMPDH, integrating structure, function, and inhibition.

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12.

Jackson, R. C.; Weber, G.; Morris, H. P. IMP dehydrogenase, an enzyme linked with proliferation and malignancy. Nature 1975, 256, 331– 333, DOI: 10.1038/256331a0

[Crossref], [PubMed], [CAS]

12.

IMP dehydrogenase, an enzyme linked with proliferation and malignancy

Jackson, Robert C.; Weber, George; Morris, Harold P.

Nature (London, United Kingdom) (1975), 256 (5515), 331-3CODEN: NATUAS; ISSN:0028-0836.

IMP dehydrogenase (EC 1.2.1.14) (I) activity was higher in rat hepatomas than in normal rat livers, a 13-fold difference occurring in the most rapidly proliferating tumors, and was correlated with growth rate. I from hepatomas and normal livers showed similar properties. I activity began to rise 6 hr after partial hepatectomy, reaching a 5-fold peak after ∼24 hr. On a per unit wet wt. basis I was almost 4 times more active in 1-day-old than in adult rats, but on a per cell basis I was only 25% more active. I in thymus, spleen, bone marrow, and testis was 10.5, 8.6, 3.3, and 2.1 times more active than liver I, but other tissues had lower activities.

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13.

Gilbert, H. J.; Lowe, C. R.; Drabble, W. T. Inosine 5′-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5′-monophosphate. Biochem. J. 1979, 183, 481– 494, DOI: 10.1042/bj1830481

[Crossref], [PubMed], [CAS]

13.

Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure, and inhibition by guanosine 5'-monophosphate

Gilbert, Harry J.; Lowe, Christopher R.; Drabble, William T.

Biochemical Journal (1979), 183 (3), 481-94CODEN: BIJOAK; ISSN:0264-6021.

Electrophoresis, N-terminal amino acid anal., and tryptic fingerprinting showed that IMP dehydrogenase (EC 1.2.1.14) (I), purified from E. coli, comprised identical subunits of mol. wt. 58,000. Trypsin and Pronase cleaved the subunit into peptides with mol. wts. 42,000 and 14,000, with a concomitant decrease in I activity. There was no cooperativity between the IMP-binding sites of I in the presence or absence of GMP, suggesting that GMP was a competitive inhibitor with respect to IMP, showing no regulatory behavior. I was purified by affinity chromatog. on immobilized nucleotides, and also by immunopptn. with monospecific antiserums. The inactive mutant enzymes from guaB strains of E. coli were also purified.

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14.

Lee, S. A.; Gallagher, L. A.; Thongdee, M.; Staudinger, B. J.; Lippman, S.; Singh, P. K.; Manoil, C. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 5189– 5194, DOI: 10.1073/pnas.1422186112

[Crossref], [PubMed], [CAS]

14.

General and condition-specific essential functions of Pseudomonas aeruginosa

Lee, Samuel A.; Gallagher, Larry A.; Thongdee, Metawee; Staudinger, Benjamin J.; Lippman, Soyeon; Singh, Pradeep K.; Manoil, Colin

Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (16), 5189-5194CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition-growth in a rich undefined medium-and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The anal. used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an av. of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metab. and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiol. as well as identifying candidate targets for new antibacterial agents.

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15.

Valentino, M. D.; Foulston, L.; Sadaka, A.; Kos, V. N.; Villet, R. A.; Santa Maria, J., Jr.; Lazinski, D. W.; Camilli, A.; Walker, S.; Hooper, D. C.; Gilmore, M. S. Genes contributing to Staphylococcus aureus fitness in abscess- and infection-related ecologies. mBio 2014, 5, e01729– 14, DOI: 10.1128/mBio.01729-14

[Crossref], [PubMed], [CAS]

15.

Genes contributing to Staphylococcus aureus fitness in abscess- and infection-related ecologies

Valentino, Michael D.; Foulston, Lucy; Sadaka, Ama; Kos, Veronica N.; Villet, Regis A.; Santa Maria, John, Jr.; Lazinski, David W.; Camilli, Andrew; Walker, Suzanne; Hooper, David C.; Gilmore, Michael S.

mBio (2014), 5 (5), e01729-14/1-e01729-14/11CODEN: MBIOCL; ISSN:2150-7511. (American Society for Microbiology)

Staphylococcus aureus is a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence of S. aureus resistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly satd. transposon insertion mutant library in the genome of S. aureus and used Tn-seq anal. to probe the entire genome, with unprecedented resoln. and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important for S. aureus fitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important for S. aureus proliferation at sites of infection, which may represent new therapeutic targets.

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16.

Sassetti, C. M.; Boyd, D. H.; Rubin, E. J. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 2003, 48, 77– 84, DOI: 10.1046/j.1365-2958.2003.03425.x

[Crossref], [PubMed], [CAS]

16.

Genes required for mycobacterial growth defined by high density mutagenesis

Sassetti, Christopher M.; Boyd, Dana H.; Rubin, Eric J.

Molecular Microbiology (2003), 48 (1), 77-84CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)

Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

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17.

Hedstrom, L. The bare essentials of antibiotic target validation. ACS Infect. Dis. 2017, 3, 2– 4, DOI: 10.1021/acsinfecdis.6b00185

[ACS Full Text ], [CAS]

17.

The Bare Essentials of Antibiotic Target Validation

Hedstrom, Lizbeth

ACS Infectious Diseases (2017), 3 (1), 2-4CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)

A review. The convergence of competitive fitness expts. and phenotypic screening would seem to be an auspicious beginning for validation of an antibacterial target. IMPDH was already identified an essential protein in Mycobacterium tuberculosis when not one, but two, groups discovered inhibitors with promising antitubercular activity. A new target appeared to be born. Surprisingly, the two groups come to completely different conclusions about the vulnerability of IMPDH and its future as a drug target. This Viewpoint discusses these papers and how to resolve this conundrum.

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18.

Petrelli, R.; Vita, P.; Torquati, I.; Felczak, K.; Wilson, D. J.; Franchetti, P.; Cappellacci, L. Novel inhibitors of inosine monophosphate dehydrogenase in patent literature of the last decade. Recent Pat. Anti-Cancer Drug Discovery 2013, 8, 103– 125, DOI: 10.2174/1574892811308020001

[Crossref], [PubMed], [CAS]

18.

Novel inhibitors of inosine monophosphate dehydrogenase in patent literature of the last decade

Petrelli, Riccardo; Vita, Patrizia; Torquati, Ilaria; Felczak, Krzysztof; Wilson, Daniel J.; Franchetti, Palmarisa; Cappellacci, Loredana

Recent Patents on Anti-Cancer Drug Discovery (2013), 8 (2), 103-125CODEN: RPADDY; ISSN:1574-8928. (Bentham Science Publishers Ltd.)

A review. Inosine monophosphate dehydrogenase (IMPDH), an NAD-dependent enzyme that controls de novo synthesis of guanine nucleotides, has received considerable interest in recent years as an important target enzyme, not only for the discovery of anticancer drugs, but also for antiviral, antiparasitic, and immunosuppressive chemotherapy. The field of IMPDH inhibitor research is highly important for providing potential therapeutics against a validated target for disease intervention. This patent review examines the chem. structures and biol. activities of recently reported IMPDH inhibitors. Patent databases SciFinder and Espacenet and Delphion were used to locate patent applications that were published between Jan. 2002 and July 2012, claiming chem. structures for use as IMPDH inhibitors. From 2002 to 2012, around 47 primary patent applications have claimed IMPDH inhibitors, which we analyzed by target and applicant. The level of newly published patent applications covering IMPDH inhibitors remains high and a diverse range of scaffolds has been claimed.

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19.

Makowska-Grzyska, M.; Kim, Y.; Wu, R.; Wilton, R.; Gollapalli, D. R.; Wang, X. K.; Zhang, R.; Jedrzejczak, R.; Mack, J. C.; Maltseva, N.; Mulligan, R.; Binkowski, T. A.; Gornicki, P.; Kuhn, M. L.; Anderson, W. F.; Hedstrom, L.; Joachimiak, A. Bacillus anthracis inosine 5′-monophosphate dehydrogenase in action: the first bacterial series of structures of phosphate ion-, substrate-, and product-bound complexes. Biochemistry 2012, 51, 6148– 6163, DOI: 10.1021/bi300511w

[ACS Full Text ], [CAS]

19.

Bacillus anthracis Inosine 5'-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes

Makowska-Grzyska, Magdalena; Kim, Youngchang; Wu, Ruiying; Wilton, Rosemarie; Gollapalli, Deviprasad R.; Wang, Ximi K.; Zhang, Rongguang; Jedrzejczak, Robert; Mack, Jamey C.; Maltseva, Natalia; Mulligan, Rory; Binkowski, T. Andrew; Gornicki, Piotr; Kuhn, Misty L.; Anderson, Wayne F.; Hedstrom, Lizbeth; Joachimiak, Andrzej

Biochemistry (2012), 51 (31), 6148-6163CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

IMP dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clin. applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have detd. three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed "apo") form and in complex with its substrate, IMP, and product, xanthosine 5'-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, contg. the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymic reaction were also detd., and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addn., the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examd. for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.

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20.

Usha, V.; Gurcha, S. S.; Lovering, A. L.; Lloyd, A. J.; Papaemmanouil, A.; Reynolds, R. C.; Besra, G. S. Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against. Microbiology 2011, 157, 290– 299, DOI: 10.1099/mic.0.042549-0

[Crossref], [PubMed], [CAS]

20.

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

Usha, Veeraraghavan; Gurcha, Sudagar S.; Lovering, Andrew L.; Lloyd, Adrian J.; Papaemmanouil, Athina; Reynolds, Robert C.; Besra, Gurdyal S.

Microbiology (Reading, United Kingdom) (2011), 157 (1), 290-299CODEN: MROBEO; ISSN:1350-0872. (Society for General Microbiology)

In contrast with most bacteria, which harbor a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB ortholog in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of di-Ph urea-based derivs., which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 μg ml-1. When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a di-Ph urea analog showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologs (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologs were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compds. in vivo.

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21.

Makowska-Grzyska, M.; Kim, Y.; Gorla, S. K.; Wei, Y.; Mandapati, K.; Zhang, M.; Maltseva, N.; Modi, G.; Boshoff, H. I.; Gu, M.; Aldrich, C.; Cuny, G. D.; Hedstrom, L.; Joachimiak, A. Mycobacterium tuberculosis IMPDH in complexes with substrates, products and antitubercular compounds. PLoS One 2015, 10, e0138976 DOI: 10.1371/journal.pone.0138976

[Crossref], [PubMed], [CAS]

21.

Mycobacterium tuberculosis IMPDH in complexes with substrates, products and antitubercular compounds

Makowska-Grzyska, Magdalena; Kim, Youngchang; Gorla, Suresh Kumar; Wei, Yang; Mandapati, Kavitha; Zhang, Minjia; Maltseva, Natalia; Modi, Gyan; Boshoff, Helena I.; Gu, Minyi; Aldrich, Courtney; Cuny, Gregory D.; Hedstrom, Lizbeth; Joachimiak, Andrzej

PLoS One (2015), 10 (10), e0138976/1-e0138976/21CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. IMP dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of IMP into xanthosine 5'-monophosphate with the concomitant redn. of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. The authors report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are detd. The authors also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.

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22.

Chen, L.; Wilson, D. J.; Xu, Y.; Aldrich, C. C.; Felczak, K.; Sham, Y. Y.; Pankiewicz, K. W. Triazole-linked inhibitors of inosine monophosphate dehydrogenase from human and. J. Med. Chem. 2010, 53, 4768– 4778, DOI: 10.1021/jm100424m

[ACS Full Text ], [CAS]

22.

Triazole-Linked Inhibitors of Inosine Monophosphate Dehydrogenase from Human and Mycobacterium tuberculosis

Chen, Liqiang; Wilson, Daniel J.; Xu, Yanli; Aldrich, Courtney C.; Felczak, Krzysztof; Sham, Yuk Y.; Pankiewicz, Krzysztof W.

Journal of Medicinal Chemistry (2010), 53 (12), 4768-4778CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

The modular nature of NAD (NAD)-mimicking inosine monophosphate dehydrogenase (IMPDH) inhibitors has prompted us to investigate novel mycophenolic adenine dinucleotides (MAD) in which 1,2,3-triazole linkers were incorporated as isosteric replacements of the pyrophosphate linker. Synthesis and evaluation of these inhibitors led to identification of low nanomolar inhibitors of human IMPDH and more importantly the first potent inhibitor of IMPDH from Mycobacterium tuberculosis (mtIMPDH). Computational studies of these IMPDH enzymes helped rationalize the obsd. structure-activity relationships. Addnl., the first cloning, expression, purifn. and characterization of mtIMPDH is reported.

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23.

Usha, V.; Hobrath, J. V.; Gurcha, S. S.; Reynolds, R. C.; Besra, G. S. Identification of novel Mt-Guab2 inhibitor series active against M. tuberculosis. PLoS One 2012, 7, e33886 DOI: 10.1371/journal.pone.0033886

[Crossref], [PubMed]

There is no corresponding record for this reference.
24.

Park, Y.; Pacitto, A.; Bayliss, T.; Cleghorn, L. A.; Wang, Z.; Hartman, T.; Arora, K.; Ioerger, T. R.; Sacchettini, J.; Rizzi, M.; Donini, S.; Blundell, T. L.; Ascher, D. B.; Rhee, K. Y.; Breda, A.; Zhou, N.; Dartois, V.; Jonnala, S. R.; Via, L. E.; Mizrahi, V.; Epemolu, O.; Stojanovski, L.; Simeons, F. R.; Osuna-Cabello, M.; Ellis, L.; MacKenzie, C. J.; Smith, A. R.; Davis, S. H.; Murugesan, D.; Buchanan, K. I.; Turner, P. A.; Huggett, M.; Zuccotto, F.; Rebollo-Lopez, M. J.; Lafuente-Monasterio, M. J.; Sanz, O.; Santos Diaz, G.; Lelievre, J.; Ballell, L.; Selenski, C.; Axtman, M.; Ghidelli-Disse, S.; Pflaumer, H.; Boesche, M.; Drewes, G.; Freiberg, G.; Kurnick, M. D.; Srikumaran, M.; Kempf, D. J.; Green, S. R.; Ray, P. C.; Read, K. D.; Wyatt, P. G.; Barry Rd, C. E.; Boshoff, H. I. Essential but not vulnerable: indazole sulfonamides targeting inosine monophosphate dehydrogenase as potential leads against. ACS Infect. Dis. 2017, 3, 18– 23, DOI: 10.1021/acsinfecdis.6b00103

[ACS Full Text ], [CAS]

24.

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis

Park, Yumi; Pacitto, Angela; Bayliss, Tracy; Cleghorn, Laura A. T.; Wang, Zhe; Hartman, Travis; Arora, Kriti; Ioerger, Thomas R.; Sacchettini, Jim; Rizzi, Menico; Donini, Stefano; Blundell, Tom L.; Ascher, David B.; Rhee, Kyu; Breda, Ardala; Zhou, Nian; Dartois, Veronique; Jonnala, Surendranadha Reddy; Via, Laura E.; Mizrahi, Valerie; Epemolu, Ola; Stojanovski, Laste; Simeons, Fred; Osuna-Cabello, Maria; Ellis, Lucy; MacKenzie, Claire J.; Smith, Alasdair R. C.; Davis, Susan H.; Murugesan, Dinakaran; Buchanan, Kirsteen I.; Turner, Penelope A.; Huggett, Margaret; Zuccotto, Fabio; Rebollo-Lopez, Maria Jose; Lafuente-Monasterio, Maria Jose; Sanz, Olalla; Diaz, Gracia Santos; Lelievre, Joel; Ballell, Lluis; Selenski, Carolyn; Axtman, Matthew; Ghidelli-Disse, Sonja; Pflaumer, Hannah; Bosche, Markus; Drewes, Gerard; Freiberg, Gail M.; Kurnick, Matthew D.; Srikumaran, Myron; Kempf, Dale J.; Green, Simon R.; Ray, Peter C.; Read, Kevin; Wyatt, Paul; Barry, Clifton E.; Boshoff, Helena I.

ACS Infectious Diseases (2017), 3 (1), 18-33CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compds. for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compd. did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochem. validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads contg. immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallog. of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compds. with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compds. in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill expts. in vitro suggest that sustained exposure to drug concns. above MIC for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis infected animals and patients revealed 0.5-2 mM concns. in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate dependent, effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

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25.

Singh, V.; Donini, S.; Pacitto, A.; Sala, C.; Hartkoorn, R. C.; Dhar, N.; Keri, G.; Ascher, D. B.; Mondesert, G.; Vocat, A.; Lupien, A.; Sommer, R.; Vermet, H.; Lagrange, S.; Buechler, J.; Warner, D. F.; McKinney, J. D.; Pato, J.; Cole, S. T.; Blundell, T. L.; Rizzi, M.; Mizrahi, V. The inosine monophosphate dehydrogenase, GuaB2, is a vulnerable new bactericidal drug target for tuberculosis. ACS Infect. Dis. 2017, 3, 5– 17, DOI: 10.1021/acsinfecdis.6b00102

[ACS Full Text ], [CAS]

25.

The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis

Singh, Vinayak; Donini, Stefano; Pacitto, Angela; Sala, Claudia; Hartkoorn, Ruben C.; Dhar, Neeraj; Keri, Gyorgy; Ascher, David B.; Mondesert, Guillaume; Vocat, Anthony; Lupien, Andreanne; Sommer, Raphael; Vermet, Helene; Lagrange, Sophie; Buechler, Joe; Warner, Digby F.; McKinney, John D.; Pato, Janos; Cole, Stewart T.; Blundell, Tom L.; Rizzi, Menico; Mizrahi, Valerie

ACS Infectious Diseases (2017), 3 (1), 5-17CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)

VCC234718, a mol. with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15,344-compd. library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary mol. target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki 100 nM and is uncompetitive with respect to IMP and NAD+. This compd. binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addn. to sensitizing Mtb to VCC2344718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concn. (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new TB drug target.

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26.

Cox, J. A.; Mugumbate, G.; Del Peral, L. V.; Jankute, M.; Abrahams, K. A.; Jervis, P.; Jackenkroll, S.; Perez, A.; Alemparte, C.; Esquivias, J.; Lelievre, J.; Ramon, F.; Barros, D.; Ballell, L.; Besra, G. S. Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen. Sci. Rep. 2016, 6, 38986, DOI: 10.1038/srep38986

[Crossref], [PubMed], [CAS]

26.

Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen

Cox, Jonathan A. G.; Mugumbate, Grace; Del Peral, Laura Vela-Glez; Jankute, Monika; Abrahams, Katherine A.; Jervis, Peter; Jackenkroll, Stefan; Perez, Arancha; Alemparte, Carlos; Esquivias, Jorge; Lelievre, Joel; Ramon, Fernando; Barros, David; Ballell, Lluis; Besra, Gurdyal S.

Scientific Reports (2016), 6 (), 38986CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)

High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of hit compds. A pre-requisite to medicinal chem. optimization programs required to improve the drug-like properties of a hit mol. is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis; however previously identified compds. lack the desired characteristics necessary for further development into lead-like mols. This study has identified 7 new chem. series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compds. were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochem. anal. The compds. were also assessed using mol. docking expts., providing a platform for their further optimization using medicinal chem. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target.

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27.

Moffat, J. G.; Vincent, F.; Lee, J. A.; Eder, J.; Prunotto, M. Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nat. Rev. Drug Discovery 2017, 16, 531– 543, DOI: 10.1038/nrd.2017.111

[Crossref], [PubMed], [CAS]

27.

Opportunities and challenges in phenotypic drug discovery: an industry perspective

Moffat, John G.; Vincent, Fabien; Lee, Jonathan A.; Eder, Jorg; Prunotto, Marco

Nature Reviews Drug Discovery (2017), 16 (8), 531-543CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)

Phenotypic drug discovery (PDD) approaches do not rely on knowledge of the identity of a specific drug target or a hypothesis about its role in disease, in contrast to the target-based strategies that have been widely used in the pharmaceutical industry in the past three decades. However, in recent years, there has been a resurgence in interest in PDD approaches based on their potential to address the incompletely understood complexity of diseases and their promise of delivering first-in-class drugs, as well as major advances in the tools for cell-based phenotypic screening. Nevertheless, PDD approaches also have considerable challenges, such as hit validation and target deconvolution. This article focuses on the lessons learned by researchers engaged in PDD in the pharmaceutical industry and considers the impact of 'omics' knowledge in defining a cellular disease phenotype in the era of precision medicine, introducing the concept of a chain of translatability. We particularly aim to identify features and areas in which PDD can best deliver value to drug discovery portfolios and can contribute to the identification and the development of novel medicines, and to illustrate the challenges and uncertainties that are assocd. with PDD in order to help set realistic expectations with regard to its benefits and costs.

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28.

Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. The challenge of new drug discovery for tuberculosis. Nature 2011, 469, 483– 490, DOI: 10.1038/nature09657

[Crossref], [PubMed], [CAS]

28.

The challenge of new drug discovery for tuberculosis

Koul, Anil; Arnoult, Eric; Lounis, Nacer; Guillemont, Jerome; Andries, Koen

Nature (London, United Kingdom) (2011), 469 (7331), 483-490CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

A review. Tuberculosis (TB) is more prevalent in the world today than at any other time in human history. Mycobacterium tuberculosis, the pathogen responsible for TB, uses diverse strategies to survive in a variety of host lesions and to evade immune surveillance. A key question is how robust are our approaches to discovering new TB drugs, and what measures could be taken to reduce the long and protracted clin. development of new drugs. The emergence of multi-drug-resistant strains of M. tuberculosis makes the discovery of new mol. scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control. Whatever the strategy used, success will depend largely on our proper understanding of the complex interactions between the pathogen and its human host. In this review, we discuss innovations in TB drug discovery and evolving strategies to bring newer agents more quickly to patients.

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29.

Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discovery 2007, 6, 29– 40, DOI: 10.1038/nrd2201

[Crossref], [PubMed], [CAS]

29.

Drugs for bad bugs: confronting the challenges of antibacterial discovery

Payne, David J.; Gwynn, Michael N.; Holmes, David J.; Pompliano, David L.

Nature Reviews Drug Discovery (2007), 6 (1), 29-40CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)

A review. The sequencing of the first complete bacterial genome in 1995 heralded a new era of hope for antibacterial drug discoverers, who now had the tools to search entire genomes for new antibacterial targets. Several companies, including GlaxoSmithKline, moved back into the antibacterials area and embraced a genomics-derived, target-based approach to screen for new classes of drugs with novel modes of action. Here, we share our experience of evaluating more than 300 genes and 70 high-throughput screening campaigns over a period of 7 years, and look at what we learned and how that has influenced GlaxoSmithKline's antibacterials strategy going forward.

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30.

Scott, D. E.; Coyne, A. G.; Hudson, S. A.; Abell, C. Fragment-based approaches in drug discovery and chemical biology. Biochemistry 2012, 51, 4990– 5003, DOI: 10.1021/bi3005126

[ACS Full Text ], [CAS]

30.

Fragment-Based Approaches in Drug Discovery and Chemical Biology

Scott, Duncan E.; Coyne, Anthony G.; Hudson, Sean A.; Abell, Chris

Biochemistry (2012), 51 (25), 4990-5003CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

A review. Fragment-based approaches to finding novel small mols. that bind to proteins are now firmly established in drug discovery and chem. biol. Initially developed primarily in a few centers in the biotech and pharma industry, this methodol. has now been adopted widely in both the pharmaceutical industry and academia. After the initial success with kinase targets, the versatility of this approach has now expanded to a broad range of different protein classes. Herein we describe recent fragment-based approaches to a wide range of target types, including Hsp90, β-secretase, and allosteric sites in human immunodeficiency virus protease and fanesyl pyrophosphate synthase. The role of fragment-based approaches in an academic research environment is also examd. with an emphasis on neglected diseases such as tuberculosis. The development of a fragment library, the fragment screening process, and the subsequent fragment hit elaboration will be discussed using examples from the literature.

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31.

Mendes, V.; Blundell, T. L. Targeting tuberculosis using structure-guided fragment-based drug design. Drug Discovery Today 2017, 22, 546– 554, DOI: 10.1016/j.drudis.2016.10.003

[Crossref], [PubMed], [CAS]

31.

Targeting tuberculosis using structure-guided fragment-based drug design

Mendes, Vitor; Blundell, Tom L.

Drug Discovery Today (2017), 22 (3), 546-554CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)

Fragment-based drug discovery is now widely used in academia and industry to obtain small mol. inhibitors for a given target and is established for many fields of research including antimicrobials and oncol. Many mols. derived from fragment-based approaches are already in clin. trials and two - vemurafenib and venetoclax - are on the market, but the approach has been used sparsely in the tuberculosis field. Here, we describe the progress of our group and others, and examine the most recent successes and challenges in developing compds. with antimycobacterial activity.

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32.

Marchetti, C.; Chan, D. S.; Coyne, A. G.; Abell, C. Fragment-based approaches to TB drugs. Parasitology 2018, 145, 184– 195, DOI: 10.1017/S0031182016001876

[Crossref], [PubMed], [CAS]

32.

Fragment-based approaches to TB drugs

Marchetti Chiara; Chan Daniel S H; Coyne Anthony G; Abell Chris

Parasitology (2018), 145 (2), 184-195 ISSN:.

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

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33.

Wei, Y.; Kuzmic, P.; Yu, R.; Modi, G.; Hedstrom, L. Inhibition of inosine-5′-monophosphate dehydrogenase from Bacillus anthracis: Mechanism revealed by pre-steady-state kinetics. Biochemistry 2016, 55, 5279– 5288, DOI: 10.1021/acs.biochem.6b00265

[ACS Full Text ], [CAS]

33.

Inhibition of Inosine-5'-monophosphate Dehydrogenase from Bacillus anthracis: Mechanism Revealed by Pre-Steady-State Kinetics

Wei, Yang; Kuzmic, Petr; Yu, Runhan; Modi, Gyan; Hedstrom, Lizbeth

Biochemistry (2016), 55 (37), 5279-5288CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

IMP dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP. IMPDH is an emerging target for antimicrobial therapy. The small mol. inhibitor, A 110, has been identified as potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent x-ray crystallog. study reported that the inhibitor binds to the NAD cofactor site and forms a ternary complex with IMP. Here, the authors report a pre-steady-state stopped-flow kinetic investigation of IMPDH of B. anthracis designed to assess the kinetic significance of the crystallog. results. Stopped-flow kinetic expts. defined 9 microscopic rate consts. and 2 equil. consts. that characterized both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results showed that the inhibitor bound with high affinity (Kd = ∼50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (Kd = ∼1 μM) was obsd. between the inhibitor, A110, and the enzyme-IMP (E-IMP) complex. Thus, the E-IMP-A110 ternary complex, obsd. by x-ray crystallog., is largely kinetically irrelevant.

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34.

Dunkern, T.; Prabhu, A.; Kharkar, P. S.; Goebel, H.; Rolser, E.; Burckhard-Boer, W.; Arumugam, P.; Makhija, M. T. Virtual and experimental high-throughput screening (HTS) in search of novel inosine 5′-monophosphate dehydrogenase II (IMPDH II) inhibitors. J. Comput.-Aided Mol. Des. 2012, 26, 1277– 1292, DOI: 10.1007/s10822-012-9615-5

[Crossref], [PubMed], [CAS]

34.

Virtual and experimental high-throughput screening (HTS) in search of novel inosine 5'-monophosphate dehydrogenase II (IMPDH II) inhibitors

Dunkern, Torsten; Prabhu, Arati; Kharkar, Prashant S.; Goebel, Heike; Rolser, Edith; Burckhard-Boer, Waltraud; Arumugam, Premkumar; Makhija, Mahindra T.

Journal of Computer-Aided Molecular Design (2012), 26 (11), 1277-1292CODEN: JCADEQ; ISSN:0920-654X. (Springer)

IMPDH (IMP dehydrogenase) catalyzes a rate-limiting step in the de novo biosynthesis of guanine nucleotides. IMPDH inhibition in sensitive cell types (e.g., lymphocytes) blocks proliferation (by blocking RNA and DNA synthesis as a result of decreased cellular levels of guanine nucleotides). This makes it an interesting target for cancer and autoimmune disorders. Currently available IMPDH inhibitors such as mycophenolic acid (MPA, uncompetitive inhibitor) and nucleoside analogs (e.g., ribavirin, competitive inhibitor after intracellular activation by phosphorylation) have unfavorable tolerability profiles which limit their use. Hence, the quest for novel IMPDH inhibitors continues. In the present study, a ligand-based virtual screening using IMPDH inhibitor pharmacophore models was performed on inhouse compd. collection. A total of 50,000 virtual hits were selected for primary screen using in vitro IMPDH II inhibition up to 10 μM. The list of 2,500 hits (with >70 % inhibition) was further subjected to hit confirmation for the detn. of IC50 values. The hits obtained were further clustered using max. common substructure based formalism resulting in 90 classes and 7 singletons. A thorough inspection of these yielded 7 interesting classes in terms of mini-SAR with IC50 values ranging from 0.163 μM to little over 25 μM. The av. ligand efficiency was found to be 0.3 for the best class. The classes thus discovered represent structurally novel chemotypes which can be taken up for further development.

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35.

Gorla, S. K.; Kavitha, M.; Zhang, M.; Chin, J. E.; Liu, X.; Striepen, B.; Makowska-Grzyska, M.; Kim, Y.; Joachimiak, A.; Hedstrom, L.; Cuny, G. D. Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase. J. Med. Chem. 2013, 56, 4028– 4043, DOI: 10.1021/jm400241j

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35.

Optimization of Benzoxazole-Based Inhibitors of Cryptosporidium parvum Inosine 5'-Monophosphate Dehydrogenase

Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D.

Journal of Medicinal Chemistry (2013), 56 (10), 4028-4043CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on IMP dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivs. with many compds. demonstrating CpIMPDH IC50 values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compds. are competitive inhibitors vs. NAD+. The SAR study reveals that pyridine and other small heteroarom. substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addn., several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochem. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine deriv. I demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. The racemic version of I, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity vs. a wild-type T. gondii strain (200-fold). No toxicity was obsd. (LD50 > 50 μM) against a panel of four mammalian cells lines.

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36.

Kitagawa, H.; Ozawa, T.; Takahata, S.; Iida, M.; Saito, J.; Yamada, M. Phenylimidazole derivatives of 4-pyridone as dual inhibitors of bacterial enoyl-acyl carrier protein reductases FabI and FabK. J. Med. Chem. 2007, 50, 4710– 4720, DOI: 10.1021/jm0705354

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36.

Phenylimidazole Derivatives of 4-Pyridone as Dual Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductases FabI and FabK

Kitagawa, Hideo; Ozawa, Tomohiro; Takahata, Sho; Iida, Maiko; Saito, Jun; Yamada, Mototsugu

Journal of Medicinal Chemistry (2007), 50 (19), 4710-4720CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

FabI and FabK are bacterial enoyl-acyl carrier protein (ACP) reductases that catalyze the final and rate-limiting step of bacterial fatty acid biosynthesis (FAS) and are potential targets of novel antibacterial agents. The authors have reported 4-pyridone deriv. 3 as a FabI inhibitor and phenylimidazole deriv. 5 as a FabK inhibitor. Here, the authors will report phenylimidazole derivs. of 4-pyridone as FabI and FabK dual inhibitors based on an iterative medicinal chem. and crystallog. study of FabK from Streptococcus pneumoniae/compd. 26. A representative compd. (I) showed strong FabI inhibitory (IC50 = 0.38 μM) and FabK inhibitory (IC50 = 0.0045 μM) activities with potent antibacterial activity against S. Pneumoniae (MIC = 0.5 μg/mL). Since elevated MIC value was obsd. against S. Pneumoniae mutant possessing one amino acid substitution in FabK, the antibacterial activity of the compd. was considered to be due to the inhibition of FabK. Moreover, this compd. showed no significant cytotoxicity (IC50 > 69 μM). These results support compd. I as a novel agent for the treatment of bacterial infections.

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37.

Soh, C. H.; Chui, W. K.; Lam, Y. An efficient and expeditious synthesis of di- and monosubstituted 2-aminoimidazoles. J. Comb. Chem. 2008, 10, 118– 122, DOI: 10.1021/cc700143n

[ACS Full Text ], [CAS]

37.

An Efficient and Expeditious Synthesis of Di- and Monosubstituted 2-Aminoimidazoles

Soh, Chai Hoon; Chui, Wai Keung; Lam, Yulin

Journal of Combinatorial Chemistry (2008), 10 (1), 118-122CODEN: JCCHFF; ISSN:1520-4766. (American Chemical Society)

A microwave-assisted protocol was developed for the construction of di- and monosubstituted 2-aminoimidazoles. The two-step reaction involves the synthesis of N-(1H-imidazol-2-yl)acetamides from readily available α-haloketones and N-acetylguanidine, followed by deacetylation. Significant rate enhancement was obsd. for both steps of the protocol, and the overall reaction time was shortened to 20 min compared to 48 h of the conventional procedures. A representative set of di- and monosubstituted 2-aminoimidazoles was prepd. using com. available parallel reactors.

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38.

Kumar, S.; Jaller, D.; Patel, B.; LaLonde, J. M.; DuHadaway, J. B.; Malachowski, W. P.; Prendergast, G. C.; Muller, A. J. Structure based development of phenylimidazole-derived inhibitors of indoleamine 2,3-dioxygenase. J. Med. Chem. 2008, 51, 4968– 4977, DOI: 10.1021/jm800512z

[ACS Full Text ], [CAS]

38.

Structure Based Development of Phenylimidazole-Derived Inhibitors of Indoleamine 2,3-Dioxygenase

Kumar, Sanjeev; Jaller, Daniel; Patel, Bhumika; LaLonde, Judith M.; DuHadaway, James B.; Malachowski, William P.; Prendergast, George C.; Muller, Alexander J.

Journal of Medicinal Chemistry (2008), 51 (16), 4968-4977CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathol. immune suppression. With the goal of developing more potent IDO inhibitors, a systematic study of 4-phenylimidazole (4-PI) derivs. was undertaken. Computational docking expts. guided design and synthesis efforts with analogs of 4-PI. In particular, three interactions of 4-PI analogs with IDO were studied: the active site entrance, the interior of the active site, and the heme iron binding. The three most potent inhibitors I (R = 4-HOC6H4, 3-HSC6H4, 4-HSC6H4) appear to exploit interactions with C129 and S167 in the interior of the active site. All three inhibitors are approx. 10-fold more potent than 4-PI. The study represents the first example of enzyme inhibitor development with the recently reported crystal structure of IDO and offers important lessons in the search for more potent inhibitors.

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39.

Jost, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragments. J. Med. Chem. 2014, 57, 7590– 7599, DOI: 10.1021/jm5006918

[ACS Full Text ], [CAS]

39.

Promiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragments

Jost Christian; Nitsche Christoph; Scholz Therese; Roux Lionel; Klein Christian D

Journal of medicinal chemistry (2014), 57 (18), 7590-9 ISSN:.

Covalent ligand-target interactions offer significant pharmacological advantages. However, off-target reactivity of the reactive groups, which usually have electrophilic properties, must be minimized, and the selectivity of irreversible inhibitors is a crucial requirement. We therefore performed a systematic study to determine the selectivity of several electrophilic groups that can be used as building blocks for covalently binding ligands. Six reactive groups with modulated electrophilicity were combined with 11 nonreactive moieties, resulting in a small combinatorial library of 72 fragment-like compounds. These compounds were screened against a group of 11 enzyme targets to assess their selectivity and their potential for promiscuous binding to proteins. The assay results showed a considerably lower degree of promiscuity than initially expected, even for those members of the screening collection that contain supposedly highly reactive electrophiles.

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40.

Fortin, S.; Moreau, E.; Lacroix, J.; Cote, M. F.; Petitclerc, E.; Gaudreault, R. C. Synthesis, antiproliferative activity evaluation and structure-activity relationships of novel aromatic urea and amide analogues of N-phenyl-N’-(2-chloroethyl)ureas. Eur. J. Med. Chem. 2010, 45, 2928– 2937, DOI: 10.1016/j.ejmech.2010.03.018

[Crossref], [PubMed], [CAS]

40.

Synthesis, antiproliferative activity evaluation and structure-activity relationships of novel aromatic urea and amide analogues of N-phenyl-N'-(2-chloroethyl)ureas

Fortin, Sebastien; Moreau, Emmanuel; Lacroix, Jacques; Cote, Marie-France; Petitclerc, Eric; C.-Gaudreault, Rene

European Journal of Medicinal Chemistry (2010), 45 (7), 2928-2937CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)

Seven subsets of arom. urea and amide analogs of N-phenyl-N'-(2-chloroethyl)ureas (CEU) have been synthesized by nucleophilic addn. of 3-chloropropylisocyanate, 2-chloroacetylisocyanate, ethylisocyanate, 2-chloroacetyl chloride, 3-chloropropanoyl chloride, 4-chlorobutanoyl chloride, and acryloyl chloride, resp., to selected anilines or benzylamines I [R1 = H, R2 = I, n = 0; R1 = H, R2 = tert-Bu, n = 0, 1; R1 = pentyl, R2 = H, n = 0; etc.] to afford 3-chloropropylureas (CPU) [II (X = NHCH2CH2CH2Cl)], 2-chloroacetylureas (CAU) [II (X = NHCOCH2Cl)], ethylureas (EU) [II (X = NHCOEt)], 2-chloroacetamides (CA) [II (X = CH2Cl)], 3-chloropropionamides (CPA) [II (X = CH2CH2Cl)], 4-chlorobutyramides (CBA) [II (X = CH2CH2CH2Cl)] and acrylamides (Acr) [II (X = CH:CH2)]. The mol. structure of these compds. has been confirmed by IR, 1H and 13C NMR, and MS spectra and their purity also confirmed by HPLC. The CEU analogs were evaluated for their antiproliferative activity against three human tumor cell lines, namely human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7. Some of CAU, CA, CPA and Acr had IC50 ranging from 1.4 to 25 μM. CAU, CA, CPA and Acr exhibited interesting antiproliferative activity through mechanism(s) of action unrelated to the acylation of glutamic acid at position 198 on β-tubulin that is characterizing CEU.

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41.

Bellina, F.; Guazzelli, N.; Lessi, M.; Manzini, C. Imidazole analogues of resveratrol: synthesis and cancer cell growth evaluation. Tetrahedron 2015, 71, 2298– 2305, DOI: 10.1016/j.tet.2015.02.024

[Crossref], [CAS]

41.

Imidazole analogues of resveratrol: synthesis and cancer cell growth evaluation

Bellina, Fabio; Guazzelli, Nicola; Lessi, Marco; Manzini, Chiara

Tetrahedron (2015), 71 (15), 2298-2305CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)

Novel trans-restricted analogs of resveratrol in which the C-C double bond of the natural deriv. was replaced by diaryl substituted imidazole analogs were designed. The syntheses of 1,4-, 2,4-, and 2,5-diarylimidazoles, in which the two aryl moieties are linked to the heteroarom. core in a 1,3 fashion to preserve the trans stereochem., were successfully carried out by regioselective sequential transition metal-catalyzed arylations of simple, com. available imidazole precursors. The anticancer activity of selected analogs was evaluated in vitro against the NCI-60 human tumor cell lines panel. From this screening, the authors were able to select a synthetic candidate that resulted more active than its natural lead.

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42.

Ascher, D. B.; Cromer, B. A.; Morton, C. J.; Volitakis, I.; Cherny, R. A.; Albiston, A. L.; Chai, S. Y.; Parker, M. W. Regulation of insulin-regulated membrane aminopeptidase activity by its C-terminal domain. Biochemistry 2011, 50, 2611– 2622, DOI: 10.1021/bi101893w

[ACS Full Text ], [CAS]

42.

Regulation of insulin-regulated membrane aminopeptidase activity by its C-terminal domain

Ascher, David B.; Cromer, Brett A.; Morton, Craig J.; Volitakis, Irene; Cherny, Robert A.; Albiston, Anthony L.; Chai, Siew Yeen; Parker, Michael W.

Biochemistry (2011), 50 (13), 2611-2622CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

The development of inhibitors of insulin-regulated aminopeptidase (IRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that assocd. with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homol. models based on the structures of recently detd. related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examd. their kinetic and biochem. properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments contg. domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that IRAP has a second Zn2+ binding site, not assocd. with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP.

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43.

Kabsch, W. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132, DOI: 10.1107/S0907444909047337

[Crossref], [PubMed], [CAS]

43.

Software XDS for image rotation, recognition and crystal symmetry assignment

Kabsch, Wolfgang

Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.

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44.

Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221, DOI: 10.1107/S0907444909052925

[Crossref], [PubMed], [CAS]

44.

PHENIX: a comprehensive Python-based system for macromolecular structure solution

Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.

Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

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45.

Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126– 2132, DOI: 10.1107/S0907444904019158

[Crossref], [PubMed], [CAS]

45.

Coot: model-building tools for molecular graphics

Emsley, Paul; Cowtan, Kevin

Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)

CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.

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46.

Jubb, H. C.; Higueruelo, A. P.; Ochoa-Montano, B.; Pitt, W. R.; Ascher, D. B.; Blundell, T. L. Arpeggio: A web server for calculating and visualising interatomic interactions in protein structures. J. Mol. Biol. 2017, 429, 365– 371, DOI: 10.1016/j.jmb.2016.12.004

[Crossref], [PubMed], [CAS]

46.

Arpeggio: A Web Server for Calculating and Visualising Interatomic Interactions in Protein Structures

Jubb, Harry C.; Higueruelo, Alicia P.; Ochoa-Montano, Bernardo; Pitt, Will R.; Ascher, David B.; Blundell, Tom L.

Journal of Molecular Biology (2017), 429 (3), 365-371CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)

Interactions between proteins and their ligands, such as small mols., other proteins, and DNA, depend on specific interat. interactions that can be classified on the basis of atom type and distance and angle constraints. Visualization of these interactions provides insights into the nature of mol. recognition events and has practical uses in guiding drug design and understanding the structural and functional impacts of mutations. The authors present Arpeggio, a web server for calcg. interactions within and between proteins and protein, DNA, or small-mol. ligands, including van der Waals', ionic, carbonyl, metal, hydrophobic, and halogen bond contacts, and hydrogen bonds and specific atom-arom. ring (cation-π, donor-π, halogen-π, and carbon-π) and arom. ring-arom. ring (π-π) interactions, within user-submitted macromol. structures. PyMOL session files can be downloaded, allowing high-quality publication images of the interactions to be generated. Arpeggio is implemented in Python and available as a user-friendly web interface at http://structure.bioc.cam.ac.uk/arpeggio/ and as a downloadable package at https://bitbucket.org/harryjubb/arpeggio.

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47.

Pires, D. E.; Blundell, T. L.; Ascher, D. B. mCSM-lig: quantifying the effects of mutations on protein-small molecule affinity in genetic disease and emergence of drug resistance. Sci. Rep. 2016, 6, 29575, DOI: 10.1038/srep29575

[Crossref], [PubMed], [CAS]

47.

mCSM-lig: quantifying the effects of mutations on protein-small molecule affinity in genetic disease and emergence of drug resistance

Pires Douglas E V; Blundell Tom L; Ascher David B; Pires Douglas E V

Scientific reports (2016), 6 (), 29575 ISSN:.

The ability to predict how a mutation affects ligand binding is an essential step in understanding, anticipating and improving the design of new treatments for drug resistance, and in understanding genetic diseases. Here we present mCSM-lig, a structure-guided computational approach for quantifying the effects of single-point missense mutations on affinities of small molecules for proteins. mCSM-lig uses graph-based signatures to represent the wild-type environment of mutations, and small-molecule chemical features and changes in protein stability as evidence to train a predictive model using a representative set of protein-ligand complexes from the Platinum database. We show our method provides a very good correlation with experimental data (up to ρ = 0.67) and is effective in predicting a range of chemotherapeutic, antiviral and antibiotic resistance mutations, providing useful insights for genotypic screening and to guide drug development. mCSM-lig also provides insights into understanding Mendelian disease mutations and as a tool for guiding protein design. mCSM-lig is freely available as a web server at http://structure.bioc.cam.ac.uk/mcsm_lig.

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48.

Pires, D. E.; Ascher, D. B. CSM-lig: a web server for assessing and comparing protein-small molecule affinities. Nucleic Acids Res. 2016, 44, W557– 61, DOI: 10.1093/nar/gkw390

[Crossref], [PubMed], [CAS]

48.

CSM-lig: a web server for assessing and comparing protein-small molecule affinities

Pires, Douglas E. V.; Ascher, David B.

Nucleic Acids Research (2016), 44 (W1), W557-W561CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

Detg. the affinity of a ligand for a given protein is a crucial component of drug development and understanding their biol. effects. Predicting binding affinities is a challenging and difficult task, and despite being regarded as poorly predictive, scoring functions play an important role in the anal. of mol. docking results. Here, we present CSM-Lig, a web server tailored to predict the binding affinity of a protein-small mol. complex, encompassing both protein and small-mol. complementarity in terms of shape and chem. via graph-based structural signatures. CSM-Lig was trained and evaluated on different releases of the PDB bind databases, achieving a correlation of up to 0.86 on 10-fold cross validation and 0.80 in blind tests, performing as well as or better than other widely used methods. The web server allows users to rapidly and automatically predict binding affinities of collections of structures and assess the interactions made. We believe CSMlig would be an invaluable tool for helping assess docking poses, the effects of multiple mutations, including insertions, deletions and alternative splicing events, in protein-small mol. affinity, unraveling important aspects that drive protein-compd. recognition.

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49.

Pires, D. E.; Blundell, T. L.; Ascher, D. B. pkCSM: Predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. J. Med. Chem. 2015, 58, 4066– 4072, DOI: 10.1021/acs.jmedchem.5b00104

[ACS Full Text ], [CAS]

49.

pkCSM: Predicting Small-Molecule Pharmacokinetic and Toxicity Properties Using Graph-Based Signatures

Pires, Douglas E. V.; Blundell, Tom L.; Ascher, David B.

Journal of Medicinal Chemistry (2015), 58 (9), 4066-4072CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

Drug development has a high attrition rate, with poor pharmacokinetic and safety properties a significant hurdle. Computational approaches may help minimize these risks. The authors have developed a novel approach (pkCSM) which uses graph-based signatures to develop predictive models of central ADMET properties for drug development. PkCSM performs as well or better than current methods. A freely accessible web server (http://structure.bioc.cam.ac.uk/pkcsm), which retains no information submitted to it, provides an integrated platform to rapidly evaluate pharmacokinetic and toxicity properties.

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50.

Singh, V.; Brecik, M.; Mukherjee, R.; Evans, J. C.; Svetlikova, Z.; Blasko, J.; Surade, S.; Blackburn, J.; Warner, D. F.; Mikusova, K.; Mizrahi, V. The complex mechanism of antimycobacterial action of 5-fluorouracil. Chem. Biol. 2015, 22, 63– 75, DOI: 10.1016/j.chembiol.2014.11.006

[Crossref], [PubMed], [CAS]

50.

The Complex Mechanism of Antimycobacterial Action of 5-Fluorouracil

Singh, Vinayak; Brecik, Miroslav; Mukherjee, Raju; Evans, Joanna C.; Svetlikova, Zuzana; Blasko, Jaroslav; Surade, Sachin; Blackburn, Jonathan; Warner, Digby F.; Mikusova, Katarina; Mizrahi, Valerie

Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (1), 63-75CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)

A combination of chem. genetic and biochem. assays was applied to investigate the mechanism of action of the anticancer drug 5-fluorouracil (5-FU), against Mycobacterium tuberculosis (Mtb). 5-FU resistance was assocd. with mutations in upp or pyrR. Upp-catalyzed conversion of 5-FU to FUMP was shown to constitute the first step in the mechanism of action, and resistance conferred by nonsynonymous SNPs in pyrR shown to be due to derepression of the pyr operon and rescue from the toxic effects of FUMP and downstream antimetabolites through de novo prodn. of UMP. 5-FU-derived metabolites identified in Mtb were consistent with the obsd. incorporation of 5-FU into RNA and DNA and the reduced amt. of mycolyl arabinogalactan peptidoglycan in 5-FU-treated cells. Conditional depletion of the essential thymidylate synthase ThyX resulted in modest hypersensitivity to 5-FU, implicating inhibition of ThyX by fluorodeoxyuridylate as a further component of the mechanism of antimycobacterial action of this drug.

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