Aminothiazole Secretase Inhibitors

 A new γ-secretase modulators with an Aminothiazole core starting from a HTS hit is disclosed .  Also, synthesis and SAR of this series are discussed.

These novel compounds demonstrate moderate to good in vitro potency in inhibiting amyloid beta (Aβ) peptide production.

Overall γ-secretase is not inhibited but the formation of the aggregating, toxic Aβ42 peptide is shifted to smaller non-aggregating Aβ peptides. Compound 15 reduced brain Aβ42 in vivo in APPSwe transgenic mice at 30 mg/kg p.o.

Dynamin GTPase Inhibitory Activity: Rhodadyns

From Rhodanine To Dynamin I GTPase Inhibitors







Six focused rhodanine-based libraries, 60 compounds in total,
were synthesized and evaluated as potential dynamin I GTPase inhibitors.

Twenty-six were more potent than the lead compound with 13 returning IC₅₀ values ≤10 μM, making the Rhodadyn series among the most active dynamin inhibitors reported.

Two analogues were highly effective at blocking receptor-mediated endocytosis: C10 and D10 with IC_50(RME) = 7.0 ± 2.2 and 5.9 ± 1.0 μM, respectively.

 These compounds are equipotent with the best reported in-cell dynamin inhibitors.

http://pubs.acs.org/doi/abs/10.1021/ml200284s

Dynamin is required for clathrin-mediated endocytosis (CME).

 Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization.

 

We have designed a series of novel pyrimidine-based “Pyrimidyn” compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency.

 

 The most potent analogue, Pyrimidyn 7, has an IC₅₀ of 1.1 μM for

dynamin I and 1.8 μM for dynamin II, making it among the most potent dynamin inhibitors identified to date.

We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin.
The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin.

Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals.

Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation.
This dual mode of action provides an important new tool for molecular dissection of dynamin’s role in endocytosis.

 

 

 

Protein Misfolding Modulators

Aldose Reductase Inhibitor can Inhibit tau Aggregation.

 

Alzheimer's disease is often referred as protein misfolding disease through peptide plaque accumulation of abnormally folded amyloid such as beta amyloid (Aβ) and tau amyloid protein in the brain system. According to the precedent reviews, some aggregation inhibitors of tau protein as well as selected amyloids are becoming a cornerstone of the capable chemotherapy for Alzheimer disease and other neurodegenerative symptoms.

 

 

 

Among the emerging small molecule tau inhibitors, particular heterocyclic structural motifs such as rhodanines and cyanine dyes are highlighted as promising and privileged key pharmacophores. Guided by these precedent findings, we are interested in the behavior and inhibitory functions of some generic (classical) drugs with traditional pigment skeletons towards tau protein aggregation. Thus, described herein is our preliminary investigation on the modulating activity of the aldose reductase inhibitor eparlestat on the tau aggregation under the in vitro experiments.

 

 

 

Rhodanine derivatives are classical heterocyclic dye materials, which recently have been investigated intensively by the German researchers towards tau interactions. Their previous results as well as commentary summary indicated the similar potency of the generic drug eparlestat, in view of the pre-existing nontoxic drugs, although no experimental results available. Thus, in order to gain a precise knowledge and evaluate the exact pleiotropic effects of eparlestat on tau aggregation, we have been investigating of some existing generic drugs along these lines, as summarized below.

 

In our preliminary in vitro experiments, three generic drugs at hands are investigated towards tau aggregation inhibitory activity along with the known methylene blue as a positive standard.

Rhodanine can Visualize Neurofibrillary Tangles

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer’s diagnosis.

A series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay.


However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays.


Probe 6 showed binding affinity (IC₅₀ = 19 nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC₅₀ values of lower than 100 nM to tau aggregates to displace Thiazine red R.


Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity.

The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72 h post fertilization.

 

 

 

 

 

 

Cyanine Dyes Can Modulate tau Aggregation.

A structure−activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay.

 

Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3′-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood−brain barrier and cell membrane penetration.

 

 

Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone.

 

 

In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity.

 

 

http://pubs.acs.org/doi/abs/10.1021/jm900116d?prevSearch=N744&searchHistoryKey=

 

 

Tranilast Binds to Aβ Monomers and Promotes Aβ Fibrillation

AD may be a potential complication for tranilast usage in elderly patients.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer’s disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role.

 

 

We used solution NMR to determine that tranilast binds to Aβ40 monomers with 300 μM affinity.

 

Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time.

 

 

Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ–tranilast interaction.

 

 

These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

http://pubs.acs.org/doi/abs/10.1021/bi400426t?prevSearch=tranilast&searchHistoryKey=

Curcuminoids Block TGF-b Sibnaling in HBC

Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated.

 

 

In vitro, transforming growth factor (TGF)-β-stimulated PTHrP secretion was inhibited by curcuminoids (IC₅₀ = 24 μM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition.


Effects on TGF-β signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-β signaling.


 In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days.


Osteolytic bone lesion area was reduced up to 51% (p < 0.01).


Consistent with specific effects on bone osteolysis, osteoclast number at the bone–tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered.


 In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment.


These data suggest that curcuminoids prevent TGF-β induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.

 

 

Novel Inhibitors of TGF Signaling

 

 

Annulated 1,4-Dihydropyridines Cardiomyogenic Compounds

as Novel Inhibitors of TGFβ Signaling

 

 

 

A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited transforming growth factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface.

 

 

Because this is an unprecedented mechanism of action, we explored the series’ structure–activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs.

 

 

We determined a pharmacophore and generated 1,4-DHPs with IC₅₀s for TGFβ inhibition in the nanomolar range (e.g., compound 28, 170 nM).

 

 

Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (−) enantiomer.

 

 

This stereopreference was not observed for the low level inhibition against Activin A signaling and was reversed for effects on calcium handling in HL-1 cells.

 

 

 

 

http://pubs.acs.org/doi/abs/10.1021/jm301144g?prevSearch=TGF%25CE%25B2%2BInhibitor&searchHistoryKey=

 

 

 

 

 

Cyanine Dye N744 Inhibits Tau Fibrillization

Cyanine Dye N744 Inhibits Tau Fibrillization by Blocking Filament Extension:  Implications for the Treatment of Tauopathic Neurodegenerative Diseases

 

 

 

 

 

 

 

Orally Bioavailable, Brain Penetrant Kinase 3 Inhibitor for Parkinsons D

Orally Bioavailable, Brain Penetrant Inhibitor of Mixed Lineage Kinase 3

 

 

 

 

 

 

 

 

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson’s disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels.

 

 

 

We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC₅₀ = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels.

 

 

 

The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC₅₀ = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

 

 

 

Fluorescence Probes for Protease and Glycosidase

We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent.

 

 

 

On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal.

 

 

 

To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG).

 

 

All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes.

 

 

We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biological and pathological investigations.

 

 

 

Adventures in Kinase Inhibitor Design and Optimization

Through the Looking Glass: Adventures in Kinase Inhibitor Design and Optimization

 

Robert D. Clark *

Department of Life Sciences, Simulations Plus, Inc., 42505 10th Street West, Lancaster, California 93534, United States

J. Med. Chem., 2013, 56 (5), pp 1796–1798

DOI: 10.1021/jm400243u

Abstract

Developing a viable new drug candidate is difficult.

Developing one that is a small molecule kinase inhibitor that binds competitively with respect to ATP with superb selectivity is even more difficult, which makes the design and optimization work described by Jimenez et al. (J. Med. Chem., DOI: 10.1021/jm301465a) particularly remarkable.

 

 

 They took a lead from a high-throughput screen against protein kinase C θ (PKCθ) through a series of optimization steps, culminating in the demonstration of in vivo activity in mice. Having identified and improved the hinge-binding “warhead” at one end of their lead molecule, they proceeded to use structure-based design tools to guide modification of the other end to enhance selectivity over a closely related isoform of the kinase. With that accomplished, they used a series of protection and deprotection maneuvers to modify the central portion of the series scaffold to further enhance potency against the target while also improving pharmacokinetic properties.

 

 

The project was a success at the preclinical level: oral administration of the ultimate analogue obtained was effective at suppressing interleukin-2 induction in mice.

 

 

 

 

Epigenetic Regulator I-BET762

Benzodiazepine & Benzoxazepine Inhibitors: 2013 JMC

Tau Aggregation Inhibitors

Agents capable of preventing the misfolding and sequestration of the microtubule-stabilizing protein tau into insoluble fibrillar aggregates hold considerable promise for the prevention and/or treatment of neurodegenerative tauopathies such as Alzheimer’s disease.

 

 

Because tauopathies are characterized by amyloidosis that is restricted to the central nervous system (CNS), plausible candidate compounds for in vivo evaluation must both prevent tau fibrillization and achieve significant brain levels.

 

 

Recently, we reported the discovery of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors

 

and now describe a series of new analogues that are both effective inhibitors of tau fibrillization and display significant brain-to-plasma exposure ratios after administration to mice.

 

 

 

Further, two of the most promising examples, 15 and 16, were found to reach significant brain exposure levels following oral administration. Taken together, these results suggest that examples from the ATPZ class hold promise as candidates for in vivo efficacy studies in animal models of neurodegenerative tauopathies.

Tau Complex Inhibition by Prototype Inhibitors

Kinetic Studies of Cdk5/p25 Kinase:

Phosphorylation of Tau and Complex Inhibition by Two Prototype Inhibitors








These studies served as a necessary kinetic backdrop for investigations of the mechanism of inhibition by prototype inhibitors :

N4-(6-aminopyrimidin-4-yl)-sulfanilamide (APS) and 1-(5-cyclobutyl-thiazol-2-yl)-3-isoquinolin-5-yl-urea (CTIU).


We found that the cdk5/p25-catalyzed phosphorylation of tau follows a rapid equilibrium, random kinetic mechanism, as evidenced by initial velocity analysis indicating sequential addition of tau and ATP, and studies of the mechanism of inhibition by substrate analogue AMP, product ADP, and analogues of peptide substrate H1P. Identical mechanistic conclusions were drawn when H1P was the phosphoryl acceptor.



Subsequent studies of inhibition by APS and CTIU revealed that
both compounds can bind to all four steady-state forms of the enzyme,
to form the complexes E:I, E:I:tau, E:I:ATP, and E:I:tau:ATP.



These results contrast with reported claims that APS and CTIU are competitive inhibitors of the binding of ATP.

Type II Kinase Inhibitors

 

 

 

A number of well-known type II inhibitors (ATP-noncompetitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson’s disease-linked mutation, G2019S.

 

 

We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between the wild type (WT) and the G2019S mutant of LRRK2.

 

 

The type II kinase inhibitors were found to work in an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected noncompetitive mechanism against WT.

 

 

 

Because the G2019S mutation lies in the DXG motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics.

 

 

 

The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form.

 

 

The conformational bias toward the active form of LRRK2 mutants has two primary consequences. (1)

 

 

 

The mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being “locked” into the activated state, and (2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP-competitive fashion.

 

 

 

Our results thus suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors because of desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant.