Pavel A. Petukhov

5-tert-butyl-N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives as novel potent inhibitors of Mycobacterium tuberculosis pantothenate synthetase: initiating a quest for new antitubercular drugs.

Pantothenate synthetase (PS) is one of the potential new antimicrobial targets that may also be useful for the treatment of the nonreplicating persistent forms of Mycobacterium tuberculosis. In this Letter we present a series of 5- tert-butyl- N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[ d]isoxazole-3-carboxamide derivatives as novel potent Mycobacterium tuberculosis PS inhibitors, their in silico molecular design, synthesis, and inhibitory activity.

Highly Potent and Specific GSK-3β Inhibitors That Block Tau Phosphorylation and Decrease α-Synuclein Protein Expression in a Cellular Model of Parkinson's Disease

Research by Klein and co‐workers suggests that the inhibition of GSK‐3β by small molecules may offer an important strategy in the treatment of a number of central nervous system (CNS) disorders including Alzheimers disease, Parkinsons disease, and bipolar disorders. Based on results from kinase‐screening assays that identified a staurosporine analogue as a modest inhibitor of GSK‐3β, a series of 3‐indolyl‐4‐indazolylmaleimides was prepared for study in both enzymatic and cell‐based assays. Most strikingly, whereas we identified ligands having poor to high potency for GSK‐3β inhibition, only ligands with a Ki value of less than 8 nM, namely maleimides 18 and 22, were found to inhibit Tau phosphorylation at a GSK‐3β‐specific site (Ser 396/404). Accordingly, maleimides 18 and 22 may protect neuronal cells against cell death by decreasing the level of α‐Syn protein expression. We conclude that the GSK‐3β inhibitors described herein offer promise in defending cells against MPP+‐induced neurotoxicity and that such compounds will be valuable to explore in animal models of Parkinsons disease as well as in other Tau‐related neurodegenerative disease states.

Phosphatidylinositol Ether Lipid Analogues That Inhibit AKT Also Independently Activate the Stress Kinase, p38α, through MKK3/6-independent and -dependent Mechanisms

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38α, was uniformly activated 2-fold. Molecular modeling of p38α revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38α-/- cells, we showed that p38α is not required for PIA-induced apoptosis but is required for H2O2- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38α-/- cells increased apoptosis. These studies indicate that p38α is activated by PIAs through a novel mechanism and show that p38α activation contributes to PIA-induced cell death. Independent modulation of Akt and p38α could account for the profound cytotoxicity of PIAs.

Design, Synthesis, Docking, and Biological Evaluation of Novel Diazide-containing Isoxazole- and Pyrazole-based Histone Deacetylase Probes

The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.

Chemical Medicine: Novel 10-Substituted Cytisine Derivatives with Increased Selectivity for α4β2 Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChR) are widely distributed in the central and peripheral nervous systems. These receptors are crucial to many normal physiological functions, and they are involved in a wide range of diseases. For these reasons nAChRs are important targets for drug discovery. Neuronal nAChRs are composed of subunits (a2-a10, b2-b4) that form pentameric ligand-gated cation channels. Different combinations of subunits define the nAChR subtypes. Among all heteromeric nAChR subtypes in the vertebrate brain, the a4b2 receptor is the predominant subtype. In contrast, the a3b4 nAChR is the major subtype in ganglia. The a4b2 receptors are involved in important brain functions, including memory, cognition, arousal, relaxation, and pleasure. 20,21] Several lines of evidence suggest that the a4b2 nAChRs mediate addiction to nicotine in tobacco smoking. In addition to acetylcholine and ( )-nicotine (1), many nAChR ligands have been discovered from natural sources, including epibatidine (2) and ( )-cytisine (3). The structures of nicotine, epibatidine, cytisine, and other natural nicotinic ligands have led to the development of a host of other synthetic compounds that act on neuronal nicotinic receptors. Although many of the synthetic analogues of these natural ligands exhibit excellent overall selectivity to neuronal nAChRs over other families of receptors, the development of ligands highly selective for a certain nAChR subtype over other subtypes has been slow because of the large number of nAChR subtypes together with the relatively subtle differences in their structures. ( )-Cytisine (3) is a plant alkaloid isolated originally from the gold chain tree (Laburnum anagyroides). This ligand binds to a4b2 nAChRs with a much higher affinity than it binds to a3b4 receptors in in vitro ligand binding assays. However, when its effects on nAChR channel function were measured in heterologous expression models, cytisine is a full or nearly full agonist at a3b4 nAChRs but a partial agonist at b2-containing receptors, stimulating less than 50% of the maximal response elicited by acetylcholine or nicotine. Cytisine has been marketed in central and eastern Europe for aiding smoking cessation for more than 40 years though its effectiveness and safety have not been conclusively established through randomized, double-blind, placebo-controlled clinical trials. As cytisine shows poor penetration of the bloodbrain barrier this may be one of the reasons that cytisine failed to show a robust effect in the clinic. In spite of these clinical shortcomings, the interesting pharmacological profile of cytisine has led to a number of medicinal chemistry efforts in recent years to create analogues with an improved subtype selectivity or PK parameters. 10,39–42] Most of these reported structural modifications involve the introduction of substituents at the alicyclic nitrogen (position 3), or at the 9or 11positions of the pyridone ring (Figure 1).

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors.

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimers disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

A Chimeric SERM–Histone Deacetylase Inhibitor Approach to Breast Cancer Therapy

Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (−)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (−) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (−) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four type I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1–3 μM potency across all targets. The superior hybrid caused significant cell death in ER (−) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.

Molecular modeling, synthesis, and activity studies of novel biaryl and fused-ring BACE1 inhibitors

A series of transition state analogues of beta-secretases 1 and 2 (BACE1, 2) inhibitors containing fused-ring or biaryl moieties were designed computationally to probe the S2 pocket, synthesized, and tested for BACE1 and BACE2 inhibitory activity. It has been shown that unlike the biaryl analogs, the fused-ring moiety is successfully accommodated in the BACE1 binding site resulting in the ligands with excellent inhibitory activity. Ligand 5b reduced 65% of Abeta40 production in N2a cells stably transfected with Swedish human APP.

Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2.

The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.

Kaempferol Exhibits Progestogenic Effects in Ovariectomized Rats

OBJECTIVE Progesterone (P4) plays a central role in womens health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri. METHODS Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro. RESULTS Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors. CONCLUSION Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation.