Characterization of Competitive Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is an enticing antimalarial drug target. Novel chemotypes are needed because existing inhibitors have safety issues that may prevent further development. This work demonstrates isoxazole-based compounds are potent ATP competitive inhibitors of PfPKG and discloses a new analogue in this series. Isoxazoles 3 and 5 had Ki values that are comparable to a known standard, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine. They also exhibited excellent selectivity for PfPKG over the human orthologue and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human orthologue. The human orthologue's larger binding site volume is predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme.

Total synthesis and evaluation of C26-hydroxyepothilone D derivatives for photoaffinity labeling of beta-tubulin.

Three photoaffinity labeled derivatives of epothilone D were prepared by total synthesis, using efficient novel asymmetric synthesis methods for the preparation of two important synthetic building blocks. The key step for the asymmetric synthesis of (S,E)-3-(tert-butyldimethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)pent-4-enal involved a ketone reduction with (R)-Me-CBS-oxazaborolidine. For the synthesis of (5S)-5,7-di[(tert-butyldimethylsilyl)oxy]-4,4-dimethylheptan-3-one an asymmetric Noyori reduction of a beta-ketoester was employed. The C26 hydroxyepothilone D derivative was constructed following a well-established total synthesis strategy and the photoaffinity labels were attached to the C26 hydroxyl group. The photoaffinity analogues were tested in a tubulin assembly assay and for cytotoxicity against MCF-7 and HCT-116 cancer cell lines. The 3- and 4-azidobenzoic acid analogues were found to be as active as epothilone B in a tubulin assembly assay, but demonstrated significantly reduced cellular cytotoxicity compared to epothilone B. The benzophenone analogue was inactive in both assays. Docking and scoring studies were conducted that suggested that the azide analogues can bind to the epothilone binding site, but that the benzophenone analogue undergoes a sterically driven ligand rearrangement that interrupts all hydrogen bonding and therefore protein binding. Photoaffinity labeling studies with the 3-azidobenzoic acid derivative did not identify any covalently labeled peptide fragments, suggesting that the phenylazido side chain was predominantly solvent-exposed in the bound conformation.

Discovery of isoxazolyl-based inhibitors of Plasmodium falciparum cGMP-dependent protein kinase.

The cGMP-dependent protein kinase in Plasmodium falciparum (PfPKG) plays multiple roles in the life cycle of the parasite. As a result, this enzyme is a potential target for new antimalarial agents. Existing inhbitors, while potent and active in malaria models are not optimal. This communication describes initial optimization of a structurally distinct class of PfPKG inhibitors.

Allosteric Indole Amide Inhibitors of p97: Identification of a Novel Probe of the Ubiquitin Pathway.

A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.

Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases.

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009-0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 cocrystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4, and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics.

A novel potent indazole carboxylic acid derivative blocks spermatogenesis and is contraceptive in rats after a single oral dose.

Women have historically been the focus for development of new contraceptive methods. The National Institutes of Health, World Health Organization, and Institute of Medicine have stressed the need to develop nonhormonal, nonsteroidal male contraceptive agents. We report results from initial dose-ranging studies of a new indazole carboxylic acid analogue, gamendazole. An infertility rate of 100% was achieved in seven out of seven proven-fertile male rats 3 wk after a single oral dose of 6 mg/kg of gamendazole. Fertility returned by 9 wk in four of seven animals, with typical numbers of normal-appearing conceptuses. A fertility rate of 100% returned in four of six animals that became infertile at a single oral dose of 3 mg/kg of gamendazole. No differences in mating behavior were observed in either of the gamendazole-treated groups versus the control (vehicle-only) group. In the animals that showed reversible infertility, a transient increase in circulating FSH levels coincided with an initial decline in inhibin B levels after administration of gamendazole, but no other significant changes in circulating reproductive hormones were observed. Gamendazole inhibited production of inhibin B by primary Sertoli cells in vitro with a median inhibitory concentration of 6.8 thorn+/- 3.0 (SEM) (3/4)x 10(-10) M, suggesting that Sertoli cells are a primary target. A biotinylated gamendazole analogue revealed cytoplasmic and perinuclear binding of gamendazole in primary Sertoli cells. Gamendazole represents the most potent new oral antispermatogenic indazole carboxylic acid to date. Our results, however, demonstrate that additional dose-finding studies are required to improve reversibility and widen the therapeutic window before more detailed drug development of this potential nonhormonal male contraceptive agent can occur.

Gamendazole, an orally active indazole carboxylic acid male contraceptive agent, targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and stimulates Il1a transcription in rat Sertoli cells.

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.