Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family.

We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family of serine/threonine kinases. Here we described the optimisation of those compounds to improve their physicochemical and ADME properties as well as reducing their off-targets activities against other kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies, advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in a concentration dependent manner.

Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family.

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75µM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.

Identification of inhibitors of the Leishmania cdc2-related protein kinase CRK3.

New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high-throughput screen of a focussed kinase set of ~3400 compounds to identify potent and parasite-selective inhibitors of an enzymatic Leishmania CRK3-cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3-CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low-nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.

High throughput screens yield small molecule inhibitors of Leishmania CRK3:CYC6 cyclin-dependent kinase.

BACKGROUND: Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites. METHODOLOGY/PRINCIPAL FINDINGS: In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major. CONCLUSIONS/SIGNIFICANCE: The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178.

The activity of cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, is regulated through post-translational mechanisms, including binding of a cyclin and phosphorylation. Previously studies have shown that Leishmania mexicana CRK3 is an essential CDK that is a functional homologue of human CDK1. In this study, recombinant histidine tagged L. mexicana CRK3 and the cyclin CYCA were combined in vitro to produce an active histone H1 kinase that was inhibited by the CDK inhibitors, flavopiridol and indirubin-3'-monoxime. Protein kinase activity was observed in the absence of phosphorylation of the T-loop residue Thr178, but increased 5-fold upon phosphorylation by the CDK activating kinase Civ1 of Saccharomyces cerevisiae. Seven recombinant L. major CRKs (1, 2, 3, 4, 6, 7 and 8) were also expressed and purified, none of which were active as monomers. Moreover, only CRK3 was phosphorylated by Civ1. HA-tagged CYCA expressed in L. major procyclic promastigotes was co-precipitated with CRK3 and exhibited histone H1 kinase activity. These data indicate that in Leishmania CYCA interacts with CRK3 to form an active protein kinase, confirm the conservation of the regulatory mechanisms that control CDK activity in other eukaryotes, but identifies biochemical differences to human CDK1.