Explorer High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK 3 : CYC 6 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 cyclindependent 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 antiparasitic 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. Citation: Walker RG, Thomson G, Malone K, Nowicki MW, Brown E, et al. (2011) High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase. PLoS Negl Trop Dis 5(4): e1033. doi:10.1371/journal.pntd. 0001033 Editor: Kiyoshi Kita, University of Tokyo, Japan Received July 13, 2010; Accepted March 10, 2011; Published April 5, 2011 Copyright: 2011 Walker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Medical Research Council (www.mrc.ac.uk; grant number G0400028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.mottram@udcf.gla. ac.uk