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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., Author Summary CRK3, a cdc2-related serine/threonine protein kinase of the CDK family, is essential for transition through the G2-M phase checkpoint of the Leishmania cell cycle. An expression and purification system has been developed to produce active L. major CRK3 in complex with a cyclin partner, CYC6. CRK3:CYC6 was used to develop an assay suitable for high throughput screening (HTS) using IMAP fluorescence polarization technology. Two compound chemical libraries were screened against CRK3:CYC6 and counter screened against a human cyclin-dependent kinase complex CDK2:CycA. Two main chemical families of inhibitors were identified that specifically inhibited the leishmanial cyclin-dependent kinase, the azapurines and the thiazoles. Structure activity relationship (SAR) analysis of the hits identified the chemical groups attached to the azapurine scaffold that are essential for the inhibition of CRK3:CYC6 protein kinase activity. The CRK3:CYC6 hits were subsequently tested against a panel of 11 mammalian kinases including human CDK1:CYCB, human CDK2:CYCA and human CDK4:CYCD1 to determine their selectivity. Compounds selective to CRK3:CYC6 were tested against Leishmania. Progress towards synthesising potent and selective derivatives of the HTS hits are discussed, with the view to evaluating their potential for the development of novel therapeutics against leishmaniasis.