High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase

Abstract

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 FindingsIn 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/SignificanceThe 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.

2-Amino-4-arylthiazole Derivatives as Anti-giardial Agents: Synthesis, Biological Evaluation and QSAR Studies

“…In the chemotherapy field of protozoan diseases, substituted 2-aminothiazoles have been investigated due to their broad anti-parasitic spectrum. These structures have been tested as pharmacophores in many synthetic compounds and have shown in vitro and in vivo activities against Trypanosoma [15], Leishmania [16], Plasmodium [17] and Toxoplasma [18], among others protozoa [19].…”

New Chemotherapeutic Strategies Against Malaria, Leishmaniasis and Trypanosomiases

“…By means of high throughput screening techniques, molecules based on azapurine and thiazole cores, inactive against human cyclin-dependent kinases but active against L. major promastigotes and amastigotes were identified. These hits are meant to be used for future hit-to-lead synthesis programs [176]. However, there are evidences that molecules highly active against L. mexicana cyclin-dependent cdc2-related serine/threonine protein kinase CRK3 do not show any leishmanicidal activity in vitro, suggesting that the activity of this specific enzyme is not indispensable for parasite survival contrary to indications provided by genetic manipulation studies [177].…”

Development of anEx VivoLymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

“…The RLUs of the signals were transformed to percentages of parasite inhibition (described previously). Hits were defined as compounds displaying Ͼ50% inhibition of light emission (15,17,25). The percentage of parasite inhibition with regard to controls was calculated as 100 Ϫ [(parasite counts in treated cells/parasite counts in untreated cells) ϫ 100].…”

“…The parasite's enzymatic repertoire and structure (e.g., membrane) have been used as drug targets, but questions have been raised as to the efficacy of these approaches compared with screenings that utilize whole parasites. Most screening methods have used promastigotes (the insect stage of Leishmania) to identify hits; however, these required subsequent validation assays using amastigote-infected macrophages (15)(16)(17).…”