Mindy I. Davis Ph.D. National Institute of Health

Bioinformatic Analyses of Parasite Genomes Predict at Least 65 Protein Kinases, but Their Biological Functions and Therapeutic Potential Were Until Now Largely Unknown

A large unmet medical need exists for effective new treatments for malaria in the developing world. The malaria parasites initially travel from the site of the infected mosquito bite to the host's liver (called liver stage) where they reproduce and then infect red blood cells (called blood stage). Taking a cue from the effective treatments for a variety of human diseases that target one or more of the ∼530 human kinases, malaria researchers are taking aim at the <100 kinases present in malaria parasites, such as Plasmodium falciparum. In this article, the authors screened 1358 small-molecule kinase inhibitors that target diverse branches of the human kinome tree to assess their effect on Plasmodium parasite load in liver cells. After this screening identified candidate compounds, the compounds were tested in blood-stage malaria. Ideally, a drug that affected both blood-stage and liver-stage malaria while leaving normal human cells unaffected was desired. For the high-throughput screen, parasites from P. berghei (containing a transgenic luciferase reporter) mosquitos were used to infect the human liver cell line HepG2, and the effects of the compounds on parasite load were assessed. Another human liver cell line, Huh-7, along with an alternate detection method (immunofluorescence detection of anti-Plasmodium fluorescent antibodies) was used as a secondary assay. Compounds with IC50<10 μM (31 total) were considered to be hits and tested against blood stage malaria (P. falciparum) and for liver cell toxicity. All of the compounds were found to have a large window between effects on the parasite assay and the liver cell viability assay. Inhibitors of the human CDK family yielded the most frequent hits in the malaria assay. The authors note that this family of inhibitors has complications with the luciferase reporter system so the immunofluorescence assay was used predominantly. For each of the CDK inhibitors identified the IC50 in the malaria assay was elevated compared to the human enzyme so the authors undertook an earlier time point (12 hours); that is, before the expected duplication of the HEPG2 cells (doubling time is ∼48 hours) to see whether there was still an effect on parasite load, which doubles at a very fast rate. The effects were still there, and this indicated that the effect did not correlate with the effect of a CDK inhibitor on the human cell host cell cycle. PfPK5 kinase in Plasmodium parasites is the kinase most similar to CDK2, the most common human target of the CDK inhibitors identified as hits. The figure below shows the docking of CDK1/2 to PfPK5. To confirm the docking prediction, all of the commercially available screening hits were tested in the commercially available competition binding assay for PfPK5 (DiscoveRx). CDK1/2, SNS-032 (currently in clinical trials for cancer indications), flavopiridol, and PIK-75 (a lipid kinase inhibitor) had Kd values for PfPK5 of <10 μM. CDK1/2, SNS-032, flavopiridol, and AT-5791 were tested and found to be safe and to inhibit liver-stage malaria in mice. SNS-032 was given prophylactically to mice prior to malaria infection and was found to reduce parasite load relative to vehicle control. The authors also tested CYC-116, an aurora inhibitor that was selective for liver-stage over blood-stage malaria, and found that it was also effective in reducing parasite load in the mouse model. It is fortunate that the predominantly human kinase KINOMEscan panel has this malaria kinase of interest here (PfPK5), and it would be helpful for the growing interest in malaria kinases if a comprehensive panel of malaria kinase assays was available as a companion to the human assay panel. In particular, it would be interesting to see how selective the above inhibitors are within the malaria kinome because many kinase inhibitors have extensive polypharmacology in humans.

Binding to PfPK5. (A) Structure of CDK 1/2 docked onto the PfPK5 crystal structure (PDB ID: 1V0O). The protein kinase (pink) and CDK 1/2 carbon (gray), hydrogen (white), nitrogen (blue), sulfur (yellow), and oxygen (red) atoms are shown. Hydrogen bonds are predicted to form (black dotted lines) between the thioamide proton of CDK 1/2 and Gln84 and between the primary amine proton on aminotriazole and Asp85 (bond lengths <2.5 Å). (B) Percentage binding of commercially available kinase inhibitors to PfPK5 at 10 μm. Significant binding (>50%) is only observed with the known CDK inhibitors, flavopiridol, SNS-032, and CDK 1/2, and with the PIK inhibitor PIK-75. (C) Competitive binding plots of CDK 1/2 (red curve), flavopiridol (black curve), and SNS-032 (blue curve) to PfPK5, where decrease in relative signal correlates with compound binding. Data in (C) were fit to a nonlinear regression equation (curves shown) to obtain Kd values.

Abstract from Chembiochem 2014; Vol. 15:1920–1930

Malaria, an infectious disease caused by eukaryotic parasites of the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1358 small-molecule kinase inhibitors to evaluate the role of both the human and the malaria kinomes in Plasmodium infection of liver cells, the parasites' obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood-stage malaria. Most of the screening hits inhibited both liver- and blood-stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests that several kinases are essential to malaria parasites, including cyclin-dependent kinases (CDKs), glycogen synthase kinases, and phosphoinositide-3-kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual-stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Chembiochem titled "Chemical interrogation of the malaria kinome." Authors of the paper are Derbyshire ER, Zuzarte-Luis V, Magalhães AD, Kato N, Sanschagrin PC, Wang J, Zhou W, Miduturu CV, Mazitschek R, Sliz P, Mota MM, Gray NS, Clardy J.

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