Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds.

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds' mechanisms of action--i.e., the specific molecular targets by which they kill the parasite--would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children's Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 µM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites.

Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

Development of potent and selective Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4) inhibitors that block the transmission of malaria to mosquitoes.

Malaria remains a major health concern for a large percentage of the world's population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitoes. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block Plasmodium falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.

A specific inhibitor of PfCDPK4 blocks malaria transmission: chemical-genetic validation.

Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147 M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that coformulation of this transmission-blocking agent with asexual stage antimalarials such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission.

A novel calcium-dependent protein kinase inhibitor as a lead compound for treating cryptosporidiosis.

Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors.

Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1-treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication.

Multiple determinants for selective inhibition of apicomplexan calcium-dependent protein kinase CDPK1.

Diseases caused by the apicomplexan protozoans Toxoplasma gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is regulated by a family of calcium-dependent protein kinases (CDPKs) that have no direct homologues in the human host. Fortuitously, CDPK1 from both parasites contains a rare glycine gatekeeper residue adjacent to the ATP-binding pocket. This has allowed creation of a series of C3-substituted pyrazolopyrimidine compounds that are potent inhibitors selective for CDPK1 over a panel of human kinases. Here we demonstrate that selectivity is further enhanced by modification of the scaffold at the C1 position. The explanation for this unexpected result is provided by crystal structures of the inhibitors bound to CDPK1 and the human kinase c-SRC. Furthermore, the insight gained from these studies was applied to transform an alternative ATP-competitive scaffold lacking potency and selectivity for CDPK1 into a low nanomolar inhibitor of this enzyme with no activity against SRC.

Development of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) inhibitors with potent anti-toxoplasma activity.

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated in part by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.