Dustin J. Maly

Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors.

New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.

Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors

Under endoplasmic reticulum (ER) stress, unfolded proteins accumulate in the ER to activate the ER transmembrane kinase/endoribonuclease (RNase)—IRE1α. IRE1α oligomerizes, autophosphorylates, and initiates splicing of XBP1 mRNA, thus triggering the unfolded protein response (UPR). Here we show that IRE1α’s kinase-controlled RNase can be regulated in two distinct modes with kinase inhibitors: one class of ligands occupy IRE1α’s kinase ATP-binding site to activate RNase-mediated XBP1 mRNA splicing even without upstream ER stress, while a second class can inhibit the RNase through the same ATP-binding site, even under ER stress. Thus, alternative kinase conformations stabilized by distinct classes of ATP-competitive inhibitors can cause allosteric switching of IRE1α’s RNase—either on or off. As dysregulation of the UPR has been implicated in a variety of cell degenerative and neoplastic disorders, small molecule control over IRE1α should advance efforts to understand the UPR’s role in pathophysiology and to develop drugs for ER stress-related diseases.

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.

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.

Combinatorial strategies for targeting protein families: application to the proteases.

Tens of thousands of proteins have been identified as a result of recent large scale genomic and proteomic efforts. With this large influx of new proteins, the formidable task of elucidating their function begins. However, this task becomes more manageable if proteins are divided into families based upon sequence homology, thereby allowing tools for their systematic study to be developed based upon their common structural and mechanistic characteristics. Combinatorial chemistry is ideally suited for the systematic study of protein families because a large amount of diversity can be readily displayed about a common scaffold designed to target a given protein family. Targeted combinatorial libraries have been particularly effective for the study of a ubiquitous family of proteins, the proteases. Substrate‐specificity profiles of many proteases have been determined by using combinatorial libraries of appropriately labeled peptides. This specificity information has been utilized to identify the physiological protein substrates of these enzymes and has facilitated inhibitor design efforts. Furthermore, combinatorial libraries of small molecules prepared with mechanism‐based scaffolds have resulted in the identification of potent, small‐molecule inhibitors of numerous proteases. Cell‐permeable small‐molecule inhibitors identified by these methods have served as powerful chemical tools to study protease function in vitro and in vivo and have served as leads for the development of therapeutic agents.

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

ABSTRACT Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.

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.

Biochemical Mechanisms of Resistance to Small-Molecule Protein Kinase Inhibitors

Protein kinases have emerged as one of the most frequently targeted families of proteins in drug discovery. While the development of small-molecule inhibitors that have the potency and selectivity necessary to be effective cancer drugs is still a formidable challenge, there have been several notable successes in this area over the past decade. However, in the course of the clinical use of these inhibitors, it has become apparent that drug resistance is a recurring problem. Because kinase inhibitors act by targeting a specific kinase or set of kinases, there is a strong selective pressure for the development of mutations that hinder drug binding but preserve the catalytic activity of these enzymes. To date, resistance mutations to clinically approved kinase inhibitors have been identified in a number of kinases. This review will highlight recent work that has been performed to understand how mutations in the kinase catalytic domain confer drug resistance. In addition, recent experimental efforts to predict potential sites of clinical drug resistance will be discussed.

Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes

The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.