Iain D. Kerr

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.

Structures of falcipain-2 and falcipain-3 bound to small molecule inhibitors: implications for substrate specificity.

Falcipain-2 and falcipain-3 are critical hemoglobinases of Plasmodium falciparum, the most virulent human malaria parasite. We have determined the 2.9 Å crystal structure of falcipain-2 in complex with the epoxysuccinate E64 and the 2.5 Å crystal structure of falcipain-3 in complex with the aldehyde leupeptin. These complexes represent the first crystal structures of plasmodial cysteine proteases with small molecule inhibitors and the first reported crystal structure of falcipain-3. Our structural analyses indicate that the relative shape and flexibility of the S2 pocket are affected by a number of discrete amino acid substitutions. The cumulative effect of subtle differences, including those at “gatekeeper” positions, may explain the observed kinetic differences between these two closely related enzymes.

Nonpeptidic Tetrafluorophenoxymethyl Ketone Cruzain Inhibitors As Promising New Leads for Chagas Disease Chemotherapy

A century after discovering that the Trypanosoma cruzi parasite is the etiological agent of Chagas disease, treatment is still plagued by limited efficacy, toxicity, and the emergence of drug resistance. The development of inhibitors of the major T. cruzi cysteine protease, cruzain, has been demonstrated to be a promising drug discovery avenue for this neglected disease. Here we establish that a nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitor substantially ameliorates symptoms of acute Chagas disease in a mouse model with no apparent toxicity. A high-resolution crystal structure confirmed the mode of inhibition and revealed key binding interactions of this novel inhibitor class. Subsequent structure-guided optimization then resulted in inhibitor analogues with improvements in potency despite minimal or no additions in molecular weight. Evaluation of the analogues in cell culture showed enhanced activity. These results suggest that nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors have the potential to fulfill the urgent need for improved Chagas disease chemotherapy.

Crystal Structures of TbCatB and Rhodesain, Potential Chemotherapeutic Targets and Major Cysteine Proteases of Trypanosoma brucei

Background Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei. Methods and Findings We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002. Conclusions The mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

A novel Entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target.

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

Novel non-peptidic vinylsulfones targeting the S2 and S3 subsites of parasite cysteine proteases.

We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of Trypanosoma brucei brucei parasites in culture. A high resolution (1.75 A) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.

Structural studies of pterin-based inhibitors of dihydropteroate synthase.

Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.