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The cysteine proteases of the trypanosomatid parasitic protozoa have been validated as targets for chemotherapy of Chagas' disease and leishmaniasis. Metal complexes of gold, platinum, iridium, palladium, rhodium and osmium have been reported to have activity against a variety of trypanosomatids, but the molecular target of these compounds has not been defined. The activity of gold(III) and palladium(II) cyclometallated complexes, and oxorhenium(V) complexes against mammalian and parasitic cysteine proteases was investigated. All gold(III) complexes (1-6) inhibited cathepsin B with IC(50) values in the range of 0.2-1.4 microM. Of the six palladium compounds, aceto[2,6-bis[(butylthio-kappa S)methyl]phenyl-kappa C]-, (SP-4-3)-palladium(II) (11) was the most potent inhibitor of cathepsin B with an IC(50) of 0.4 microM. A clear structure-activity relationship was observed with the oxorhenium(V) complexes with chloro[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium(V) (16) being the most potent inhibitor of cathepsin B with an IC(50) of 0.009 microM. Six complexes were further tested against the parasite cysteine proteases, cruzain from T. cruzi, and cpB from L. major; the most potent inhibitors were the two rhenium complexes (2(1H)-pyridinethionato-kappa S(2))[2,6-bis[(mercapto-kappa S)methyl]pyridine-kappa N(1)] oxorhenium(V) (15) and chloro[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium(V) (16). The compounds were also evaluated in assays for parasite growth. Two oxorhenium(V) compounds ((p-methoxyphenylthiolato-S)[2,6-bis[(mercapto-kappa S)methyl]pyridine-kappa N(1)] oxorhenium(V) (14) and (methanethiolato)[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium (V) (18)) and the palladium compound 11 inhibited T. cruzi intracellular growth, and compound 11 inhibited promastigote growth in three Leishmania species. In conclusion this preliminary data indicates that metal complexes targeted at parasite cysteine proteases show promise for the treatment of both Chagas' disease and leishmaniasis.

Cruzain is the major cysteine protease of Trypanosoma cruzi, which is the causative agent of Chagas disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the substrate activity screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored beta-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group p K a, with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second-order inactivation constant of 147,000 s (-1) M (-1). This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas disease.

A systematic study of P2 and P3 substitution in a series of vinyl sulfone cysteine protease inhibitors is described. The introduction of a methyl substituent in the P2 phenylalanine aryl ring had a favorable effect on protease inhibition and conferred modest selectivity for rhodesain over cruzain. Rhodesain selectivity could be enhanced further by combining these P2 modifications with certain P3 amide substituents.

We have synthesized a library of thiosemicarbazones and screened them against three parasitic cysteine proteases, cruzain, falcipain-2, and rhodesain, and against the respective parasite sources of these three proteases, Trypanosoma cruzi, Plasmodium falciparum, and Trypanosoma brucei. The screens identified compounds that were effective against the enzymes and the parasites but also some compounds that were parasiticidal despite a lack of activity against the proteases. Several compounds were effective in killing all tested parasites. These promising lead compounds were tested for general toxicity in mice, and only one produced observable toxicity after 62 h. Our results suggest that thiosemicarbazones represent validated drug leads that kill several species of protozoan parasites through the inhibition of cysteine proteases as well as other novel targets.

Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.

Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas’ disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.

Promastigotes of all Leishmania species, except most Leishmania major stocks, synthesize and secrete extracellular soluble acid phosphatases [SAcP(s)] which are capable of dephosphorylating a wide range of substrates and, consequently, of modifying the parasite environment. To assess their antigenic/structural relatedness, the SAcP(s) from various species were compared immunochemically using both a monospecific rabbit antiserum and several monoclonal antibodies made against the purified promastigote SAcP of a cloned line of Leishmania donovani donovani. Results obtained from antibody-bridged enzyme activity assays demonstrated that these reagents quantitatively immunoprecipitated the enzymatic activities of SAcP(s) from 18 different W.H.O. reference stocks of Leishmania, including two L. major strains. Those results showed, for the first time, that all SAcP(s) possessed some common cross-reactive antigenic epitopes. Immunoprecipitates obtained from [35S]methionine, metabolically labeled promastigote culture supernatants of the various species, were analyzed by SDS-PAGE/fluorography. These analyses showed that marked differences existed among the SAcP(s) both in their relative mobilities and the number of constituent bands resolved. Tunicamycin treatment resulted in a significant reduction in the apparent molecular weights of SAcP(s) from three different isolates, confirming the presence of N-linked carbohydrate side chains in these enzymes. In addition, the SAcP released by L. donovani intracellular amastigotes was also immunoprecipitated with the monospecific rabbit antisera from the culture supernatant of infected macrophages. Cumulatively, these data demonstrate that despite individual species variations, the SAcP(s) from all Leishmania tested have retained certain common antigenic/structural epitopes and functional protein domains. Such conservation among SAcP(s) suggests that this enzyme must play an essential role in the survival of promastigotes and amastigotes of all Leishmania species.

Background Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas' disease. Methodology/Principal Findings WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 Å crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported. Conclusions We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas' disease., Author Summary Current drugs for Chagas' disease, caused by Trypanosoma cruzi infection, are limited in efficacy and are severely toxic. Hence the development of novel chemotherapeutic agents targeting T. cruzi infections is an important undertaking. In recent years, there has been considerable interest in cruzain, the major protease in T. cruzi, as a target to treat Chagas' disease. Herein, we present the synthesis of WRR-483, a small molecule designed as an irreversible cysteine protease inhibitor, and an assessment of its biological activity against cruzain and T. cruzi infection. This compound displays pH-dependent affinity for cruzain and highly effective trypanocidal activity in both cell cuture and a mouse model of acute Chagas' disease. The crystal structure of WRR-483 bound to cruzain elucidates the details of inhibitor binding to the enzyme. Based on these results, this inhibitor is a promising compound for the development of therapeutics for Chagas' disease.

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.

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas' disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival: cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a cocrystal was obtained of our most potent analogue bound to cruzain.

Parasitic diseases are of enormous public health significance in developing countries—a situation compounded by the toxicity of and resistance to many current chemotherapeutics. We investigated a focused library of 18 structurally diverse bis-acridine compounds for in vitro bioactivity against seven protozoan and one helminth parasite species and compared the bioactivities and the cytotoxicities of these compounds toward various mammalian cell lines. Structure-activity relationships demonstrated the influence of both the bis-acridine linker structure and the terminal acridine heterocycle on potency and cytotoxicity. The bioactivity of polyamine-linked acridines required a minimum linker length of approximately 10 Å. Increasing linker length resulted in bioactivity against most parasites but also cytotoxicity toward mammalian cells. N alkylation, but less so N acylation, of the polyamine linker ameliorated cytotoxicity while retaining bioactivity with 50% effective concentration (EC 50 ) values similar to or better than those measured for standard drugs. Substitution of the polyamine for either an alkyl or a polyether linker maintained bioactivity and further alleviated cytotoxicity. Polyamine-linked compounds in which the terminal acridine heterocycle had been replaced with an aza-acridine also maintained acceptable therapeutic indices. The most potent compounds recorded low- to mid-nanomolar EC 50 values against Plasmodium falciparum and Trypanosoma brucei ; otherwise, low-micromolar potencies were measured. Importantly, the bioactivity of the library was independent of P. falciparum resistance to chloroquine. Compound bioactivity was a function of neither the potential to bis-intercalate DNA nor the inhibition of trypanothione reductase, an important drug target in trypanosomatid parasites. Our approach illustrates the usefulness of screening focused compound libraries against multiple parasite targets. Some of the bis-acridines identified here may represent useful starting points for further lead optimization.

American trypanosomiasis, or Chagas' disease, is the leading cause of heart disease in Latin America. Currently there is an urgent need to develop antitrypanosomal therapy due to the toxicity of existing agents and emerging drug resistance. A novel series of potent thio semicarbazone small-molecule inhibitors of the Trypanosoma cruzi cysteine protease cruzain have been identified. Some of these inhibitors have been shown to be trypanocidal. We initially discovered that 3'-bromopropiophenone thio semicarbazone (1i) inhibited cruzain and could cure mammalian cell cultures infected with T. cruzi. 3'-Bromopropiophenone thio semicarbazone showed no toxicity for mammalian cells at concentrations that were trypanocidal. Following this lead, more than 100 compounds were designed and synthesized. A specific structure-activity relationship (SAR) was established, and many potent analogues with IC(50) values in the low nanomolar range were identified. Eight additional analogues were trypanocidal in a cell culture assay, and this indicates that aryl thio semicarbazone is a productive scaffold for killing the parasites. Kinetic studies show that these are time-dependent inhibitors. Molecular modeling studies of the enzyme-inhibitor complex have led to a proposed mechanism of interaction as well as insight into the SAR of the thio semicarbazone series. The nonpeptide nature of this series, small size, and extremely low cost of production suggest this is a promising direction for the development of new antitrypanosome chemotherapy.

All parasitic protozoa examined to date are incapable of de novo synthesis of purine nucleotides and rely on salvage mechanisms for survival. We have identified hypoxanthine, guanine, xanthine phosphoribosyl-transferase activities in crude cell-free extracts of Cryptosporidium sporulated oocysts utilizing radiolabeled substrates. Guanine, hypoxanthine, and xanthine were converted to their corresponding mononucleotides with specific activities of 346, 280, and 108 nmol/min/mg protein, respectively. The conversion of the radiolabeled purines was examined in the presence of another, unlabeled, purine base. These competition assays showed that both hypoxanthine and guanine were capable of inhibiting conversion of hypoxanthine, guanine, and xanthine to the corresponding nucleotides. Xanthine had a much lower inhibitory effect on the conversion of guanine and hypoxanthine to the nucleotides, whereas adenine had no effect at all. Autoradiographic studies of Cryptosporidium-infected Madin-Darby canine kidney (MDCK) cells showed that radiolabeled hypoxanthine, guanine, and adenine were primarily incorporated by intracellular Cryptosporidium as well as by MDCK nuclei. No apparent incorporation of xanthine by either host cells or intracellular parasites occurred. Radiolabeled glycine and formate were incorporated only into the nuclei of MDCK cells, suggesting a lack of de novo synthesis of purine nucleotides in Cryptosporidium. Radiolabeled hypoxanthine and guanine were also incorporated by excysting Cryptosporidium sporozoites. Altogether, our results indicate the presence of HPRTase, GPRTase, and XPRTase activities. These activities may play an important role in purine salvage, and may localize to a single HGXPRTase enzyme, as in the case of Eimeria, Toxoplasma, and Plasmodium.

Leishmania donovani donovani amastigotes, isolated from spleens of infected hamsters or axenically cultured, and promastigotes were comparatively examined for the expression of lipophosphoglycans (LPG). Parasites were metabolically labeled with [32p]-phosphate, [3H]galactose, or [3H]mannose. Radiolabeled material was extracted with water/ethanol/diethylether/pyridine/NH40H and purified further by gel filtration and hydrophobic column chromatographies. These radiolabeled compounds were identified as phosphorylated lipid-containing glycoconjugates. Mild acid treatment resulted in degradation of the glycolipids into low-molecular-weight fragments. All glycolipids isolated were susceptible to nitrous acid and phosphatidylinositol-specific phospholipase C treatments, as has been reported for the L. donovani promastigote LPG. Moreover, glycoconjugates purified from the three Leishmania stocks were not susceptible to trypsin treatment. Acid hydrolysis of promastigote LPG resulted in a predominant [P04-6galactose(beta1,4)mannose(alpha)1] fragment. In contrast, the main radiolabeled anionic fragments isolated from splenic and axenic amastigotes differed from that of promastigotes, as evidenced by the elution profiles obtained by HPLC anion-exchange chromatography. These cumulative results indicate that lipophosphoglycan molecules, structurally distinct from the previously characterized LPG of the promastigote stage, are being expressed by L. donovani splenic and axenic amastigotes.

Erratum to ‘‘Metal compounds for the treatment of parasitic diseases” [J. Inorg. Biochem. 102 (2008) 1839–1845] Simon P. Fricker *, Renee M. Mosi , Beth R. Cameron , Ian Baird , Youngbao Zhu , Virginia Anastassov , Jennifer Cox , Patricia S. Doyle , Elizabeth Hansell , Gloria Lau , Jonathan Langille , Micki Olsen , Ling Qin , Renato Skerlj , Rebecca S.Y. Wong , Zefferino Santucci , James H. McKerrow c

Epimastigote cultures of the two cloned Trypanosoma cruzi stocks, CA-I/72 and HO-3/15, grown under identical conditions, differ both qualitatively and quantitatively in their cytochrome content. The CA-I/72 stock has a four-fold higher cytochrome b content (19.2 nM (mg protein)-1) than the HO-3/15 stock (4.9 nM (mg protein)-1). Cytochrome o is present at 29 nM (mg protein)-1 in the CA-I/72 stock but is below detectable limits in the HO-3/15 stock. There is no inter-stock difference in oxygen utilization (12-15 nM O2 min-1 (mg protein)-1) during exponential growth. However, stationary phase CA-I/72 epimastigotes utilize twice as much oxygen as HO-3/15 epimastigotes. Oxygen utilization by HO-3/15 epimastigotes incubated in Dulbecco's phosphate buffer solution (starvation conditions), was stimulated earlier and to higher levels by the addition of glucose than by CA-I/72 epimastigotes under identical conditions. Under starvation conditions and with the cytochrome chain partially inhibited by antimycin A,(anti-A) the addition addition of glucose also increased oxygen utilization by CA-I/72 epimastigotes. In contrast, anti-A did not influence glucose-stimulated oxygen utilization by HO-3/15 epimastigotes. Following partial inhibition with anti-A, salicylhydroxamic acid produced an additional 50% inhibition in oxygen utilization in both stocks irrespective of the growth phase of the organisms. These data indicate that marked intra-specific differences in oxidative metabolism exist within the T. cruzi population and that an alpha-glycerophosphate oxidase or similar salicylhydroxamic acid-inhibitable compound may be present in the organism.

Trypomastigote cells of Trypanosoma cruzi incubated with [U- 14 C]glucose accumulated dolichol- P - P -linked Man 2 GlcNAc 2 and Man 9 GlcNAc 2 . Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man 9 GlcNAc 2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man 9 GlcNAc 2 and Man 8 GlcNAc 2 occurred in epimastigotes and trypomastigotes but the structure of the main Man 7 GlcNAc 2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man 7 GlcNAc 2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N -glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.