Parasite Proteases Research Articles

A cysteine protease activity from Plasmodium falciparum cleaves human erythrocyte ankyrin

The malaria parasite Plasmodium falciparum undergoes distinct morphologic changes during its 48-h life cycle inside human red blood cells. Parasite proteinases appear to play important roles at all stages of the erythrocytic cycle of human malaria. Proteases involved in erythrocyte rupture and invasion are possibly required to breakdown erythrocyte membrane skeleton. To identify such proteases, soluble cytosolic extract of isolated trophozoites/schizonts was incubated with erythrocyte membrane ghosts or spectrin-actin depleted inside-out vesicles, which were then analyzed by SDS-PAGE. In both cases, a new protein band of 155 kDa was detected. The N-terminal peptide sequencing established that the 155 kDa band represents truncated ankyrin. Immunoblot analysis using defined monoclonal antibodies confirmed that ankyrin was cleaved at the C-terminus. While the enzyme preferentially cleaved ankyrin, degradation of protein 4.1 was also observed at high concentrations of the enzyme. The optimal activity of the purified enzyme, using ankyrin as substrate, was observed at pH 7.0–7.5, and the activity was strongly inhibited by standard inhibitors of cysteine proteinases (cystatin, NEM, leupeptin, E-64 and MDL 28 170), but not by inhibitors of aspartic (pepstatin) or serine (PMSF, DFP) proteinases. Furthermore, we demonstrate that protease digestion of ankyrin substantially reduces its interaction with ankyrin-depleted membrane vesicles. Ektacytometric measurements showed a dramatic increase in the rate of fragmentation of ghosts after treatment with the protease. Although the role of ankyrin cleavage in vivo remains to be determined, based on our findings we postulate that the parasite-derived cysteine protease activity cleaves host ankyrin thus weakening the ankyrin-band 3 binding interactions and destabilizing the erythrocyte membrane skeleton, which, in turn, facilitates parasite release. Further characterization of the enzyme may lead to the development of novel antimalarial drugs.

Partial Characterization of a Novel Cathepsin L-like Protease from Fasciola hepatica

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CHCH2-CO2R (where R = Me/Et/But). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.

Aryl ureas represent a new class of anti-trypanosomal agents

Background: The trypanosomal diseases including Chagas’ disease, African sleeping sickness and Nagana have a substantial impact on human and animal health worldwide. Classes of effective therapeutics are needed owing to the emergence of drug resistance as well as the toxicity of existing agents. The cysteine proteases of two trypanosomes, Trypanosoma cruzi (cruzain) and Trypanosoma brucei (rhodesain), have been targeted for a structure-based drug design program as mechanistic inhibitors that target these enzymes are effective in cell-based and animal models of trypanosomal infection. Results: We have used computational methods to identify new lead scaffolds for non-covalent inhibitors of cruzain and rhodesain, have demonstrated the efficacy of these compounds in cell-based and animal assays, and have synthesized analogs to explore structure activity relationships. Nine compounds with varied scaffolds identified by DOCK4.0.1 were found to be active at concentrations below 10 μM against cruzain and rhodesain in enzymatic studies. All hits were calculated to have substantial hydrophobic interactions with cruzain. Two of the scaffolds, the urea scaffold and the aroyl thiourea scaffold, exhibited activity against T. cruzi in vivo and both enzymes in vitro. They also have predicted pharmacokinetic properties that meet Lipinski’s ‘rule of 5’. These scaffolds are synthetically tractable and lend themselves to combinatorial chemistry efforts. One of the compounds, 5′(1-methyl-3-trifluoromethylpyrazol-5-yl)-thiophene 3′-trifluoromethylphenyl urea ( D16) showed a 3.1 μM IC 50 against cruzain and a 3 μM IC 50 against rhodesain. Infected cells treated with D16 survived 22 days in culture compared with 6 days for their untreated counterparts. The mechanism of the inhibitors of these two scaffolds is confirmed to be competitive and reversible. Conclusions: The urea scaffold and the thiourea scaffold are promising leads for the development of new effective chemotherapy for trypanosomal diseases. Libraries of compounds of both scaffolds need to be synthesized and screened against a series of homologous parasitic cysteine proteases to optimize the potency of the initial leads.

The Substrate Specificity of a Recombinant Cysteine Protease fromLeishmania mexicana: Application of a Combinatorial Peptide Library Approach

Rapid determination of the substrate specificity of a parasite cysteine protease was accomplished with a solid-phase “one-bead-one-compound” assay. The synthesis of >105 fluorescence-quenched substrates on enzyme-permeable polyethylene glycol–acrylamide copolymer (PEGA) support and processing with overexpressed Leishmania protease gave a range of active substrates (see scheme) whose sequence determination revealed the protease specificity. F = fluorescent group, Q = quenching group.

Partial characterization of serine proteinases secreted by adult Trichinella spiralis.

Serine proteinases secreted by adult Trichinella spiralis were isolated from excretory/secretory products (ES) of in-vitro-cultured parasites by affinity chromatography with p-benzamidine-celite. The purified enzymes had molecular weights of approximately 18, 40, and 50 kDa and displayed enzyme activity against a range of low-molecular-weight substrates, gelatin, and azocasein. The antigenicity of these parasite proteinases was demonstrated by the inhibition of enzymatic activity with IgG purified from infected hosts. The inactivation of major secreted proteinases of adult T. spiralis by immune antibody could presumably contribute to impairment of the survival of the parasite in sensitized hosts.

Proteases involved in erythrocyte invasion by the malaria parasite: function and potential as chemotherapeutic targets.

Malaria places an increasing burden on global public health resources. In the face of growing resistance of the malaria parasite to available antimalarial drugs, there is a need for new drugs and the identification of new chemotherapeutic targets. The malaria parasite has a complex life cycle which includes a number of obligate intracellular stages. Clinical malaria results from cyclic asexual replication of the blood-stage parasite in circulating erythrocytes of the human host. Erythrocyte entry and host cell rupture require the activity of parasite proteases, and these enzymes are, therefore, attractive targets for rational approaches to new drug development. Malarial proteases play a role in at least two distinct aspects of host cell invasion; modification of parasite proteins involved in host cell recognition and entry; and restructuring of the host cell itself, during and following invasion, and in order to allow parasite release from the host cell. This review details recent advances in the identification of these proteases, describes current understanding of their activation and functional role, and discusses their potential as targets for protease inhibitor-based drugs.

Host specificity in blood feeding parasites: a defining contribution by haemoglobin-degrading enzymes?

A hypothesis is presented that proposes that the compatibility between species-specific variants of haemoglobin-degrading proteases of blood-feeding parasites (e.g. hookworms, schistosomes, malarial parasites, etc.), and their natural substrates, i.e. haemoglobins from diverse species of mammals, has influenced to evolution of the host range of these parasites. Support for the hypothesis was drawn from molecular modelling studies of the three dimensional structure of an aspartic protease, Acasp, from the canine hookworm Ancylostoma caninum, and models of canine and human haemoglobins docked with the active site of Acasp. The molecular modelling suggested that Acasp, from a canine-specific hookworm, would have a higher substrate affinity for canine haemoglobin than for human haemoglobin.

Proteases in helminthic parasites

Proteases catalyse the cleavage of internal peptide bonds within peptides and proteins. They are classified into four major classes and are involved in a broad range of eukaryotic processes. Proteases have also been found to play a number of critical roles in the virulence of pathogenic agents, particularly of nematode parasites. Parasitic proteases are involved in different aspects of host-parasite interactions. They facilitate the invasion of host tissues and allow nutrition as well as the survival of the parasite in its host. Proteases also participate in the parasite's evasion from the host's immune response. The functional diversity and complexity of these enzymes are described in this review, with a particular focus on the principally identified proteases of four helminths: Schistosoma sp., Fasciola sp., Taenia sp. and Haemonchus sp. Some of these proteases, especially the cysteine proteases secreted by the parasitic trematode Fasciola hepatica, have been successfully tested in experimental immunodiagnosis. Proteases identified in different parasites are currently under study for a use as recombinant vaccines. In this respect, proteases are proposed as major potential targets for immunotherapy and chemotherapy against parasitic diseases.

Development of cysteine protease inhibitors as chemotherapy for parasitic diseases: insights on safety, target validation, and mechanism of action

Cysteine proteases have been identified as promising targets for the development of antiparasitic chemotherapy. An attractive aspect of these enzymes is their widespread importance in both protozoan and helminth parasites of domestic animals and humans. Concerns about the ability to selectively inhibit parasite proteases without affecting host homologues have been addressed in recent studies of Trypanosoma cruzi and Plasmodium falciparum. Significant data on half-life, metabolism, pharmacokinetics and safety have been accumulated. Differential uptake of proteases by parasitic organisms versus host cells, and relatively less redundancy in parasite protease gene families, may be two factors which contribute to the successful treatment of animal models of infection.

Cysteine protease inhibitors as chemotherapy for parasitic infections

Analysis of the evolution, localization and biologic function of papain family cysteine proteases in metazoan and protozoan parasites has provided important and often surprising insights into the biochemistry and cellular function of this diverse enzyme family. Furthermore, the relative lack of redundancy of cysteine proteases in parasites compared to their mammalian hosts makes them attractive targets for the development of new antiparasitic chemotherapy. The treatment of experimental models of parasitic diseases with cysteine protease inhibitors has provided an important ‘proof of concept’ for the use of cysteine protease inhibitors in vivo. Evidence has now accumulated that cysteine protease inhibitors can selectively arrest replication of a microbial pathogen without untoward toxicity to the host. Furthermore, this can be achieved with reasonable dosing schedules and oral administration of the drug. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. Work on Trypanosoma brucei, the agent of African trypanosomiasis, is preliminary but also promising. Target validation studies have shown that biotinylated or radiolabeled irreversible inhibitors specifically bind to the cysteine protease targets thought to represent the major activity within the parasite. In the case of T. cruzi, the effect of inhibitors appears to be predominantly in blocking protease processing. Transfection studies using variant constructs have supported this model. Finally, the generation of null mutants for the multiple protease genes in Leishmania mexicana has provided the first genetic support for the key role of this enzyme family in parasite virulence. Safety studies in rodents and analysis of uptake of inhibitors by parasites and host cells suggest that the selectivity of inhibitors for the parasite targets may reside in the lack of redundancy of parasite proteases, the higher concentration of host proteases in intracellular compartments, and differential uptake of inhibitors by parasites. Attempts to elicit resistance to cysteine protease inhibitors in parasite cultures suggest that mechanisms of induced resistance are independent of resistance to the traditional antiparasitic agents. This suggests that cysteine protease inhibitors may provide an alternative to traditional therapy in drug-resistant organisms. ©

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH 2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide susbstrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5′ end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S 1 and S 2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (r Tc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to r Tc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of r Tc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

Cysteine Protease Activities during Maturation Stages of Paragonimus westermani

In mature Paragonimus westermani, specific activity of parasitic cysteine protease declines. To clarify which of the known 17-, 27-, and 28-kDa enzyme activities is decreased, the cysteine proteases were purified from the crude extracts of metacercariae, 4- and 7-wk juveniles, and 16-wk adults by gel filtration, ion-exchange, and affinity matrix chromatographies; the enzyme activity was monitored with the fluorogenic substrate, Cbz-phe-arg-AMC. In addition to 3 known enzymes, 2 other cysteine proteases at 15 and 53 kDa were identified in juveniles and adults and were purified. The 2 novel enzymes were most active in 0.1 M ionic strength and pH 5-6 and were inhibited by N-(N-[L-3-transcarboxyrane-2-carbonyl]-L-leucyl)agamatine, iodoacetamide, and leupeptin. Of the 5 enzymes, specific activities of metacercarial 27- and 28-kDa enzymes were lowered from metacercaria to 16 wk. Between 4 and 16 wk, activities of 3 cysteine proteases of juveniles and adults were additionally exhibited. The activity changes of 5 different cysteine proteases may be associated with migration and immune evasion during the maturation stage of P. westermani when the parasite environment is changing.

Kininogenase Activity by the Major Cysteinyl Proteinase (Cruzipain) from Trypanosoma cruzi

The major isoform of Trypanosoma cruzi cysteinyl proteinase (cruzipain) has generated Lys-bradykinin (Lys-BK or kallidin), a proinflammatory peptide, by proteolysis of kininogen. The releasing of this peptide was demonstrated by mass spectrometry, radioimmunoassay, and ileum contractile responses. The kinin-releasing activity was immunoabsorbed selectively by monoclonal antibodies to the characteristic COOH-terminal domain of cruzipain. To determine the hydrolysis steps that account for the kininogenase activity of cruzipain, we synthesized a fluorogenic peptide (o-aminobenzoyl-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-S er-Pro-Phe-Arg389-Ser390-Ser-Arg-Ile-NH2) based on the sequence Leu373 to Ile393 of the human high molecular weight kininogen. The hydrolysis products from this peptide were isolated by high performance liquid chromatography, and Lys-BK was characterized as the major released kinin by mass spectrometry. Intramolecularly quenched fluorogenic peptides spanning the Met379-Lys380 and Arg389-Ser390 bradykinin-flanking sequences were then used to assess the substrate specificity requirements of the parasite-derived protease compared with two COOH-terminal truncated recombinant isoforms (cruzain and cruzipain 2). In contrast to the high catalytic efficiency of parasite-derived cruzipain, the recombinant proteinases cleaved the bradykinin-flanking sites at markedly different rates. In addition, we also demonstrated that cruzipain activates plasmatic prekallikrein, which would be a second and indirect way of the parasite protease to release bradykinin.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low K i values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2′. Substrates having the sequence VGGP from P2 to P2′ were hydrolysed by both congopain and cruzipain with a k cat/ K m greater than 4.10 3 mM −1 s −1. Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Parasite proteinases and amino acid metabolism: possibilities for chemotherapeutic exploitation

Parasite enzymes involved in proteolysis and amino acid metabolism have attracted considerable attention over the last decade. Nevertheless, current knowledge is extensive for just a few parasites and several enzymes. Most enzymes remain largely unexplored. This review concentrates upon a selection of the better studied enzymes and the potentially valuable approaches now being adopted in their study. We present a personal view on the most suitable strategies for exploiting this area of parasite biochemistry with novel antiparasite drugs. The content of the review reflects our own work and interests, but we have aimed to include a sufficiently broad range of topics so that this overview serves as a useful introduction for those new to the subject.There have been several reviews that provide good coverage of the appropriate literature (Barrett, 1991; McKerrow et al. 1993; North & Lockwood, 1995; Sakanari et al. 1995; Robertson et al. 1996; Vial, 1996; Coombs & Mottram, 1997; Walker & Barrett, 1997), therefore we detail here just some of the publications and refer readers to the reviews quoted for further information. This treatise mainly highlights progress made in studies with parasitic protozoa. Parasitic worms present more difficult problems for drug designers and there has been only limited progress to date in this area of biochemistry; we include here mention of just some of the more exciting advances so far.

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl- l-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 μM, none at 28 μM) even though the addition of 2.8 μM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH 2OCO-(2,6,-(CF 3) 2)Ph, at 16 μM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 μM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.

Antimalarial chemotherapy based on proteinase inhibitors

During the erythrocytic phase of malaria, parasite proteinases are involved in the breakdown of haemoglobin, the re-invasion of erythrocytes by merozoites, and antigen maturation. Two factors are important in the design of compounds to inhibit them: (1) any parasite proteinase acting in the host's plasma must have a narrow substrate specificity if it is to escape the activities of the numerous proteinase inhibitors naturally present in the blood; and (2) inhibitors of parasite proteinase acting on intracellular targets must be able to enter host cells. By using serum-free medium supplemented with human, high-density lipoproteins in re-invasion experiments, a neutral cysteine proteinase, previously described as the merozoite proteinase for erythrocyte invasion (MPEI), was detected, both in free merozoites and the culture medium. Of various pseudopeptides tested as inhibitors of this enzyme, GlcA-Val-Leu-Gly- Lysψ [CH2-CO]C2H5 was the most efficient. This was used in the design of macromolecular inhibitors ...

Enzymatic effect of hypodermin A, a parasite protease, on bovine lymphocyte membrane antigens.

The protease hypodermin A (HA) is produced by the parasitic warble-fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti-lymphocyte receptor antibodies tested. Anti-BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti-MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 micrograms/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)-induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi.

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin-peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

Structure-based design of parasitic protease inhibitors

To streamline the preclinical phase of pharmaceutical development, we have explored the utility of structural data on the molecular target and synergy between computational and medicinal chemistry. We have concentrated on parasitic infectious diseases with a particular emphasis on the development of specific noncovalent inhibitors of proteases that play a key role in the parasites' life cycles. Frequently, the structure of the enzyme target of pharmaceutical interest is not available. In this setting we have modeled the structure of the relevant enzyme by virtue of its sequence similarity with proteins of known structure. For example, we have constructed a homology-based model of falcipain, the trophozoite cysteine protease, and used the computational ligand identification algorithm DOCK to identify in compuo enzyme inhibitors including oxalic bis(2-hydroxy-1-naphthylmethylene)hydrazide ( 1) [Ring, C.S.; Sun, E.; McKerow, J.H.; Lee, G.; Rosenthal, P.J., Kuntz, I.D.; Cohen, F.E., Proc. Natl Acad. Sci. U.S.A. 1993, 90, 3583]. Compound 1 inhibits falcipain (IC 50 6 μM) and the organism in vitro as judged by hypoxanthine uptake (IC 50 7 μM). Following this lead, to date, we have identified potent bis arylacylhydrazides (IC 50 150 nM) and chalcones (IC 50 200 nM) that are active against both chloroquine-sensitive and chloroquine-resistant strains of malaria. In a second example, cruzain, the crystallographically determined structure of a papain-like cysteine protease, resolved to 2.35 Å, was available. Aided by DOCK, we have identified a family of bis-arylacylhydrazides that are potent inhibitors of cruzain (IC 50 600 μM). These compounds represent useful leads for pharmaceutical development over strict enzyme inhibition criteria in a structure-based design program.