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Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S 2 subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola . An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P 2 proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P 2 proline, further highlighting the importance of Y69 for FhCatL2 P 2 proline acceptance. Finally, the P 1 –P 4 specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P 2, which is unique amongst Fasciola cathepsins examined to date.

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

Cysteine proteinases are key virulence factors of the protozoan parasiteEntamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of theE. histolyticagenome project has revealed more than 40 genes encoding cysteine proteinases. We have focused onE. histolyticacysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasiveE. histolyticaand is highly expressed and released. Recombinant EhCP1 was expressed inEscherichia coliand refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.

Asparaginyl endopeptidases, or ‘legumains’ have been identified and characterized in plants, the blood fluke parasite Schistosoma , and mammals. The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues. Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positionalscanning synthetic combinatorial library (PS-SCL). We first screened Sm32 using a P1-diverse library. This library demonstrated the absolute specificity of Sm32 for asparagine at P1. To determine the P2/P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2/P3 positions randomized. The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi. The schistosome legumain showed a preference for P3: Thr � /Ala � /Val � /Ile, and P2: Ala� /Thr� /Val� /Asn, with an overall broader specificity at P3 than at P2. Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3. However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn. Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays. # 2002 Published by Elsevier Science B.V.

Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTRprot− (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine α-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTRprot−, was measured. Cells grown with TTRprot− displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a high rate of mortality. The SARS-associated coronavirus (SARS-CoV) has been identified as the etiological agent of the disease. Although public health procedures have been effective in combating the spread of SARS, concern remains about the possibility of a recurrence. Various approaches are being pursued for the development of efficacious therapeutics. One promising approach is to develop small molecule inhibitors of the essential major polyprotein processing protease 3Clpro. Here we report a complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160,000 possible naturally occurring tetrapeptides. The substrate specificity data show the expected P1-Gln P2-Leu specificity and elucidate a novel preference for P1-His containing substrates equal to the expected preference for P1-Gln. These data were then used to develop optimal substrates for a high-throughput screen of a 2000 compound small-molecule inhibitor library consisting of known cysteine protease inhibitor scaffolds. We also report the 1.8 A X-ray crystal structure of 3Clpro bound to an irreversible inhibitor. This inhibitor, an alpha,beta-epoxyketone, inhibits 3Clpro with a k3/Ki of 0.002 microM(-1) s(-1) in a mode consistent with the substrate specificity data. Finally, we report the successful rational improvement of this scaffold with second generation inhibitors. These data provide the foundation for a rational small-molecule inhibitor design effort based upon the inhibitor scaffold identified, the crystal structure of the complex, and a more complete understanding of P1-P4 substrate specificity.

Caspase-14 is a cysteine endoproteinase that is expressed in the epidermis and a limited number of other tissues. It is activated during keratinocyte differentiation by zymogen processing, but its precise function is unknown. To obtain caspase-14 for functional studies, we engineered and expressed a constitutively active form of human caspase-14 (Rev-hC14) in Escherichia coli and cultured mammalian cells. Rev-hC14 required no proteolytic processing for activity, showed strong activity against the caspase substrate WEHD, and was inhibited by the pan-caspase inhibitor zVAD-fmk. Mammalian cells that expressed active caspase-14 showed normal cell adherence and morphology. Using positional scanning of synthetic tetrapeptide libraries, we determined the substrate preference of human caspase-14 to be W (or Y)-X-X-D. These studies affirm that caspase-14 has a substrate specificity similar to the group I caspases, and demonstrate that it functions in a distinct manner from executioner caspases to carry out specific proteolytic events during keratinocyte differentiation.

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity (kcat/Km increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.

A new method was developed to identify nonpeptidic metalloproteinase inhibitors with novel zinc binding groups. Application of this method to matrix metalloproteinase-9 resulted in the identification of aminomethyl benzimidazole analogue 7a with an IC(50)=13 microM.

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.

Water borne cercaria(ae) of the trematode genus Schistosoma rapidly penetrate host skin. A single serine protease activity, cercarial elastase, is deposited in advance of the invading parasite by holocytosis of vesicles from ten large acetabular gland cells. Cercarial elastase activity is a composite of multiple isoforms. Genes coding for the isoforms can be divided into two classes by amino acid and promoter sequence homology. Two of the five genes identified in Schistosoma mansoni account for over 90% of the activity and protein released. The remaining genes produce little protein or are silent. Positional scanning synthetic combinatorial substrate libraries demonstrate that the two major isoforms have similar substrate specificities and are, therefore, isoenzymes. The closely related Schistosoma hematobium and the distantly related Schistosomatium douthitti also contain multiple orthologous cercarial elastase genes suggesting that gene duplication may have occurred after speciation in Schistosoma evolution and that this duplication has been conserved.

Background Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive. Findings We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5–7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively. Conclusion VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol., Author Summary Plasmodium vivax affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel P. vivax CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate Plasmodium species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of P. vivax.

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 P(1) - P(4) amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2) position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18)O 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 P(2) 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.