Antimalarial Synergy of Cysteine and Aspartic Protease Inhibitors

It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease inhibitors exhibited synergistic effects in blocking parasite metabolism and development. The inhibitors also demonstrated apparent synergistic inhibition of plasmodial hemoglobin degradation both in culture and in a murine malaria model. When evaluated for the treatment of murine malaria, a combination of cysteine and aspartic protease inhibitors was much more effective than higher concentrations of either compound used alone. These results support a model whereby plasmodial cysteine and aspartic proteases participate in the degradation of hemoglobin, and they suggest that combination antimalarial therapy with inhibitors of the two classes of proteases is worthy of further study.Malaria is one of the most important infectious diseases in the world. Infections with Plasmodium falciparum, the most virulent human malaria parasite, are responsible for hundreds of millions of illnesses and over a million deaths per year (22). A major reason for the continued severity of the worldwide malaria problem is the increasing resistance of malaria parasites to available drugs (12). Thus, it is important to identify new targets for antimalarial therapy and to evaluate new modes of therapy directed against these targets.Potential new targets for antimalarial chemotherapy include parasite enzymes required for the degradation of hemoglobin. Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis (reviewed in references 5 and 16). The food vacuole of P. falciparum contains the cysteine protease falcipain and the aspartic proteases plasmepsin I and plasmepsin II (7, 8, 15). Each of these proteases degrades hemoglobin in vitro, and it has been proposed that the enzymes act in a concerted manner to hydrolyze globin to small peptides or free amino acids (5, 16). In a number of in vitro studies, inhibitors of both cysteine and aspartic proteases had potent effects against cultured malaria parasites (1, 4, 11, 14, 15, 17, 18, 20). In an in vivo study utilizing a murine malaria model, a peptidyl cysteine protease inhibitor cured Plasmodium vinckei-infected mice (14). However, high doses of this inhibitor (200 to 400 mg/kg of body weight/day) were required for a pronounced antimalarial effect.As cysteine and aspartic proteases appear to act cooperatively to degrade hemoglobin, and as inhibitors of both classes of proteases have antimalarial effects, it may be appropriate to use combinations of inhibitors to treat malaria. Such combination therapy might improve efficacy and also slow the development of resistance to new agents. We now report an evaluation of the in vitro and in vivo antimalarial effects of combinations of peptidyl cysteine and aspartic protease inhibitors. These combinations had strong, apparently synergistic inhibitory effects on plasmodial development and hemoglobin degradation in both cultured parasites and in a murine malaria model.     

Structure-activity relationships for inhibition of cysteine protease activity and development of Plasmodium falciparum by peptidyl vinyl sulfones

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemoglobin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors. Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P(2) leucine moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.     

Inhibition of Plasmodium falciparum oocyst production by membrane-permeant cysteine protease inhibitor E64d

During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.     

Antimalarial effects of vinyl sulfone cysteine proteinase inhibitors.

We evaluated the antimalarial effects of vinyl sulfone cysteine proteinase inhibitors. A number of vinyl sulfones strongly inhibited falcipain, a Plasmodium falciparum cysteine proteinase that is a critical hemoglobinase. In studies of cultured parasites, nanomolar concentrations of three vinyl sulfones inhibited parasite hemoglobin degradation, metabolic activity, and development. The antimalarial effects correlated with the inhibition of falcipain. Our results suggest that vinyl sulfones or related cysteine proteinase inhibitors may have promise as antimalarial agents.     

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.     

Quinoline-triazole hybrids inhibit falcipain-2 and arrest the development of Plasmodium falciparum at the trophozoite stage

Falcipain-2 (FP2) is a papain family cysteine protease and a key member of the hemoglobin degradation pathway, a process that is required at erythrocytic stages of Plasmodium falciparum to obtain amino acids. In this study, we report a set of 10 quinoline-triazole-based compounds (T1–T10) which exhibit a good binding affinity for FP2, inhibit its catalytic activity at micromolar concentrations and thereby arrest the parasite growth. Compounds T4 and T7 inhibited FP2 with IC₅₀ values of 16.16 μM and 25.64 μM respectively. Both the compounds T4 and T7 arrested the development of P. falciparum at the trophozoite stage with an EC₅₀ value 21.89 μM and 49.88 μM. These compounds also showed morphological and food-vacuole abnormalities like E-64, a known inhibitor of FP2. Our results thus identify the quinoline-triazole-based compounds as a probable starting point for the design of FP2 inhibitors and they should be further investigated as potential antimalarial agents.

The present study involves development of novel quinoline triazole-containing cysteine protease inhibitors which arrest the development of P. falciparum at the trophozoite stage.     

The Proteasome Inhibitor Epoxomicin Has Potent Plasmodium falciparum Gametocytocidal Activity

Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester and N-acetyl-l-leucyl-l-leucyl-l-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum.The current recommended treatments for malaria caused by Plasmodium falciparum, including artemisinin combination therapy, eliminate intraerythrocytic asexual parasites that are responsible for the clinical symptoms. However, these treatments do not kill mature intraerythrocytic gametocytes that are required for the transmission of the parasite (24). In contrast to the 2-day asexual cycle of P. falciparum, the production of a mature stage V gametocyte takes 10 to 12 days. Once mature gametocytes are taken up by a mosquito during a blood meal, fertilization is stimulated. The resulting zygotes develop into oocysts where thousands of sporozoites are produced that can be transmitted to humans during a subsequent blood meal. The prolonged period required for P. falciparum gametocyte maturation in the human host suggests that malaria can be transmitted for several weeks after asexual parasites are eliminated (23). Thus, the development of drugs that are effective against both asexual-stage parasites and gametocytes may directly decrease malaria morbidity and mortality and reduce the spread of the disease.Cysteine protease and proteasome inhibitors have been found to affect asexual intraerythrocytic parasites and are being evaluated as possible antimalarial agents (4, 7, 10, 15, 19-21, 25). However, their effect on the 10- to 12-day course of intraerythrocytic gametocyte development has not been reported. Proteasome inhibitors also have not been tested on parasites taken up by a mosquito, while cysteine protease inhibitors have been shown to significantly decrease P. falciparum gamete surface antigen processing, oocyst production, and sporozoite maturation (7, 10). The dual cysteine and serine protease inhibitors l-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) also have been reported to reduce P. falciparum exflagellation and the transmission of Plasmodium berghei to mosquitoes (22, 26).Genes predicted to code for cysteine proteases and the proteasome are expressed throughout gametocytogenesis, providing targets for both classes of compounds (12, 28). Falcipain 1 and the P. berghei orthologs of PfSERA8 (PbECP1) and metacaspase 1 (PbMC1) are the only proteases whose function has been studied directly during gametocytogenesis by targeted gene disruption (3, 9, 14). The disruption of falcipain 1 and PfECP1 affected oocyst production in the mosquito midgut but not the asexual or sexual intraerythrocytic stage, while no stage of the life cycle was affected by the PbMC1 knockout. The work described here evaluates the effect of cysteine and proteasome inhibitors during P. falciparum sexual differentiation and development in the mosquito midgut.     

Proteases and protease inhibitors in infectious diseases

There are numerous proteases of pathogenic organisms that are currently targeted for therapeutic intervention along with many that are seen as potential drug targets. This review discusses the chemical and biological makeup of some key druggable proteases expressed by the five major classes of disease causing agents, namely bacteria, viruses, fungi, eukaryotes, and prions. While a few of these enzymes including HIV protease and HCV NS3‐4A protease have been targeted to a clinically useful level, a number are yet to yield any clinical outcomes in terms of antimicrobial therapy. A significant aspect of this review discusses the chemical and pharmacological characteristics of inhibitors of the various proteases discussed. A total of 25 inhibitors have been considered potent and safe enough to be trialed in humans and are at different levels of clinical application. We assess the mechanism of action and clinical performance of the protease inhibitors against infectious agents with their developmental strategies and look to the next frontiers in the use of protease inhibitors as anti‐infective agents.     

Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.     

Antimalarial activities of novel synthetic cysteine protease inhibitors

Among promising new targets for antimalarial chemotherapy are the cysteine protease hemoglobinases falcipain-2 and falcipain-3. We evaluated the activities of synthetic peptidyl aldehyde and alpha-ketoamide cysteine protease inhibitors against these proteases, against cultured Plasmodium falciparum parasites, and in a murine malaria model. Optimized compounds inhibited falcipain-2 and falcipain-3, blocked hemoglobin hydrolysis, and prevented the development of P. falciparum at nanomolar concentrations. The compounds were equally active against multiple strains of P. falciparum with varied sensitivities to standard antimalarial agents. The peptidyl inhibitors were consistently less active against vinckepain-2, the putative falcipain-2 and falcipain-3 ortholog of the rodent malaria parasite Plasmodium vinckei. The lead compound morpholinocarbonyl-leucine-homophenylalanine aldehyde, which blocked P. falciparum development at low nanomolar concentrations, was tested in a murine P. vinckei model. When infused continuously at a rate of 30 mg/kg of body weight/day, the compound delayed the progression of malaria but did not eradicate infections. Our data demonstrate the potent antimalarial activities of novel cysteine protease inhibitors. Additionally, they highlight the importance of consideration of the specific enzyme targets of animal model parasites. In the case of falcipains, differences between P. falciparum and rodent parasites complicate the use of the rodent malaria model in the drug discovery process.     

Comparison of efficacies of cysteine protease inhibitors against five strains of Plasmodium falciparum

Falcipain-2, a cysteine protease and essential hemoglobinase of Plasmodium falciparum, is a potential antimalarial drug target. We compared the falcipain-2 sequences and sensitivities to cysteine protease inhibitors of five parasite strains that differ markedly in sensitivity to established antimalarial drugs. The sequence of falcipain-2 was highly conserved, and the sensitivities of all of the strains to falcipain-2 inhibitors were very similar. Thus, cross-resistance between cysteine protease inhibitors and other antimalarial agents is not expected in parasites that are now circulating and falcipain-2 remains a promising chemotherapeutic target.     

Hybrid molecules with potential in vitro antiplasmodial and in vivo antimalarial activity against drug-resistant Plasmodium falciparum

Malaria is a tropical disease, leading to around half a million deaths annually. Antimalarials such as quinolines are crucial to fight against malaria, but malaria control is extremely challenged by the limited pipeline of effective pharmaceuticals against drug-resistant strains of Plasmodium falciparum which are resistant toward almost all currently accessible antimalarials. To tackle the growing resistance, new antimalarial drugs are needed urgently. Hybrid molecules which contain two or more pharmacophores have the potential to overcome the drug resistance, and hybridization of quinoline privileged antimalarial building block with other antimalarial pharmacophores may provide novel molecules with enhanced in vitro and in vivo activity against drug-resistant (including multidrug-resistant) P falciparum. In recent years, numerous of quinoline hybrids were developed, and their activities against a panel of drug-resistant P falciparum strains were screened. Some of quinoline hybrids were found to possess promising in vitro and in vivo potency. This review emphasized quinoline hybrid molecules with potential in vitro antiplasmodial and in vivo antimalarial activity against drug-resistant P falciparum, covering articles published between 2010 and 2019.     

Role of Plasmodium falciparum Digestive Vacuole Plasmepsins in the Specificity and Antimalarial Mode of Action of Cysteine and Aspartic Protease Inhibitors

Hemoglobin (Hb) degradation is essential for the growth of the intraerythrocytic stages of malarial parasites. This process, which occurs inside an acidic digestive vacuole (DV), is thought to involve the action of four aspartic proteases, termed plasmepsins (PMs). These enzymes have received considerable attention as potential antimalarial drug targets. Leveraging the availability of a set of PM-knockout lines generated in Plasmodium falciparum, we report here that a wide range of previously characterized or novel aspartic protease inhibitors exert their antimalarial activities independently of their effect on the DV PMs. We also assayed compounds previously shown to inhibit cysteine proteases residing in the DV. The most striking observation was a ninefold increase in the potency of the calpain inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) against parasites lacking all four DV PMs. Genetic ablation of PM III or PM IV also decreased the level of parasite resistance to the β-hematin binding antimalarial chloroquine. On the basis of the findings of drug susceptibility and isobologram assays, as well as the findings of studies of the inhibition of Hb degradation, morphological analyses, and stage specificity, we conclude that the DV PMs and falcipain cysteine proteases act cooperatively in Hb hydrolysis. We also identify several aspartic protease inhibitors, designed to target DV PMs, which appear to act on alternative targets early in the intraerythrocytic life cycle. These include the potent diphenylurea compound GB-III-32, which was found to be fourfold less potent against a P. falciparum line overexpressing plasmepsin X than against the parental nontransformed parasite line. The identification of the mode of action of these inhibitors will be important for future antimalarial drug discovery efforts focusing on aspartic proteases.Plasmodium falciparum malaria continues to exert a tremendous burden on communities in tropical and subtropical regions of the world. Efforts to control this disease have been stymied by the acquisition of resistance by this parasite to key antimalarials, including chloroquine (CQ) and pyrimethamine-sulfadoxine (60). Hemoglobin (Hb) degradation plays an essential role in malarial parasite development within infected red blood cells. As such, the parasite proteases involved in Hb degradation appear to be attractive targets for the development of novel antimalarials that are unaffected by the existing mechanisms of drug resistance.Hb digestion takes place in an acidic compartment, referred to as the digestive vacuole (DV; also known as the food vacuole) (23). This degradative process provides a source of amino acids and is thought to help maintain intracellular osmolarity during rapid parasite growth (40). Biochemical studies have implicated the DV aspartic proteases, termed plasmepsin (PMs), and the cysteine proteases, termed falcipains, as key mediators of this degradative process (24, 51, 55). Because of their ability to initiate the degradation of native Hb, PMs have long been considered important candidate drug targets (4).The four highly homologous DV PMs (PMs I, II, III [histidine aspartic protease {HAP}], and IV; gene identifiers, PF14_0076, PF14_0077, PF14_0078, and PF14_0075, respectively) are located as a contiguous set on chromosome 14. PM IV is the sole DV-specific PM found in all Plasmodium species sequenced and is the original DV PM ortholog that gave rise to P. falciparum paralogs PM I, II, and III through gene duplications (13, 16). PMs are translated as type II integral membrane proenzymes (5). Following trafficking to the DV, the N-terminal prodomain, which encompasses the cytosolic and transmembrane regions, is cleaved to release the mature enzyme into the DV lumen (25). This process has been shown to be inhibited by the calpain inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), suggesting that calpain might act as a maturase for PM activation (3). Drew et al. (18) recently showed that PM activation could occur via the falcipains. At low pH, PM processing can also occur autocatalytically (3, 18, 32).Two separate studies have now shown that no single DV PM is essential for the in vitro propagation of P. falciparum intraerythrocytic stages (38, 48). However, some of the PM-disrupted lines displayed reduced growth rates when they were cultured in complete medium. This was particularly pronounced with the PM IV-knockout (KO) line (38, 48). The slow growth of these slow-growth phenotypes was exacerbated in amino acid-limited medium (38, 39). Recently, a double-crossover integration strategy was successfully used to obtain parasites lacking PM I, PM II, and PM III (the triple-PM-KO line, denoted Δ3pfpm) or all four DV PMs (the quadruple-PM-KO line, denoted Δ4pfpm) (8). The Δ3pfpm parasite showed small decreases in growth rates and susceptibility to inhibitors compared to those of parental strain 3D7. Greater differences were observed with the Δ4pfpm parasite line, which had a lower rate of growth in complete medium, was severely hindered in its growth in amino acid-limited medium, and produced less of the Hb degradation by-product hemozoin. Δ4pfpm parasites also demonstrated condensed DVs by microscopic analysis (8; P. Moura, unpublished observations). Those parasites also contained numerous multimembrane vesicles in their DVs, suggesting that the growth defect of these parasites might have an etiology beyond impaired Hb digestion.In the study described here, we have leveraged the availability of these DV PM-KO lines to assess the specificity of putative DV protease inhibitors and probe their modes of action. Our data reveal that the disruption of PMs significantly enhances parasite susceptibility to several cysteine protease inhibitors and can also influence the potency of CQ. We also found that several PM aspartic protease inhibitors, previously thought to be specific to PM I or PM II, do not appear to act primarily on those targets in cultured parasites and appear to kill early-stage parasites prior to DV formation. The data suggest that these aspartic protease inhibitors exert their antimalarial activities primarily on one or more non-DV PMs and secondarily on the DV PMs.     

Plasmepsins as potential targets for new antimalarial therapy

Malaria is one of the major diseases in the world. Due to the rapid spread of parasite resistance to available antimalarial drugs there is an urgent need for new antimalarials with novel mechanisms of action. Several promising targets for drug intervention have been revealed in recent years. This review addresses the parasitic aspartic proteases termed plasmepsins (Plms) that are involved in the hemoglobin catabolism that occurs during the erythrocytic stage of the malarial parasite life cycle. Four Plasmodium species are responsible for human malaria; P. vivax, P. ovale, P. malariae, and P. falciparum. This review focuses on inhibitors of the haemoglobin-degrading plasmepsins of the most lethal species, P. falciparum; Plm I, Plm II, Plm IV, and histo-aspartic protease (HAP). Previously, Plm II has attracted the most attention. With the identification and characterization of new plasmepsins and the results from recent plasmepsin knockout studies, it now seems clear that in order to achieve high-antiparasitic activities in P. falciparum-infected erythrocytes it is necessary to inhibit several of the haemoglobin-degrading plasmepsins. Herein we summarize the structure-activity relationships of the Plm I, II, IV, and HAP inhibitors. These inhibitors represent all classes which, to the best of our knowledge, have been disclosed in journal articles to date. The 3D structures of inhibitor/plasmepsin II complexes available in the protein data bank are briefly discussed and compared.     

Antimalarial effects of human immunodeficiency virus type 1 protease inhibitors differ from those of the aspartic protease inhibitor pepstatin

Human immunodeficiency virus type 1 protease inhibitors (HIVPIs) and pepstatin are aspartic protease inhibitors with antimalarial activity. In contrast to pepstatin, HIVPIs were not synergistic with a cysteine protease inhibitor or more active against parasites with the cysteine protease falcipain-2 knocked out than against wild-type parasites. As with pepstatin, HIVPIs were equally active against wild-type parasites and against parasites with the food vacuole plasmepsin aspartic proteases knocked out. The antimalarial mechanism of HIVPIs differs from that of pepstatin.     

Inhibition of Protein Synthesis and Malaria Parasite Development by Drug Targeting of Methionyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS^cyt and PfMRS^api. Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS^cyt) or proteobacteria/primitive bacteria (PfMRS^api). We show that PfMRS^cyt localizes in parasite cytoplasm, while PfMRS^api localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several “hit” compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with ³⁵S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs.     

Potato tuber proteins efficiently inhibit human faecal proteolytic activity: implications for treatment of peri-anal dermatitis

Background Frequent diarrhoea after intestinal resections and faecal incontinence in healthy infants may lead to perianal injury. A causative agent may be a high concentration of pancreatic proteases in faeces. The aim of the present study was to assess whether protease inhibitors are applicable for treating and preventing peri‐anal dermatitis by inhibiting the initial cause of the inflammation, the faecal proteases. Design Proteolytic activity was estimated in faeces of subjects frequently suffering from peri‐anal dermatitis: patients with intestinal resections and healthy infants. The development of perianal dermatitis was studied after the construction of a reservoir with ileoanal anastomosis. The inhibitory effect of crude and partly purified potato juice on proteolytic activity of faecal output from patients with intestinal resections and healthy infants was investigated in vitro and in vivo (skin tests). Results Faecal protease activity in faeces from patients with intestinal resections and healthy infants was found to be significantly higher than in healthy adults. After the construction of an ileum reservoir, 46 of 48 patients developed a protease‐related peri‐anal dermatitis. The partly purified protein fraction from potatoes inhibited the larger part of faecal proteases in vitro and completely prevented skin irritation by pancreatic proteases dissolved in sterilized faecal fluid, in a 24‐h skin test, on the back of healthy human volunteers. Conclusions Potato proteins contain protease inhibitors, which suppress almost the complete proteolytic activity in faeces. Topical application of potato protease inhibitors might be a novel approach in preventing protease‐induced peri‐anal dermatitis, and therapeutic studies are needed to confirm our results.     

Discovery,mechanisms of action and combination therapy of artemisinin

Despite great international efforts, malaria still inflicts an enormous toll on human lives, especially in Africa. Throughout history, antimalarial medicines have been one of the most powerful tools in malaria control. However, the acquisition and spread of parasite strains that are resistant to multiple antimalarial drugs have become one of the greatest challenges to malaria treatment, and are associated with the increase in morbidity and mortality in many malaria-endemic countries. To deal with this grave situation, artemisinin-based combinatory therapies (ACTs) have been introduced and widely deployed in malarious regions. Artemisinin is a new class of antimalarial compounds discovered by Chinese scientists from the sweet wormwood Artemisia annua. The potential development of resistance to artemisinins by Plasmodium falciparum threatens the usable lifespan of ACTs, and therefore is a subject of close surveillance and extensive research. Studies at the Thai–Cambodian border, a historical epicenter of multidrug resistance, have detected reduced susceptibility to artemisinins as manifested by prolonged parasite-clearance times, raising considerable concerns on resistance development. Despite this significance, there is still controversy on the mode of action of artemisinins. Although a number of potential cellular targets of artemisinins have been proposed, they remain to be verified experimentally. Here, we review the history of artemisinin discovery, discuss the mode of action and potential drug targets, and present strategies to elucidate resistance mechanisms.     

Colorimetric High-Throughput Screen for Detection of Heme Crystallization Inhibitors

Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay''s reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.Malaria poses an enormous public health burden, causing over 1 million fatalities annually worldwide, with the majority of morbidity and mortality attributed to Plasmodium falciparum malaria. Chloroquine (CQ) served as the main chemotherapeutic for several decades, but the emergence and spread of drug resistance has limited its current usefulness. After the introduction of every new antimalarial drug, with the exception of artemisinin, resistant malaria parasites have emerged (25). Hence, the continued development of new antimalarial drugs is necessary to continue to successfully treat malaria infection.The malaria parasite cycles between two hosts, namely, mosquitoes (Anopheles sp.) and humans. In the human host, after a brief liver stage, P. falciparum resides exclusively inside red blood cells, where it feeds on hemoglobin, reproduces, and then releases progeny, which invade new red blood cells. During this intraerythrocytic stage, proteases digest hemoglobin within the food vacuole, a lysosome-like organelle (9). As hemoglobin is digested, heme molecules, containing redox-active iron centers, are released. The parasite overcomes the oxidative stress thus produced through the crystallization of free heme molecules into hemozoin (20). Hemozoin crystals are formed in an enzyme-independent reaction that is essential for parasite survival and therefore an excellent target for antimalarial chemotherapy. CQ, the most successful antimalarial agent to date, accumulates in the food vacuole, where it inhibits heme crystallization and prevents parasite proliferation (18). Small-molecule disruption of hemozoin formation has been proposed as a mechanism of action of many other antimalarial agents, including mefloquine (MQ), amodiaquine (AMQ), quinine, and quinidine, since each of these drugs successfully inhibits heme crystallization in in vitro assays, and they are all structurally related to CQ.We believe that the development of antimalarial agents based on the physicochemical process of heme crystallization could identify molecules that are less likely to generate resistance, like CQ. Drug resistance usually entails expression changes or mutations in the target protein or similar changes in pumps in order to expel the antimalarial agent (22, 26). Since heme crystallization inhibitors do not target a protein but a physicochemical process, resistance can occur only through the latter approach. For example, CQ resistance is achieved through multiple mutations in the P. falciparum CQ resistance transporter 1 (pfcrt1) allele, which encodes a transmembrane protein that, when mutated, significantly reduces the concentration of CQ in the food vacuole (8). Since the heme crystallization pathway remains unaltered in resistant parasites, it is still possible to exploit parasite heme crystallization while avoiding cross-resistance with CQ.In this study, we have adapted the pyridine hemichrome inhibition of β-hematin (Phiβ) assay described by Ncokazi and Egan for high-throughput screening (HTS) (17). This assay recapitulates in vivo heme crystallization by solubilizing hematin and then allowing it to spontaneously form crystalline β-hematin (synthetically identical to hemozoin) within a 384-well plate (1). Pyridine is used as a developing reagent to monitor heme crystallization, as pyridine molecules coordinated with the iron centers of free heme molecules produce a concentration-dependent color change, with a strong absorption at 405 nm. The optimized cell-free heme crystallization screen (CFHCS) was used to verify the mode of action of known antimalarials and to identify new chemical entities that inhibit hemozoin crystal formation.