Analysis of Antimalarial Synergy between Bestatin and Endoprotease Inhibitors Using Statistical Response-Surface Modelling

Abstract

The pathway of hemoglobin degradation by erythrocytic stages of the human malarial parasite Plasmodium falciparum involves initial cleavages of globin chains, catalyzed by several endoproteases, followed by liberation of amino acids from the resulting peptides, probably by aminopeptidases. This pathway is considered a promising chemotherapeutic target, especially in view of the antimalarial synergy observed between inhibitors of aspartyl and cysteine endoproteases. We have applied response-surface modelling to assess antimalarial interactions between endoprotease and aminopeptidase inhibitors using cultured P. falciparum parasites. The synergies observed were consistent with a combined role of endoproteases and aminopeptidases in hemoglobin catabolism in this organism. As synergies between antimicrobial agents are often inferred without proper statistical analysis, the model used may be widely applied in studies of antimicrobial drug interactions.Malaria remains one of the world's most important infectious diseases, and new antimalarial drugs are urgently needed, especially in areas where drug-resistant strains of the most lethal human malarial parasite, Plasmodium falciparum, are prevalent (21,22,30). The pathway of hemoglobin degradation by intraerythrocytic stages of P. falciparum has received a lot of attention as a potential therapeutic target (5). The parasite ingests large quantities of erythrocyte cytosol, polymerizing the heme moiety of hemoglobin into harmless crystalline inclusions (hemozoin) and digesting the globin to provide many of the amino acids required for protein synthesis. To date, most models have proposed that aspartyl proteases (plasmepsins I and II), cysteine protease (falcipain), and metalloproteases (falcilysin) are involved in hemoglobin degradation within a unique organelle, the digestive (food) vacuole (8,10,13,14,17,25,29). The growth-inhibitory actions of certain combinations of endoprotease inhibitors, especially those specific for aspartyl and cysteine protease classes, are synergistic on cultured parasites and possibly in animal models of malaria (1,25,27). The mechanism of synergy is unclear but may be related to the idea that endoproteases act sequentially in the same catabolic pathway. Accordingly, the possibility of developing combination therapy to target concomitantly more than one protease of the hemoglobinolytic pathway has become attractive.The aminopeptidase-specific inhibitors bestatin and nitrobestatin block malarial parasite growth in culture (20), and it is thought that one or more Plasmodium aminopeptidases are required for the terminal stages of hemoglobin breakdown, exoproteolytically cleaving globin-derived peptides to liberate free amino acids for incorporation into parasite proteins (7,12,17). Therefore, the aim of the present study was to investigate whether aminopeptidase and endoprotease inhibitors would act synergistically on the growth of cultured P. falciparum.A serious deficiency of the analysis of many published antimicrobial synergy and antagonism d...

Downstream effects of haemoglobinase inhibition in Plasmodium falciparum-infected erythrocytes

“…2), indicating 312 that the effects of the haemoglobinase inhibitors on protein synthesis were unlikely to be 313 merely secondary consequences of growth inhibition by some other mechanism. 314 315 Plasmepsin and falcipain inhibitors have been reported to display strong synergistic 316 effects on growth of P. falciparum [26, [45][46][47]. The data in Fig.…”

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

“…In addition, information gleaned from our preliminary SAR and crystallography efforts may provide a jumping off point for future medicinal chemistry efforts against both enzymes. Our data here suggest that combination therapy involving endopeptidase inhibitors, such as those for falcipains, and PfA-M1-specific inhibitors might provide an opportunity for a synergistic drug combination (52). Ultimately, this strategy may represent a good way to reduce the chance of parasite resistance.…”

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

“…We have demonstrated that the aminopeptidase inhibitor bestatin, an antibiotic and natural analogue of the dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents P. falciparum malaria growth in culture (13,14). More recently, it was shown not only that synthetic phosphinate dipeptide analogues that inhibit metallo-aminopeptidases prevented the growth of wildtype and the chloroquine-resistant parasites in culture but also that one compound, hPheP[CH 2 ]Phe (termed compound 4, Co4), reduced a murine infection of Plasmodium chabaudi chabaudi by 92% compared with controls (15,16).…”