Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Abstract

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, Pf A-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric Pf A-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the Pf A-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to Pf A-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in Pf A-M17 and one in Pf A-M1) in both substrate and drug binding. Our detailed understanding of the Pf A-M1 and Pf A-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.drug design | malaria | protease | structural biology | neutral aminopeptidases

Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions

“…The active site of PfA-M1 is enclosed deep within the catalytic domain and is accessed by putative substrate entry and exit channels [9]. In contrast, the six active sites of PfA-M17 are located on the edge of the catalytic domain, and exposed to solvent in the interior cavity of the hexameric assembly [12]. Despite these major differences between PfA-M1 and PfA-M17, the enzymes share similar architecture within the active sites ( Fig.…”

“…1A and 1B). These similarities prompted suggestions that a single compound capable of inhibiting both enzymes could be developed [12]. A drug that can effectively inhibit more than one target could potentially slow the emergence of drug resistance parasites.…”

“…PfA-M1 possesses one catalytic zinc ion (Zn 2+ ), whereas PfA-M17 has two Zn 2+ and a catalytic carbonate atom. Previous inhibition studies show that cross inhibition of PfA-M1/PfA-M17 is achievable by targeting these catalytic zinc ion/s [9,10,[12][13][14][15], and led us to develop ((4-(1H-pyrazol-1-yl)phenyl)(amino)methyl)phosphonic acid (1) (Fig. 1C) (PfA-M1 K i = 104 µM and PfA-M17 K i = 0.011 µM), which binds within the S1 pocket of both enzymes [16].…”

“…The production of recombinant malaria neutral aminopeptidases PfA-M1 and PfA-M17 was undertaken in Escherichia coli and proteins purified using a two-step purification process of Ni-NTAagarose column followed by size exclusion chromatography on a Superdex 200 16/60 using an AKTAxpress high throughput chromatography system (http://proteinexpress.med.monash.edu.au/index.htm) as described previously [9,12]. Biochemical analysis indicated that kinetic parameters (k cat , K m , k cat /K m ) of material purified and used in subsequent crystallization trials were the same as published.…”

Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials

“…Table 1) [33], with the most lipophilic, 8c, being also the most active anti-plasmodial; amino acid and dipeptide esters are known to cross cell membranes by passive diffusion [38], so it is possible that the presence of the dipeptide spacer in HEDICINs augments cell permeation of these compounds by such process; b) recognition of the dipeptide motif by specific transporters from the P. falciparum ABC transporter super-family, many of which have been taken as putative drug transporting proteins [39] and found to be directly implicated in drugresistance mechanisms [40,41]; c) increased permeability of dipeptide derivatives due to the new permeation pathways created in P. falciparum-infected RBCs to promote uptake of nutrients such as sugars or nucleosides [42], as well as amino acids and oligopeptides [31]; II. Alternatively, a difference in the respective mechanism of action (MOA) may be operating in addition, or in alternative, to a difference in uptake of HEDICINs versus HECINs: a) due to their dipeptide moiety, HEDICINs might be able to inhibit plasmodial cytosolic proteases like aminopeptidases PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for parasite growth and development inside the red blood cell [43,44]; b) HEDICINs anti-plasmodial activity could otherwise be related to the cinnamoyl moiety, as it has been shown that cinnamic acid derivatives inhibit the growth of intraerythrocytic P. falciparum in culture by inhibiting monocarboxylate (e.g. lactate) transport and translocation of other nutrients, as carbohydrates and amino acids required for parasite nourishment [45]; c) finally, HEDICINs anti-plasmodial activity could as well be due to inhibition of the new permeation pathways; meaning that HEDICINs could eventually block the new permeation pathways created in infected RBC (see above, I.c), hampering the uptake of molecules essential to the parasite; it is interesting to notice that amongst our most active molecules as antiplasmodials are those bearing a nitro-aryl moiety, also present in potent new permeation pathways inhibitors such as 5-nitro-2-(phenylalkylamino) benzoic acid derivatives [31].…”