Design, synthesis and antimalarial activity of novel, quinoline-Based, zinc metallo-aminopeptidase inhibitors

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Abstract

PfA-M1, a neutral zinc aminopeptidase of Plasmodium falciparum, is a new potential target for the discovery of antimalarials. The design and synthesis of a library of 45 quinoline-based inhibitors of PfA-M1 is reported. The best inhibitor displays an IC50 of 854 nM. The antimalarial activity on a CQ-resistant strain and the specificity towards mammalian aminopeptidase N are also discussed.

Abstract

A new series of antimalarials consisting in 45 potential inhibitors of PfA-M1 is described.

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Introduction

Malaria, caused by the parasite Plasmodium falciparum, is still prevalent and lethal in many countries.1 Chloroquine (CQ), exerting its antimalarial activity by inhibiting haemozoin formation in the food vacuole of the parasite, has been the standard drug for many decades. Unfortunately, the spread of resistant P. falciparum to this molecule has created an urgent need to develop new antimalarial treatments.2 In a project to find new quinoline-based antimalarials that would not induce resistance, unlike CQ, we have designed several compounds displaying a piperazine linker. Several of these compounds were active on the CQ-resistant strain FcB1.3

The recent publication of the genome of the parasite opens the opportunities of a better understanding of the biology of the parasite.4 This will hopefully result in the discovery of new specific targets to tackle with drugs. Proteases expressed in the erythrocytic stage of P. falciparum, for example, can be considered as interesting targets for the design of antimalarials.5, 6

In particular, PfA-M1,7 a neutral zinc-aminopeptidase (M1 family), inhibited by the classical aminopeptidase inhibitor bestatin, has been proposed:

(1) to hydrolyze the small peptides generated in the food vacuole, during hemoglobin digestion by acidic endopeptidases (aspartyl, cysteine and metallo-endopeptidases), into amino-acids at the level of the cytoplasm, and: (2) to play a role in the erythrocyte re-invasion by the parasite.8 PfA-M1 shares a maximal homology in the active site region (44%) with two human proteases: the human aminopeptidase-N and leukotriene A4 hydrolase.9

It is the only aminopeptidase of P. falciparum that has been purified and biochemically characterized. This enables the design and testing of potential inhibitors. Such compounds would help understanding the role of this enzyme and validate it as a therapeutic target.

In a project aiming at decreasing the potential for resistance occurrence of our quinoline-based antimalarials, we have designed dual inhibitors that would inhibit both the haemozoin formation and one of the proteases potentially involved in globin digestion. Such a strategy has been developed by Avery for the design of malarial cysteine proteases inhibitors based on mefloquine and chloroquine.10 The screening of several of our in-house quinoline-based compounds allowed us to identify compound 1 as an interesting inhibitor of PfA-M1, that served as a starting point for analoguing (Fig. 1).

We report here the design and parallel synthesis of a library of 45 non-peptidic analogues of 1, and its biological evaluation on PfA-M1. Preliminary results on the antimalarial activity on FcB1 strain of the most active enzyme inhibitors, and preliminary specificity data on mammalian aminopeptidase N, are presented and discussed.

Section snippets

Chemistry

PfA-M1 is a zinc-metalloprotease, of the M1 family, preferably cleaving basic, hydrophobic, as well as aromatic amino-acids.8 Zn-chelating groups are critical for the inhibition of such enzymes.11 To fulfill this function, we incorporated at least a carboxylic acid or an hydroxamate group in our molecules. In order to mimic the side chain of a Leucine residue, an isobutyl group (Ibu), analogue of the methyl-cyclopropyl moiety in 1, was also added in our potential inhibitors.

The 45 analogues (5

Biological Assays

The analogues and the deprotected precursors were screened for their ability to inhibit PfA-M1 at 10 μM.14 Crude products displaying an inhibition percentage above 50% were selected for re-synthesis and IC50 determination on fully purified and controlled samples. The selected compounds were then tested for their ability to inhibit FcB1 parasite growth.15 The specificity against other aminopeptidases was evaluated in a model of mammalian aminopeptidase-N from porcine kidney.16

Conclusion

We have successfully designed non-peptidic inhibitors of PfA-M1, an aminopeptidase of P. falciparum. The quinoline moiety allows these compounds also to be inhibitors of haem detoxification, making these compounds potentially dual inhibitors of hemoglobin digestion. Yet the relative part of each mode of action needs to be determined. The three selected inhibitors 3335 are active against CQ-resistant strain, thus validating their ability to cross membranes. The activity on PfA-M1, and the

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