Aminobenzosuberone derivatives as PfA-M1 inhibitors: Molecular recognition and antiplasmodial evaluation

Highlights

• Antiplasmodial activity of racemic aminobenzosuberone derivatives.

• Crystal structure analysis of protein - ligand complexes.

• ADME-Tox and pharmacokinetic studies.

Abstract

Aminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC₅₀ values of 6.5–11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein.

Introduction

Despite encouraging wide-scale reduction in malaria incidence and mortality rates, malaria is still one of the most severe public health problems worldwide. It is a leading cause of death and disease in many developing countries particularly in young children. According to the WHO world malaria report 2019, there were an estimated 228 million cases of malaria and 405 000 related deaths in 2018 [1]. The regular emergence of antimalarial drugs resistance threatens the global malaria control and elimination effort, especially since artemisinin-resistant strains of Plasmodium falciparum were observed in the Greater Mekong region [2]. There is an urgent need for the discovery of novel Plasmodium molecular targets and original antimalarial agents with new modes of action to combat malaria.

P. falciparum proteases have appeared as promising targets for the development of new classes of therapeutic agents against malaria. They are involved in a number of vital pathways which are essential for parasite survival: red blood cell (RBC) invasion, hemoglobin (Hb) degradation, egress [3]. Among these pathways, Hb degradation provides amino acids necessary to the metabolism and survival of the parasites inside erythrocytes [4], [5], [6], [7], and also reduces the colloid-osmotic pressure within the host RBCs and thus preventing their premature lysis [8], [9].

One of these attractive protease targets is the Plasmodium falciparum M1 aminopeptidase, PfA-M1. This zinc-dependent metallo-aminopeptidase, belonging to the M1 family of aminopeptidases, catalyzes the cleavage of amino-acids at the N-terminus of peptides and plays a major role in the Plasmodium life cycle [10], [11], [12], [13], [14], [15], [16], [17], [18]. It has been mainly involved during asexual blood stage development in the last step of hemoglobin breakdown by hydrolyzing dipeptides into essential amino-acids. Contrary to other endopeptidases also involved in this Hb digestion, PfA-M1 is encoded by a single copy gene (MAL13P1.56 or PF3D7_1311800) [19], [20], [21] and possesses non-redundant function with no overlapping substrate specificity with other hemoglobinases [7], [22], [23]. The disruption of PfA-M1 gene has been unsuccessful until now, suggesting that this protease is important for parasite growth/multiplication [18], [19], [24], [25]. Moreover, in P. berghei most proteases involved in the Hb degradation pathway could be deleted except the orthologues of P. falciparum falcilysin and PfA-M1 [26]. According to omics data (Table S1), PfA-M1 has been reported in other developmental stages of the parasite’s life cycle, as it is expressed in merozoite, gametocyte and insect stages [15], [27], [28], [29], [30], [31], [32], [33], [34], [35]. Besides its involvement in Hb digestion, PfA-M1 might thus also play alternative roles in RBC invasion, egress or during gametogenesis [10], [12], [13], [18]. Currently considered as a promising drug target candidate, PfA-M1 was also recently proposed as a possible diagnostic biomarker for the presence of malaria parasite in young patients, displaying immunologic characteristics similar to those of some vaccine candidates (AMA1, MSP1, CSP) [36].

Given these results, PfA-M1 seems to be an essential protein and hence, a very attractive validated target for the development of novel antiplasmodial drugs [3], [19], [37], [38]. In addition, blocking PfA-M1 catalytic activity with low molecular weight inhibitors is fatal for the parasite without major toxicities for the host [16], [39].

Most of the PfA-M1 inhibitors (Table 1) consist of tetrahedral intermediate mimics, such as the well-known aminopeptidase inhibitor bestatin 1, or as phosphinic acid 2, or zinc-chelating group inserted in a peptide-like scaffold, such as hydroxamic acids 3–5. Despite demonstrating nanomolar to submicromolar activities in vitro on the enzyme, these compounds were usually 100 to 1000 fold less potent on in cellulo parasite growth inhibition assay (Table 1). Only, a handful of PfA-M1 inhibitors (1, 2 and 4) have been evaluated in vivo and demonstrated a reduction or suppression of the parasitaemia in murine models [24], [40]. However, their frequent lack of selectivity regarding other metallo-aminopeptidase families remains problematic. Indeed, most inhibit more potently PfA-M17, a bimetallic aminopeptidase, which is also considered as a promising antimalarial target [38], [39], [41], [42]. This dual activity is a valuable asset in today’s malaria eradication context, but it questions the relative involvement and importance of each enzyme in the parasite biology.

In this context, our research group has previously described the development of potent and selective aminobenzosuberone-based inhibitors of APN/CD13 [43], [44], [45], a human M1 aminopeptidase involved in angiogenesis and tumor metastasis [46], [47].

Recently, we reported that our scaffold 7c demonstrated potent in vitro inhibitory activity and was selective for rPfA-M1 over rPfA-M17 [48]. This compound was progressed to a 4-day suppressive test of Peters using a non-lethal parasite model P.c.chabaudi and was able to reduce the parasitaemia by 44% with a daily dose of 12 mg/kg, in four daily intraperitoneal injections, further strengthening the potential of PfA-M1 as a target for antimalarial research [49].

We report herein our initial research effort towards the identification and selection of the 4-phenyl aminobenzosuberone derivative 7c for in vivo efficacy assay. We also describe the crystal structure of rPfA-M1 in complex with aminobenzosoberone derivatives 7c and 7h.