Synthesis of New (−)-Bestatin-Based Inhibitor Libraries Reveals a Novel Binding Mode in the S1 Pocket of the Essential Malaria M1 Metalloaminopeptidase

Abstract

The malarial PfA-M1 metallo-aminopeptidase is considered a putative drug target. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1. Herein we present a new, efficient and high-yielding protocol for the synthesis of bestatin derivatives from natural and unnatural N-Boc-D-amino acids. A diverse library of bestatin derivatives was synthesized with variants at the sidechain of either the α-hydroxy-β-amino acid (P1) or the adjacent natural α-amino acid (P1’). Surprisingly, we found that extended aromatic sidechains at the P1 position resulted in potent inhibition against PfA-M1. To understand these data, we determined the X-ray co-crystal structures of PfA-M1 with two derivatives having either a Tyr(OMe) 15 or Tyr(OBzl) 16 at the P1 position and observed substantial inhibitor-induced rearrangement of the primary loop within the PfA-M1 pocket that interacts with the P1 sidechain. Our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme that may eventually lead to new therapies for malaria.

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

“…2E). Although aminopeptidases, including PfA-M1, are known to be capable of substantial active site flexibility [9,22], such main chain movements are likely to come at an energetic cost, which may account for the loss in PfA-M1 inhibitory activity of 9f compared to 9b. Despite the kinetic liability of the biphenyl group to both the 9f and 10f, its incorporation allows us access to the hydrophilic end of the pocket, which is occupied by ordered water molecules in 9f:PfA-M1 (Fig.…”

Development of Plasmodium falciparum Protease Inhibitors in the Past Decade (2002–2012)

“…PfA-M1 is found in both trophozoites and schizonts, being involved in hemoglobin degradation and erythrocyte reinvasion [142]. Therefore, design and synthesis of potential inhibitors of the PfA-M1 activity can lead to the discovery of new antimalarials [141,143,144]. For example, Deprez-Poulain's group first identified three non peptidic and non-selective inhibitors of this protease, and further optimized their results, and found the desired specificity against mammalian neutral aminopeptidases (APN), the host M1 family prototype [141].…”

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

“…[41][42][43][44][45] Recombinant expression in E. coli and/or insect cells was achieved by optimizing the gene codon usage, deleting regions with low complexity (including the trans-membrane portion in the case of PfM1APP) and removal of potential N-glycosylation sites. [41][42][43] Consistent with their mammalian homologues, rPfM1AAP was shown to be monomeric whereas rPfM17LAP is a homo-hexamer of approximately 320 kDa.…”

“…Using our recombinant proteins (see above) McGowan et al 42,43 reported the X-ray crystal structures of the monomeric PfM1AAP (lacking the first 194 residues which contains the trans-membrane domain) 42 and the hexameric leucine PfM17LAP, 43 alone and in complex with bestatin and the phosphinate analogue hPheP[CH 2 ]Phe. More recently, the same recombinant proteins were employed in the SAR studies of novel bestatin-based inhibitor libraries and activity-based probes by Valmourougane et al 44 and Harbut et al 45 The PfM1AAP consists of four domains; the highly-conserved zinc-binding H 496 EYFHX 17 KE 519 motif and the substrate recognition G 460 AMEN motif are contained within domain IV, or the catalytic domain ( Figure 2). These zinc binding and substrate coordinating motifs lie within the buried active site pocket that has access to the external milieu via two openings.…”