br Conclusion We have identified three non

Conclusion We have identified three non-competitive inhibitors of the human and porcine APN with Ki values in the μM range, by combining virtual screening and kinetic assays. Molecular docking simulations suggest these novel inhibitors block APN activity by an alternative mechanism to Zn coordination. Of note, these compounds also inhibited the porcine aminopeptidase A (pAPA) by a competitive inhibition mode. This indicated differences in the binding mode of these compounds with APN and APA. Based on sequence and structural analyses we predict that targeting human APN residues: A351, R442, A474, F896 and N900 may lead to the design of more selective APN inhibitors. Remarkably, compounds BTB07018 and JFD00064 inhibited APN activity in the brain, liver and kidney of rats indicating a good bio-distribution in vivo. Compound BTB07018 seemed to be the most promising lead among the identified inhibitors due to its cLogP <4. This work reinforces the recent idea of designing novel APN inhibitors based on lead compounds without ZBG.
Notes
Introduction M1-Aminopeptidases constitute a diverse and widely distributed family of zinc-dependent hydrolytic enzymes [1]. They are strict exopeptidases that catalyze the hydrolysis of the amino-terminal residue from a peptide substrate. We are interested in understanding the biological roles of the M1-family aminopeptidase termed “PfA-M1” [2] during the pathogenic asexual intraerythrocytic stage of the malaria parasite Plasmodium falciparum. PfA-M1 exhibits a complex subcellular distribution in the parasite, residing in the food vacuole, the nucleus and the parasitophorous vacuole [3], [4], [5]. The best-characterized function of PfA-M1 is the catabolism of hemoglobin-derived peptides in the lumen of the acidic food vacuole. During its residence in the erythrocyte, P. falciparum ingests and degrades to ABT-888 up to 75% of soluble host cell cytosol, which consists mostly of hemoglobin [6]. A series of cleavages by vacuolar endo- and exo-peptidases results in the generation of large quantities of sequence-diverse short peptides [7]. By virtue of its vacuolar localization and its catalytic activity at the acidic pH of the vacuole [5], PfA-M1 is positioned to be a key catalyst for degrading these short globin peptides to individual amino acids. Treatment of parasites with a PfA-M1-selective inhibitor resulted in swelling of the food vacuole, accumulation of globin peptides, and, importantly, parasite death [8], which together with the biochemical evidence cited above support an essential role for PfA-M1 in vacuolar peptide catabolism. The inability to disrupt the gene encoding PfA-M1 in the haploid blood stage parasite is further evidence of its importance [4]. The essential role of PfA-M1 in disease-causing, asexual parasites has stimulated efforts to develop PfA-M1 inhibitors into novel anti-malarials [9], [10]. The natural product bestatin, a Phe-Leu analog with an N-terminal α-hydroxy-β-amino acid [11], is a sub-micromolar inhibitor of PfA-M1 [5], [12], [13] that has provided a convenient scaffold for optimization of inhibitor potency and selectivity [8], [14]. Phosphonate analogs of amino acids have also been explored as transition state mimics [15], [16]. The hydroxamate moiety, which chelates the catalytic zinc atom, has been successfully exploited to generate potent PfA-M1 inhibitors [17], [18], [19], [20]. To better understand the biological functions of PfA-M1 and to facilitate the design of potent inhibitors, we are interested in elucidating principles of substrate selection and catalysis. The S1 subsite is a well-defined, cylindrical pocket that receives the substrate’s P1 residue sidechain [13]. The S1 subsite plays a major role in substrate selection: basic and non-β-branched, non-polar P1 sidechains such as those of Leu, Met, Phe, Arg and Lys are accommodated, whereas substrates with acidic, β-branched or cyclic P1 residues (Asp, Glu, Val, Ile, Pro) are not efficiently hydrolyzed [2], [13], [21], [22]. The structure of the PfA-M1 S1 subsite occupied by the sidechain of the Phe-Leu analog bestatin, a competitive inhibitor of PfA-M1, is shown in Fig. 1[13]. At the bottom is the catalytic zinc atom liganded by three enzyme sidechains. The largely non-polar “walls” of the cylinder are formed by four amino acid sidechains: E319 (β- and γ-methylene groups), V459, M462 and Y575. As the cylinder wall residues define the shape and polarity of the base of the S1 subsite, they likely contribute to the selection against substrates with acidic or β-branched sidechains; however, they cannot explain how the subsite can tolerate both positively charged and large non-polar P1 sidechains.