An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling, inhibitory studies and molecular modeling

Abstract

Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria. To explore the possibility of NmAPN being a potential drug target for inhibition and development of novel therapeutic agents, the specificity of the S1 and S1′ binding sites were explored using an integrated approach. Initially, an extensive library consisting of almost 100 fluorogenic substrates derived from both natural and unnatural amino acids, were used to obtain a detailed substrate fingerprint of the S1 pocket of NmAPN. A broad substrate tolerance of NmAPN was revealed, with bulky basic and hydrophobic ligands being the most favored substrates. Additionally, the potency of a set of organophosphorus inhibitors of neutral aminopeptidases, amino acid and dipeptide analogues was determined. Inhibition constants in the nanomolar range, determined for phosphinic dipeptides, proves the positive increase in inhibition impact of the P1′ ligand elongation. The results were further verified via molecular modeling and docking of canonical aminopeptidase phosphinic dipeptide inhibitors in the NmAPN active site. These studies present comprehensive characterization of interactions responsible for specific ligand binding. This knowledge provides invaluable insight into understanding of the enzyme and development of novel NmAPN inhibitors.

A structural insight into the P1 S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases

“…Most probably these plausible interactions are not essential effects, as 1-amino-3-phenylpropylphosphonic acid (the homophenylalanine analogue), the compound missing the β-amino group, is bound in a quite similar manner (Fig. S2) but exhibits an improved activity ( K i = 1.26 μM [1]).…”

“…A detailed substrate fingerprint study [1] has recently revealed a broad substrate tolerance of the S1 pocket of the M1 alanine metalloaminopeptidase from Neisseria meningitidis ( Nm APN) – a gram-negative diplococcus bacterium that is considered the major causative agent of meningitis and other meningococcal diseases [2-4]. Fluorogenic substrates, derived from unnatural α-amino acids that comprise bulky hydrophobic P1 side-chain residues bearing a fragment of a basic character, were ranked among the most favored ligands.…”

“…For Nm APN, we recently postulated that an additional heteroatom group located in the proximity of the side chain is able to enhance interactions with the glutamate-rich anionic site that is formed by Glu117, Glu260 and Glu316 and typically dedicated to the binding the N-terminus of peptidic substrates (Fig. 1, panel A ) [1]. …”

Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library

“…3) [21,22,25,26]. The application of 100 mM inhibitor resulted in a 95% decrease in activity, while 200 mM completely inhibited substrate hydrolysis.…”

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

“…In this manner, the sets of data that may be useful for design and synthesis of selective inhibitors are obtained (Drąg et al 2010;Kasperkiewicz et al 2012;Poras et al 2011Poras et al , 2013Węglarz-Tomczak et al 2013;Poręba et al 2014).…”