title: 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 was 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 analogs 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.
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Węglarz-Tomczak
authors.forename: Marcin
authors.initials: M
authors.lastname: Poręba
authors.forename: Anna
authors.initials: A
authors.lastname: Byzia
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Berlicki
authors.forename: Bogusław
authors.initials: B
authors.lastname: Nocek
authors.forename: Rory
authors.initials: R
authors.lastname: Mulligan
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.forename: Marcin
authors.initials: M
authors.lastname: Drąg
authors.forename: Artur
authors.initials: A
authors.lastname: Mucha
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D018826:CD13 Antigens / Q000737:chemistry*; D002863:Chromogenic Compounds / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000737:chemistry / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry*; D011485:Protein Binding; D021281:Quantitative Structure-Activity Relationship; D011994:Recombinant Proteins / Q000737:chemistry; D054852:Small Molecule Libraries; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D004151:Dipeptides; D008024:Ligands; D009943:Organophosphorus Compounds; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D018826:CD13 Antigens