Structure-Guided, Single-Point Modifications in the Phosphinic Dipeptide Structure Yield Highly Potent and Selective Inhibitors of Neutral Aminopeptidases

Seven crystal structures of alanyl amino-peptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorous compounds were resolved to determine the optimal inhibitor-enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the PI amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hphe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constituion of P1 extended structures was rationally designed and the lead, phosphinic dipeptide hPheP psi[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom based fragment to either the P1 or P1' residue required new synthetic pathways . The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a resonable selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which wre promising for inhibitor development, were identified, including a pi-pi stacking interaction between a pyridine ring and Tyr372.