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

Abstract

Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor–enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 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 constitution of P1′-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[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 reasonable 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 were promising for inhibitor development, were identified, including a π–π stacking interaction between a pyridine ring and Tyr372.

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

“…As evidenced by previous studies on organophosphorus inhibitors, more potent inhibitors of aminopeptidases are those extended to the P1' fragment rather than simple amino acid analogues designed to bind only within the S1 region [11,25,27,34]. This is not the case in the current study.…”

“…2, panel A for N' -cyclohexyl and panel B for N' -benzyl derivative). The crystals of the recombinant enzyme soaked with an inhibitor do not show significant changes in the overall binding site architecture compared with the ligand-unbound protein [24], nor to those visualized for Nm APN complexed with 1-aminoalkylphosphonic acids [25]. Two negatively charged phosphonic oxygen atoms coordinate the catalytic zinc ion (2.41 and 2.23 Å for 1h and 2.07 and 2.20 Å for 1n ), mimicking the gem -diolate transition state of the scissile peptide bond.…”

“…One of them also interacts with the hydroxyl group of Tyr377 (2.55 Å for 1h and 2.52 Å for 1 n ), the key residue for the hydrolytic mechanism. Nevertheless, the phosphonate of 1,2-diaminophosphonic acids seems to be buried somewhat shallower than in the 1-amino analogues for which the corresponding distances are slightly shorter, typically, O-Zn below 2.1 Å and O-Tyr377 below 2.5 Å [25]. The fundamental contacts (the salt bridges) of the α-amino group with the glutamates responsible for the binding of the N-terminus are also well reproduced, in particular for the N' -benzyl derivative 1n , with distances 2.65 Å to Glu117 and Glu260.…”

“…A careful and thorough optimization of the P1 and P1' substituent structure (modification/extension) in phosphorus-based transition state inhibitors and the examination of their interactions with the corresponding sub-sites has led to the discovery of very potent and selective inhibitors of metallo-dependent aminopeptidases, including APNs [25,27,34]. Although synthetically challenging, this method seems to be a rational and attractive approach for the construction of new ligands and an alternative to the elongation of pseudopeptidic sequences [35].…”

“…Typically, tightening organophosphorus ligand-APN interactions by P1'-S1' contacts provides at least a one to two order of magnitude gain in the binding constants [11,25]. Molecular modeling has somewhat clarified this issue.…”