Structure − Activity Relationship Study of Opiorphin , a Human Dual Ectopeptidase Inhibitor with Antinociceptive Properties

Toward developing new potential analgesics, this first structure−activity relationship study of opiorphin (HGln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe derivatives showed that replacing L-Phe for D-Phe increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH−π stacking interactions between the aromatic ring of D-Phe and the Hγ protons of Arg. This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues. ■ INTRODUCTION The need for compounds with intermediate analgesic activity between nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids has fueled the interest in enzyme inhibitors that protect endogenous opioid neuropeptides. One goal has been to potentiate the natural analgesic effect of the endogenous peptides Metand Leu-enkephalin by inhibiting their enzymatic degradation. Owing to the complementary roles of both neprilysin [neutral endopeptidase (NEP), EC 3.4. 24.11] and aminopeptidase N (AP-N; EC 3.4.11.2) in enkephalin inactivation, the design of dual inhibitors of these ectopeptidases seems an ideal approach. Both NEP and AP-N are membrane-bound zinc metalloenzymes that belong to the M13 (NEP) and M1 (AP-N) peptidase families. Although they share a common catalytic domain, differences in the spatial arrangement of the catalytic residues and of their surroundings cause NEP to function as a carboxypeptidase that cleaves substrates at the N-terminal side of hydrophobic amino acids such as Phe, Leu, and Met, while AP-N releases the N-terminal amino acids from peptides that are not substituted α-amino acids. To date, both enzymes have escaped full experimental structural elucidation. Thus, the X-ray crystal structure of the extracellular domain (Asp52−Trp749) complexed with six different small-molecule inhibitors is the only structural information available for NEP. Conversely, the structural information available for AP-N is more limited and reduced to three X-ray crystal structures corresponding to complexes of a small-molecule inhibitor with an AP-N variant from Escherichia coli. Regrettably, human AP-N has an overall sequence identity of only 13.6% with the one fom E. coli. Even though homology models of human AP-N have been recently developed for designing nonpeptidic inhibitors, the authors accept that authentic structural information on the human variant would be necessary for building more accurate and useful models. In spite of these caveats, the analogies of the well-studied bacterial protease thermolysin with NEP have long been used for the early designs of selective as well as dual and transition-state inhibitors of NEP and AP-N using secondary structure prediction and molecular modeling algorithms. One of the first examples of such compounds is the selective NEP inhibitor thiorphan, which has led to drugs with antisecretory activity such as the antidiarrheal racecadotril (acetorphan). The difficulty in formulating a dual inhibitor arises from the obvious necessity to find optimal interactions for two zinc peptidases that, despite their similarity, show different specificities. Nevertheless, mixed inhibitory products such as RB101, which is prepared by covalently linking a potent AP-N Received: September 13, 2011 Published: January 8, 2012 Article