Utilization of a peptide lead for the discovery of a novel PTP1B-binding motif

Examination of the PTP1B inhibitory potency of an extensive series of phosphotyrosyl (pTyr) mimetics (Xxx) expressed in the EGFr-derived hexapeptide platform Ac-Asp-Ala-Asp-Xxx-Leu-amide previously led to the finding of high inhibitory potency when Xxx = 4-(phosphono-difluoromethyl)phenylalanyl (F(2)Pmp) (K-i = 0.2 muM) and when Xxx = 3-carboxy-4-carboxy-methyloxyphenylalanyl (K-i = 3.6 muM). In the first instance, further work led from the F(2)Pmp-containing peptide to monomeric inhibitor, 6-(phosphonodifluoromethyl)-2-naphthoic acid (Ki = 22 muM), and to the pseudo-dipeptide mimetic, N-[6-(phosphonodifluoromethyl)-2-naphthoyl]-glutamic acid (K-i = 12 muM). In the current study, a similar approach was applied to the 3-carboxy-4-carboxymethyloxyphenylalanyl-containing peptide, which led to the preparation of monomeric 5-carboxy-6-carboxymethyloxy-2-naphthoic acid (K-i = 900 muM). However, contrary to expectations based on the aforementioned F(2)Pmp work, incorporation of this putative pTyr mimetic into the pseudo-dipeptide, N-[5-carboxy-6-carboxymethyloxy-2-naphthoyl]-glutamic acid, resulted in a substantial loss of binding affinity. A reevaluation of binding orientation for 5-carboxy-6-carboxymethyloxy-2-naphthoic acid was therefore undertaken, which indicated a 180 degrees reversal of the binding orientation within the PTP1B catalytic site. In the new orientation, the naphthyl 2-carboxyl group, and not the o-carboxy carboxymethyloxy groups, mimics a phosphoryl group. Indeed, when 5-carboxy-2-naphthoic acid itself was examined at neutral pH for inhibitory potency, it was found to have K-i = 31 +/- 7 muM, which is lower than parent 5-carboxy-6-carboxymethyloxy-2-naphthoic acid. In this fashion, 5-carboxy-2-naphthoic acid (or more appropriately, 6-carboxy-1-naphthoic acid) has been identified as a novel PTP1B binding motif.