C-alpha methylation in molecular recognition - Application to substance P and the two neurokinin-1 receptor binding sites

Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associ ated with the neurokinin-1 receptor. For the first time with a bioactive pe ptide, the C-alpha methylation constraint, shown to be a helix stabiliser i n model peptides, was systematically used to probe the molecular requiremen ts of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven C-alpha methylated analogu es of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. T he consequences of C-alpha methylation on the structure of SP have been ana lysed by circular dichroism and nuclear magnetic resonance combined with re strained molecular dynamics. The decreased potencies of six out of these se ven C-alpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two bindin g sites. Strikingly, the most active analogue, [alpha MeMet5]SP, leads to v ariable subnanomolar affinity and potency when interacting with the NK-1m b inding site. The conformational analyses show that the structural consequen ces associated with C-alpha methylation of SP are sequence dependent. Moreo ver, a single C-alpha methylation is not sufficient by itself to drasticall y stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha -aminoisobutyric acid. Furthermore, C-alpha methylation of residues 5 and 6 of SP in the middle of the postulated heli x does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other poten t SP analogues.