A SINGLE-POINT SLIGHT ALTERATION SET AS A TOOL FOR STRUCTURE-ACTIVITYRELATIONSHIP STUDIES OF OVINE CORTICOTROPIN-RELEASING FACTOR

In order to determine which amino acid side chains of ovine corticotro pin releasing factor (oCRF) are most sensitive to alterations with res pect to receptor binding and activation, we synthesized a single-point replacement set by replacing each residue by a similar, preferably pr oteinogenic amino acid, maintaining a minimal change of character at e ach position (Ser by Thr, Gln by Asn, Glu by Asp, Arg by Lys, and vice versa, Pro by N-MeAla, Ile by Leu, Leu by Nle, Phe by Trp, His by Ala , Val by Leu, Met by Nle, Ala by Leu). In general, any loss in the bio logical potency by a single-point substitution in oCRF parallels a dec rease in receptor binding, indicating that, in contrast to previous su ggestions,(1) there is no specific side chain in the peptide that is m ore responsible for receptor activation than for receptor binding. In addition to Arg(16), Ala(31), and Arg(35), amino acid residues in the N-terminal sequence (5-14) were found to be sensitive to alteration, d emonstrating their particular importance for the receptor interaction of CRF agonists. Most of the analogs tested exhibited agonistic potenc ies in an in vitro pituitary cell culture assay at a concentration of 0.3 nM, and all analogs showed full agonistic potency at 1 mu M. In co ntrast to the results of an alanine replacement study,(2) the stronges t-decrease in receptor binding and biological potency was observed for analogs with substitutions of hydrophilic amino acids Ser(7), Arg(16) , Glu(17), or Asn(34). In the case of Ser(7) and Arg(16), side chain s pecific interactions with the receptor may be required for high affini ty. Alanine replacements at positions 17 or 34 resulted in analogs tha t were as potent as oCRF, while replacement of Glu(17) by Asp or Asn(3 4) by Gln caused a dramatic loss in potency, thereby suggesting an imp ortant effect at sterically or conformationally sensitive positions. I n contrast to corresponding alanine analogs which exhibited a signific ant loss in biological potency,(2) slight alterations of lipophilic si de chains at positions 6, 12, or 38 did not-cause-a-significant reduct ion of receptor binding and activation, indicating that it is not spec ific side chains but rather lipophilicity which is essential at these positions. Indeed, replacement of Phe(12) by Trp provides an agonist w ith significantly increased receptor binding and biological potency.