CONFORMATIONAL-ANALYSIS OF CYCLIC HEXAPEPTIDES DESIGNED AS CONSTRAINED LIGANDS FOR THE SH2 DOMAIN OF THE P85 SUBUNIT OF PHOSPHATIDYLINOSITOL-3-OH KINASE

The structures of the cyclic hexapeptide cycle (-Gly-Tyr-Val-Pro-Met-L eu-) (1) and its phosphotyrosyl (pTyr) derivative cycle [-Gly-Tyr(PO3H 2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology, 2 (SH2) region of the p85 subun it of phosphatidylinositol-3-ON kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was f ound to exist as a 2:1 mixture of isomers about the Val-Pro bond (tran s and cis prolyl) between 292-330 K in 75% CD3O(D,H)/ (D,H)(2)O soluti ons. A third species of undetermined undetermined structure (etc. 5%) was also observed Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformat ional isomerism. When either compound was dissolved in pure solvent [i .e., 100% CD3O(H,D) or (H.D)(2)O] the ratio of cis to trans isomers wa s ca 1:1. A battery of one- and two-dimensional nmr experiments at dif ferent temperatures and solvent compositions allowed a complete assign ment of both the cis and trans forms of 1 and indicated the trans comp ound to be the major. isomer. The spectral properties of the phophoryl ated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectros ropy data, coupling constants, amide proton temperature dependence and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations us ing the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-me mbered ring separated by a distance of 16-18 Angstrom, in contrast wit h the cis isomer where the side chains of these residues are much clos er in space (7-14 Angstrom). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical reco gnition elements for pTyr-peptide binding to the PI-3 kinase SH2 domai n. Such cyclic structures may offer appropriate scaffolding for positi oning important amino acid side chains of pTyr containing peptides as a means of increasing their binding affinities to SH2 domains, and in trim provide a conceptual approach toward the design of SH2 domain dir ected peptidomimetics. (C) 1996 John Wiley & Sons, Inc.