NMR-derived model of interconverting conformations of an ICAM-1 inhibitory cyclic nonapeptide

: We have produced by phage‐display a disulfide‐linked cyclic nonapeptide (inhibitory peptide‐01, IP01), CLLRMRSIC, that binds to intracellular adhesion molecule‐1 (ICAM‐1) and blocks binding to its counter‐structure, leukocyte functional antigen‐1 (LFA‐1). As a first step towards improving its pharmacologic properties, we have performed a structural and functional analysis of this peptide inhibitor to determine the features relevant to ICAM‐1 binding. We report here the solution model of our initial product, IP01, as derived from two‐dimensional nuclear magnetic resonance (NMR) restraints and molecular modeling. Distance and dihedral angle restraints, generated from nuclear Overhauser effect spectroscopy (NOESY) and one‐dimensional‐NMR experiments respectively, were used to generate an ensemble of structures using distance geometry and simulated annealing. Molecular dynamic simulations produced three interconverting conformational families consistent with the NMR‐derived constraints. We describe these conformations and their mechanism of interconversion. Furthermore, we have measured the IC50 s of a series of inhibitors generated from IP01 through alanine substitution of each residue. These results show that the L2‐L3‐R4‐M5‐R6 segment is functionally active, conformationally flexible, and contains a β‐turn involving residues R4‐S7, while the C1‐C9‐I8‐S7 segment is less functionally‐active but adopts a more defined solution conformation, consistent with a scaffolding function. This model will be useful for designing nonpeptide‐based organic inhibitors with improved pharmacologic properties.