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

Abstract

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 beta-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.

Exhaustive Conformational Sampling of Complex Fused Ring Macrocycles Using Inverse Kinematics

“…For IP01, BRIKARD generated low-energy conformations within 0.83–0.97 Å of all four major conformations identified by NMR 17 in 0.14 wallclock hours. The torsional angles of residue 4 (ARG) are distinct for each NMR state, so we use them to visualize the extent of conformational sampling in Figure 5.…”

Design and Structure of Peptide and Peptidomimetic Antagonists of Protein- Protein Interaction

“…Other laboratories [85][86][87][88][89][90] have synthesized interface peptides based on the binding sequence for ICAM-1 derived from the crystal structure of the complex [91]. We have used NMR to determine the solution structure of our peptides [92], and have docked the most active molecules onto the surface of ICAM-1 in a manner which reveals additional details relevant for the understanding of the relationship between peptide structure and activity [93], particularly the importance of amino acid residues enriched at the sites of protein-protein interaction. Other β2 integrin inhibitors are being pursued after the discovery of an LLG interface peptide sequence [94].…”

Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the β 3 -Integrin Receptor

“…The peptides that we identified in this study may serve as lead compounds for further optimization. The optimization of peptide antagonist activity through alanine and homologous amino acid substitution has resulted in 2-to 3-log improvements in inhibitory activity in other systems and may be justified in this system as well (35,37). Finally, structural information about peptides may be used as a guide to create orally available nonpeptide organics.…”

“…In fact, 10 of 11 peptides (group 4) with the greatest inhibition of SNV infectivity did not have significant sequence relatedness to SNV glycoproteins. Identification of inhibitory peptides that do not have significant sequence relatedness to native ligands has been described in other systems (35)(36)(37), and a large amount of work has indicated that nonhomologous amino acids may mimic the binding activities of each other (reviewed in reference 35). In addition, a comparison of the peptide sequences homologous with SNV to HTNV and PHV glycoproteins did not clearly identify possible contact points of FIG.…”

“…We chose this approach since we had previously used it successfully to identify inhibitory peptides of other integrin-ligand pairs (35)(36)(37). It is unclear what epitope on ␤ 3 integrin is recognized by ReoPro, and it is likely that antibodies are large enough to prevent interactions with regions well beyond the antibody's own epitope or the virus binding site on ␤ 3 .…”