Novel cyclic peptide inhibits intercellular adhesion molecule‐1‐mediated cell aggregation

Abstract

Leukocyte adherence mediated by intercellular adhesion molecule-1 (ICAM-1) binding to leukocyte function-associated antigen (LFA-1) is required for proper inflammatory and immune function. Inhibition of ICAM-1\LFA-1 binding using monoclonal antibodies (mAb) has been shown to be efficacious at inhibiting lymphoma metastasis as well as leukocyte emigration into tissue in a number of inflammatory diseases such as ischemia-reperfusion injury, septic shock and rheumatoid arthritis. In this report, we describe the development and characterization of a small peptide antagonist of ICAM-1-dependent cell aggregation. By using repeated selection of a cyclic nonapeptide phage display library on purified ICAM-1, we identified phage that were competitively eluted with anti-ICAM-1 mAb. The peptide sequences were determined by nucleotide sequencing, and the peptide sequence (C*LLRMRSIC*) (IP01) that occurred most frequently was chosen for further study. Phage expressing this peptide sequence specifically bound ICAM-1 over a range of 5 x 10(6) to 1 x 10(8) phage/microL. A cyclic IP01 peptide, linear IP01 peptide, a cyclic nonapeptide with a scrambled IP01 sequence, and a random, cyclic nonapeptide were synthesized. The cyclic and linear IP01 peptides were able to inhibit ICAM-1-mediated cell aggregation at a concentration of 1 mM, whereas the random and scrambled peptide sequences did not alter aggregation. Cyclic IP01 had a half-maximal inhibitory concentration of approximately 970 microM. Cyclic IP01 did not inhibit cellular aggregation that was dependent on ICAM-2 or ICAM-3. Alanine substitutions in the cyclic IP01 identified at least four amino acids necessary for inhibition of ICAM-1 dependent cell aggregation; leucine 2, leucine 3, methionine 5, and arginine 6. Finally, we showed that cyclic IP01 can inhibit firm adhesion of neutrophils to endothelium, a critical event in inflammatory diseases, in an assay that recapitulates physiologic flow conditions. Homology of IP01 with the primary amino acid sequences of the alpha or beta subunit of LFA-1 was not identified. Thus, we identified a unique molecule that inhibits ICAM-1 dependent cell adhesion, but is not related to the primary sequence of the ICAM-1 ligand LFA-1. Due to the small size and ability to block cell-cell adhesion, IP01 may serve as a useful tool for study of ICAM-1 and LFA-1 biology as well as for the development of small molecule therapeutics.

Multivalent Presentation of Antihantavirus Peptides on Nanoparticles Enhances Infection Blockade

“…Furthermore, peptides can act as lead compounds by providing a structural basis for the identification of nonpeptide mimetics when oral availability and increased potency are required. Peptides have been identified that can inhibit a number of cellular targets, including angiotensin-converting enzyme 2 (17), melanoma inhibitor of apoptosis (8), vascular endothelial growth factor (14), and intracellular adhesion molecule 1 (44), to name but a few. Likewise, in addition to a large number of naturally occurring antimicrobial peptides, a number of additional peptides, either novel or based on cellular components, have been identified, including a 36-amino-acid peptide called enfuvirtide (Fuzeon; Roche) currently marketed to prevent HIV-1 infection of T cells (7,27).…”

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 .…”

The signature molecular descriptor

“…Recently, identification of cell surface adhesion molecules that mediate the adhesion of leukocytes to the endothelium, such as leukocyte functional antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1), have allowed investigation into leukocyte trafficking [3][4][5]. Collaborators at the University of New Mexico Health Sciences Center developed a novel antagonist of ICAM-1 that has in-vivo efficacy [6][7][8][9], and Kelly and coworkers [10,11] have developed a small molecule antagonist of LFA-1, although toxicity effects have limited its development as an in-vivo inhibitor. Using our technique, we can predict compounds that provide the proper inhibitory effect, some of which hopefully lack any adverse toxicity effects.…”