Identification of New RGD-containing Cyclic Peptides As avb3 Integrin Antagonists
Introduction
Integrins are receptors for many extracellular matrix proteins which participate in multiple physiological processes. As a member of the integrin superfamily, αvβ3 regulates cell adhesion to extracellular matrix by binding with the conserved RGD-sequence motif. The αvβ3 integrin is known to be overexpressed in many cancers and in tumor vasculature, therefore making it a cancer target for cancer therapy and imaging. Many efforts have been on developing small molecular antagonists against the integrin. For example, a series of RGD-containing cyclic octapeptides- LXW analogues were screened using one-bead-one-compound (OBOC) combinatorial technology. However, these peptide antagonists show dramatically different binding affinity to the integrin and their structure-activity relationships (SAR) remain elusive. Furthermore, this in vitro screening procedure requires considerable efforts such as synthesis of OBOC libraries, on-bead whole-cell screening assays, etc., which is often time-consuming and costly. To develop and apply a rapid, low-cost in silico screening method combining with selective in vitro validation is needed for the screening purpose.
Methods
NMR structural biology, molecular modeling and computational docking approaches were applied for the systematic SAR studies of LXW cyclic peptides and αvβ3 integrin. A list of new peptide hits was virtually screened and the peptides were validated using the competition binding assay on αvβ3 integrin-transfected cells (K562/αvβ3+).
Results
The solution structures of representative LXW cyclic octapeptides determined by NMR and the structural modeling of these peptides in complex with the integrin revealed that the hydrophobicity and aromaticity of the residue at position 7 (X7) in the peptide amino acid sequence plays a critical role in binding with the integrin. This important structural clue was used for peptide optimization to increase their integrin binding potency, which allowed the screening of new peptide hits. The new peptides were examined by in vitro experiments in cell culture and exhibited stronger binding affinity than the original lead. As a result, new αvβ3 integrin antagonists were identified.
Conclusion
The SAR studies of LXW-analogous cyclic octapeptides and αvβ3 integrin were conducted showing that the hydrophobicity and aromaticity of the X7 amino acid in LXW-analogous sequence is important for integrin binding. New LXW-analogous peptides were identified by in silico screening. One of the peptides - LXZ2 was arbitrarily chosen and verified by cell-based competitive binding assays. This new αvβ3 antagonist was identified through a brief and comparatively inexpensive screening procedure. LXZ2 as a new αvβ3 integrin antagonist, can be used a vehicle for delivery of cytotoxic payload to tumors and tumor blood vessels with overexpressing αvβ3 integrin. These results provide new insight into the ligand recognition specificity of integrin, and valuable clues for rational design of novel αvβ3 integrin antagonists.