Protein Structure-based Peptide Design, Synthesis And Biological Characterization

Structure-based molecule design strongly relies on knowledge of thethree dimensional structure of the biological target obtained throughmethods such as x-ray crystallography or NMR spectroscopy. Using thestructure of the biological target, candidate drugs that are predicted tobind with high affinity and selectivity to the target may be designed usinginteractive graphics and the intuition of a medicinal chemist. We use twodifferent proteins as models, WDR5and CXCR4, which represent themembrane proteins and cytoplasmic proteins, to investigate the highaffinity, specificity ligand design.As members of the GPCR superfamily, chemokine receptors such asCXCR4play critical roles in normal physiology as well as the pathologyof many human diseases including cancer, inflammation, autoimmunediseases, and human immunodeficiency virus (HIV) infection. Here we report the discovery and study of a series of novel peptide ligands ofCXCR4using bivalent ligand approach. These peptides show very highaffinity for CXCR4with an IC50of4-200nM in anti-CXCR4mAbmonoclonal antibody (mAb)12G5competitive assay, which is evenbetter than full length natural ligand SDF-1α even though the peptide isless than half of the number of residues of SDF-1α. The antagonistpeptides can block the calcium influx stimulated by SDF-1α and inhibitcancer cell migration in vitro via CXCR4, while the agonist peptides caninduce the receptor phosphorylation and cell migration. One peptide,DV1-K-(SDF-1-8), shows anti-HIV-1infection activity in vitro. With itshigh receptor affinity these peptides may be a new probe of CXCR4functions in physiology and pathology and promising lead for therapeuticdevelopment. The MLL (Mixed Lineage Leukemia) protein is a histone3lysine4(H3K4) methyltransferase. Abnormal activities of H3K4methylatingenzymes have been observed in various cancers and particularly inleukemias. The catalytic activity of MLL is regulated by a core complexthat consists of MLL, WDR5, RbBP5, and Ash2L. The H3K4methyltransferase activity depends on the interaction between WDR5andMLL. Interruption of this interaction would therefore be a valuablestrategy for cancer therapy. We used purified WDR5protein as a targetfor screening two phage-display random peptide libraries (12-mer linearand7-mer cyclic) and we obtained a number of phage clones that couldbind specifically to WDR5. The corresponding peptides were synthesizedand several showed inhibition of the WDR5-MLL3interaction in afluorescence polarization-based competitive assay with low micro-molarrange. These peptides also effectively inhibited the dimethylation activity catalyzed by the MLL1core complex in vitro. Determination of co-crystal structure of WDR5with peptide No.3and No.5gave us thestructural basis for further peptide modification.