High affinity CXCR4 inhibitors generated by linking low affinity peptides.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• G-protein coupled receptors (GPCRs) are crucial drug targets, but developing effective peptide inhibitors is challenging due to low affinity of peptide fragments.
• Current peptide fragments derived from GPCR ligands often lack structural integrity and exhibit weak receptor binding, limiting their therapeutic potential.

Purpose of the Study:

• To investigate a novel combinational strategy for generating high-affinity peptide inhibitors of GPCRs.
• To demonstrate proof of concept using the chemokine receptor CXCR4 (a GPCR) and viral peptide fragments.

Main Methods:

• Designed and synthesized two peptides (AR5 and AR6) by covalently linking fragments from viral CXCR4 ligands (vMIP-II and HIV-1 gp120).
• Assessed binding affinity, inhibition of HIV-1 entry via CXCR4, and downstream signaling inhibition.
• Employed western blotting, site-directed mutagenesis, molecular dynamics simulations, and binding free energy calculations for mechanistic studies of AR6.

Main Results:

• The synthesized peptides AR5 and AR6 exhibited nanomolar binding affinity for CXCR4, a significant improvement over the micromolar affinity of individual fragments.
• Both peptides effectively inhibited HIV-1 entry mediated by CXCR4.
• Mechanistic studies elucidated the interaction of AR6 with CXCR4 and its inhibition of downstream signaling.

Conclusions:

• A combinational approach of synthetically linking peptide fragments is effective for creating high-affinity GPCR inhibitors.
• This strategy yields potent CXCR4 inhibitors with nanomolar activity, applicable to drug discovery for various GPCRs.