High affinity CXCR4 inhibitors generated by linking low affinity peptides

Abstract

G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and

Designing drugs and chemical probes with the dualsteric approach

“…This method 54,57,76,84,86,88,117,121,126,129,132,134,136,160,161,171,252 is based on the theory that if a residue has a significant contribution ligand binding, the overall binding affinity might be remarkably changed when it is mutated into another amino acid (e.g., alanine). Therefore, we could perform site-directed mutagenesis to residues in both orthosteric and allosteric sites and to see whether the ligand binding is influenced in both the Fig.…”

Discoveries and developments of CXCR4-targeted HIV-1 entry inhibitors

“…64,65 The wise application of a combination of chemical, molecular modeling, mutagenesis, and biological approaches has led to the continuous discovery and development of more potent cyclic, bivalent, and multivalent analogs (Table 2). 52,53,56,57,59,60,62–69 The molecular modeling of CXCR4 dimer–synthetic ligand interactions based on the crystal structure of CXCR4 has demonstrated that synthetic dimeric ligands are capable of interacting with the CXCR4 dimeric structure by allowing the essential amino acid residues to interact with binding or signaling pockets of CXCR4 molecules. The use of different linkers, including polyethylene glycol, poly(L-proline), or Ahx, could allow the maintenance of an appropriate distance between the two binding sites of the ligands, consistent with that of CXCR4 dimers.…”

“…The use of different linkers, including polyethylene glycol, poly(L-proline), or Ahx, could allow the maintenance of an appropriate distance between the two binding sites of the ligands, consistent with that of CXCR4 dimers. 66–68 This part of research will surely be expanded in the future, not only to develop drug leads, but also to elucidate how the lead ligands interact with the amino acid residues of CXCR4.…”

“…Some of these new inhibitors show promise as therapeutic candidates for drug development and clinical applications, not only limited to inhibit HIV-1 entry, but also hinder HIV-1-induced actin rearrangement (which facilitates viral genome integration into host chromosome), and induce apoptosis of latently infected resting memory T cells. 32,68,70,71 The use of physiological ligand sequences, the viral ligand sequences, and conjugation strategies has led to a serious of peptide–peptide, peptide–small-molecule, or small molecule–small-molecule conjugates that possess bivalent or multivalent properties with enhanced or improved CXCR4 binding and anti-HIV-1 entry activities against either sole-tropic or dual-tropic HIV-1 isolates. 32,63,68 The use of natural protease-resistant D-peptides can be advantageous for drug development, because these peptides are highly stable and resistant to proteolytic degradation.…”

“…32,68,70,71 The use of physiological ligand sequences, the viral ligand sequences, and conjugation strategies has led to a serious of peptide–peptide, peptide–small-molecule, or small molecule–small-molecule conjugates that possess bivalent or multivalent properties with enhanced or improved CXCR4 binding and anti-HIV-1 entry activities against either sole-tropic or dual-tropic HIV-1 isolates. 32,63,68 The use of natural protease-resistant D-peptides can be advantageous for drug development, because these peptides are highly stable and resistant to proteolytic degradation. 72 Some of these peptides have potent anti-HIV-1 activities that are comparable to that of enfuvirtide (T20), a marketed drug targeting HIV-1 gp41 and entry.…”