Unique Ligand Binding Sites on CXCR4 Probed by a Chemical Biology Approach: Implications for the Design of Selective Human Immunodeficiency Virus Type 1 Inhibitors

Abstract

The direct fusion of viral and target cell membranes required for human immunodeficiency virus type 1 (HIV-1) entry is initiated by the primary receptor, CD4, and a chemokine receptor, usually CXCR4 or CCR5. Chemokine receptors are members of the G-protein-coupled receptor (GPCR) superfamily that possess seven transmembrane (TM) domains. Because of its importance in the development of AIDS, CXCR4 has been explored as a new target for drug discovery to combat the AIDS epidemic (3,8,10). As the natural ligands of chemokine receptors, chemokines are small soluble proteins of about 70 amino acid residues that play prominent roles in leukocyte activation and inflammation (5, 11). Most of the known human chemokines are broadly categorized into the CXC and CC chemokines based on the position of two conserved cysteine residues in their amino (N)-terminal domains (3, 11). The natural chemokines of CXCR4 or CCR5 can inhibit HIV-1 infection (4, 13) by blocking HIV-1 gp120 binding sites (2, 14) and/or inducing receptor internalization (1, 9).Despite their important roles in the pathogenesis of AIDS and other human diseases, the lack of receptor selectivity of natural chemokines has made their direct clinical applications problematic. It is common knowledge that a chemokine receptor can often be recognized by multiple ligands, while a chemokine ligand binds to several different receptors (15), illustrating the apparent redundancy and the lack of selectivity in the chemokine ligand-receptor interaction network. As such, we have been working toward the development of a systematic chemical biology approach based on chemokine protein structures and chemistry to generate synthetically and modularly modified (SMM) chemokines that have higher receptor binding selectivity and improved pharmacological profiles compared with natural chemokines. This SMM chemokine approach was recently applied to generate novel ligands selective for CXCR4 or CCR5 by modifying the N-terminal (1-10) sequence module of viral macrophage inflammatory protein II (vMIP-II) or stromal cell-derived factor 1␣ (SDF-1␣) (unpublished data). Importantly, some of these SMM chemokines,

Neuronal Apoptotic Signaling Pathways Probed and Intervened by Synthetically and Modularly Modified (SMM) Chemokines

“…Note that we have tested various strains of gp120, including both X4-and R5-preferring gp120, and found that many of them caused neurotoxicity in RCCs. 3 Although the possibility remains that these CXCR4-specific SMM-chemokines could activate other known or unknown signaling pathways, the importance of steric hindrance in their neuroprotective mechanism is further supported by our mutational analysis of their binding sites, which shows overlap in the CXCR4 binding residues of these SMM-chemokines with X4-preferring gp120 but not with SDF-1␣ (48,49). Another mechanism whereby CCR5-selective ligands could suppress the neuroprotective action of these new CXCR4 antagonists would be heterologous desensitization (50,51), but such an effect need not be invoked here because the CCR5 ligands RCP188, RCP189, and gp120 BAL are neurotoxic on their own.…”

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

“…Three of these critical residues are buried directly below the CXCR4 signal initiator residues discussed above. These residues are F292 7.43 , which has been previously shown to be important for CXCR4 activity (17,19), A291 7.42 , which has a known role in introducing constitutive activity in CCR5 (27), and W252 6.48 , which is part of the well-characterized CWxP rotamer motif (28,29). Homologous positions in the A2AR structure (H278 7.43 and S277 7.42 ) have been shown to move closer to helix III upon agonist binding (30), supporting an important role for these residues in GPCR signal transmission.…”

“…3B, blue). These engagement residues include D97 2.63 at the top of helix II and D187 ECL2 in ECL2, both of which have previously been implicated in binding CXCL12 (9,17,22,23). On the other side of the pocket, D262 6.58 toward the top of helix VI (consistent with previous studies, ref.…”

“…CXCR4 residues involved in chemokine engagement. The interaction of CXCL12 with CXCR4 is known to be mediated by two distinct epitopes (1,8,12,(16)(17)(18)(19)(20)(21)(22). In the first [chemokine recognition site 1 (CRS1)], the unstructured N terminus of CXCR4 interacts with the globular core of the chemokine, specifically with the N loop and the 40s loop of CXCL12.…”

Inhibition of CXCR4 with the novel RCP168 peptide overcomes stroma-mediated chemoresistance in chronic and acute leukemias

“…It has been reported that RCP168 and SDF-1a bind overlapping yet distinct sites on the CXCR4 receptor. This pattern of RCP168 binding disrupts HIV infection much more potently than SDF-1a signaling through CXCR4 (nanomolar versus micromolar concentrations), albeit both effects are mediated by modulating the function of this receptor (29). RCP112 is an analogue of viral macrophage inflammatory protein II in which the first 10 amino acids in the NH 2 terminus have been deleted; it has shown a significantly reduced affinity to CXCR4 (30).…”