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sequence module of viral macrophage inflammatory protein II (vMIP-II) or stromal cell-derived factor 1␣ (SDF-1␣) (unpublished data).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,
The chemokine receptor CXCR4 is one of two principal coreceptors for HIV-1 entry into target cells. CXCR4 is known to form homodimers. We previously demonstrated that the amino (N)-terminus of viral macrophage protein (vMIP)-II is the major determinant for CXCR4 recognition, and that V1 peptide derived from the N-terminus of vMIP-II (1-21 residues) showed significant CXCR4 binding. Interestingly, an all-D-amino acid analog of V1 peptide, DV1 peptide, displayed even higher binding affinity and strong antiviral activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. In the present study, we synthetically linked two DV1 peptides with the formation of a disulfide bond between the two cysteine residues present in the peptide sequence to generate a dimeric molecule potentially capable of interacting with two CXCR4 receptors. DV1 dimer showed enhanced binding affinity and antiviral activity compared with DV1 monomer. Ligand binding site mapping experiments showed that DV1 dimer overlaps with HIV-1 gp120 on CXCR4 binding sites, including several transmembrane (TM) residues located close to the extracellular side and the N-terminus of CXCR4. This finding was supported by the molecular modeling of CXCR4 dimer–DV1 dimer interaction based on the crystal structure of CXCR4, which showed that DV1 dimer is capable of interacting with the CXCR4 dimeric structure by allowing the N-terminus of each DV1 monomer to reach into the binding pocket of CXCR4 monomer. The development of this bivalent ligand provides a tool to further probe the functions of CXCR4 dimerization and to study CXCR4 heterodimerization with other receptors.
Human immunodeficiency virus type 1 (HIV-1) uses a chemokine receptor, usually CXCR4 or CCR5, for entry into the target cells. Here, we used a chemical biology approach to demonstrate that binding and signaling domains in CXCR4 are possibly distinct and separate, as the new analogue, D(1-10)-vMIP-II-(9-68)-SDF-1alpha (RCP222), could not activate CXCR4 despite the fact that its binding activity was comparable to that of stromal cell-derived factor (SDF)-1alpha, the only natural ligand of CXCR4.
As the main coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, CXCR4 and CCR5 play important roles in HIV-associated dementia (HAD). HIV-1 glycoprotein gp120 contributes to HAD by causing neuronal damage and death, either directly by triggering apoptotic pathways or indirectly by stimulating glial cells to release neurotoxins. Here, to understand the mechanism of CXCR4 or CCR5 signaling in neuronal apoptosis associated with HAD, we have applied synthetically and modularly modified (SMM)-chemokine analogs derived from natural stromal cell-derived factor-1␣ or viral macrophage inflammatory protein-II as chemical probes of the mechanism(s) whereby these SMM-chemokines prevent or promote neuronal apoptosis. We show that inherently neurotoxic natural ligands of CXCR4, such as stromal cell-derived factor-1␣ or viral macrophage inflammatory protein-II, can be modified to protect neurons from apoptosis induced by CXCR4-preferring gp120 IIIB , and that the inhibition of CCR5 by antagonist SMM-chemokines, unlike neuroprotective CCR5 natural ligands, leads to neurotoxicity by activating a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Furthermore, we discover distinct signaling pathways activated by different chemokine ligands that are either natural agonists or synthetic antagonists, thus demonstrating a chemical biology strategy of using chemically engineered inhibitors of chemokine receptors to study the signaling mechanism of neuronal apoptosis and survival.
Chemokines and their receptors play important roles in normal physiological functions and the pathogeneses of a wide range of human diseases, including the entry of human immunodeficiency virus type 1 (HIV-1). However, the use of natural chemokines to probe receptor biology or to develop therapeutic drugs is limited by their lack of selectivity and the poor understanding of mechanisms in ligand-receptor recognition. We addressed these issues by combining chemical and structural biology in research into molecular recognition and inhibitor design. Specifically, the concepts of chemical biology were used to develop synthetically and modularly modified (SMM) chemokines that are unnatural and yet have properties improved over those of natural chemokines in terms of receptor selectivity, affinity, and the ability to explore receptor functions. This was followed by using structural biology to determine the structural basis for synthetically perturbed ligand-receptor selectivity. As a proof-of-principle for this combined chemical and structural-biology approach, we report a novel D-amino acid-containing SMM-chemokine designed based on the natural chemokine called viral macrophage inflammatory protein II (vMIP-II). The incorporation of unnatural D-amino acids enhanced the affinity of this molecule for CXCR4 but significantly diminished that for CCR5 or CCR2, thus yielding much more selective recognition of CXCR4 than wild-type vMIP-II. This D-amino acid-containing chemokine also showed more potent and specific inhibitory activity against HIV-1 entry via CXCR4 than natural chemokines. Furthermore, the high-resolution crystal structure of this D-amino acid-containing chemokine and a molecular-modeling study of its complex with CXCR4 provided the structure-based mechanism for the selective interaction between the ligand and chemokine receptors and the potent anti-HIV activity of D-amino acid-containing chemokines.Protein-protein interactions play important roles in a wide variety of physiological and pathological processes. The inhibition or promotion of these interactions, by either small or relatively large synthetic molecules, is of great interest for understanding the mechanism of biological recognition and developing novel therapeutic agents. In this regard, much progress has been made in recent years (3,8,29). This type of chemical research in protein-protein interactions is becoming increasingly important, especially in the postgenomic era, as chemically synthesized regulators of protein-protein interactions can be used to study the functions of new proteins uncovered by genomic-research efforts.One of the most important and challenging questions in the field of protein-protein interactions and development of intervening agents is selectivity in protein-protein interactions. Specifically, what are the mechanisms that dictate how one protein recognizes another out of a myriad of biological molecules, especially when the interacting partners in the same protein family share structural or functional homology? Alternativ...
The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1α (SDF-1α) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure–function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1α analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1α play a role in SDF-1α signaling via CXCR4 and/or receptor internalization. The results show that SDF-1α analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1α to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1α. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.
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