Journal of Molecular Biology
Involvement of the Second Extracellular Loop and Transmembrane Residues of CCR5 in Inhibitor Binding and HIV-1 Fusion: Insights into the Mechanism of Allosteric Inhibition
Introduction
C-C chemokine receptor 5 (CCR5) is a member of G-protein-coupled seven-transmembrane segment receptors (GPCRs), which comprise the largest superfamily of proteins in the body.1 In 1996, it was revealed that CCR5 represents one of the two essential coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into human CD4 + cells, thereby serving as an attractive target for possible intervention for HIV-1 infection using CCR5 as a coreceptor (R5-HIV-1).2, 3, 4, 5 The second extracellular loop (ECL2) of GPCRs is known to play a critical role in ligand binding and ensuing signal transduction. The ECL2 of CCR5 is also thought to play an important role in CCR5 interactions with HIV-1 envelope. To date, scores of newly designed and synthesized CCR5 inhibitors have been reported to be potent against R5-HIV-1;6, 7, 8, 9, 10, 11, 12, 13, 14, 15 one such inhibitor, maraviroc (MVC),11, 15 has recently been approved by the US Food and Drug Administration for treatment of HIV-1-infected individuals who do not respond to any existing antiretroviral regimen.
HIV-1 gp120 interacts with CCR5 following its binding to CD4, and such an interaction is thought to involve the V3 region of gp120 and the N-terminus and extracellular loops of CCR5.16, 17 Recent reports18, 19, 20, 21 have determined the orientation and location of CCR5 inhibitors within CCR5 and have shown that those inhibitors are all located in a hydrophobic cavity formed by the transmembrane domains of CCR5. In fact, earlier reports demonstrated that mutations in the extracellular loops did not have any effect on the binding of CCR5 inhibitors SCH-C and TAK-779.19, 22, 23 Taking these observations together, the binding sites in CCR5 for CCR5 inhibitors distinctly differ from the binding sites in CCR5 for HIV-1 gp120, strongly suggesting that CCR5 inhibitors block the interactions of CCR5 with HIV-1 gp120 by eliciting allosteric changes in extracellular loop structures.9, 22, 23
We previously reported a small-molecule CCR5 inhibitor, aplaviroc (APL; 4-[4-[(3R)-1-butyl-3-[(1R)cyclohexylhydroxymethyl]-2,5-dioxo-1,4,9-triazaspiro [5.5] undec-9 ylmethyl] phenoxy] benzoic acid hydrochloride), which has a high affinity for CCR5 (Kd values of 3 nM) and exerts potent activity against a wide spectrum of R5-HIV-1 isolates, including multidrug-resistant R5-HIV-1 strains.14, 24 APL significantly reduced viremia in patients with HIV-1 infection, as examined in a phase 2a clinical trial in the United States. However, in phase 2b clinical trials enrolling about 300 patients, four individuals receiving APL developed grade 3 or greater treatment-emergent elevations in ALT; in late 2005, the clinical development of APL was terminated. However, using APL as a specific probe, we further conducted structural analyses of CCR5 inhibitor interactions with CCR5, employing homology modeling, robust structure refinement, and molecular docking based on site-directed-mutagenesis-based saturation binding assay data of CCR5 inhibitors.22
In the current study, we determined the structural and molecular interactions of two novel CCR5 inhibitors, AK530 [(3S)-1-but-2-yn-1-yl-3-[(1S)-cyclohexyl-hydroxymethyl]-9 (3,5-dimethyl-1-phenyl-1H-pyrazol-4-ylmethyl)-1,4,9-triazaspiro [5.5] undecane 2,5-dione dihydrochloride] and AK317 [4-(4-{[(3S)-1-butyl-3-(cyclohexylmethyl)-2,5-dioxo-1,4,9-triazaspiro[5.5] undec-9-yl] methyl} phenoxy) benzoic acid hydrochloride] (Fig. 1), both of which contain a novel spirodiketopiperazine (SDP) scaffold. We found that these two inhibitors lodge in a hydrophobic cavity located between the upper transmembrane domain and the ECL2 of CCR5. We found substantial differences between the two molecules: AK530 had a 10-fold-greater CCR5-binding affinity (Kd = 1.4 nM) than AK317 (Kd = 16.7 nM), while their antiviral potencies were virtually identical [IC50 = 2.1 nM (AK530) and 1.5 nM (AK317)]. Modeling analysis showed that AK530 has the least interactions with S180 and K191 of ECL2, with which AK317 has a close association, suggesting that the interaction profile of the inhibitors with ECL2 residues is one of the important determinants of antiviral potency. We also found that the hairpin motif in the N-terminal half of ECL2 is critical for HIV-1-envelope-elicited fusion event. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits or substantially delays the emergence of HIV-1 that utilizes the “inhibitor-bound” CCR5 for cellular entry—a mechanism of HIV-1's resistance to CCR5 inhibitors. We also carried out molecular dynamics simulations of unbound CCR5 and compared the conformation with inhibitor-bound CCR5. Critical interhelix hydrogen bond interactions and interactions between the helices and the ECL2 seen in the unbound CCR5 were lost when transmembrane helix residues rearranged to accommodate AK530 and AK317 in the binding pocket. These observations add considerable insights into the mechanism of the allosteric inhibition of CCR5–gp120 interaction by CCR5 inhibitors.
Section snippets
Structural modeling of unliganded human CCR5
It is thought that the ECL2 of human CCR5 (Fig. 2) plays an important role in the binding of CC chemokines to CCR5, as well as in the binding of HIV-1-gp120/CD4 complex to CCR5 in the cellular entry of HIV-1.25, 26 On the other hand, certain amino acid residue substitutions such as Y108A, Y251A, and E283A, all of which are located in the transmembrane domain (Fig. 2a), significantly reduce both HIV-1-gp120/CD4 complex binding to CCR5 and HIV-1 susceptibility of CCR5-expressing cells, as
Discussion
In the present study, we demonstrated that two novel CCR5 inhibitors, AK530 and AK317, are lodged in a hydrophobic cavity located between the upper transmembrane domain and ECL2. CCR5 is a member of GPCRs, the largest superfamily of proteins in the body. Understanding the structure of human GPCRs would be invaluable in elucidating their roles in a number of biological processes and should also greatly aid in designing therapeutics. In particular, the elucidation of the detailed structure of
Reagents
Three SDP derivatives, APL,14, 46 AK530, and AK317, are discussed in the present report. The methods for the synthesis and physicochemical profiles of AK530 and AK317 will be described elsewhere. Tritiation of these three CCR5 inhibitors was conducted as previously reported.14 The structures of these three CCR5 inhibitors are illustrated in Fig. 1.
Cells, viruses, and anti-HIV-1 assay
CHO cells expressing wild-type CCR5 (CCR5WT-CHO cells) or mutant CCR5 (CCR5MT-CHO cells)22 were maintained in Ham's F-12 medium (Invitrogen,
Acknowledgements
The authors thank David A. Davis and Yasuhiro Koh for critical reading of the manuscript. This work was supported, in part, by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH, and in part by a Grant for the Promotion of AIDS Research from the Ministry of Health, Welfare, and Labor of Japan, and the Grant to the Cooperative Research Project on Clinical and Epidemiological Studies of Emerging and Reemerging Infectious Diseases (Renkei Jigyo: No.
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