Small Molecule CCR5 Antagonists Research Articles

Abstract 2731: CCR5-001, a novel drug Fc-conjugate (DFC) targeting CCR5, demonstrates potent efficacy in a colorectal cancer mouse model

Abstract Background The approval of immune checkpoint inhibitors (ICI) such as PD-(L)1 inhibitors has revolutionized cancer therapy and established a role for immune effector cells, most notably CD8+ T cells, in tumor control and destruction. While anti-PD-(L)1 therapy has demonstrated durable responses, only a small subset of patients benefit and respond to therapy. CCR5 is expressed on multiple immune cells (e.g. myeloid-derived suppressor cells) and CCR5-dependent migration of these cells contribute to an immune-suppressive tumor microenvironment (TME) thereby limiting response to ICI therapy. In addition, expression of CCR5 on cancer cells has been linked to increased metastasis and resistance to ICI therapy. Upregulation of CCL5/CCR5 has been associated with poor outcomes in multiple solid tumor indications, including colorectal cancer (CRC). Therefore, targeting CCR5 has the potential to increase response rates to ICI therapy and improve outcomes. Herein, we describe a first-in-class CCR5 targeting DFC, CCR5-001, a multivalent conjugate of a potent, small molecule CCR5 antagonist conjugated to an immune-silent proprietary human IgG1 Fc. This CCR5 targeting DFC has the potential to improve responses to ICI therapy by preventing pro-tumorigenic reprogramming of the TME induced by CCL5/CCR5 expressing tumor and myeloid cells. Methods Binding of CCR5-001 to hCCR5 was measured by surface plasmon resonance (SPR) and to CCR5+ PathHunter cells (DiscoverX) by flow cytometry. Functional activity was determined in CCR5+ PathHunter β-arrestin assay with CCL3 to induce CCR5 signaling according to manufacturer’s instructions. Efficacy was determined in an immune-competent syngeneic mouse model using the CRC cell line, MC38. CCR5-001 was dosed at 20 mg/kg twice per week for two weeks. Tumor volumes were recorded and statistical analysis was conducted by t-test (Mann-Whitney) or two-way ANOVA in Prism. Results CCR5-001 displayed a KD of 4.2 nM to immobilized hCCR5 by SPR and IC50 of 0.63-0.98 nM for binding to engineered CCR5+ PathHunter cells. This high affinity binding translated to potent functional inhibition of CCL3-induced CCR5 signaling with an IC50 of 12.7 nM in CCR5+ cell-based assay. CCR5-001 demonstrated potent, statistically significant (p = 0.0052) tumor growth inhibition of 76% by day 13 in a syngeneic mouse model. Conclusions CCR5-001 demonstrated potent CCR5 binding and functional inhibition of CCR5 signaling in cell-based assays. The in vitro activity translated to robust efficacy as a monotherapy in a syngeneic mouse model. Based on these results and other emerging data, CCR5-001 represents an exciting new avenue for targeting CCR5 with DFCs in solid cancers where the pathology is driven by the CCR5/CCL5 axis. Citation Format: Simon Döhrmann, James Levin, Nicholas Dedeic, Amanda Almaguer, Doug Zuill, Elizabeth Abelovski, Jeffrey B. Locke, Joanne Fortier, Maria Hernandez, Qiping Zhao, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Grayson Hough, Thomas P. Brady, Allen Borchardt, Jason N. Cole, Leslie W. Tari. CCR5-001, a novel drug Fc-conjugate (DFC) targeting CCR5, demonstrates potent efficacy in a colorectal cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2731.

Multitargeted drug design strategy for discovery of short-peptide-based HIV-1 entry inhibitors with high potency

The development of short-peptide-based inhibitors to prevent HIV-1 entry into the host cell has been rewarded with limited success. Herein, we report a multitarget-directed ligand strategy to generate a series of short-peptide HIV-1 entry inhibitors that integrated the pharmacological activities of a peptide fusion inhibitor able to disrupt HIV-1 gp41 glycoprotein hexameric coiled-coil assembly and a small-molecule CCR5 antagonist that blocks the interaction between HIV-1 and its coreceptor. Among these inhibitors, dual-target 23-residue peptides SP12T and SP12L displayed dramatically increased inhibitory activities against HIV-1 replication as compared to the marketed 36-residue peptide T20. Moreover, results suggested that SP12T and SP12L successfully performed a dual-targeting mechanism. It can be concluded that these short-peptide-based HIV-1 entry inhibitors have potential for further development as candidates for a novel multitarget therapy to treat HIV-1 infection.

CCR5-Mediated Signaling is Involved in Invasion of Glioblastoma Cells in Its Microenvironment

The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.

Human CCL3L1 copy number variation, gene expression, and the role of the CCL3L1-CCR5 axis in lung function.

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc,and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5del32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5del32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Human CCL3L1 copy number variation, gene expression, and the role of the CCL3L1-CCR5 axis in lung function

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc, and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5d32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5d32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Human CCL3L1 copy number variation, gene expression, and the role of the CCL3L1-CCR5 axis in lung function

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc, and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5del32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5del32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Short Communication: Limited Anti-HIV-1 Activity of Maraviroc in Mucosal Tissues.

The potential of maraviroc (MVC), a small-molecule CCR5 antagonist, as a candidate to prevent HIV-1 sexual transmission by oral or topical dosing has not yet been completely established. Using relevant cellular and mucosal tissue explant models, we show partial antiviral activity of MVC when tested in multiple preclinical dosing strategies.

Temporary CXCR3 and CCR5 antagonism following vaccination enhances memory CD8 T cell immune responses.

Although current vaccination strategies have been successful at preventing a variety of human diseases, attempts at vaccinating against some pathogens such as AIDS and tuberculosis (TB) have been more problematic, largely in that abnormally high numbers of antigen specific CD8+ T cells are required for protection. This study assessed the effect of temporarily dampening the chemokine receptor CXCR3 and CCR5 after vaccination on host immune responses by the administration of TAK-779, a small molecule CXCR3 and CCR5 antagonists commonly used to inhibit HIV infection. Our results showed that the use of TAK-779 enhanced memory CD8+ T cell immune responses both qualitatively and quantitatively. Treatment with TAK-779 following vaccination of an influenza virus antigen resulted in enhanced memory generation with more CD8+CD127+ memory precursor and fewer terminally differentiated effector CD8+CD69+ T cells. These memory T cells were able to become IFN-γ-secreting effector cells when re-encountered the same antigen, which can further enhance the efficacy of vaccination. The mice vaccinated in the presence of TAK-779 were better protected upon influenza virus challenge than the control. These results showed that vaccination while temporarily inhibiting chemokine receptor CXCR3 and CCR5 by TAK-779 could be a promising strategy to generate large number of protective memory CD8+ T cells.

Medicinal chemistry of small molecule CCR5 antagonists for blocking HIV-1 entry: a review of structural evolution.

CCR5, a member of G protein-coupled receptors superfamily, plays an important role in the HIV-1 entry process. Antagonism of this receptor finally leads to the inhibition of R5 strains of HIV entry into the human cells. The identification of CCR5 antagonists as antiviral agents will provide more option for HAART. Now, more than a decade after the first small molecule CCR5 inhibitor was discovered, great achievements have been made. In this article, we will give a brief introduction of several series of small molecule CCR5 antagonists, focused on their appealing structure evolution, essential SAR information and thereof the enlightenment of strategies on CCR5 inhibitors design.

Generation of an HIV Resistant T-cell Line by Targeted “Stacking” of Restriction Factors

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

Differential Use of CCR5 by HIV-1 Clinical Isolates Resistant to Small-Molecule CCR5 Antagonists

How HIV-1 resistant to small-molecule CCR5 antagonists uses the coreceptor for entry has been studied in a limited number of isolates. We characterized dependence on the N terminus (NT) and the second extracellular loop (ECL2) of CCR5 of three vicriviroc (VCV)-resistant clinical isolates broadly cross-resistant to other CCR5 antagonists. Pseudoviruses were constructed to assess CCR5 use by VCV-sensitive and -resistant envelopes of subtype B and C viruses. We determined the extent of entry inhibition by monoclonal antibodies (MAbs) directed against the NT and ECL2 in the presence and absence of VCV and the capacity of these pseudoviruses to use CCR5 mutants that contained scanning alanine substitutions in the CCR5 NT and ECL2 domains. Sensitive and resistant viruses were completely and competitively inhibited by the ECL2-specific MAb 2D7, whereas the NT-specific MAb CTC5 led to partial noncompetitive inhibition. VCV-resistant clones showed greater sensitivity to 2D7 than VCV-sensitive clones, but in the presence of saturating VCV concentrations, the 2D7 susceptibilities of two VCV-resistant viruses were similar to that of VCV-sensitive virus. The entry of VCV-sensitive and -resistant isolates was impaired to differing degrees by alanine mutations in CCR5; substitutions in NT had the greatest effect on viral entry. HIV-1 clinical isolates broadly resistant to CCR5 antagonists demonstrated significant heterogeneity in their use of CCR5. This heterogeneity makes it difficult to draw general conclusions about the relationship between patterns of CCR5 antagonist resistance and the use of specific CCR5 domains for entry.

Recent Progress in Small Molecule CCR5 Antagonists as Potential HIV-1 Entry Inhibitors

Currently the long-term usage of traditional anti-HIV drugs, such as nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs), eventually leads to the emergence of drug resistance and severe side effects. Thus it is imperative to design and develop more promising HIV-1 inhibitors to overcome these drawbacks. Fortunately, with the identification of some fascinating targets in the entry process of viral life cycle, HIV-1 entry inhibitors (EIs) with low cytotoxicity and mild side effects turned out as novel and effective anti-HIV agents. Especially, of these potent EIs, small molecule CCR5 antagonist maraviroc was approved by US FDA in 2007, which significantly increased the therapeutic options for the clinical treatment of HIV-infected patients. Subsequently, as promising anti-HIV drug candidates, kinds of small molecule CCR5 antagonists have been synthesized and evaluated in clinical trials. In this article, current progress in the development of novel small molecule CCR5 antagonists will be reviewed on the basis of their chemical structures with a special attention to their discovery stories. Simultaneously, binding mode analysis based on molecular modeling studies will also be introduced.

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C- and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell β-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either population-derived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance.

Allosteric and Orthosteric Sites in CC Chemokine Receptor (CCR5), a Chimeric Receptor Approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.

Renal Insufficiency Has No Effect on the Pharmacokinetics of Vicriviroc in a Ritonavir-Containing Regimen

Vicriviroc is a small-molecule CCR5 antagonist currently in development for the treatment of HIV in patients on a regimen containing a ritonavir-boosted protease inhibitor. As renal disease and renal dysfunction are prevalent in the HIV-infected population, patients with varying degrees of renal insufficiency may receive vicriviroc, which is metabolized by cytochrome P450 (CYP) 3A4. The present study therefore examined the impact of renal insufficiency on the pharmacokinetics and safety of vicriviroc alone and in the presence of ritonavir, a strong CYP3A4 inhibitor. This study was an open-label, randomized, two-treatment crossover trial conducted in HIV-negative subjects with haemodialysis-dependent end-stage renal disease (ESRD) and healthy subjects with normal renal function matched by age, height, bodyweight and sex. Subjects received a single dose of vicriviroc 75 mg alone in one period, and in another period they received a single dose of vicriviroc 15 mg after 4 days of ritonavir 100 mg once daily. Ritonavir treatment was then continued for an additional 13 days. The two trial periods were separated by an interval of at least 3 weeks. The primary endpoints were the log-transformed area under the plasma concentration-time curve (AUC) and the maximum plasma concentration (C(max)), and the 90% confidence intervals (CIs) of the mean differences between subjects with ESRD and matched healthy subjects. The protocol provided the option of dose modification and further study if the vicriviroc C(max) and AUC values were at least twice as high in subjects with ESRD compared with healthy subjects, or if warranted by other safety and tolerability observations. Twelve subjects (six with ESRD, six healthy) completed the study. When vicriviroc was administered alone, the mean vicriviroc C(max) and AUC ratio estimates (90% CI) for subjects with ESRD versus healthy subjects were 74% (53, 103) and 84% (49, 145), respectively. When ritonavir was added to the regimen, the ratio estimates (90% CI) were 81% (59, 111) and 134% (105, 171), respectively. Ritonavir plasma concentrations were substantially higher in subjects with ESRD than in healthy subjects. Treatment-emergent adverse events considered possibly or probably related to treatment occurred only during the ritonavir period of the study and in one healthy subject and two subjects with ESRD; all were of mild or moderate severity. ESRD had no clinically relevant impact on exposure of vicriviroc when vicriviroc was administered alone or in the presence of ritonavir. In this single-dose study, vicriviroc was well tolerated both by healthy subjects and by those with ESRD. Dose adjustment of vicriviroc is therefore not necessary in renally impaired populations.

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 + T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL, with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function.

Adaptive Mutations in a Human Immunodeficiency Virus Type 1 Envelope Protein with a Truncated V3 Loop Restore Function by Improving Interactions with CD4

We previously reported that a human immunodeficiency virus type 1 (HIV-1) clade B envelope protein with a severely truncated V3 loop regained function after passage in tissue culture. The adapted virus, termed TA1, retained the V3 truncation, was exquisitely sensitive to neutralization by the CD4 binding site monoclonal antibody b12 and by HIV-positive human sera, used CCR5 to enter cells, and was completely resistant to small molecule CCR5 antagonists. To examine the mechanistic basis for these properties, we singly and in combination introduced each of the 5 mutations from the adapted clone TA1 into the unadapted envelope. We found that single amino acid changes in the C3 region, the V3 loop, and in the fusion peptide were responsible for imparting near-normal levels of envelope function to TA1. T342A, which resulted in the loss of a highly conserved glycosylation site in C3, played the primary role. The adaptive amino acid changes had no impact on CCR5 antagonist resistance but made virus more sensitive to neutralization by antibodies to the CD4 binding site, modestly enhanced affinity for CD4, and made TA1 more responsive to CD4 binding. Specifically, TA1 was triggered by soluble CD4 more readily than the parental Env and, unlike the parental Env, could mediate entry on cells that express low levels of CD4. In contrast, TA1 interacted with CCR5 less efficiently and was highly sensitive to antibodies that bind to the CCR5 N terminus and ECL2. Therefore, enhanced utilization of CD4 is one mechanism by which HIV-1 can overcome mutations in the V3 region that negatively affect CCR5 interactions.

CCR5 monoclonal antibodies for HIV-1 therapy

To summarize emerging clinical and preclinical data pertaining to the use of CCR5 monoclonal antibodies (mAbs) as therapies for HIV-1 infection. The epitope specificity of CCR5 mAbs is discussed in relation to its critical impact on antiviral activity and CCR5 antagonism. We compare and contrast mAbs and small-molecule CCR5 antagonists in terms of their binding and antiviral properties. Two CCR5 mAbs have entered clinical testing and have successfully completed proof-of-concept studies in HIV-infected individuals, providing initial information on the potential therapeutic utility of these agents. New studies support the view that the most potent antiviral CCR5 mAbs recognize the second extracellular loop of CCR5 either exclusively or in combination with the amino terminus. Studies have revealed fundamental differences in how mAbs and small molecules bind CCR5 and inhibit HIV-1. CCR5 mAbs and small-molecule CCR5 antagonists have demonstrated consistent antiviral synergy and limited or no viral cross-resistance in independent studies. Single intravenous infusions of CCR5 mAbs significantly reduced HIV-1 RNA levels in infected individuals for 2-3 weeks without appreciable toxicity. CCR5 mAbs have demonstrated broad and potent antiviral activity in vitro. Clinical studies have established CCR5 mAbs as potent antiretroviral agents with prolonged activity following a single dose. CCR5 mAbs represent both a distinct class of CCR5 inhibitor and a novel approach to HIV-1 therapy.

Discovery of a Novel CCR5 Antagonist Lead Compound Through Fragment Assembly

CCR5, as the major co-receptor for HIV-1 entry, is an attractive novel target for the pharmaceutical industry in the HIV-1 therapeutic area. In this study, based on the structures of maraviroc and 1,4-bis(4-(7-chloroquinolin-4-yl)piperazin-1-yl)butane-1,4- dione (1), which was identified using structure-based virtual screening in conjunction with a calcium mobilization assay, a series of novel small molecule CCR5 antagonists have been designed and synthesized through fragment assembly. Preliminary SARs were obtained, which are in good agreement with the molecular binding model and should prove helpful for future antagonist design. The novel scaffold presented here might also be useful in the development of maraviroc-derived second generation CCR5 antagonists.

Maraviroc: A Ccr5 Antagonist

HIV requires binding to both the CD4 molecule and a coreceptor to enable entry into the cell. CCR5 is a chemokine receptor that is utilized as a coreceptor by the majority of virus in early asymptomatic HIV infection. Maraviroc is a novel small molecule CCR5 antagonist which, in Phase IIb/III clinical trials up to 48 weeks, has been shown to be efficacious as part of an optimized antiretroviral regimen against CCR5 tropic HIV-1 in treatment-experienced patients. A further trial has demonstrated its noninferiority to efavirenz in achieving a HIV viral load less than 400 copies/ml as part of highly active antiretroviral therapy in treatment-naive individuals. It has recently received regulatory approval for use in North America and Europe in treatment-experienced patients. With increasing use, the role of maraviroc in the treatment of HIV-infected patients will be more clearly defined.