Penicillixanthone A, a marine-derived dual-coreceptor antagonist as anti-HIV-1 agent.

Marine micro-organisms have been proven to be excellent sources of bioactive compounds against HIV-1. Several natural products obtained from marine-derived Aspergillus fungi were screened for their activities to inhibit HIV-1 infection. Penicillixanthone A (PXA), a natural xanthone dimer from jellyfish-derived fungus Aspergillus fumigates, displayed potent anti-HIV-1 activity by inhibiting infection against CCR5-tropic HIV-1 SF162 and CXCR4-tropic HIV-1 NL4-3, with IC50 of 0.36 and 0.26 µM, respectively. Molecular docking study was conducted to understand the possible binding mode of PXA with the CCR5/CXCR4. The results revealed that, the marine-derived PXA, as a CCR5/CXCR4 dual-coreceptor antagonist, presents a new type of potential lead product for the development of anti-HIV therapeutics.

Cenicriviroc inhibits trans-endothelial passage of monocytes and is associated with impaired E-selectin expression.

Incidences of cardiovascular diseases (CVD) are high among virologically suppressed HIV-infected individuals. Monocyte activation and trafficking are key mechanisms in the evolution of CVD. We studied the ability of cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and CCR5 antagonist, to influence the migration of monocytes from HIV-infected individuals on antiretroviral therapy (ART). Monocytes were derived from 23 ART-suppressed HIV-infected and 16 HIV-uninfected donors. In a trans-endothelial migration model, monocytes, and human aortic endothelial cells (HAoECs) were exposed to cenicriviroc and migrated monocytes, quantified. Expression of CCR2 and CCR5 on monocytes and adhesion molecules (E-selectin, ICAM-1, VCAM-1, PECAM-1, and CD99) on HAoECs were measured. The single antagonists, BMS-22 (CCR2), and maraviroc (CCR5), served as controls. When both HAoECs and monocytes together were exposed to the antagonists, cenicriviroc led to a greater decrease in monocyte migration compared to BMS-22 or vehicle in both HIV-infected and HIV-uninfected groups (P < 0.05), with maraviroc having no inhibitory effect. Cenicriviroc treatment of HAoECs alone decreased monocyte migration in the HIV-infected group when compared to vehicle (P < 0.01). Inhibition of migration was not evident when monocytes alone were exposed to cenicriviroc, BMS-22 or maraviroc. Incubation of HAoECs with cenicriviroc decreased E-selectin expression (P = 0.045) but had limited effects on the other adhesion molecules. Cenicriviroc inhibits monocyte trans-endothelial migration more effectively than single chemokine receptor blockade, which may be mediated via disruption of monocyte-endothelial tethering through reduced E-selectin expression. Cenicriviroc should be considered as a therapeutic intervention to reduce detrimental monocyte trafficking.

[Tat-based cell-cell fusion method for screening HIV-1 fusion inhibitors].

An HIV-1 cell-cell fusion system was developed to screen HIV-1 entry inhibitors that block cell-cell fusion. In this system, the pEGFP-Tat plasmid was constructed and cotransfected into effector cells (HEK-293T) with HIV-1 envelope plasmid. TZM-bl cell, a genetically engineered cell line that expresses CD4, CXCR4, CCR5 as well as Tat-inducible ß-galactosidase and luciferase reporter gene, was used as target cell. Thus, the co-culture of target cells and effector cells allows the cell fusion via Env and the activity of the fusion inhibitor can be quantified by measuring the reporter protein expression. The experimental parameters were optimized and 11 anti-HIV-1 agents including CCR5 antagonist maraviroc, reverse transcription inhibitor zidovudine (AZT) and integrase inhibitor raltegravir were tested. The result showed that the system exhibited high specificity and sensitivity. Two of eight tested anti-HIV-1 agents were found to block the cell-cell fusion. The system is suitable for efficient screening of HIV-1 cell-cell fusion inhibitors.

Cell-Delivered Entry Inhibitors for HIV-1: CCR5 Downregulation and Blocking Virus/Membrane Fusion in Defending the Host Cell Population.

HIV-1 infection requires the presence of the CD4 receptor on the target cell surface and a coreceptor, predominantly CC-chemokine receptor 5 (CCR5). It has been shown that individuals who are homozygous for a defective CCR5 gene are protected from HIV-1 infection. A novel self-inactivating lentiviral vector LVsh5/C46 (Cal-1) has been engineered to block HIV-1 infection with two viral entry inhibitors, conferring resistance to HIV-1 infection from both CCR5 and CXCR4 tropic strains. Cal-1 encodes a short hairpin RNA (sh5) to downregulate CCR5 and C46, an HIV-1 fusion inhibitor. Gene therapy by Cal-1 is aimed at transducing CD4+ T cells and CD34+ hematopoietic stem/progenitor cells in an autologous transplant setting. Pre-clinical safety and efficacy studies in vitro and in vivo (humanized mouse model and nonhuman primates) have shown that Cal-1 is safe with no indication of any toxicity risk and acts to decrease viral load and increase CD4 counts. Two clinical trials are underway using Cal-1: a phase I/II study to assess safety and feasibility in an adult HIV-1-positive population not on antiretroviral therapy (ART); and a second Fred Hutchinson Investigator Initiated phase I study to assess safety and feasibility in adults with HIV-1-associated non-Hodgkin or Hodgkin lymphoma.

Screening HIV-1 fusion inhibitors based on capillary electrophoresis head-end microreactor targeting to the core structure of gp41.

In this paper, we design a microreactor based on electrophoretically mediated microanalysis (EMMA) with capillary electrophoresis (CE) for screening HIV-1 inhibitors that bind to the N-terminal heptad repeat (NHR, N36) region. Initially, a test sample plug is loaded into a capillary filled with buffer solution followed by N36 peptide solution, and the two solutions simultaneously mix by diffusion. Then, voltage is applied, and the sample molecules pass through the N36 peptide zone. The active compounds combine with N36, leading to a loss in the peak height of the active compound. More than 100 traditional Chinese medicine extracts (TCME) were screened, and an extract of Pheretima aspergillum (E. Perrier) (L5) was identified as having potent inhibitory activity. The results showed that L5 could significantly inhibit the HIV-1JR-FL pseudotyped virus infection; the 50% effective concentration (EC50) of L5 was approximately 32.1±1.2µg/mL, and the 50% cytotoxicity concentration (CC50) value of L5 was 146.9±4.4µg/mL, suggesting that L5 had low in vitro cytotoxicity on U87-CD4-CCR5 cells. The new method is simple and rapid, is free of antibodies, and does not require tedious processes.

Maraviroc modifies gut microbiota composition in a mouse model of obesity: a plausible therapeutic option to prevent metabolic disorders in HIV-infected patients.

INTRODUCTION: The proportion of HIV-infected patients with overweight/obesity has increased in recent years. These patients have an increased metabolic/cardiovascular risk compared with non-obese patients. Modulation of gut microbiota composition arises as a promising tool to prevent the development of obesity and associated disorders. The aim of this study was to investigate the impacts of maraviroc (MVC), a CCR5 antagonist approved for clinical use in HIV-infected patients, on gut microbiota composition in a mouse model of obesity. METHODS: Thirty two male C57BL/6 mice were assigned to:a) Control (chow diet), b) MVC (chow diet plus 300 mg/L MVC), c) High-fat diet (HFD) or d) HFD/MVC (HFD plus 300 mg/L MVC) groups. Body weight and food intake was recorded every 2-3 days. Mice were euthanized after 16 weeks of treatment and cecal contents were removed to analyse by real-time PCR four bacterial orders from the most dominant phyla in gut. RESULTS: Mice fed with a HFD showed a significant increase in Enterobacteriales (p<0.001 vs. control). MVC treatment induced a significant decrease in control (p<0.05) and HFD fed mice (p<0.001). Interestingly, this order was positively associated with body weight gain, insulin resistance and fatty liver. HFD induced a significant decrease in Bacteroidales and Clostridiales levels (p<0.05 and p<0.01, respectively). MVC decreased the presence of Bacteroidales (p<0.05 vs. control) while an increase was observed in HFD/MVC mice (p=0.01 vs. HFD). No direct effects of MVC were observed on Clostridiales and Lactobacillales. CONCLUSIONS: MVC may constitute a new therapeutic option to prevent obesity and related disorders in HIV-infected patients.

Preclinical discovery and development of maraviroc for the treatment of HIV.

INTRODUCTION: Maraviroc is a first-in-class antiretroviral (ARV) drug acting on a host cell target (CCR5), which blocks the entry of the HIV virus into the cell. Maraviroc is currently indicated for combination ARV treatment in adults infected only with CCR5-tropic HIV-1. AREAS COVERED: This drug discovery case history focuses on the key studies that led to the discovery and approval of maraviroc, as well as on post-launch clinical reports. The article is based on the data reported in published preclinical and clinical studies, conference posters and on drug package data. EXPERT OPINION: The profound understanding of HIV's entry mechanisms has provided a strong biological rationale for targeting the chemokine receptor CCR5. The CCR5-antagonist mariviroc, with its unique mode of action and excellent safety profile, is an important therapeutic option for HIV patients. In general, the authors believe that targeting host factors is a useful approach for combating new and re-emerging transmissible diseases, as well as pathogens that easily become resistant to common antiviral drugs. Maraviroc, offering a potent and safe cellular receptor-mediated pharmacological response to HIV, has paved the way for the development of a new generation of host-targeting antivirals.

Targeting CCR5 for anti-HIV research.

Highly active antiretroviral therapy (HAART) is the only approach for human immunodeficiency virus (HIV) infection treatment at present. Although HAART is effective in controlling the progression of infection, it is impossible to eradicate the virus from patients. The patients have to live with the virus. Alternative ways for the cure of HIV infection have been investigated. As the major co-receptor for HIV-1 infection, C-C motif chemokine receptor 5 (CCR5) is naturally an ideal target for anti-HIV research. The first CCR5 antagonist, maraviroc, has been approved for the treatment of HIV infection. Several other CCR5 antagonists are in clinical trials. CCR5 delta32 is a natural genotype, conferring resistance to CCR5 using HIV-1 strains. Gene therapy research targeting this mutant has been conducted for HIV infection treatment. A Berlin patient has been cured of HIV infection by the transplantation of stem cells from a CCR5 delta32 genotype donor. The infusion of an engineered zinc finger nuclease (ZFN)-modified autologous cluster of differentiation 4 (CD4) T cells has been proved to be a promising direction recently. In this study, the anti-HIV research targeting CCR5 is summarized, including CCR5 antagonist development, stem cell transplantation, and gene therapy.

DT-13, a saponin of dwarf lilyturf tuber, exhibits anti-cancer activity by down-regulating C-C chemokine receptor type 5 and vascular endothelial growth factor in MDA-MB-435 cells.

AIM: To investigate the anticancer activity of DT-13 under normoxia and determine the underlying mechanisms of action. METHODS: MDA-MB-435 cell proliferation, migration, and adhesion were performed to assess the anticancer activity of DT-13, a saponin from Ophiopogon japonicus, in vitro. In addition, the effects of DT-13 on tumor growth and metastasis in vivo were evaluated by orthotopic implantation of MDA-MB-435 cells into nude mice; mRNA levels of vascular endothelial growth factor (VEGF), C-C chemokine receptor type 5 (CCR5) and hypoxia-inducible factor 1α (HIF-1α) were evaluated by real-time quantitative PCR; and CCR5 protein levels were detected by Western blot assay. RESULTS: At 0.01 to 1 µmol·L(-1), DT-13 inhibited MDA-MB-435 cell proliferation, migration, and adhesion significantly in vitro. DT-13 reduced VEGF and CCR5 mRNAs, and decreased CCR5 protein expression by down-regulating HIF-1α. In addition, DT-13 inhibited MDA-MB-435 cell lung metastasis, and restricted tumor growth slightly in vivo. CONCLUSION: DT-13 inhibited MDA-MB-435 cell proliferation, adhesion, and migration in vitro, and lung metastasis in vivo by reducing VEGF, CCR5, and HIF-1α expression.

DT-13, a saponin of dwarf lilyturf tuber, exhibits anti-cancer activity by down-regulating C-C chemokine receptor type 5 and vascular endothelial growth factor in MDA-MB-435 cells / 中国天然药物

AIM@#To investigate the anticancer activity of DT-13 under normoxia and determine the underlying mechanisms of action.@*METHODS@#MDA-MB-435 cell proliferation, migration, and adhesion were performed to assess the anticancer activity of DT-13, a saponin from Ophiopogon japonicus, in vitro. In addition, the effects of DT-13 on tumor growth and metastasis in vivo were evaluated by orthotopic implantation of MDA-MB-435 cells into nude mice; mRNA levels of vascular endothelial growth factor (VEGF), C-C chemokine receptor type 5 (CCR5) and hypoxia-inducible factor 1α (HIF-1α) were evaluated by real-time quantitative PCR; and CCR5 protein levels were detected by Western blot assay.@*RESULTS@#At 0.01 to 1 μmol·L(-1), DT-13 inhibited MDA-MB-435 cell proliferation, migration, and adhesion significantly in vitro. DT-13 reduced VEGF and CCR5 mRNAs, and decreased CCR5 protein expression by down-regulating HIF-1α. In addition, DT-13 inhibited MDA-MB-435 cell lung metastasis, and restricted tumor growth slightly in vivo.@*CONCLUSION@#DT-13 inhibited MDA-MB-435 cell proliferation, adhesion, and migration in vitro, and lung metastasis in vivo by reducing VEGF, CCR5, and HIF-1α expression.

Identifying chemicals with potential therapy of HIV based on protein-protein and protein-chemical interaction network.

Acquired immune deficiency syndrome (AIDS) is a severe infectious disease that causes a large number of deaths every year. Traditional anti-AIDS drugs directly targeting the HIV-1 encoded enzymes including reverse transcriptase (RT), protease (PR) and integrase (IN) usually suffer from drug resistance after a period of treatment and serious side effects. In recent years, the emergence of numerous useful information of protein-protein interactions (PPI) in the HIV life cycle and related inhibitors makes PPI a new way for antiviral drug intervention. In this study, we identified 26 core human proteins involved in PPI between HIV-1 and host, that have great potential for HIV therapy. In addition, 280 chemicals that interact with three HIV drugs targeting human proteins can also interact with these 26 core proteins. All these indicate that our method as presented in this paper is quite promising. The method may become a useful tool, or at least plays a complementary role to the existing method, for identifying novel anti-HIV drugs.

Progress in the discovery of CC chemokine receptor 2 antagonists, 2009 - 2012.

INTRODUCTION: CC chemokine receptor 2 (CCR2) is a key mediator of the activation and migration of inflammatory monocytes. As such, it has been investigated extensively as a target for therapeutic intervention in a diverse range of diseases. AREAS COVERED: This article reviews both the patent and peer-reviewed literature on the discovery of CCR2 antagonists from January 2009 to December 2012. Developments have occurred within each of the major chemical families of CCR2 antagonists, and are framed in that context. As has been true historically, a number of the compound families also exhibit substantial activity against the related CC chemokine receptor 5 (CCR5), making them formally CCR2/5-dual antagonists. EXPERT OPINION: Significant progress continues to be made in identifying novel, potent CCR2 antagonists. In addition, researchers have had success in addressing issues related to selectivity, cardiac safety, and preclinical pharmacokinetics. Establishing proof-of-concept in clinical trials remains the primary challenge for the field.

CCR5 as a natural and modulated target for inhibition of HIV.

Human immunodeficiency virus type 1 (HIV-1) infection of target cells requires CD4 and a co-receptor, predominantly the chemokine receptor CCR5. CCR5-delta32 homozygosity results in a truncated protein providing natural protection against HIV infection-this without detrimental effects to the host-and transplantation of CCR5-delta32 stem cells in a patient with HIV ("Berlin patient") achieved viral eradication. As a more feasible approach gene-modification strategies are being developed to engineer cellular resistance to HIV using autologous cells. We have developed a dual therapeutic anti-HIV lentiviral vector (LVsh5/C46) that down-regulates CCR5 and inhibits HIV-1 fusion via cell surface expression of the gp41-derived peptide, C46. This construct, effective against multiple strains of both R5- and X4-tropic HIV-1, is being tested in Phase I/II trials by engineering HIV-resistant hematopoietic cells.

A potential in vitro and in vivo anti-HIV drug screening system for Chinese herbal medicines.

Chinese herbal medicines are often applied as an alternative therapy for viral diseases. However, the development of anti-HIV herbal drugs has proceeded slowly, partly because of the lack of a high-throughput system for screening these drugs. The present study evaluated 16 herbal medicines for anti-HIV activities in vitro and in vivo. Herbal medicines were first screened for the ability to regulate C-X-C receptor 4 (CXCR4) and C-C receptor 5 (CCR5) promoter activities. A single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms. CD4+ T cells from healthy volunteers were examined for changes in CXCR4 and CCR5 levels. HIV-1 replication was evaluated by ELISA. Spica Prunellae and Herba Andrographitis were found to down-regulate the activities of both the CXCR4 and CCR5 promoters. Also, Spica Prunellae and Herba Andrographitis (>1000 µM) inhibited HIV-1 in a dose-dependent manner. CXCR4 and CCR5 levels were reduced in CD4+ T cells from healthy volunteers (p<0.05). Spica Prunellae and Herba Andrographitis (EC50: 3.18 and 5.49 µg/mL, respectively) could suppress cell fusion and decrease p24 antigen. In conclusion, the data demonstrated that Spica Prunellae and Herba Andrographitis possessed anti-HIV-1 capabilities, perhaps through the inhibition of the CXCR4 and CCR5 promoters and HIV-1 replication.

Rescue of HIV-1 long-time archived X4 strains to escape maraviroc.

Entry of Human Immunodeficiency Virus type 1 (HIV-1) into target cells is mediated by the CD4 receptor and a coreceptor, CCR5 or CXCR4. Maraviroc interferes with HIV entry by binding the CCR5 coreceptor. Virological failure to maraviroc-containing regimens can occur through the emergence of resistance, or through tropism evolution and broadened coreceptor usage. In the latter case, the physiological relevance of minority strains is a major concern. Here we report a retrospective analysis of coreceptor-usage and evolution based on 454-ultra-deep-sequencing of plasma and Peripheral Blood Mononuclear Cell (PBMC)-derived envelope V3-loops, accounting for coreceptor usage, from a patient who failed a maraviroc-containing regimen through the emergence of X4 strains. The X4 maraviroc-escape variant resulted from recombination between a long time archived proviral sequence from 2003 (5'-portion, including the V3-loop) and the dominant R5 strains circulating in plasma at the time of maraviroc-treatment initiation (3'-portion). Phylogenetic analyses and BEAST modeling highlighted that an early diverse viral quasispecies underwent a severe bottleneck following reinitiation of HAART and repeated IL-2 cycles between 1999 and 2001, leading to the transient outgrowth and archiving of one highly homogeneous X4 population from plasma, and to the expansion in plasma of one PBMC-derived R5 strain. Under maraviroc selective pressure, the early, no longer detectable X4 strains archived in PBMC were partially rescued to provide X4-determinants to the main circulating strain.

Discovery of 4-[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenoxy]benzoic acid hydrochloride: a highly potent orally available CCR5 selective antagonist.

Based on the original spirodiketopiperazine design framework, further optimization of an orally available CCR5 antagonist was undertaken. Structural hybridization of the hydroxylated analog 4 derived from one of the oxidative metabolites and the new orally available non-hydroxylated benzoic acid analog 5 resulted in another potent orally available CCR5 antagonist 6a as a clinical candidate. Full details of a structure-activity relationship (SAR) study and ADME properties are presented.

Baseline CD4(+) T-cell counts and weighted background susceptibility scores strongly predict response to maraviroc regimens in treatment-experienced patients.

BACKGROUND: Maraviroc-containing regimens are known to achieve virological suppression in many treatment-experienced patients. This study aimed to evaluate a more rigorous methodological approach to resistance-response analysis in large clinical studies and to better establish which subpopulations of patients were most likely to benefit from maraviroc by refining and extending previous subgroup analyses from the MOTIVATE studies. METHODS: Individual weighted optimized background therapy (OBT) susceptibility scores were calculated by combining genotypic or phenotypic resistance testing with prior drug use information. Virological response (HIV-1 RNA<50 copies/ml at week 48) using each of these methods was compared with a commonly used method of counting active drugs. Baseline predictors of virological response, including weighted or unweighted scoring, maraviroc use, baseline CD4(+) T-cell count, HIV-1 plasma viral load and tropism, were assessed by logistic regression modelling. RESULTS: Genotypic or phenotypic weighted methods were similarly predictive of virological response and better than counting active drugs. Weighted scoring and baseline CD4(+) T-cell count were the strongest predictors of virological response (P<0.0001): ≈70% of maraviroc patients with a weighted score ≥2 had a virological response, rising to ≈80% when the baseline CD4(+) T-cell count was ≥50 cells/mm(3). CONCLUSIONS: Approximately 80% of patients with a CD4(+) T-cell count ≥50 cells/mm(3) receiving maraviroc with the equivalent of at least two fully active agents achieved HIV-1 RNA<50 copies/ml at week 48 in the MOTIVATE studies. Genotypic and phenotypic weighted scores were similarly predictive of virological response.

Spirodiketopiperazine-based CCR5 antagonist: discovery of an antiretroviral drug candidate.

Following the discovery that hydroxylated derivative 3 (Fig. 1) was one of the oxidative metabolites of the original lead 1, it was found that hydroxylated compound 4 possesses higher in vitro anti-HIV potency than the corresponding non-hydroxylated compound 2. Structural hybridation of 4 with the orally available analog 5 resulted in another orally-available spirodiketopiperazine CCR5 antagonist 6a that possesses more favorable pharmaceutical profile for use as a drug candidate.

Discovery of N-benzyl-N'-(4-pipyridinyl)urea CCR5 antagonists as anti-HIV-1 agents (I): optimization of the amine portion.

Several series of carbamate, urea and carboxamide-based CCR5 antagonists have been discovered via optimizations at the amine portion of lead compound 2. All compounds were evaluated for their antiviral activities. Lead urea 29 showed good pharmacokinetic properties, justifying further development of this series.

Discovery of N-benzyl-N'-(4-pipyridinyl)urea CCR5 antagonists as anti-HIV-1 agents (II): modification of the acyl portion.

Modification of the acyl moiety in the CCR5 lead molecule 2 led to identification of several new classes of CCR5 antagonists. Antiviral activity and pharmacokinetic properties of the synthesized compounds were evaluated. Structure-activity relationship (SAR) derived from these studies further guided the optimization efforts, ultimately leading to the discovery of 36 with an acceptable drug-like profile.