Organotropic dendrons with high potency as HIV-1, HIV-2 and EV-A71 cell entry inhibitors

Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science 307: 1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.

HIV fusion and entry are two steps in the viral lifecycle that can be targeted by several classes of antiviral drugs. The discovery of chemokines focused the attention on cellular co-receptors used by the virus for entering cells, and on the various steps of such processes that are subject to interactions with small molecules. Intense research has led to a wide range of effective compounds that are able to inhibit these initial steps of viral replication. All steps in the process of HIV entry into the cell may be targeted by specific compounds, grouped into three main classes (attachment inhibitors, co-receptor binding inhibitors and fusion inhibitors), which may be developed as novel antiretrovirals. Thus, several inhibitors of the gp120–CD4 interaction have been discovered (e.g., zintevir and BMS-378806). Small molecule chemokine receptor antagonists acting as HIV entry inhibitors have also been described recently, including those which interact with both the CXCR4 co-receptor (e.g., AMD3100, AMD3465, ALX40-4C, T22, T134 and T140) and CCR5 co-receptor antagonists (TAK-779, TAK-220, E913, AK-602 and NSC 651016 in clinical trials). Recently, a third family of antivirals started to be used clinically (in addition to reverse transcriptase and protease inhibitors), with the advent of enfuvirtide (T20), the first fusion inhibitor to be approved as an anti-HIV agent. Some of these compounds demonstrated in vitro synergism with other classes of antivirals, thus offering the rationale for their combination in therapies for HIV-infected individuals. Many HIV entry and fusion inhibitors are currently being investigated in controlled clinical trials, and a number of them are bioavailable as oral formulations. In 2007, the US FDA approved maraviroc as an anti-HIV agent. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin. Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. Furthermore, in October 2007, the FDA announced the approval of raltegravir for the treatment of HIV-1 infection as part of combination antiretroviral therapy in treatment-experienced patients with evidence of HIV-1 replication despite optimized background antiretroviral therapy. At present, raltegravir is the only drug in the integrase inhibitor class approved for clinical use. With the approval of raltegravir, oral agents targeting all three constitutive viral enzymes, reverse transcriptase, protease and integrase, are now represented in FDA-approved therapies.

Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science307<b>:</b>1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.

HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-β conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-β variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (∼350 Å(2)) positively charged surface in the all-β XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors. The host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of noncytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8(+) T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-β fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-β XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

Considerable advances have been made in the last years in the design of derivatives acting as inhibitors of HIV entry and fusion. The discovery of chemokines focused the attention on cellular coreceptors used by the virus for entering within cells, and consequently the various steps of such processes have been characterized in detail. Intense research led to a wide range of effective compounds that are able to inhibit the initial steps of HIV life cycle. All steps in the process of HIV entry into the cell may be targeted by specific compounds that may be developed as novel types of antiretrovirals. Thus, several inhibitors of the gp120-CD4 interaction have been detected so far (zintevir, FP-21399 and BMS-378806 in clinical trials). Small molecule chemokine receptor antagonists acting as HIV entry inhibitors also were described in the last period, which interact both with the CXCR4 coreceptor (such as AMD3100; AMD3465; ALX40-4C; T22, T134 and T140), or which are antagonist of the CCR5 coreceptor (TAK-779, TAK-220, SCH-C, SCH-D, E913, AK-602, UK-427857 and NSC 651016 in clinical trials), together with new types of fusion inhibitors possessing the same mechanism of action as enfuvirtide (such as T1249). Recently, a third family of antivirals started to be used clinically (in addition to the reverse transcriptase and protease inhibitors), with the advent of enfuvirtide (T20), the first fusion inhibitor to be approved as an anti-HIV agent. Some of these compounds demonstrated in vitro synergism with other classes of antivirals, offering thus the rationale for their combination in therapies for HIV-infected individuals. Many HIV entry and fusion inhibitors are currently being investigated in controlled clinical trials, and a number of them is bioavailable as oral formulations. This is an essential feature for an extended use of these compounds with the purpose of ameliorating adherence of patients to these medications and preventing the development of drug resistance.

HIV entry and fusion are two steps in the viral life cycle that can be targeted by several classes of antiviral drugs. The discovery of chemokines focused the attention on cellular coreceptors used by the virus for entering within cells, and to the various steps of such processes which are subject to interactions with small molecules. Intense research led to a wide range of effective compounds that are able to inhibit these initial steps of viral replication. All steps in the process of HIV entry into the cell may be targeted by specific compounds that may be developed as novel types of antiretrovirals. Thus, several inhibitors of the gp120-CD4 interaction have been detected so far (zintevir, FP-21399 and BMS-378806 in clinical trials). Small molecule chemokine receptor antagonists acting as HIV entry inhibitors also were described in the last period, which interact both with the CXCR4 coreceptor (such as AMD3100; AMD3465; ALX40-4C; T22, T134 and T140), or which are antagonist of the CCR5 coreceptor (TAK-779, TAK-220, SCH-C, SCH-D, E913, AK-602 and NSC 651016 in clinical trials), together with new types of fusion inhibitors possessing the same mechanism of action as enfuvirtide (such as T1249). Recently, a third family of antivirals started to be used clinically (in addition to the reverse transcriptase and protease inhibitors), with the advent of enfuvirtide (T20), the first fusion inhibitor to be approved as an anti-HIV agent. Some of these compounds demonstrated in vitro synergism with other classes of antivirals, offering thus the rationale for their combination in therapies for HIV-infected individuals. Many HIV entry and fusion inhibitors are currently investigated in controlled clinical trials, and there are a number of them that is bioavailable as oral formulations. This is an essential feature for an extended use of these compounds with the purpose of ameliorating adherence of patients to these medications and preventing the development of drug resistance.

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

GB virus C/hepatitis G virus (GBV-C/HGV) is the most closely related human virus to hepatitis C virus (HCV). GBV-C is lymphotropic and not associated with any known disease, although it is associated with improved survival in HIV-infected individuals. In peripheral blood mononuclear cells, GBV-C induces the release of soluble ligands for HIV entry receptors (RANTES, MIP-1a, MIP-1b and SDF-1), suggesting that GBV-C may interact with lymphocytes to induce a chemokine and/or cytokine milieu that is inhibitory to HIV infection. Expression of GBV-C envelope glycoprotein E2 in CD4+ T cells or addition of recombinant E2 to CD4 cells recapitulates the HIV inhibition seen with GBV-C infection. Like HCV E2, GBV-C E2 is predicted to be post-translationally processed in the endoplasmic reticulum and is involved with cell binding. The C-termini of GBV-C E1 and E2 proteins contain predicted transmembrane domains sharing features with HCV TM domains. To date, cellular receptor(s) for GBV-C E2 have not been identified. GBV-C E2-mediated HIV inhibition is dose-dependent and HIV replication is blocked at the binding and/or entry step. In addition, a putative GBV-C E2 fusion peptide interferes with HIV gp41 peptide oligomerization required for HIV-1 fusion, further suggesting that GBV-C E2 may inhibit HIV entry. Additional work is needed to identify the GBV-C E2 cellular receptor, characterize GBV-C E2 domains responsible for HIV inhibition, and to examine GBV-C E2-mediated fusion in the context of the entire envelope protein or viral-particles. Understanding the mechanisms of action may identify novel approaches to HIV therapy.

HIV-1 cell entry and advances in viral entry inhibitor therapy.

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187-11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors.

The use of synthetic peptides as HIV-1 inhibitors has been the object of research over recent years. A large number of peptides that affect different stages of the HIV-1 life cycle have been and continue to be studied due to their possible clinical application in the fight against HIV-1 infection. The main advantages of synthetic peptides as therapeutic agents are their low systemic toxicity, the fact that structural modifications can be made to them and their resulting capacity to mimic certain substrates or epitopes. HIV-1-inhibiting peptides have been identified and/or developed using different methods. Some therapeutic peptides such as enfuvirtide-already approved for clinical use-are derived from HIV-1 itself. Others are natural peptides such as chemokines, defensins or the "virus inhibitory peptide"; while still others have been designed and synthesized based on crystallographic data on HIV-1 proteins or from peptide libraries. Initial attempts at therapeutic applications focused on HIV-coded enzymes (reverse transcriptase, protease and, more recently, integrase). However, structural HIV proteins and, more specifically, the mechanisms that involve the virus in cell infection and replication are now also considered therapeutic targets. Several chemical strategies to improve both the stability of peptides and their pharmacokinetics, including prolonging their half-life, have recently been described in the literature. There is growing an interest in inhibitors that prevent HIV entry into the host cell (fusion inhibitors) which could lead to the development of new antiviral agents. Knowledge of the mechanism of action of fusion inhibitors is essential not only for the development of future generations of entry inhibitors, but also to gain an understanding of the form and kinetics of membrane fusion induced by the virus. The physico-chemical processes involved at the interface between the lipid surface of cells and enveloped viruses (such as HIV-1) are essential to the action of peptides that prevent HIV-1 entry into the host cell. The interaction of these peptides with biological membranes may be related to their inhibition efficiency and to their mechanism of action, as the HIV-1 gp41 glycoprotein is bound and confined between the cellular membrane and the viral envelope.

Background: The gp41 C34 peptide, which is part of the HIV envelope glycoprotein, is crucial for HIV entry into host cells because it facilitates membrane fusion and serves as a biomarker for viral replication. Lipoproteins, including HDL, LDL, IDL, VLDL, and chylomicrons, affect HIV infection via their cholesterol levels and particle sizes, but their causal relationships with HIV remain unclear. Methods: Utilizing the Mendelian randomization (MR) approach to infer causality, this study leverages genetic data from the UK Biobank (115,082 individuals) and the KORA cohort (997 individuals) to explore the causal relationships between 39 lipoprotein traits (cholesterol levels and subtype concentrations of different particle sizes) and gp41 C34 expression. The primary MR method employed was the random-effect inverse variance weighted (IVW) approach. To ensure robust and reliable causal inference, multiple sensitivity analyses, including weighted median, MR‒Egger regression, simple mode, weighted mode, and leave-one-out analyses, were conducted. Results: Elevated HDL cholesterol levels were significantly associated with reduced gp41 C34 expression (IVW: β = -0.61, SE = 0.186, p = 1.25e-4, FDR = 0.004), suggesting a protective role of HDL cholesterol in HIV infection. Higher HDL particle concentrations were also inversely associated with gp41 C34 expression (IVW: β = -0.549, SE = 0.202, p = 0.007, FDR = 0.032). Increased cholesterol levels in large HDL particles were significantly inversely related to gp41 C34 expression (IVW: β = -0.46, SE = 0.16, p = 0.004, FDR = 0.03). Similarly, higher concentrations of medium HDL particles were linked to lower gp41 C34 expression (IVW: β = -0.473, SE = 0.166, p = 0.005, FDR = 0.028). No significant causal relationships were found between gp41 C34 expression and the cholesterol levels or sizes of IDL, LDL, or VLDL particles or chylomicrons. Consequently, these lipoprotein particles are unlikely to influence gp41 C34 expression and HIV cell entry. Conclusion: HDL cholesterol and HDL particle concentrations, particularly large and medium HDL particles, play a protective role against HIV cell entry by reducing gp41 C34 expression. Other lipoprotein particles do not show significant causal relationships, indicating that specific lipid traits modulate HIV entry mechanisms. These findings enhance our understanding of the influence of lipoprotein traits on HIV infection and persistence.

Considerable advances have been made on compounds that are active as inhibitors of HIV entry and fusion. The discovery of chemokines a few years ago focused the attention on coreceptor inhibitors in addition to fusion and attachment blockers. During the last 5 years, there has been an intense research activity from both private companies and academic institutions to find effective compounds that are capable of inhibiting the initial steps in the HIV life cycle. Some of the presented compounds demonstrated in vitro synergism, thus there is the rationale of their combined use in HIV-infected individuals. Many entry and fusion inhibitors of HIV are being investigated in controlled clinical trials and there are a number of them that are bioavailable as oral formulations. This is an essential feature for an extended use of these compounds with the purpose of ameliorating patients’ adherence to medications; therefore, preventing the development of drug resistance. Among the many compounds that are being investigated, some are in the preclinical arena and others are more advanced in development stages. Overall, the main aim is to establish the action of these compounds on the immune system (e.g., the balance of the system after shutting off CCR5 or CXCR4 coreceptors) and the possible burden of unexplained side effects. This review focuses on the recent developments in this field with a particular attention on promising compounds in preclinical and clinical trials.

BackgroundHighly active anti-retroviral therapy (HAART) is the current HIV/AIDS treatment modality. Despite the fact that HAART is very effective in suppressing HIV-1 replication and reducing the mortality of HIV/AIDS patients, it has become increasingly clear that HAART does not offer an ultimate cure to HIV/AIDS. The high cost of the HAART regimen has impeded its delivery to over 90% of the HIV/AIDS population in the world. This reality has urgently called for the need to develop inexpensive alternative anti-HIV/AIDS therapy. This need has further manifested by recent clinical trial failures in anti-HIV-1 vaccines and microbicides. In the current study, we characterized a panel of extracts of traditional Chinese medicinal herbal plants for their activities against HIV-1 replication.MethodsCrude and fractionated extracts were prepared from various parts of nine traditional Chinese medicinal herbal plants in Hainan Island, China. These extracts were first screened for their anti-HIV activity and cytotoxicity in human CD4+ Jurkat cells. Then, a single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms of these extracts.ResultsTwo extracts, one from Euphorbiaceae, Trigonostema xyphophylloides (TXE) and one from Dipterocarpaceae, Vatica astrotricha (VAD) inhibited HIV-1 replication and syncytia formation in CD4+ Jurkat cells, and had little adverse effects on host cell proliferation and survival. TXE and VAD did not show any direct inhibitory effects on the HIV-1 RT enzymatic activity. Treatment of these two extracts during the infection significantly blocked infection of the reporter virus. However, pre-treatment of the reporter virus with the extracts and treatment of the extracts post-infection had little effects on the infectivity or gene expression of the reporter virus.ConclusionThese results demonstrate that TXE and VAD inhibit HIV-1 replication likely by blocking HIV-1 interaction with target cells, i.e., the interaction between gp120 and CD4/CCR5 or gp120 and CD4/CXCR4 and point to the potential of developing these two extracts to be HIV-1 entry inhibitors.

HIV-1 entry into human podocytes is mediated through lipid rafts