Potent D-peptide inhibitors of HIV-1 entry

During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (D-peptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (IC(50) = 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS.     

HIV-1进入细胞机制及进入抑制剂的研究进展

关于HIV-1进入抑制剂的研究是HIV研究领域近年来的一个新进展，为人类深入了解HIV发病机理，开发新的抗病毒治疗药物，并最终战胜HIV带来了新的曙光。处于临床应用和试验阶段的HIV-1进入抑制剂包括：阻断gpl20与CD4连接的抑制剂；作用于gpl20-CCR5或gpl20-CXCR4的抑制剂；直接作用于gp41介导的膜融合抑制剂。最近发现一种细胞表面蛋白即细胞表面蛋白即二硫化异构酶（PDI）在HIV-1进人过程中起着重要作用。PDI参与病毒进入的3个步骤：1）表面PDI活化；2）PDI与CD4连接；3）CD4-PDI进入gpl20二硫键。新型PDI抑制剂主要针对这3个步骤，也是这类药物与以往药物作用信点不同的地方。     

HIV-1 entry inhibitors: classes, applications and factors affecting potency

Antiviral agents targeting human immunodeficiency virus type-1 (HIV-1) attachment, co-receptor engagement and fusion, collectively referred to as entry inhibitors, are emerging as promising therapeutic agents in the treatment of HIV-1 infection. Viral evolution and concomitant emergence of resistant strains will continue to be an important consideration in the development of any new therapeutic against HIV-1. However, unique challenges facing the development of entry inhibitors center around the highly variable and flexible nature of the HIV-1 envelope protein (Env). For example, the evolution of Env during the course of HIV-1 infection increases the efficiency of Env-CCR5 interactions, which consequently increases Env-mediated fusogenicity and decreases sensitivity to entry inhibitors. This points to a relationship between co-receptor interactions and fusogenicity that merits further consideration in the design of HIV-1 entry inhibitors. It also underscores the importance of considering the biological properties of late-emerging HIV-1 variants in the design of new therapeutics. This review examines the various entry inhibitors that are undergoing preclinical or clinical testing or which are in the early stages of clinical use, their applications in a clinical setting and possible factors that may affect potency against HIV-1.     

Design of potent inhibitors of HIV-1 entry from the gp41 N-peptide region

The HIV-1 gp41 envelope glycoprotein promotes fusion of the virus and cell membranes through the formation of a trimer-of-hairpins structure, in which the amino- and carboxyl-terminal regions of the gp41 ectodomain are brought together. Synthetic peptides derived from these two regions (called N and C peptides, respectively) inhibit HIV-1 entry. In contrast to C peptides, which inhibit in the nanomolar range, N peptides are weak inhibitors with IC(50) values in the micromolar range. To test the hypothesis that the weak inhibition of N peptides results from their tendency to aggregate, we have constructed chimeric variants of the N-peptide region of gp41 in which soluble trimeric coiled coils are fused to portions of the gp41 N peptide. These molecules, which present the N peptide in a trimeric coiled-coil conformation, are remarkably more potent inhibitors than the N peptides themselves and likely target the carboxyl-terminal region of the gp41 ectodomain. The best inhibitors described here inhibit HIV-1 entry at nanomolar concentrations.     

Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins

When interacting with the CD4 receptor, the HIV gp120 envelope glycoprotein undergoes conformational changes that allow binding to the chemokine receptor. Receptor binding is proposed to lead to conformational changes in the gp41 transmembrane envelope glycoprotein involving the creation and/or exposure of a coiled coil consisting of three heptad repeat (HR) sequences. The subsequent interaction of the HR2 region of gp41 with this coiled coil results in the assembly of a six-helix bundle that promotes the fusion of the viral and target cell membranes. Here we show that CD4 binding to gp120 induces the formation and/or exposure of the gp41 HR1 coiled coil in a process that does not involve gp120 shedding and that depends on the proteolytic maturation of the gp160 envelope glycoprotein precursor. Importantly, BMS-806 and related HIV-1 entry inhibitors bind gp120 and block the CD4 induction of HR1 exposure without significantly affecting CD4 binding. Moreover, these compounds do not disrupt gp120-chemokine receptor binding or the HR1-HR2 interaction within gp41. These studies thus define a receptor-induced conformational rearrangement of gp120-gp41 that is important for both CD4-dependent and CD4-independent HIV-1 entry and is susceptible to inhibition by low-molecular-weight compounds.     

A mouse model for HIV-1 entry

Passive transfer of neutralizing antibodies against HIV-1 can prevent infection in macaques and seems to delay HIV-1 rebound in humans. Anti-HIV antibodies are therefore of great interest for vaccine design. However, the basis for their in vivo activity has been difficult to evaluate systematically because of a paucity of small animal models for HIV infection. Here we report a genetically humanized mouse model that incorporates a luciferase reporter for rapid quantitation of HIV entry. An antibody’s ability to block viral entry in this in vivo model is a function of its bioavailability, direct neutralizing activity, and effector functions.HIV-1 (HIV), the causative agent of AIDS, represents a formidable global challenge, with the development of an effective vaccine being of paramount importance (1–4). Rapid progress in this area has been hindered in part by lack of a widely available small animal model for HIV entry. Currently available animal models include nonhuman primates and immunodeficient humanized mice, neither of which is readily available or amenable to genetic modifications (5, 6).Some viral pathogens exhibit a narrow host range, one of those being HIV. HIV’s entry into target cells is mediated by binding of its trimeric envelope spike (gp160) to human CD4 (hCD4) (7) and subsequently to a coreceptor such as human CXCR4 (8) or human CCR5 (hCCR5) (9–11). hCCR5 is of particular interest because it seems to be the primary coreceptor used for transmission (12, 13), as evidenced by the finding that homozygous deletion in the CCR5 allele confers resistance against HIV-1 acquisition (14, 15) and can also lead to long-term control of HIV after stem cell transplantation (16). Finally, HeLa cells and other HIV-resistant cells, including mouse cells, support viral entry when they are engineered to express hCD4/hCCR5/hCXCR4 (17–19).Here, we describe a hCCR5- and hCD4-expressing luciferase reporter mouse that can be used to measure HIV pseudovirus entry and antibody-mediated protection against initial infection in vivo.     

Short constrained peptides that inhibit HIV-1 entry

Peptides corresponding to the C-terminal heptad repeat of HIV-1 gp41 (C-peptides) are potent inhibitors of HIV-1 entry into cells. Their mechanism of inhibition involves binding in a helical conformation to the central coiled coil of HIV-1 gp41 in a dominant-negative manner. Short C-peptides, however, have low binding affinity for gp41 and poor inhibitory activity, which creates an obstacle to the development of small drug-like C-peptides. To improve the inhibitory potency of short C-peptides that target the hydrophobic pocket region of gp41, we use two strategies to stabilize the C-peptide helix: chemical crosslinking and substitution with unnatural helix-favoring amino acids. In this study, the short linear peptide shows no significant inhibitory activity, but a constrained peptide (C14linkmid) inhibits cell-cell fusion at micromolar potency. Structural studies confirm that the constrained peptides bind to the gp41 hydrophobic pocket. Calorimetry reveals that, of the peptides analyzed, the most potent are those that best balance the changes in binding enthalpy and entropy, and surprisingly not those with the highest helical propensity as measured by circular dichroism spectroscopy. Our study reveals the thermodynamic basis of inhibition of an HIV C-peptide, demonstrates the utility of constraining methods for a short antiviral peptide inhibitor, and has implications for the future design of constrained peptides.     

The theta-defensin, retrocyclin, inhibits HIV-1 entry

Retrocyclin is a circular antimicrobial 18-residue peptide encoded in the human genome by a theta-defensin pseudogene. In the human genome, the gene for retrocyclin is inactivated by an in-frame stop codon in its signal sequence but its mature coding sequence is intact. The peptide corresponding to the processed human retrocyclin, generated by solid phase peptide synthesis, inhibited replication of R5 and X4 strains of HIV-1 in human cells. Luciferase reporter virus and Vpr-BLaM entry assays were used to demonstrate that retrocyclin specifically blocked R5 and X4 HIV-1 replication at entry. Surface plasmon resonance demonstrated that retrocyclin bound to soluble CD4 and gp120, but gp120 cell-binding assays revealed that retrocyclin did not fully inhibit the binding of soluble CD4 to gp120. A fluorescent retrocyclin congener localized in cell-surface patches either alone or colocalized with CD4, CXCR4, and CCR5. In the aggregate, these results suggest that retrocyclin blocks an entry step in HIV-1 replication. Retrocyclin represents a new class of small molecule HIV-1 entry inhibitors.     

alpha-adrenergic antagonists as possible calcium channel inhibitors.

The effects of various organic Ca2+ channel inhibitors were investigated on the binding of the alpha 1-antagonist 3H-labeled 2-[(2',6'-dimethoxyphenoxyethyl)aminomethyl]-1,4-benzodioxane ([3H]WB-4101) to membranes from rat brain and neuroblastoma-glioma hybrid cells (NG108-15). As found by monitoring binding of [3H]WB-41-1, the Ca2+ channel inhibitors methoxyverapamil (D600), verapamil, and the nifedipine analogue YC-93 bind to two different sites in rat brain: a high-affinity site (dissociation constant Kd = 2.9 nM and binding capacity B = 360 fmol/mg of protein) and a low-affinity site (Kd = 260 nM and B = 2700 fmol/mg of protein). In NG108-15 cells, where no alpha 1 receptors were detected with [3H]WB-4101, the Ca2+ antagonists were found to bind to nonadrenergic sites in the membrane with a capacity B = 976 fmol/mg of protein. The binding of Ca2+ antagonists to [3H]WB-41-1 sites led to the investigation of WB-4101 as a Ca2+ inhibitor by electrophysiological techniques. WB-4101 depressed the amplitude and reduced the rate of rise of the CA2+ spike with an affinity slightly greater than that observed for D600. The concentration for 50% inhibition of the Ca2+ spike amplitude was 48 microM for WB-4101 and 80 microM for D600. The WB-4101-induced blockade of the Ca2+ spike was antagonized by high Ca2+ concentrations, indicating a common site for Ca2+ and the alpha-antagonist. D600 and WB-4101 also inhibited voltage-dependent Na+ and K+ conductances. The results suggest that Ca2+ channels can account for a fraction of the sites labeled with [3H]WB-4101 in membrane preparations from brain and NG108-15 cells.     

Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity,receptor density,and fusion kinetics

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.     

Variability of critical epitopes within HIV-1 heptad repeat domains for selected entry inhibitors in HIV-infected populations worldwide [corrected

BACKGROUND: Two of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. OBJECTIVES: To examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. METHODS: A total of 150 specimens representing HIV-1 group M subtypes (A-G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. RESULTS: In general, both HR1 (a.a. 540-593) and HR2 (a.a. 628-673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547-549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix interaction include residues at positions 628W, 631W, 635I, 638Y, 642I, 645L, 649S, 652Q, 656N, and 659E. Analysis of the 150 specimens revealed that all had identical residues at six of these positions, whereas two positions had minor variations (635 and 649) and two (645 and 659) appeared to have subtype-specific substitutions. CONCLUSIONS: This data indicates that there is limited resistance to T-20 in these worldwide populations and that the critical epitopes for effective 5-helix binding are highly conserved across all subtypes. Taken together, these data suggest that T-20 and 5-helix should provide useful additives to current antiretroviral therapy for clinical management of HIV disease.     

Protease inhibitors work against HIV-1 subtype C

Stanford University researchers have determined that protease inhibitors are effective against HIV-1 subtype C infections. HIV-1 subtype C, while common in Africa and India, is not prevalent in the United States, and the drugs previously had not been tested for their effectiveness against that strain. All HIV subtypes are expected to be susceptible to highly active antiretroviral therapies (HAART) which are used to treat subtype B, the strain most common in the United States, Europe, and Australia. In Africa, however, economics interferes with the ability of HIV-infected individuals to get access to anti-HIV drugs. Several studies on the effects of anti-HIV treatment and the prevention of transmission of the disease from pregnant women to their babies are described.