Joel R. Courter

Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions

HIVs shape-shifting envelope protein HIVs envelope protein (Env) coats virus particles and allows HIV to enter host cells. HIV entry is highly dynamic. Env proteins work in groups of three (called trimers), which bind to the viral receptor and co-receptor, both expressed by host cells. Viral receptor binding causes a structural rearrangement in the trimer that allows for co-receptor binding and finally, viral entry. To visualize dynamic changes in Env conformation during viral entry, Munro et al. added differently colored fluorescent tags to two different regions of individual HIV trimers. Single-molecule fluorescence resonance entry transfer revealed three distinct Env conformations before cell entry. Occupation of particular conformations depended on host receptor binding. Science, this issue p. 759 Single-molecule fluorescence studies reveal how HIV’s viral spike protein varies its conformation as it binds to receptors. The HIV-1 envelope (Env) mediates viral entry into host cells. To enable the direct imaging of conformational dynamics within Env, we introduced fluorophores into variable regions of the glycoprotein gp120 subunit and measured single-molecule fluorescence resonance energy transfer within the context of native trimers on the surface of HIV-1 virions. Our observations revealed unliganded HIV-1 Env to be intrinsically dynamic, transitioning between three distinct prefusion conformations, whose relative occupancies were remodeled by receptor CD4 and antibody binding. The distinct properties of neutralization-sensitive and neutralization-resistant HIV-1 isolates support a dynamics-based mechanism of immune evasion and ligand recognition.

Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state.

Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus–cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high–potential-energy viral entry machines that are normally activated by receptor binding.

Small-Molecule CD4 Mimics Interact with a Highly Conserved Pocket on HIV-1 gp120

Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogs and gp120 mutants suggest that (1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4-bound conformation; (2) the NBD-556 phenyl ring projects into the Phe 43 cavity; (3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and (4) increased affinity of NBD-556 analogs for gp120 improves antiviral potency during infection of CD4-expressing cells.

Contribution of intrinsic reactivity of the HIV-1 envelope glycoproteins to CD4-independent infection and global inhibitor sensitivity.

Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the “intrinsic reactivity” of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant (“Tier 2-like”) viruses, globally sensitive (“Tier 1”) viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of inhibitor/antibody binding to the envelope glycoprotein trimer and by envelope glycoprotein reactivity to the inhibitor/antibody binding event. Quantitative differences in intrinsic reactivity contribute to HIV-1 strain variability in global susceptibility to neutralization and explain the long-observed relationship between increased inhibitor sensitivity and decreased entry requirements for target cell CD4.

Structure-Based Design, Synthesis, and Characterization of Dual Hotspot Small-Molecule HIV-1 Entry Inhibitors.

Cellular infection by HIV-1 is initiated with a binding event between the viral envelope glycoprotein gp120 and the cellular receptor protein CD4. The CD4-gp120 interface is dominated by two hotspots: a hydrophobic gp120 cavity capped by Phe43(CD4) and an electrostatic interaction between residues Arg59(CD4) and Asp368(gp120). The CD4 mimetic small-molecule NBD-556 (1) binds within the gp120 cavity; however, 1 and related congeners demonstrate limited viral neutralization breadth. Herein, we report the design, synthesis, characterization, and X-ray structures of gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm for inhibiting the CD4-gp120 protein-protein interaction.

CD4 mimetics sensitize HIV-1-infected cells to ADCC

Significance The prevention of HIV-1 transmission and progression likely requires approaches that can specifically eliminate HIV-1-infected cells. Rationally designed CD4-mimetic compounds (CD4mc) have been shown to efficiently inhibit viral entry and sensitize HIV-1 particles to neutralization by otherwise nonneutralizing CD4-induced antibodies. Here we found that CD4mc can also sensitize HIV-1-infected cells to antibody-dependent cell-mediated cytotoxicity (ADCC). Indeed, CD4mc induced the CD4-bound conformation of HIV-1 envelope glycoproteins, exposing CD4-induced epitopes recognized by easy-to-elicit antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we provide evidence that CD4mc can efficiently sensitize primary CD4 T cells from HIV-1-infected individuals to ADCC mediated by autologous sera and effector cells. Therefore, CD4mc might represent an attractive approach to prevent and control HIV-1 infection. HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

Design, synthesis and biological evaluation of small molecule inhibitors of CD4-gp120 binding based on virtual screening.

The low-molecular-weight compound JRC-II-191 inhibits infection of HIV-1 by blocking the binding of the HIV-1 envelope glycoprotein gp120 to the CD4 receptor and is therefore an important lead in the development of a potent viral entry inhibitor. Reported here is the use of two orthogonal screening methods, gold docking and ROCS shape-based similarity searching, to identify amine-building blocks that, when conjugated to the core scaffold, yield novel analogs that maintain similar affinity for gp120. Use of this computational approach to expand SAR produced analogs of equal inhibitory activity but with diverse capacity to enhance viral infection. The novel analogs provide additional lead scaffolds for the development of HIV-1 entry inhibitors that employ protein-ligand interactions in the vestibule of gp120 Phe 43 cavity.

Tetrazine Phototriggers: Probes for Peptide Dynamics†

Ultrafast photochemical triggers hold the promise of providing information on the dynamics of peptide and protein folding.[1] Prior to photolysis, bonding to the trigger constrains the peptide to have a narrow structure distribution. Photochemical triggering releases the constraints, permitting the molecule to evolve to a different equilibrium distribution. The structure evolution, even when ultrafast, can be followed by infrared probe or two-dimensional infrared spectroscopy. Fast phototriggering can thus reveal early kinetic events in protein dynamics by providing a means to explore the free energy landscape of folding and misfolding. Several phototriggers have been developed for this purpose,[1,2] but there remain significant challenges. For example, disulfide bonds in peptides can be used as a phototriggers. Deep UV light severs the disulfide bond and releases the structural constraints; such experiments have been carried out in short helical peptides,[1a,b] cyclic peptides,[1d] and β hairpins.[1c] Although disulfide photolysis offers the capability of initiating ultrafast structure equilibration, limitations preclude broad generality of this technique. Homolytic S–S bond scission reveals two reactive radicals that can undergo geminate recombination, as well as reactions with protein sidechains. Moreover, the UV excitation required to dissociate the disulfide bond also excites the peptide backbone. Another example is azobenzene which undergoes fast, reversible photoisomerization. When designed into a peptide a light pulse can be used to cause the system to reversibly shift between significantly different equilibrium configurations.[3]

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

ABSTRACT Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.

Structure-based design, synthesis and validation of CD4-mimetic small molecule inhibitors of HIV-1 entry: conversion of a viral entry agonist to an antagonist.

Conspectus This Account provides an overview of a multidisciplinary consortium focused on structure-based strategies to devise small molecule antagonists of HIV-1 entry into human T-cells, which if successful would hold considerable promise for the development of prophylactic modalities to prevent HIV transmission and thereby alter the course of the AIDS pandemic. Entry of the human immunodeficiency virus (HIV) into target T-cells entails an interaction between CD4 on the host T-cell and gp120, a component of the trimeric envelope glycoprotein spike on the virion surface. The resultant interaction initiates a series of conformational changes within the envelope spike that permits binding to a chemokine receptor, formation of the gp41 fusion complex, and cell entry. A hydrophobic cavity at the CD4–gp120 interface, defined by X-ray crystallography, provided an initial site for small molecule antagonist design. This site however has evolved to facilitate viral entry. As such, the binding of prospective small molecule inhibitors within this gp120 cavity can inadvertently trigger an allosteric entry signal. Structural characterization of the CD4–gp120 interface, which provided the foundation for small molecule structure-based inhibitor design, will be presented first. An integrated approach combining biochemical, virological, structural, computational, and synthetic studies, along with a detailed analysis of ligand binding energetics, revealed that modestly active small molecule inhibitors of HIV entry can also promote viral entry into cells lacking the CD4 receptor protein; these competitive inhibitors were termed small molecule CD4 mimetics. Related congeners were subsequently identified with both improved binding affinity and more potent viral entry inhibition. Further assessment of the affinity-enhanced small molecule CD4 mimetics demonstrated that premature initiation of conformational change within the viral envelope spike, prior to cell encounter, can lead to irreversible deactivation of viral entry machinery. Related congeners, which bind the same gp120 site, possess different propensities to elicit the allosteric response that underlies the undesired enhancement of CD4-independent viral entry. Subsequently, key hotspots in the CD4–gp120 interface were categorized using mutagenesis and isothermal titration calorimetry according to the capacity to increase binding affinity without triggering the allosteric signal. This analysis, combined with cocrystal structures of small molecule viral entry agonists with gp120, led to the development of fully functional antagonists of HIV-1 entry. Additional structure-based design exploiting two hotspots followed by synthesis has now yielded low micromolar inhibitors of viral entry.