The molecular immune modulator adenosine deaminase-1 enhances HIV specific humoral and cellular responses to a native-like HIV envelope trimer DNA vaccine.

There is currently no prophylactic vaccine available for human immunodeficiency virus (HIV). Research efforts have resulted in improved immunogens that mimic the native envelope (Env) glycoprotein structure. Recently, a novel triple tandem trimer (TTT) platform has been used to generate a plasmid encoding Env immunogen (pBG505-TTT) that expresses only as trimers, making it more suitable for nucleic acid vaccines. We have previously demonstrated that adenosine deaminase-1 (ADA-1) is critical to the T follicular helper (TFH) function and improves vaccine immune responses in vivo. In this study, we demonstrate that co-delivery of plasmid-encoded adenosine deaminase 1 (pADA) with pBG505-TTT enhances the magnitude, durability, isotype switching and functionality of HIV-specific antibodies in a dose-sparing manner. Co-delivery of the molecular immune modulator ADA-1 also enhances HIV-specific T cell polyfunctionality, activation, and degranulation as well as memory B cell responses. These data demonstrate that pADA enhances HIV-specific cellular and humoral immunity, making ADA-1 a promising immune modulator for HIV-targeting vaccines.

Injectable Granular Hydrogels Enable Avidity-Controlled Biotherapeutic Delivery.

Protein therapeutics represent a rapidly growing class of pharmaceutical agents that hold great promise for the treatment of various diseases such as cancer and autoimmune dysfunction. Conventional systemic delivery approaches, however, result in off-target drug exposure and a short therapeutic half-life, highlighting the need for more localized and controlled delivery. We have developed an affinity-based protein delivery system that uses guest-host complexation between ß-cyclodextrin (CD, host) and adamantane (Ad, guest) to enable sustained localized biomolecule presentation. Hydrogels were formed by the copolymerization of methacrylated CD and methacrylated dextran. Extrusion fragmentation of bulk hydrogels yielded shear-thinning and self-healing granular hydrogels (particle diameter = 32.4 ± 16.4 µm) suitable for minimally invasive delivery and with a high host capacity for the retention of guest-modified proteins. Bovine serum albumin (BSA) was controllably conjugated to Ad via EDC chemistry without affecting the affinity of the Ad moiety for CD (KD = 12.0 ± 1.81 µM; isothermal titration calorimetry). The avidity of Ad-BSA conjugates was directly tunable through the number of guest groups attached, resulting in a fourfold increase in the complex half-life (t1/2 = 5.07 ± 1.23 h, surface plasmon resonance) that enabled a fivefold reduction in protein release at 28 days. Furthermore, we demonstrated that the conjugation of Ad to immunomodulatory cytokines (IL-4, IL-10, and IFNγ) did not detrimentally affect cytokine bioactivity and enabled their sustained release. Our strategy of avidity-controlled delivery of protein-based therapeutics is a promising approach for the sustained local presentation of protein therapeutics and can be applied to numerous biomedical applications.

Irreversible Inactivation of SARS-CoV-2 by Lectin Engagement with Two Glycan Clusters on the Spike Protein.

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins cyanovirin-N (CV-N) and griffithsin (GRFT). We discovered in this study that CV-N not only inhibits SARS-CoV-2 infection but also leads to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with CV-N and then washed to remove all soluble lectin did not recover infectivity. The infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition: one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable spike before host cell encounter.

Pharmacophore Variants of the Macrocyclic Peptide Triazole Inactivator of HIV-1 Env.

Previously we established a family of macrocyclic peptide triazoles (cPTs) that inactivate the Env protein complex of HIV-1, and identified the pharmacophore that engages Env's receptor binding pocket. Here, we examined the hypothesis that the side chains of both components of the triazole Pro - Trp segment of cPT pharmacophore work in tandem to make intimate contacts with two proximal subsites of the overall CD4 binding site of gp120 to stabilize binding and function. Variations of the triazole Pro R group, which previously had been significantly optimized, led to identification of a variant MG-II-20 that contains a pyrazole substitution. MG-II-20 has improved functional properties over previously examined variants, with Kd for gp120 in the nM range. In contrast, new variants of the Trp indole side chain, with either methyl- or bromo- components appended, had disruptive effects on gp120 binding, reflecting the sensitivity of function to changes in this component of the encounter complex. Plausible in silico models of cPT:gp120 complex structures were obtained that are consistent with the overall hypothesisof occupancy by the triazole Pro and Trp side chains, respectively, into the ß20/21 and Phe43 sub-cavities. The overall results strengthen the definition of the cPT-Env inactivator binding site and provide a new lead composition (MG-II-20) as well as structure-function findings to guide future HIV-1 Env inactivator design.

Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

Peptide Triazole Inhibitors of HIV-1: Hijackers of Env Metastability.

With 1.5 million new infections and 690,000 AIDS-related deaths globally each year, HIV- 1 remains a pathogen of significant public health concern. Although a wide array of effective antiretroviral drugs have been discovered, these largely target intracellular stages of the viral infectious cycle, and inhibitors that act at or before the point of viral entry still require further advancement. A unique class of HIV-1 entry inhibitors, called peptide triazoles (PTs), has been developed, which irreversibly inactivates Env trimers by exploiting the protein structure's innate metastable nature. PTs, and a related group of inhibitors called peptide triazole thiols (PTTs), are peptide compounds that dually engage the CD4 receptor and coreceptor binding sites of Env's gp120 subunit. This triggers dramatic conformational rearrangements of Env, including the shedding of gp120 (PTs and PTTs) and lytic transformation of the gp41 subunit to a post-fusion-like arrangement (PTTs). Due to the nature of their dual receptor site engagement, PT/PTT-induced conformational changes may elucidate mechanisms behind the native fusion program of Env trimers following receptor and coreceptor engagement, including the role of thiols in fusion. In addition to inactivating Env, PTT-induced structural transformation enhances the exposure of important and conserved neutralizable regions of gp41, such as the membrane proximal external region (MPER). PTT-transformed Env could present an intriguing potential vaccine immunogen prototype. In this review, we discuss the origins of the PT class of peptide inhibitors, our current understanding of PT/PTT-induced structural perturbations and viral inhibition, and prospects for using these antagonists for investigating Env structural mechanisms and for vaccine development.

Improved Durability to SARS-CoV-2 Vaccine Immunity following Coimmunization with Molecular Adjuvant Adenosine Deaminase-1.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have demonstrated strong immunogenicity and protection against severe disease, concerns about the duration and breadth of these responses remain. In this study, we show that codelivery of plasmid-encoded adenosine deaminase-1 (pADA) with SARS-CoV-2 spike glycoprotein DNA enhances immune memory and durability in vivo. Coimmunized mice displayed increased spike-specific IgG of higher affinity and neutralizing capacity as compared with plasmid-encoded spike-only-immunized animals. Importantly, pADA significantly improved the longevity of these enhanced responses in vivo. This coincided with durable increases in frequencies of plasmablasts, receptor-binding domain-specific memory B cells, and SARS-CoV-2-specific T follicular helper cells. Increased spike-specific T cell polyfunctionality was also observed. Notably, animals coimmunized with pADA had significantly reduced viral loads compared with their nonadjuvanted counterparts in a SARS-CoV-2 infection model. These data suggest that pADA enhances immune memory and durability and supports further translational studies.

Altered Env conformational dynamics as a mechanism of resistance to peptide-triazole HIV-1 inactivators.

BACKGROUND: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. RESULTS: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. CONCLUSIONS: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.

Peptide Triazole Thiol Irreversibly Inactivates Metastable HIV-1 Env by Accessing Conformational Triggers Intrinsic to Virus-Cell Entry.

KR13, a peptide triazole thiol previously established to inhibit HIV-1 infection and cause virus lysis, was evaluated by flow cytometry against JRFL Env-presenting cells to characterize induced Env and membrane transformations leading to irreversible inactivation. Transiently transfected HEK293T cells were preloaded with calcein dye, treated with KR13 or its thiol-blocked analogue KR13b, fixed, and stained for gp120 (35O22), MPER (10E8), 6-helix-bundle (NC-1), immunodominant loop (50-69), and fusion peptide (VRC34.01). KR13 induced dose-dependent transformations of Env and membrane characterized by transient poration, MPER exposure, and 6-helix-bundle formation (analogous to native fusion events), but also reduced immunodominant loop and fusion peptide exposure. Using a fusion peptide mutant (V504E), we found that KR13 transformation does not require functional fusion peptide for poration. In contrast, simultaneous treatment with fusion inhibitor T20 alongside KR13 prevented membrane poration and MPER exposure, showing that these events require 6-helix-bundle formation. Based on these results, we formulated a model for PTT-induced Env transformation portraying how, in the absence of CD4/co-receptor signaling, PTT may provide alternate means of perturbing the metastable Env-membrane complex, and inducing fusion-like transformation. In turn, the results show that such transformations are intrinsic to Env and can be diverted for irreversible inactivation of the protein complex.

The Case for S2: The Potential Benefits of the S2 Subunit of the SARS-CoV-2 Spike Protein as an Immunogen in Fighting the COVID-19 Pandemic.

As COVID-19 cases continue to rise, it is imperative to learn more about antibodies and T-cells produced against the causative virus, SARS-CoV-2, in order to guide the rapid development of therapies and vaccines. While much of the current antibody and vaccine research focuses on the receptor-binding domain of S1, a less-recognized opportunity is to harness the potential benefits of the more conserved S2 subunit. Similarities between the spike proteins of both SARS-CoV-2 and HIV-1 warrant exploring S2. Possible benefits of employing S2 in therapies and vaccines include the structural conservation of S2, extant cross-reactive neutralizing antibodies in populations (due to prior exposure to common cold coronaviruses), the steric neutralization potential of antibodies against S2, and the stronger memory B-cell and T-cell responses. More research is necessary on the effect of glycans on the accessibility and stability of S2, SARS-CoV-2 mutants that may affect infectivity, the neutralization potential of antibodies produced by memory B-cells, cross-reactive T-cell responses, antibody-dependent enhancement, and antigen competition. This perspective aims to highlight the evidence for the potential advantages of using S2 as a target of therapy or vaccine design.

HIV-1 Env-Dependent Cell Killing by Bifunctional Small-Molecule/Peptide Conjugates.

A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW ("Trp3"), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.

Identification of a glycan cluster in gp120 essential for irreversible HIV-1 lytic inactivation by a lectin-based recombinantly engineered protein conjugate.

We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.

Roles of variable linker length in dual acting virucidal entry inhibitors on HIV-1 potency via on-the-fly free energy molecular simulations.

The Dual-Acting Virolytic Entry Inhibitors, or DAVEI's, are a class of recombinant chimera fusion proteins consisting of a lectin, a flexible polypeptide linker, and a fragment of the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEIs trigger virolysis of HIV-1 virions through interactions with the trimeric envelope glycoprotein complex (Env), though the details of these interactions are not fully determined as yet. The purpose of this work was to use structural modeling to rationalize a dependence of DAVEI potency on the molecular length of the linker connecting the two components. We used temperature accelerated molecular dynamics and on-the-fly parameterization to compute free energy versus end-to-end distance for two different linker lengths, DAVEI L0 (His6 ) and DAVEI L2 ([Gly4 Ser]2 His6 ). Additionally, an envelope model was created based on a cryo-electron microscopy-derived structure of a cleaved, soluble Env construct, with high-mannose glycans added which served as putative docking locations for the lectin, along with MPER added that served as a putative docking location for the MPER region of DAVEI (MPERDAVEI ). Using MD simulation, distances between the lectin C-terminus and Env gp41 MPER were measured. We determined that none of the glycans were close enough to gp41 MPER to allow DAVEI L0 to function, while one, N448, will allow DAVEI L2 to function. These findings are consistent with the previously determined dependence of lytic function on DAVEI linker lengths. This supports the hypothesis that DAVEI's engage Env at both glycans and the Env MPER in causing membrane poration and lysis.

The HIV-1 Env gp120 Inner Domain Shapes the Phe43 Cavity and the CD4 Binding Site.

The HIV-1 envelope glycoproteins (Env) undergo conformational changes upon interaction of the gp120 exterior glycoprotein with the CD4 receptor. The gp120 inner domain topological layers facilitate the transition of Env to the CD4-bound conformation. CD4 engages gp120 by introducing its phenylalanine 43 (Phe43) in a cavity ("the Phe43 cavity") located at the interface between the inner and outer gp120 domains. Small CD4-mimetic compounds (CD4mc) can bind within the Phe43 cavity and trigger conformational changes similar to those induced by CD4. Crystal structures of CD4mc in complex with a modified CRF01_AE gp120 core revealed the importance of these gp120 inner domain layers in stabilizing the Phe43 cavity and shaping the CD4 binding site. Our studies reveal a complex interplay between the gp120 inner domain and the Phe43 cavity and generate useful information for the development of more-potent CD4mc.IMPORTANCE The Phe43 cavity of HIV-1 envelope glycoproteins (Env) is an attractive druggable target. New promising compounds, including small CD4 mimetics (CD4mc), were shown to insert deeply into this cavity. Here, we identify a new network of residues that helps to shape this highly conserved CD4 binding pocket and characterize the structural determinants responsible for Env sensitivity to small CD4 mimetics.

Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors.

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.

Pharmacokinetic stability of macrocyclic peptide triazole HIV-1 inactivators alone and in liposomes.

Previously, we reported the discovery of macrocyclic peptide triazoles (cPTs) that bind to HIV-1 Env gp120, inhibit virus cell infection with nanomolar potencies, and cause irreversible virion inactivation. Given the appealing virus-killing activity of cPTs and resistance to protease cleavage observed in vitro, we here investigated in vivo pharmacokinetics of the cPT AAR029b. AAR029b was investigated both alone and encapsulated in a PEGylated liposome formulation that was designed to slowly release inhibitor. Pharmacokinetic analysis in rats showed that the half-life of FITC-AAR029b was substantial both alone and liposome-encapsulated, 2.92 and 8.87 hours, respectively. Importantly, liposome-encapsulated FITC-AAR029b exhibited a 15-fold reduced clearance rate from serum compared with the free FITC-cPT. This work thus demonstrated both the in vivo stability of cPT alone and the extent of pharmacokinetic enhancement via liposome encapsulation. The results obtained open the way to further develop cPTs as long-acting HIV-1 inactivators against HIV-1 infection.

Mechanical characterization of HIV-1 with a solid-state nanopore sensor.

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo-type human immunodeficiency virus type 1 at sub-micron scale. Non-infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.

Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus-Cell Interface.

To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of the HIV-1 envelope (Env) gp120 protein and the host-cell coreceptor (CoR) protein can be covalently joined into bifunctional synergistic combinations with improved antiviral capabilities. A synthetic protocol was established to covalently combine a CCR5 small-molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components and, at the same time, exhibited low to subnanomolar potencies in inhibiting cell infection by different pseudoviruses, which were substantially greater than those of a noncovalent mixture of the individual components. The results demonstrate that targeting the virus-cell interface with a single molecule can result in improved potencies and also the introduction of new phenotypes to the chimeric inhibitor, such as the irreversible inactivation of HIV-1.

Roles of conserved tryptophans in trimerization of HIV-1 membrane-proximal external regions: Implications for virucidal design via alchemical free-energy molecular simulations.

The Dual-Action Virolytic Entry Inhibitors, or "DAVEI's," are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15-residue fragment from the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEI's trigger rupture of HIV-1 virions, and the interaction site between DAVEI MPER and HIV-1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free-energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER3 . We found that a W666A mutation had a large positive ΔΔG for all three protomers, while W672A had a large positive ΔΔG for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER3 stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER3 . Our findings are consistent with our previous experimental study of W-to-A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV-1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.