HIV-1 Envelope Glycoprotein Gp120 Research Articles

HIV-1 gp120 amplifies astrocyte elevated gene-1 activity to compromise the integrity of the outer blood-retinal barrier.

This study aims to investigate the functions and mechanistic pathways of Astrocyte Elevated Gene-1 (AEG-1) in the disruption of the blood-retinal barrier (BRB) caused by the HIV-1 envelope glycoprotein gp120. We utilized ARPE-19 cells challenged with gp120 as our model system. Several analytical techniques were employed to decipher the intricate interactions at play. These included PCR, Western blot, and immunofluorescence assays for the molecular characterization, and transendothelial electrical resistance (TEER) measurements to evaluate barrier integrity. We observed that AEG-1 expression was elevated, whereas the expression levels of tight junction proteins ZO-1, Occludin, and Claudin5 were downregulated in gp120-challenged cells. TEER measurements corroborated these findings, indicating barrier dysfunction. Additional mechanistic studies revealed that the activation of NFκB and MMP2/9 pathways mediated the AEG-1-induced barrier destabilization. Through the use of lentiviral vectors, we engineered cell lines with modulated AEG-1 expression levels. Silencing AEG-1 alleviated gp120-induced downregulation of tight junction proteins and barrier impairment while concurrently inhibiting the NFκB and MMP2/9 pathways. Conversely, overexpression of AEG-1 exacerbated these pathological changes, further compromising the integrity of the BRB. Gp120 upregulates the expression of AEG-1 and activates the NFκB and MMP2/9 pathways. This in turn leads to the downregulation of tight junction proteins, resulting in the disruption of barrier function.

Development of a potent and infection-resistant anti-HIV CAR-T cells therapy

Abstract Acquired immune deficiency syndrome (AIDS) caused by HIV infection is a serious global public health issue. The current combined antiretroviral therapy (ART) effectively controls HIV replication but fails to eliminate HIV reservoir cells, which allows a persistent viral infection. HIV-specific chimeric antigen receptor (CAR) T-cells have been designed to eliminate HIV-infected cells. The broad anti-viral activity, resistance to HIV infection and immune suppressive microenvironment determine a successful CAR-T therapy for HIV infection. In this study, we developed a potent CD4-based CAR-T cell with herpes virus entry mediator (HVEM) costimulatory domain. We intruded a S60C mutation in CD4 extracellular domain, which mediates a higher binding affinity to HIV envelope glycoprotein gp120 while preventes gp41-induced fusion between viral envelope and cell membrane. We demonstrated that CD4-based CAR-T cells bearing this 2C mutation were resistant to HIV infection. In addition, the HVEM costimulatory domain (CSD) mediated a superior effector T cell function than CD28 or 4-1BB-derived CSD did in CAR-T cells, showed by either cytotoxicity effect or cytokines response in contact with the HIV Envelope-transduced K562 cells in vitro. Furthermore, HVEM-CAR-T showed a better therapeutic efficacy than CD28 or 4-1BB CAR-T in humanized mouse model in vivo. Overall, we designed and developed a potent infection-resistant anti-HIV CAR-T cell by incorporating a mutant CD4 extracellular domain and HVEM-derived CSD.

Structure and Carbohydrate Recognition by the Nonmitogenic Lectin Horcolin.

Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on the surface of viruses such as HIV-1. However, their therapeutic potential has been compromised by their mitogenicity that stimulates uncontrolled division of T-lymphocytes. Horcolin, a member of the jacalin family of lectins, tightly binds the HIV-1 envelope glycoprotein gp120 and neutralizes HIV-1 particles but is nonmitogenic. In this report, we combine X-ray crystallography and NMR spectroscopy to obtain atomic-resolution insights into the structure of horcolin and the molecular basis for its carbohydrate recognition. Each protomer of the horcolin dimer adopts a canonical β-prism I fold with three Greek key motifs and carries two carbohydrate-binding sites. The carbohydrate molecule binds in a negatively charged pocket and is stabilized by backbone and side chain hydrogen bonds to conserved residues in the ligand-binding loop. NMR titrations reveal a two-site binding mode and equilibrium dissociation constants for the two binding sites determined from two-dimensional (2D) lineshape modeling are 4-fold different. Single-binding-site variants of horcolin confirm the dichotomy in binding sites and suggest that there is allosteric communication between the two sites. An analysis of the horcolin structure shows a network of hydrogen bonds linking the two carbohydrate-binding sites directly and through a secondary binding site, and this coupling between the two sites is expected to assume importance in the interaction of horcolin with high-mannose glycans found on viral envelope glycoproteins.

Physicochemical characterization of the recombinant lectin scytovirin and microbicidal activity of the SD1 domain produced in rice against HIV-1

Key messageRice-produced SD1 retains its physicochemical properties and provides efficient pre-exposure HIV-1 prophylaxis against infection in vitro.Scytovirin (SVN) is an HIV-neutralizing lectin that features two structural domains (SD1 and SD2) that bind to HIV-1 envelope glycoproteins. We expressed SD1 in rice seeds as a potential large-scale production platform and confirmed that rice-derived SD1 binds the HIV-1 envelope glycoprotein gp120 in vitro. We analyzed the thermodynamic properties of SD1 compared to full-size SVN (produced in E. coli) by isothermal titration and differential scanning calorimetry to characterize the specific interactions between SVN/SD1 and gp120 as well as to high-mannose oligosaccharides. SVN bound with moderate affinity (Kd = 1.5 µM) to recombinant gp120, with 2.5-fold weaker affinity to nonamannoside (Kd of 3.9 µM), and with tenfold weaker affinity to tetramannoside (13.8 µM). The melting temperature (Tm) of full-size SVN was 59.1 °C and the enthalpy of unfolding (ΔHunf) was 16.4 kcal/mol, but the Tm fell when SVN bound to nonamannoside (56.5 °C) and twice as much energy was required for unfolding (ΔHunf = 33.5 kcal/mol). Interestingly, binding to tetramannoside destabilized the structure of SD1 (ΔTm ~ 11.5 °C) and doubled the enthalpy of unfolding, suggesting a dimerization event. The similar melting phenomenon shared by SVN and SD1 in the presence of oligomannose confirmed their conserved oligosaccharide-binding mechanisms. SD1 expressed in transgenic rice was able to neutralize HIV-1 in vitro. SD1 expressed in rice, therefore, is suitable as a microbicide component.

In silico Design of Glyco-D,L-Peptide Antiviral Molecules

Background: Most licensed antiviral drugs are nucleoside analogs. A recent research focuses on blocking a virus from entering the cells in the viral cell adsorption/entry stage. In this entry mechanism, the glycans present on the viral surface play a fundamental role. Homochiral L-peptides acting this fusion mechanism have shown some inhibition of viral infection. Peptides with regularly alternating enantiomeric sequence (L,D-peptides) can assume structures that are not accessible to the corresponding homochiral molecules. Furthermore, L,D-peptides are less sensitive to enzymatic digestion. Aim: In silico design of an L, D-peptide with a high affinity for the viral surface glycans and consequently able to interfere with its fusion mechanism. Methods: We used a [Formula: see text]-Mannopentaose (3-6MP) molecule to simulate a viral surface glycan. We performed molecular dynamics (MD) simulations of 3-6MP and D,L-peptide in water using the force field AMBER12-GLYCAM06i. We evaluated the binding constant from trajectories. The D,L-peptide molecule was modified over the sequence, the length, the terminals and finally glycosylated to attain a very high binding constant value for 3-6MP. In addition, we studied the specific interaction between T lymphocyte CD4 glycoprotein and HIV envelope glycoprotein gp120 through MD simulations between a D,L-peptide, bounded to a typical CD4 glycan, and a highly conserved HIV gp120 glycan. Results: Concerning the interaction with 3-6MP molecule, the very effective molecule obtained was H-d-Trp-l-Pro-d-Asn-l-Pro-d-Trp-l-Pro-d-Asn-l-Pro-OH where the Asn residues in position 3 and 7 are glycosylated with alpha-D-Mannopyranosyl-([Formula: see text])-[alpha-D-mannopyranosyl-([Formula: see text])]-alpha-D-mannopyranosyl-([Formula: see text])-N-acetyl-beta-D-glucopyranosyl-([Formula: see text])-N-acetyl-beta-D-glucopyranosyl-1-OH. As far as the interaction with HIV envelope is considered instead, the very effective molecule obtained, able to antagonize the CD4 glycoprotein, was H-d-Trp-l-Pro-d-Asn-l-Pro-d-Trp-l-Pro-d-Asn-l-Pro-OH where the Asn residue in position 3 is glycosylated with alpha-D-galactopyranosyl-([Formula: see text])- N-acetyl-beta-D-glucopyranosyl-([Formula: see text])-alpha-D-Mannopyranosyl-([Formula: see text])- [alpha-D-galactopyranosyl-([Formula: see text])- N-acetyl-beta-D-glucopyranosyl-([Formula: see text])- alpha-D-Mannopyranosyl-([Formula: see text])]- beta-D-mannopyranosyl-([Formula: see text])-N-acetyl-beta-D-glucopyranosyl-([Formula: see text])-N-acetyl-beta-D-glucopyranosyl-1-OH Conclusion: The above optimized glycosylated D,L-peptide molecules could be very promising representatives of a new powerful class of antiviral agents.

From Initiation to Maintenance: HIV-1 Gp120-induced Neuropathic Pain Exhibits Different Molecular Mechanisms in the Mouse Spinal Cord Via Bioinformatics Analysis Based on RNA Sequencing.

Human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), remains one of the most diverse crucial health and development challenges around the world. People infected with HIV constitute a large patient population, and a significant number of them experience neuropathic pain. To study the key mechanisms that mediate HIV-induced neuropathic pain (HNP), we established an HNP mouse model via intrathecal injection of the HIV-1 envelope glycoprotein gp120. The L3~L5 spinal cord was isolated on postoperative days 1/12 (POD1/12), 1 (POD1), and 14 (POD14) for RNA sequencing to investigate the gene expression profiles of the initiation, transition, and maintenance stages of HNP. A total of 1682, 430, and 413 differentially expressed genes were obtained in POD1/12, POD1, and POD14, respectively, and their similarity was low. Bioinformatics analysis confirmed that POD1/12, POD1, and POD14 exhibited different biological processes and signaling pathways. Inflammation, oxidative damage, apoptosis, and inflammation-related signaling pathways were enriched on POD1/12. Inflammation, chemokine activity, and downstream signaling regulated by proinflammatory cytokines, such as the MTOR signaling pathway, were enriched on POD1, while downregulation of ion channel activity, mitochondrial damage, endocytosis, MAPK and neurotrophic signaling pathways developed on POD14. Additionally, we screened key genes and candidate genes, which were verified at the transcriptional and translational levels. Our results suggest that the initiation and maintenance of HNP are regulated by different molecular mechanisms. Therefore, our research may yield a fresh and deeper understanding of the mechanisms underlying HNP, providing accurate molecular targets for HNP therapy.

A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration

Despite a reduction in global HIV prevalence the development of a pipeline of new therapeutics or pre-exposure prophylaxis to control the HIV/AIDS epidemic are of high priority. Antibody-based therapies offer several advantages and have been shown to prevent HIV-infection. Plant-based production is efficient for several biologics, including antibodies. We provide a short review on the work by Singh et al., 2020 who demonstrated the transient production of potent CAP256-VRC26 broadly neutralizing antibodies. These antibodies have engineered posttranslational modifications, namely N-glycosylation in the fragment crystallizable region and O-sulfation of tyrosine residues in the complementary-determining region H3 loop. The glycoengineered Nicotiana benthamiana mutant (ΔXTFT) was used, with glycosylating structures lacking β1,2-xylose and/or α1,3-fucose residues, which is critical for enhanced effector activity. The CAP256-VRC26 antibody lineage targets the first and second variable region of the HIV-1 gp120 envelope glycoprotein. The high potency of this lineage is mediated by a protruding O-sulfated tyrosine in the CDR H3 loop. Nicotiana benthamiana lacks human tyrosyl protein sulfotransferase 1, the enzyme responsible for tyrosine O-sulfation. The transient coexpression of the CAP256-VRC26 antibodies with tyrosyl protein sulfotransferase 1 in planta had restored the efficacy of these antibodies through the incorporation of the O-sulfation modification. This approach demonstrates the strategic incorporation of posttranslational modifications in production systems, which may have not been previously considered. These plant-produced CAP256-VRC26 antibodies have therapeutic as well as topical and systemic pre-exposure prophylaxis potential in enabling the empowerment of young girls and women given that gender inequalities remain a major driver of the epidemic.

Chemoenzymatic Synthesis and Antibody Binding of HIV-1 V1/V2 Glycopeptide-Bacteriophage Qβ Conjugates as a Vaccine Candidate.

The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qβ conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qβ), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qβ-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qβ conjugates synthesized represent a promising candidate of HIV-1 vaccine.

HIV envelope antigen valency on peptide nanofibers modulates antibody magnitude and binding breadth

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Development of a platform-based approach for the clinical production of HIV gp120 envelope glycoprotein vaccine candidates

Preclinical development of vaccine candidates is an important link between the discovery and manufacture of vaccines for use in human clinical trials. Here, an exploratory clinical study utilizing multiple gp120 envelope proteins as vaccine antigens was pursued, which required a harmonized platform development approach for timely and efficient manufacture of the combined HIV vaccine product.Development of cell lines, processes, and analytical methods was initiated with a transmitted founder envelope protein (CH505TF), then applied to produce three subsequent gp120 Env (envelope) variants. Cell lines were developed using the commercially available Freedom CHO DG44 kit (Life Technologies). The fed-batch cell culture production process was based on a commercially-available medium with harmonized process parameters across the variants. A platform purification process was developed utilizing a mixed mode chromatography capture step, with ceramic hydroxyapatite and ion exchange polishing steps. A suite of analytical methods was developed to establish and monitor the Quality Target Profile (QTP), release and long-term stability testing of the vaccine products.The platform development strategy was successfully implemented to produce four gp120 envelope protein variants. In some cases, minor changes to the platform were required to optimize for a particular variant; however, baseline conditions for the processes (cell line type, media & feed system, chromatography resins, and analytical approaches) remained constant, leading to successful transfer and manufacture of all four proteins in a cGMP facility.This body of work demonstrates successful pursuit of a platform development approach to manufacture important vaccine candidates and can be used as a model for other vaccine glycoproteins, such as HIV gp140 trimers or other viral glycoproteins with global health implications.Clinical trial identifier.NCT03220724, NCT03856996.

Enhanced Humoral Immune Response by High Density TLR Agonist Presentation on Hyperbranched Polymers

AbstractProphylactic vaccines often exploit adjuvants to drive potent and directed immune responses. Small‐molecule toll‐like receptor (TLR) agonists are promising adjuvant candidates; however, the poor pharmacokinetics and rapid distribution of these relatively hydrophilic small molecules results in systemic side effects that have limited their usage to topical application. In this study, TLR7/8 agonists are covalently attached to the highly dense end‐groups of hyperbranched polymers synthesized by controlled radical polymerization techniques. Subcutaneous administration of these adjuvants results in a potent and prolonged type I interferon response. When co‐administered in a model subunit vaccine, with ovalbumin or the HIV envelope glycoprotein gp120, these hyperbranched polymeric adjuvants potentiate an enhanced immunoglobulin response and near‐immediate type‐switching to IgG2c, suggesting they promote the type of strong T helper cell 1 immune response that is desirable for the treatment of intracellular pathogens such as mycobacterial and viral infections. Controlling the pharmacokinetics of potent TLR agonists through conjugation to hyperbranched polymers, therefore, enables the development of potent adjuvants in vaccines to drive durable and high‐quality humoral immunity.

Exploring Viral Interference Using Peptides: Molecular Determinants of HIV-1 Inhibition by a Peptide Derived from Human Pegivirus-1 Envelope Protein E2.

Co‐infection with the human pegivirus 1 (HPgV‐1) often has a beneficial effect on disease progression in HIV‐1‐infected individuals. Several HPgV‐1 proteins and peptides, including a 20‐mer peptide (P6‐2) derived from the N‐terminal region of the HPgV‐1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C‐terminal negative charge as key factors for the HIV‐1 inhibitory activity of P6‐2. Analysis of mutations in P6‐2‐resistant HIV‐1 indicated a binding site for the peptide in the HIV‐1 envelope glycoprotein gp120. In fact, P6‐2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV‐1 inhibitory activity of P6‐2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6‐2 with the gp120 V3 loop.

The analysis of the viral transmission and structural interactions between the HIV-1 envelope glycoprotein and the lymphocyte receptor integrin α4β7

The Human Immunodeficiency Virus (HIV) infects approximately 40 million people globally, and one million people die every year from Acquired Immune Deficiency Syndrome (AIDS)-related illnesses. The HIV-1 virus proliferates, infects people, and kills those who host the virus. Unfortunately, there is little research on the structural interactions between HIV-1 proteins and human cell receptors. This study examined the interactions between the HIV-1 envelope glycoprotein gp120 and the human lymphocyte receptor integrin α4β7, the putative first long-range receptor for the envelope glycoprotein of the virus in mucosal tissues. The specific site of binding between the glycoprotein and the receptor has not been determined. However, discovering the binding site can open the field towards the research of molecules that could potentially block this interaction and prevent the initial binding of the HIV1 virus. We hypothesized that the V1 and V2 loops of the envelope glycoprotein of HIV-1 are involved in the binding between the HIV-1 virus and the human lymphocyte receptor α4β7. Using structural analysis software, we analyzed the electrostatics and structural interactions between the glycoprotein and receptor. We report structural insights into the interactions between α4β7 and envelope glycoprotein gp120. Our data support the claim that the V1 loop is involved in the binding between α4β7 and the HIV-1 envelope glycoprotein through molecular dockings.

Concerted Interactions between Multiple gp41 Trimers and the Target Cell Lipidome May Be Required for HIV-1 Entry.

The HIV-1 envelope glycoprotein gp41 mediates the fusion between viral and host cell membranes leading to virus entry and target cell infection. Despite years of research, important aspects of this process such as the number of gp41 trimers involved and how they orchestrate the rearrangement of the lipids in the apposed membranes along the fusion pathway remain obscure. To elucidate these molecular underpinnings, we performed coarse-grained molecular dynamics simulations of HIV-1 virions pinned to the CD4 T cell membrane by different numbers of gp41 trimers. We built realistic cell and viral membranes by mimicking their respective lipid compositions. We found that a single gp41 was inadequate for mediating fusion. Lipid mixing between membranes, indicating the onset of fusion, was efficient when three or more gp41 trimers pinned the membranes. The gp41 trimers interacted strongly with many different lipids in the host cell membrane, triggering lipid configurational rearrangements, exchange, and mixing. Simpler membranes, comprising fewer lipid types, displayed strong resistance to fusion, revealing the crucial role of the lipidomes in HIV-1 entry. Performing simulations at different temperatures, we estimated the free energy barrier to lipid mixing, and hence membrane stalk formation, with three and four tethering gp41 trimers to be ∼6.2 kcal/mol, a >4-fold reduction over estimates without gp41. Together, these findings present molecular-level, quantitative insights into the early stages of gp41-mediated HIV-1 entry. Preventing the requisite gp41 molecules from tethering the membranes or altering membrane lipid compositions may be potential intervention strategies.

Binding Thermodynamics to Intrinsically Disordered Protein Domains.

Many proteins are intrinsically disordered or contain one or more disordered domains. These domains can participate in binding interactions with other proteins or small ligands. Binding to intrinsically disordered protein domains requires the folding or structuring of those regions such that they can establish well-defined stoichiometric interactions. Since, in such a situation binding is coupled to folding, the energetics of those two events is reflected in the measured binding thermodynamics. In this protocol, we illustrate the thermodynamic differences between binding coupled to folding and binding independent of folding for the same protein. As an example, we use the HIV-1 envelope glycoprotein gp120 that contains structured as well as disordered domains. In the experiments presented, the binding of gp120 to molecules that bind to disordered regions and trigger structuring (CD4 or MAb 17b) and to molecules that bind to structured regions and do not induce conformational structuring (MAb b12) is discussed.

Probing intrinsic dynamics and conformational transition of HIV gp120 by molecular dynamics simulation.

The HIV envelope glycoprotein gp120 has evolved two distinct conformational states to balance viral infection and immune escape. One is a closed state resistant to most neutralization antibodies, and the other is an open state responsible for the binding of the receptor and coreceptors. Although the structures of gp120 in these two conformational states have been determined, a detailed molecular mechanism involving intrinsic dynamics and conformational transition is still elusive. In this study, μs-scale molecular dynamics simulation is performed to probe molecular dynamics and conformational transition away from the open state and approach the closed state. Our results reveal that open gp120 shows a larger structural deviation, higher conformational flexibility, and more conformational diversity than the form in the closed state, providing a structural explanation for receptor or coreceptor affinity at the open state and the neutralization resistance of closed conformation. Seven regions with greatly decreased coupled motions in the open states have been observed by dynamic cross-correlation analysis, indicating that conformational transition can be mainly attributed to the relaxation of intrinsic dynamics. Three conformations characterized by the structural orientations of the V1/V2 region and the V3 loop, suggesting gp120 is intrinsically dynamic from the open state to the closed state. Taken together, these findings shed light on the understanding of the conformational control mechanism of HIV.

Serine substitutions are linked to codon usage and differ for variable and conserved protein regions

Serine is the only amino acid that is encoded by two disjoint codon sets (TCN & AGY) so that a tandem substitution of two nucleotides is required to switch between the two sets. We show that these codon sets underlie distinct substitution patterns at positions subject to purifying and diversifying selections. We found that in humans, positions that are conserved among ~100 vertebrates, and thus subjected to purifying selection, are enriched for substitutions involving serine (TCN, denoted S′), proline, and alanine, (S′PA). In contrast, the less conserved positions are enriched for serine encoded with AGY codons (denoted S″), glycine and asparagine, (GS″N). We tested this phenomenon in the HIV envelope glycoprotein (gp120), and the V-gene that encodes B-cell receptors/antibodies. These fast evolving proteins both have hypervariable positions, which are under diversifying selection, closely adjacent to highly conserved structural regions. In both instances, we identified an opposite abundance of two groups of serine substitutions, with enrichment of S′PA in the conserved positions, and GS″N in the hypervariable regions. Finally, we analyzed the substitutions across 60,000 individual human exomes to show that, when serine has a specific functional constraint of phosphorylation capability, S′ codons are 32-folds less prone than S″ to substitutions to Threonine or Tyrosine that could potentially retain the phosphorylation site capacity. Combined, our results, that cover evolutionary signals at different temporal scales, demonstrate that through its encoding by two codon sets, serine allows for the existence of alternating substitution patterns within positions of functional maintenance versus sites of rapid diversification.

Tenofovir disoproxil fumarate induces peripheral neuropathy and alters inflammation and mitochondrial biogenesis in the brains of mice

Mounting evidence suggests that antiretroviral therapy (ART) drugs may contribute to the prevalence of HIV-associated neurological dysfunction. The HIV envelope glycoprotein (gp120) is neurotoxic and has been linked to alterations in mitochondrial function and increased inflammatory gene expression, which are common neuropathological findings in HIV+ cases on ART with neurological disorders. Tenofovir disproxil fumarate (TDF) has been shown to affect neurogenesis in brains of mice and mitochondria in neurons. In this study, we hypothesized that TDF contributes to neurotoxicity by modulating mitochondrial biogenesis and inflammatory pathways. TDF administered to wild-type (wt) and GFAP-gp120 transgenic (tg) mice caused peripheral neuropathy, as indicated by nerve conduction slowing and thermal hyperalgesia. Conversely TDF protected gp120-tg mice from cognitive dysfunction. In the brains of wt and gp120-tg mice, TDF decreased expression of mitochondrial transcription factor A (TFAM). However, double immunolabelling revealed that TFAM was reduced in neurons and increased in astroglia in the hippocampi of TDF-treated wt and gp120-tg mice. TDF also increased expression of GFAP and decreased expression of IBA1 in the wt and gp120-tg mice. TDF increased tumor necrosis factor (TNF) α in wt mice. However, TDF reduced interleukin (IL) 1β and TNFα mRNA in gp120-tg mouse brains. Primary human astroglia were exposed to increasing doses of TDF for 24 hours and then analyzed for mitochondrial alterations and inflammatory gene expression. In astroglia, TDF caused a dose-dependent increase in oxygen consumption rate, extracellular acidification rate and spare respiratory capacity, changes consistent with increased metabolism. TDF also reduced IL-1β-mediated increases in IL-1β and TNFα mRNA. These data demonstrate that TDF causes peripheral neuropathy in mice and alterations in inflammatory signaling and mitochondrial activity in the brain.

High affinity CXCR4 inhibitors generated by linking low affinity peptides

G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model of GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and binding free energy calculation to determine how the peptide interacts with CXCR4 and inhibits its downstream signaling. These results demonstrate that this combinational approach is effective for generating nanomolar active inhibitors of CXCR4 and may be applicable to other GPCRs.

Synthesis, Antiviral Activity, and Structure–Activity Relationship of 1,3‐Benzodioxolyl Pyrrole‐Based Entry Inhibitors Targeting the Phe43 Cavity in HIV‐1 gp120

The pathway by which HIV-1 enters host cells is a prime target for novel drug discovery because of its critical role in the life cycle of HIV-1. The HIV-1 envelope glycoprotein gp120 plays an important role in initiating virus entry by targeting the primary cell receptor CD4. We explored the substitution of bulky molecular groups in region I in the NBD class of entry inhibitors. Previous attempts at bulky substituents in that region abolished antiviral activity, even though the binding site is hydrophobic. We synthesized a series of entry inhibitors containing the 1,3-benzodioxolyl moiety or its bioisostere, 2,1,3-benzothiadiazole. The introduction of the bulkier groups was well tolerated, and despite only minor improvements in antiviral activity, the selectivity index of these compounds improved significantly.