HIV-1 Entry Inhibitors Research Articles

Fusion Proteins CLD and CLDmut Demonstrate Potent and Broad Neutralizing Activity against HIV-1.

HIV-1 envelope glycoprotein (Env) interacts with cellular receptors and mediates virus entry into target cells. Blocking Env-receptor interactions represents an effective interventional strategy for developing HIV-1 entry inhibitors. We previously designed a panel of CD4-linker-DC-SIGN (CLD) constructs by fusing the extracellular CD4 and DC-SIGN domains with various linkers. Such CLDs produced by the prokaryotic system efficiently inhibited HIV-1 infection and dissemination in vitro and ex vivo. In this study, following the construction and identification of the most promising candidate with a linker of 8 Gly4Ser repeats (named CLD), we further designed an improved form (named CLDmut) by back mutating Cys to Ser at amino acid 60 of CD4. Both CLD and CLDmut were produced in mammalian (293F) cells for better protein translation and modification. The anti-HIV-1 activity of CLD and CLDmut was assessed against the infection of a range of HIV-1 isolates, including transmitted and founder (T/F) viruses. While both CLD and CLDmut efficiently neutralized the tested HIV-1 isolates, CLDmut demonstrated much higher neutralizing activity than CLD, with an IC50 up to one log lower. The neutralizing activity of CLDmut was close to or more potent than those of the highly effective HIV-1 broadly neutralizing antibodies (bNAbs) reported to date. Findings in this study indicate that mammalian cell-expressed CLDmut may have the potential to be used as prophylaxis or/and therapeutics against HIV-1 infection.

Design of a Bispecific HIV Entry Inhibitor Targeting the Cell Receptor CD4 and Viral Fusion Protein Gp41.

Given the high variability and drug-resistance problem by human immunodeficiency virus type 1 (HIV-1), the development of bispecific or multi-specific inhibitors targeting different steps of HIV entry is highly appreciated. We previously generated a very potent short-peptide–based HIV fusion inhibitor 2P23. In this study, we designed and characterized a bifunctional inhibitor termed 2P23-iMab by genetically conjugating 2P23 to the single-chain variable fragment (scFv) of ibalizumab (iMab), a newly approved antibody drug targeting the cell receptor CD4. As anticipated, 2P23-iMab could bind to the cell membrane through CD4 anchoring and inhibit HIV-1 infection as well as viral Env-mediated cell–cell fusion efficiently. When tested against a large panel of HIV-1 pseudoviruses with different subtypes and phenotypes, 2P23-iMab exhibited dramatically improved inhibitory activity than the parental inhibitors; especially, it potently inhibited the viruses not being susceptible to iMab. Moreover, 2P23-iMab had a dramatically increased potency in inhibiting two panels of HIV-1 mutants that are resistant to T-20 or 2P23 and the infections of HIV-2 and simian immunodeficiency virus (SIV). In conclusion, our studies have provided new insights into the design of novel bispecific HIV entry inhibitors with highly potent and broad-spectrum antiviral activity.

Design, synthesis, and bio-evaluation of novel triterpenoid derivatives as anti-HIV-1 compounds

Two betulinic acid derivatives, RPR103611 (2) and IC9564 (3) were previously reported to be potent HIV-1 entry inhibitors. In this current study, a SAR study of the triterpenoid moiety of 2 and 3 has been performed and an oleanolic acid derivative (4) was identified as a novel HIV-1 entry inhibitor. In addition, the combination of 4 with several-type of HIV-1 neutralizing antibodies provided significant synergistic effects. The synthetic utility of the CC double bond in the C-ring of 4 was also demonstrated to develop the 12-keto-type oleanolic acid derivative (5) as a potent anti-HIV compound. This simple transformation led to a significantly increased anti-HIV activity and a reduced cytotoxicity of the compound.

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

We have recently described a novel family of compounds of reduced size and dual anti-HIV and anti-EV71 activity that encompasses tripodal and tetrapodal derivatives. The tripodal prototype, AL-470, has a nitro group at the focal point of the central scaffold and three attached tryptophan residues, each of which bearing an isophthaloyl moiety at the C2 position of the indole ring. A nitro to amino substitution has allowed us now to introduce a chemically addressable functionality to perform further structural modifications consisting of both direct and linker-mediated attachment of several aromatic groups, including the fluorescent dye Alexa Fluor 647 and the antibody-recruiting 2,4-dinitrophenyl motif. Some of the derivatives turned out to be more potent and selective than AL-470 against HIV-1, HIV-2 and EV-A71. The fluorescent probe demonstrated a specific tropism for intestines and lungs, two important niches for the human microbiome in health and disease.

Design of artificial α-helical peptides targeting both gp41 deep pocket and subpocket as potent HIV-1 fusion inhibitors

Both the deep pocket region and its neighboring subpocket site on the N-trimer of HIV-1 gp41 protein can serve as targets for the development of HIV-1 entry inhibitors. Pocket-binding domain (PBD)-containing peptides with the potential to inhibit HIV-1 fusion through targeting the deep pocket have been extensively exploited. However, using an artificial peptide strategy, we herein report the design of α-helical lipopeptides with non-native protein sequences as HIV-1 fusion inhibitors that can occupy both gp41 deep cavity and subpocket sites. The most active compound, PP24C, inhibited HIV-1 replication, including T20-resistant HIV-1 mutants, at low nanomolar level. Biophysical approaches revealed that both the artificial α-helical peptide P35A4 and its cholesterol-tagged peptide PP24C could bind to T21 peptide used as a target surrogate comprising both pockets. Our study offers a new template for the design of artificial anti-HIV-1 therapeutics and highlights the novel concept of peptide secondary structure-based virus fusion inhibitors.

Two cationic meso-thiophenium porphyrins and their zinc-complexes as anti-HIV-1 and antibacterial agents under non-photodynamic therapy (PDT) conditions

The anti-HIV-1 and antimicrobial activities of novel cationic meso-thiophenium porphyrins and their zinc-complex are reported under in vitro non-photodynamic (PDT) conditions. While all the cationic porphyrins led to the inhibition of de novo virus infection, the Zn(II)-complexes of T2(OH)2M (A2B2-type) and T(OH)3M (AB3-type) displayed potent inhibition of HIV-1 entry with T2(OH)2MZn displaying maximal anti-HIV activity. The Zinc complex of both the thiophenium porphyrins T2(OH)2M and T(OH)3M also depicted antibacterial activities against Escherichia coli (ATCC 25922) and more prominently against Staphylococcus aureus (ATCC 25923). Again, the antibacterial activity was more potent for T2(OH)2MZn. Our study highlighted that the presence of two thiophenium groups at the meso-positions of the A2B2-type porphyrins along with zinc strongly enhanced anti-HIV and antimicrobial properties of these novel thiophenium porphyrins under non-PDT conditions.

Sendai virus particles carrying target virus glycoproteins for antibody induction

Induction of antibodies targeting viral glycoproteins is a key for the development of a vaccine against enveloped virus infection. Glycoproteins on the virion exhibiting native multimer structure may be a good immunogen to present antibody epitopes, but it is often difficult to prepare immunogenic inactivated virions. Preparation of soluble glycoprotein multimers has been attempted, while virus-like particles carrying target glycoproteins can be a more immunogenic antigen. In the present study, a target glycoprotein-embedded Sendai virus (SeV) particle was developed for induction of anti-virus antibodies. We constructed a chimeric antigen, HIV-1 EnvF, consisting of HIV-1 Env ectodomain and SeV F transmembrane-cytoplasmic domain, which was shown to be efficiently incorporated into the SeV virion. EnvF was recognized by anti-HIV-1 broadly-neutralizing monoclonal antibodies (bnAbs) including 35O22 that targets an Env trimer-dependent epitope. Analysis revealed that HIV-1AD8 EnvF can mediate viral entry into the cells, which is inhibited by anti-HIV-1 bnAbs and HIV-1 entry inhibitors, suggesting that the EnvF exhibits an HIV-1 Env native-like functional structure to present bnAb epitopes. Immunization of mice with replication-defective SeVs expressing HIV EnvF and non-infectious SeV particles (NVP) carrying HIV EnvF efficiently induced anti-HIV Env antibodies. HTLV-1 EnvF also showed the potential to efficiently induce anti-HTLV-1 Env antibodies. These results indicate that SeV particles carrying EnvF can be a promising vaccine platform for induction of antibodies targeting enveloped virus glycoproteins.

In Search of HIV Entry Inhibitors Using Molecular Docking, ADME, and Toxicity Studies of Some Thiazolidinone-Pyrazine Derivatives Against CXCR4 Co-receptor.

Entry inhibitors prevent the binding of human immunodeficiency virus protein to the chemokine receptor CXCR4 and are used along with conventional anti-HIV therapy. They aid in restoring immunity and can prevent the development of HIV-TB co-infection. In the present study, various thiazolidinone-pyrazine derivatives earlier studied for NNRT inhibition activity were gauged for their entry inhibitor potential. The objective of the study is to perform molecular docking, ADME, toxicity studies of some thiazolidinone-pyrazine derivatives as entry inhibitors targeting CXCR4 co-receptors. In-silico docking studies were performed using AutoDock Vina software and compounds were further studied for ADME and toxicity using SwissADME and pkCSM software, respectively. Taking into consideration the docking results, pharmacokinetic behaviour and toxicity profile, four molecules (compounds 1, 9, 11, and 16) have shown potential as entry inhibitors. These compounds have shown potential as both NNRTI and entry inhibitors and hence can be used in management of immune compromised diseases like TB-HIV coinfection.

Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

BackgroundThe chemokine receptor CCR5 is the major coreceptor for HIV-1 cell entry. We previously observed that not all CCR5 mAbs reduce HIV-1 infection, suggesting that only some CCR5 populations are permissive for HIV-1 entry. This study aims to better understand the relevant conformational states of the cellular coreceptor, CCR5, involved in HIV entry. We hypothesized that CCR5 assumes multiple configurations during normal cycling on the plasma membrane, but only particular forms facilitate HIV-1 infection.MethodsTo this end, we quantified different CCR5 populations using six CCR5 monoclonal antibodies (mAbs) with different epitope specificities and visualized them with super-resolution microscopy. We quantified each surface CCR5 population before and after HIV-1 infection.ResultsBased on CCR5 conformational changes, down-modulation, and trafficking rates (internalization and recycling kinetics), we were able to distinguish among heterogeneous CCR5 populations and thus which populations might best be targeted to inhibit HIV-1 entry. We assume that a decreased surface presence of a particular CCR5 subpopulation following infection means that it has been internalized due to HIV-1 entry, and that it therefore represents a highly relevant target for future antiviral therapy strategies. Strikingly, this was most true for antibody CTC8, which targets the N-terminal region of CCR5 and blocks viral entry more efficiently than it blocks chemokine binding.ConclusionsDefining the virus-host interactions responsible for HIV-1 transmission, including specific coreceptor populations capable of establishing de novo infections, is essential for the development of an HIV-1 vaccine. This study hopefully will facilitate further development of inhibitors to block CCR5 usage by HIV-1, as well as inform future HIV-1 vaccine design.

Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Endogenous Peptide Inhibitors of HIV Entry.

The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The knowledge that RANTES, MlP-1α, and MlP-1β specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.

Peptide-Based Dual HIV and Coronavirus Entry Inhibitors.

The continued HIV/AIDS epidemic worldwide and the battle against emerging infectious diseases caused by coronaviruses underscore the need for the development of an ever-expanding repertoire of antiviral drugs. Entry inhibitors are of particular interest because of their potential to be used as therapeutic or prophylactic treatments for blocking viral invasion. HIV and coronaviruses utilize class I fusion proteins to facilitate their entry and membrane fusion. Discovery of a common hexameric coiled-coil fusion complex resulting from the packing of three C-terminal heptad repeat region from the fusion-mediating subunit of viral fusion proteins against trimeric coiled-coil made up by their N-terminal heptad repeat prompted the search for peptides mimicking the heptad repeat regions that could potentially inhibit viral entry. This has led to the development of effective peptides that are specific to the virus that is developed for. In this review, we focus on peptide-based entry dual inhibitors that block fusion process not only of HIV but also coronaviruses through interrupting their fusogenic six-helical bundle core and which hopefully will help to gain insight into the α-helical secondary structure- and coiled-coil superstructure-based strategies to design entry inhibitors with broad-spectrum antiviral activity against enveloped viruses with class I fusion proteins.

Peptide-Based HIV Entry Inhibitors.

The development of peptide-based HIV entry inhibitors has made an important contribution to the stock of anti-HIV drugs. In particular, the peptide-based anti-HIV drugs enfuvirtide and albuvirtide were approved for clinical use by the U.S. FDA and CFDA in 2003 and 2018, respectively. Peptide-based HIV entry inhibitors exert antiviral activity by targeting the early stage of viral infection, i.e., binding of a viral surface protein to the receptor(s) on the host cell and the subsequent fusion between the viral and host cell membranes. Therefore, they are particularly useful for HIV-infected patients who have failed to respond to the highly active antiretroviral drugs (ARD) targeting the late stage of HIV replication, such as reverse transcriptase inhibitors and protease inhibitors. In this chapter, we will focus on the past, current, and future trends in research and development of peptide-based HIV entry inhibitors.

Hybrids of Small CD4 Mimics and Gp41-Related Peptides as Dual-Target HIV Entry Inhibitors

Hybrids of Small CD4 Mimics and Gp41-Related Peptides as Dual-Target HIV Entry Inhibitors

Small-Molecule HIV Entry Inhibitors Targeting gp120 and gp41.

Interrupting early events in the virus life cycle, such as those prior to the formation of provirus, could effectively prevent HIV-1 infection. HIV-1 entry into host cells is mediated by the envelope glycoprotein (Env) trimer, which is composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Hence, the development of novel inhibitors targeting these two glycoproteins could hold the key to early inhibition of HIV-1 infection. Small-molecule entry inhibitors targeting early events in the virus life cycle comprise a well-established class of useful drugs. Many libraries of small-molecule inhibitors have been established to screen potential drug candidates for a variety of targets based on computer docking, FRET, or peptide-linked assay. This chapter reviews the mechanisms of some small-molecule inhibitors targeting HIV-1 gp120 and gp41 and corresponding high-efficiency screening strategies for potential small-molecule inhibitors.

Design of gp120 HIV-1 entry inhibitors by scaffold hopping via isosteric replacements.

We present the development of alternative scaffolds and validation of their synthetic pathways as a tool for the exploration of new HIV gp120 inhibitors based on the recently discovered inhibitor of this class, NBD-14136. The new synthetic routes were based on isosteric replacements of the amine and acid precursors required for the synthesis of NBD-14136, guided by molecular modeling and chemical feasibility analysis. To ensure that these synthetic tools and new scaffolds had the potential for further exploration, we eventually tested few representative compounds from each newly designed scaffold against the gp120 inhibition assay and cell viability assays.

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

BackgroundWe 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. ResultsHIV-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.ConclusionsTogether, 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.

Design and Identification of Potential HIV-1 Entry Inhibitors Using In Silico Click Chemistry and Molecular Modeling Methods

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to computer-aided design of potential HIV-1 inhibitors able to block the membrane-proximal external region (MPER) of HIV-1 gp41, which plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds exhibiting high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Roles of Virion-Incorporated CD162 (PSGL-1), CD43, and CD44 in HIV-1 Infection of T Cells.

Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4+ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4+ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4+ T cells.

The Role of Inflammasome Activation in Early HIV Infection.

The inflammasome pathway is an important arm of the innate immune system that provides antiviral immunity against many viruses. The main pathways involved in virus infections include the NLRP3, IFI16, and AIM2 pathways. However, a succinct understanding of its role in HIV is not yet well elucidated. In this review, we showed that NLRP3 inflammasome activation plays a vital role in inhibiting HIV entry into target cells via the purinergic pathway; IFI16 detects intracellular HIV ssDNA, triggers interferon I and III production, and inhibits HIV transcription; and AIM2 binds to HIV dsDNA and triggers acute inflammation and pyroptosis. Remarkably, by understanding these mechanisms, new therapeutic strategies can be developed against the disease.