HIV-1 Entry Inhibitors Research Articles

Adaptation of the REINVENT neural network architecture to generate potential HIV-1 entry inhibitors

Objectives. The main purpose of this work is to adapt the architecture of the REINVENT neural network to generate potential inhibitors of the HIV-1 envelope protein gp120 using in the learning process with reinforcement of molecular docking on GPUs.Methods. To modify the initial network model, molecular docking on GPUs implemented in the learning process with reinforcement was used, and an algorithm was developed that allows converting the representations of connections generated by the SMILES network into the PDBQT format necessary for docking. To accelerate the learning of the neural network in the modified version of the REINVENT model, the AutoDock-Vina-GPU-2.1 docking program was used, and to clarify the results of its work, the procedure for revaluing the affinity of compounds to the target using the RFScore-4 evaluation function was used.Results. Using a modified version of the REINVENT model, more than 60,000 compounds were obtained, of which about 52,000 molecules have a binding energy value to the HIV-1 gp120 protein comparable to the value calculated for the HIV-1 inhibitor NBD-14204, used in calculations as a positive control. Of the 52,000 compounds selected, about 34,000 molecules satisfy the restrictions imposed on a potential drug to ensure its bioavailability when taken orally.Conclusion. The results obtained allow us to demonstrate the effectiveness of an adapted neural network by the example of designing new potential inhibitors of the gp120 HIV-1 protein capable of blocking the CD4- binding site of the gp120 virus envelope protein and preventing its penetration into host cells.

A New Chimeric Antibody against the HIV-1 Fusion Inhibitory Peptide MT-C34 with a High Affinity and Fc-Mediated Cellular Cytotoxicity.

Peptides from heptad repeat (HR1 and HR2) regions of gp41 are effective inhibitors of HIV-1 entry that block the fusion of viral and cellular membranes, but the generation of antibodies highly specific for these peptides is challenging. We have previously described a mouse hybridoma that recognizes MT-C34-related peptides derived from HR2. It was used for the selection of HIV-1-resistant CD4 lymphocytes engineered to express the MT-C34 peptide via a CRISPR/Cas9-mediated knock-in into the CXCR4 locus. In this study, we cloned variable domains of this antibody and generated a recombinant chimeric antibody (chAb) by combining it with the constant regions of the humanized antibody Trastuzumab. The new chAb displayed a high specificity and two-fold higher level of affinity than the parental mouse monoclonal antibody. In addition, chAb mediated up to 27-43% of the antibody-dependent cellular cytotoxicity towards cells expressing MT-C34 on their surface. The anti-MT-C34 chAb can be easily generated using plasmids available for the research community and can serve as a valuable tool for the detection, purification, and even subsequent elimination of HIV-1-resistant CD4 cells or CAR cells engineered to fight HIV-1 infection.

Characterization of a CXCR4 antagonist TIQ-15 with dual tropic HIV entry inhibition properties.

The chemokine co-receptors CXCR4 and CCR5 mediate HIV entry and signal transduction necessary for viral infection. However, to date only the CCR5 antagonist maraviroc is approved for treating HIV-1 infection. Given that approximately 50% of late-stage HIV patients also develop CXCR4-tropic virus, clinical anti-HIV CXCR4 antagonists are needed. Here, we describe a novel allosteric CXCR4 antagonist TIQ-15 which inhibits CXCR4-tropic HIV-1 infection of primary and transformed CD4 T cells. TIQ-15 blocks HIV entry with an IC50 of 13 nM. TIQ-15 also inhibits SDF-1α/CXCR4-mediated cAMP production, cofilin activation, and chemotactic signaling. In addition, TIQ-15 induces CXCR4 receptor internalization without affecting the levels of the CD4 receptor, suggesting that TIQ-15 may act through a novel allosteric site on CXCR4 for blocking HIV entry. Furthermore, TIQ-15 did not inhibit VSV-G pseudotyped HIV-1 infection, demonstrating its specificity in blocking CXCR4-tropic virus entry, but not CXCR4-independent endocytosis or post-entry steps. When tested against a panel of clinical isolates, TIQ-15 showed potent inhibition against CXCR4-tropic and dual-tropic viruses, and moderate inhibition against CCR5-tropic isolates. This observation was followed by a co-dosing study with maraviroc, and TIQ-15 demonstrated synergistic activity. In summary, here we describe a novel HIV-1 entry inhibitor, TIQ-15, which potently inhibits CXCR4-tropic viruses while possessing low-level synergistic activities against CCR5-tropic viruses. TIQ-15 could potentially be co-dosed with the CCR5 inhibitor maraviroc to block viruses of mixed tropisms.

Effective in vivo reactivation of HIV-1 latency reservoir via oral administration of EK-16A-SNEDDS

The latent reservoir of human immunodeficiency virus (HIV) is a major obstacle in the treatment of acquired immune deficiency syndrome (AIDS). The “shock and kill” strategy has emerged as a promising approach for clearing HIV latent reservoirs. However, current latency-reversing agents (LRAs) have limitations in effectively and safely activating the latent virus and reducing the HIV latent reservoirs in clinical practice. Previously, EK-16A was extracted from Euphorbia kansui, which had the effect of interfering with the HIV-1 latent reservoir and inhibiting HIV-1 entry. Nevertheless, there is no suitable and efficient EK-16A oral formulation for in vivo delivery and clinical use. In this study, an oral EK-16A self-nanoemulsifying drug delivery system (EK-16A-SNEDDS) was proposed to “shock” the HIV-1 latent reservoir. This system aims to enhance the bioavailability and delivery of EK-16A to various organs. The composition of EK-16A-SNEDDS was optimized through self-emulsifying grading and ternary phase diagram tests. Cell models, pharmacokinetic experiments, and pharmacodynamics in HIV-1 latent cell transplant animal models suggested that EK-16A-SNEDDS could be absorbed by the gastrointestinal tract and enter the blood circulation after oral administration, thereby reaching various organs to activate latent HIV-1. The prepared EK-16A-SNEDDS demonstrated safety and efficacy, exhibited high clinical experimental potential, and may be a promising oral preparation for eliminating HIV-1 latent reservoirs.

Porphyrins with combinations of 4-carboxyphenyl and 4-hydroxyphenyl substituents in meso-positions as anti-HIV-1 agents

A series of 4-carboxyphenyl/4-hydroxyphenyl meso-substituted porphyrins were synthesized, purified, and characterized. The compounds exhibited anti-HIV-1 activities, in vitro, under both non-photodynamic (non-PDT) and photodynamic (PDT) conditions. Specifically, the porphyrins inhibited HIV-1 virus entry, with c-PB2(OH)2 and PB(OH)3 showing significant anti-HIV-1 activity. All of the porphyrins inhibited HIV-1 subtype B and C virus entry under PDT conditions. Our study demonstrated that the compounds bearing combinations of 4-carboxyphenyl/4-hydroxyphenyl moieties were not toxic even at higher concentrations, as compared to the reference porphyrins 5,10,15,20-tetra-(4-carboxyphenyl)porphyrin (TCPP) and 5,10,15,20-tetra-(4-hydroxyphenyl)porphyrin (THPP), under PDT conditions. This study underscores the promising potential of these compounds as HIV entry inhibitors in both non-PDT and PDT scenarios.

Using a Drug Repurposing Strategy to Virtually Screen Potential HIV-1 Entry Inhibitors That Block the NHR Domain of the Viral Envelope Protein gp41

Using a drug repurposing strategy, virtual screening of potential inhibitors of the NHR domain of the HIV-1 gp41 protein, a conserved region critical for the virus-cell membrane fusion and viral infectivity, was carried out. The used computational approach included: (1) molecular docking of this functionally significant region of the HIV-1 envelope with compounds from a library of bioactive molecules containing clinically approved drugs, experimental drugs, and investigational drug candidates; (2) assessing the binding affinity of these compounds to the therapeutic target; (3) molecular dynamics simulations of ligand/NHR-gp41 complexes; (4) calculations of the binding free energy followed by the analysis of molecular dynamics trajectories and selection of compounds promising to test for anti-HIV-1 activity. As a result, six compounds that exhibited the high binding affinity to the NHR domain of the HIV-1 gp41 protein and showed acceptable pharmacological properties were identified. The predicted compounds are assumed to form a promising basis for the development of new, effective and safe broad-spectrum antiviral agents able to inhibit the HIV-1 entry into the host cell.

ADS-J21 is a novel HIV-1 entry inhibitor targeting gp41

HIV-1 envelope glycoprotein gp41 mediates fusion between HIV-1 and host cell membranes, making inhibitors of gp41 attractive anti-HIV drugs. We previously reported an efficient HIV-1 fusion inhibitor, ADS-J1, with a Y-shaped structure. Here, we discovered a new compound, ADS-J21, with a Y-shaped structure similar to that of ADS-J1 but with a lower molecular weight. Moreover, ADS-J21 exhibited effective anti-HIV-1 activity against divergent HIV-1 strains in vitro, including several HIV-1 laboratory-adapted strains and primary isolates with different subtypes (clades A to F) and tropisms (X4 or R5). Mechanistic studies have demonstrated that ADS-J21 blocks the formation of the gp41 six-helix bundle (6-HB) by targeting conserved amino acids Lys35 and Trp32. These findings suggest that ADS-J21 can be used as a new lead compound for further optimization in the development of a small-molecule fusion inhibitor.

Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir

The HIV-1 entry inhibitor temsavir prevents the viral receptor CD4 (cluster of differentiation 4) from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this, temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveals that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad antiviral activity.

The Antiviral Compound PSP Inhibits HIV-1 Entry via PKR-Dependent Activation in Monocytic Cells.

Actin depolymerization factor (ADF) cofilin-1 is a key cytoskeleton component that serves to lessen cortical actin. HIV-1 manipulates cofilin-1 regulation as a pre- and post-entry requisite. Disruption of ADF signaling is associated with denial of entry. The unfolded protein response (UPR) marker Inositol-Requiring Enzyme-1α (IRE1α) and interferon-induced protein (IFN-IP) double-stranded RNA- activated protein kinase (PKR) are reported to overlap with actin components. In our published findings, Coriolus versicolor bioactive extract polysaccharide peptide (PSP) has demonstrated anti-HIV replicative properties in THP1 monocytic cells. However, its involvement towards viral infectivity has not been elucidated before. In the present study, we examined the roles of PKR and IRE1α in cofilin-1 phosphorylation and its HIV-1 restrictive roles in THP1. HIV-1 p24 antigen was measured through infected supernatant to determine PSP's restrictive potential. Quantitative proteomics was performed to analyze cytoskeletal and UPR regulators. PKR, IRE1α, and cofilin-1 biomarkers were measured through immunoblots. Validation of key proteome markers was done through RT-qPCR. PKR/IRE1α inhibitors were used to validate viral entry and cofilin-1 phosphorylation through Western blots. Our findings show that PSP treatment before infection leads to an overall lower infectivity. Additionally, PKR and IRE1α show to be key regulators in cofilin-1 phosphorylation and viral restriction.

Multitargeted drug design strategy for discovery of short-peptide-based HIV-1 entry inhibitors with high potency

The development of short-peptide-based inhibitors to prevent HIV-1 entry into the host cell has been rewarded with limited success. Herein, we report a multitarget-directed ligand strategy to generate a series of short-peptide HIV-1 entry inhibitors that integrated the pharmacological activities of a peptide fusion inhibitor able to disrupt HIV-1 gp41 glycoprotein hexameric coiled-coil assembly and a small-molecule CCR5 antagonist that blocks the interaction between HIV-1 and its coreceptor. Among these inhibitors, dual-target 23-residue peptides SP12T and SP12L displayed dramatically increased inhibitory activities against HIV-1 replication as compared to the marketed 36-residue peptide T20. Moreover, results suggested that SP12T and SP12L successfully performed a dual-targeting mechanism. It can be concluded that these short-peptide-based HIV-1 entry inhibitors have potential for further development as candidates for a novel multitarget therapy to treat HIV-1 infection.

Insertion of an Amphipathic Linker in a Tetrapodal Tryptophan Derivative Leads to a Novel and Highly Potent Entry Inhibitor of Enterovirus A71 Clinical Isolates.

AL-471, the leading exponent of a class of potent HIV and enterovirus A71 (EV-A71) entry inhibitors discovered in our research group, contains four l-tryptophan (Trp) units bearing an aromatic isophthalic acid directly attached to the C2 position of each indole ring. Starting from AL-471, we (i) replaced l-Trp with d-Trp, (ii) inserted a flexible linker between C2 and the isophthalic acid, and (iii) substituted a nonaromatic carboxylic acid for the terminal isophthalic acid. Truncated analogues lacking the Trp motif were also synthesized. Our findings indicate that the antiviral activity seems to be largely independent of the stereochemistry (l- or d-) of the Trp fragment and also that both the Trp unit and the distal isophthalic moiety are essential for antiviral activity. The most potent derivative, 23 (AL-534), with the C2 shortest alkyl urea linkage (three methylenes), showed subnanomolar potency against different EV-71 clinical isolates. This finding was only observed before with the early dendrimer prototype AL-385 (12 l-Trp units) but remained unprecedented for the reduced-size prototype AL-471. Molecular modeling showed the feasibility of high-affinity binding of the novel l-Trp-decorated branches of 23 (AL-534) to an alternative site on the VP1 protein that harbors significant sequence variation among EV-71 strains.

In Vitro Anti-HIV-1 Activity of Chitosan Oligomers N-Conjugated with Asparagine and Glutamine.

Chitosan oligomers (COS) are polysaccharides obtained by the hydrolyzation of chitosan. They are water-soluble, biodegradable, and have a wide range of beneficial properties for human health. Studies have shown that COS and its derivatives possess antitumor, antibacterial, antifungal, and antiviral activities. The goal of the current study was to investigate the anti-human immunodeficiency virus-1 (HIV-1) potential of amino acid-conjugated COS compared to COS itself. The HIV-1 inhibitory effects of asparagine-conjugated (COS-N) and glutamine-conjugated (COS-Q) COS were evaluated by their ability to protect C8166 CD4+ human T cell lines from HIV-1 infection and infection-mediated death. The results show that the presence of COS-N and COS-Q was able to prevent cells from HIV-1-induced lysis. Additionally, p24 viral protein production was observed to be suppressed in COS conjugate-treated cells compared to COS-treated and untreated groups. However, the protective effect of COS conjugates diminished by delayed treatment indicated an early stage inhibitory effect. COS-N and COS-Q did not show any inhibitory effect on the activities of HIV-1 reverse transcriptase and protease enzyme. The results suggest that COS-N and COS-Q possess an HIV-1 entry inhibition activity compared to COS and further studies to develop different peptide and amino acid conjugates containing N and Q amino acids might yield more effective compounds to battle HIV-1 infection.

Structure-Based Identification of Natural-Product-Derived Compounds with Potential to Inhibit HIV-1 Entry.

Broadly neutralizing antibodies (bNAbs) are potent in neutralizing a wide range of HIV strains. VRC01 is a CD4-binding-site (CD4-bs) class of bNAbs that binds to the conserved CD4-binding region of HIV-1 envelope (env) protein. Natural products that mimic VRC01 bNAbs by interacting with the conserved CD4-binding regions may serve as a new generation of HIV-1 entry inhibitors by being broadly reactive and potently neutralizing. This study aimed to identify compounds that mimic VRC01 by interacting with the CD4-bs of HIV-1 gp120 and thereby inhibiting viral entry into target cells. Libraries of purchasable natural products were virtually screened against clade A/E recombinant 93TH057 (PDB: 3NGB) and clade B (PDB ID: 3J70) HIV-1 env protein. Protein-ligand interaction profiling from molecular docking and dynamics simulations showed that the compounds had intermolecular hydrogen and hydrophobic interactions with conserved amino acid residues on the CD4-binding site of recombinant clade A/E and clade B HIV-1 gp120. Four potential lead compounds, NP-005114, NP-008297, NP-007422, and NP-007382, were used for cell-based antiviral infectivity inhibition assay using clade B (HXB2) env pseudotype virus (PV). The four compounds inhibited the entry of HIV HXB2 pseudotype viruses into target cells at 50% inhibitory concentrations (IC50) of 15.2 µM (9.7 µg/mL), 10.1 µM (7.5 µg/mL), 16.2 µM (12.7 µg/mL), and 21.6 µM (12.9 µg/mL), respectively. The interaction of these compounds with critical residues of the CD4-binding site of more than one clade of HIV gp120 and inhibition of HIV-1 entry into the target cell demonstrate the possibility of a new class of HIV entry inhibitors.

Identification of terpenoids from Withania somnifera as a HIV-1 entry inhibitors that prevent gp120 binding to CD4 using In Silico Approach

Identification of terpenoids from Withania somnifera as a HIV-1 entry inhibitors that prevent gp120 binding to CD4 using In Silico Approach

Targeting a Conserved Lysine in the Hydrophobic Pocket of HIV-1 gp41 Improves Small Molecule Antiviral Activity.

Human Immunodeficiency virus (HIV-1) fusion is mediated by glycoprotein-41, a protein that has not been widely exploited as a drug target. Small molecules directed at the gp41 ectodomain have proved to be poorly drug-like, having moderate efficacy, high hydrophobicity and/or high molecular weight. We recently investigated conversion of a fairly potent hydrophobic inhibitor into a covalent binder, by modifying it to react with a lysine residue on the protein. We demonstrated a 10-fold improvement in antiviral efficacy. Here, we continue this study, utilizing instead molecules with better inherent drug-like properties. Molecules possessing low to no antiviral activity as equilibrium binders were converted into µM inhibitors upon addition of an electrophilic warhead in the form of a sulfotetrafluorophenyl (STP) activated ester. We confirmed specificity for gp41 and for entry. The small size of the inhibitors described here offers an opportunity to expand their reach into neighboring pockets while retaining drug-likeness. STP esterification of equilibrium binders is a promising avenue to explore for inhibiting HIV-1 entry. Many gp41 targeting molecules studied over the years possess carboxylic acid groups which can be easily converted into the corresponding STP ester. It may be worth the effort to evaluate a library of such inhibitors as a way forward to small molecule inhibition of fusion of HIV and possibly other enveloped viruses.

Hybrids of small CD4 mimics and gp41-related peptides as dual-target HIV entry inhibitors

Hybrid molecules containing small CD4 mimics and gp41-C-terminal heptad repeat (CHR)-related peptides have been developed. A YIR-821 derivative was adopted as a CD4 mimic, which inhibits the interaction of gp120 with CD4. SC-peptides, SC34 and SC22EK, were also used as CHR-related peptides, which inhibit the interaction between the N-terminal heptad repeat (NHR) and CHR and thereby membrane fusion. Therefore, these hybrid molecules have dual-targets of gp120 and gp41. In the synthesis of the hybrid molecules of CD4 mimic-SC-peptides with different lengths of linkers, two conjugating methods, Cu-catalyzed azide-alkyne cycloaddition and direct cysteine alkylation, were performed. The latter reaction caused simpler operation procedures and higher synthetic yields than the former. The synthesized hybrid molecules of CD4 mimic-SC22EK have significantly higher anti-HIV activity than each sole agent. The present data should be useful in the future design of anti-HIV agents as dual-target entry inhibitors.

Computational study of the structural ensemble of CC chemokine receptor type 5 (CCR5) and its interactions with different ligands.

CC Chemokine receptor 5 (CCR5), a member of the Superfamily of G Protein-Coupled Receptors (GPCRs), is an important effector in multiple physiopathological processes such as inflammatory and infectious entities, including central nervous system neuroinflammatory diseases such as Alzheimer’s disease, recovery from nervous injuries, and in the HIV-AIDS infective processes. Thus, CCR5 is an attractive target for pharmacological modulation. Since maraviroc was described as a CCR5 ligand that modifies the HIV-AIDS progression, multiple efforts have been developed to describe the functionality of the receptor. In this work, we characterized key structural features of the CCR5 receptor employing extensive atomistic molecular dynamics (MD) in its apo form and in complex with an endogenous agonist, the chemokine CCL5/RANTES, an HIV entry inhibitor, the partial inverse agonist maraviroc, and the experimental antagonists Compound 21 and 34, aiming to elucidate the structural features and mechanistic processes that constitute its functional states, contributing with structural details and a general understanding of this relevant system.

SERINC5 restricts influenza virus infectivity.

SERINC5 is a multi-span transmembrane protein that is incorporated into HIV-1 particles in producing cells and inhibits HIV-1 entry. Multiple retroviruses like HIV-1, equine infectious anemia virus and murine leukemia virus are subject to SERINC5 inhibition, while HIV-1 pseudotyped with envelope glycoproteins of vesicular stomatitis virus and Ebola virus are resistant to SERINC5. The antiviral spectrum and the underlying mechanisms of SERINC5 restriction are not completely understood. Here we show that SERINC5 inhibits influenza A virus infection by targeting virus-cell membrane fusion at an early step of infection. Further results show that different influenza hemagglutinin (HA) subtypes exhibit diverse sensitivities to SERINC5 restriction. Analysis of the amino acid sequences of influenza HA1 strains indicates that HA glycosylation sites correlate with the sensitivity of influenza HA to SERINC5, and the inhibitory effect of SERINC5 was lost when certain HA glycosylation sites were mutated. Our study not only expands the antiviral spectrum of SERINC5, but also reveals the role of viral envelope glycosylation in resisting SERINC5 restriction.

Enzyme Responsive Delivery of Anti-Retroviral Peptide via Smart Hydrogel.

In response to an urgent need for advanced formulations for the delivery of anti-retrovirals, a stimuli-sensitive hydrogel formulation that intravaginally delivers HIV-1 entry inhibitor upon being exposed to a specific protease was developed. The hydrogel formulation consists of PEG-azide and PEG-DBCO covalently linked to the entry inhibitor peptide, enfuvirtide, via substrate linker that is designed to undergo proteolysis by prostate specific antigen (PSA) present in seminal fluid and release innate enfuvirtide. Of the tested PSA substrate linkers (HSSKLQYY, GISSFYSSK, AYLMYY, and AYLMGRR), HSSKLQ was found to be an optimal candidate for PEG-based hydrogel with kcat/KM of 2.2M-1s-1. The PEG-based hydrogel displayed a pseudoplastic, thixotropic behavior with overall viscosity varying between 1516 and 2.2Pa.s, within the biologically relevant shear rates of 0.01-100s-1. It also exhibited viscoelastic properties appropriate for uniform spreading and being retained in vagina. PEG-based hydrogels were loaded with N3-HSSKLQ-enfuvirtide (HF42) that is customarily synthesized enfuvirtide prodrug with its N-terminus connected to HSSKLQ linker. The stimuli-sensitive PEG-based hydrogel formulations upon being exposed to PSA released 36.5 ± 4.8% of enfuvirtide over 24h in human ejaculate mimic of vaginal simulant fluid and seminal simulant fluid mixed in 1:3 ratio, which is significantly greater than its IC50. The PEG-based hydrogel was non-cytotoxic to both vaginal epithelial cells (VK2/E6E7) and murine macrophages (RAW 264.7) and did not significantly induce the production of nitric oxide, an inflammatory mediator. The PEG-based hydrogel is found to have suitable physicochemical properties for an intravaginal formulation of the PSA substrate-linked anti-retrovirals and is safe towards vaginal epithelium. It is capable of delivering enfuvirtide with effective concentrations to prevent women from HIV-1 infection.

Characterization of Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Variants Selected for Resistance to a CD4-Mimetic Compound.

Binding to the host cell receptors CD4 and CCR5/CXCR4 triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a short-lived prehairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs like BNM-III-170 inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. In cell culture, we selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred an ~5-fold resistance to BNM-III-170 with minimal fitness cost. A third change (E64G) in layer 1 of the gp120 inner domain resulted in ~100-fold resistance to BNM-III-170, ~2- to 3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170-induced Env activation and shedding of gp120. Together, the S375N and I424T changes, but not the E64G change, conferred >100-fold and 33-fold resistance to BMS-806 and BMS-529 (temsavir), respectively, potent HIV-1 entry inhibitors that block Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCE CD4-mimetic compounds (CD4mcs) and conformational blockers like BMS-806 and BMS-529 (temsavir) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and conformational blockers inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor CD4 and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of a CD4mc and conformational blockers. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.