HIV-1 Entry Inhibitors Research Articles

Importance of structure-based studies for the design of a novel HIV-1 inhibitor peptide

Based on the structure of an HIV-1 entry inhibitor peptide two stapled- and a retro-enantio peptides have been designed to provide novel prevention interventions against HIV transmission. The three peptides show greater inhibitory potencies in cellular and mucosal tissue pre-clinical models than the parent sequence and the retro-enantio shows a strengthened proteolytic stability. Since HIV-1 fusion inhibitor peptides need to be embedded in the membrane to properly interact with their viral target, the structural features were determined by NMR spectroscopy in micelles and solved by using restrained molecular dynamics calculations. Both parent and retro-enantio peptides demonstrate a topology compatible with a shared helix–turn–helix conformation and assemble similarly in the membrane maintaining the active conformation needed for its interaction with the viral target site. This study represents a straightforward approach to design new targeted peptides as HIV-1 fusion inhibitors and lead us to define a retro-enantio peptide as a good candidate for pre-exposure prophylaxis against HIV-1.

Opening the HIV envelope: potential of CD4 mimics as multifunctional HIV entry inhibitors.

Close to 2 million individuals globally become infected with HIV-1 each year and just over two-thirds will have access to life-prolonging antivirals. However, the rapid development of drug resistance creates challenges, such that generation of more effective therapies is not only warranted but a necessary endeavour. This review discusses a group of HIV-1 entry inhibitors known as CD4 mimics which exploit the highly conserved relationship between the HIV-1 envelope glycoprotein and the receptor, CD4. We review the structure/function guided evolution of these inhibitors, vital mechanistic insights that underpin broad and potent functional antagonism, recent evidence of utility demonstrated in animal and physiologically relevant in-vitro models, and current progress towards effective new-generation inhibitors. The current review highlights the promising potential of CD4 mimetics as multifunctional therapeutics.

Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent d-peptide HIV entry inhibitor

Cholesterol-PIE12-trimer (CPT31) is a potent d-peptide HIV entry inhibitor that targets the highly conserved gp41 N-peptide pocket region. CPT31 exhibited strong inhibitory breadth against diverse panels of primary virus isolates. In a simian-HIV chimeric virus AD8 (SHIVAD8) macaque model, CPT31 prevented infection from a single high-dose rectal challenge. In chronically infected animals, CPT31 monotherapy rapidly reduced viral load by ∼2 logs before rebound occurred due to the emergence of drug resistance. In chronically infected animals with viremia initially controlled by combination antiretroviral therapy (cART), CPT31 monotherapy prevented viral rebound after discontinuation of cART. These data establish CPT31 as a promising candidate for HIV prevention and treatment.

Trehalose Inhibits Human Immunodeficiency Virus Type 1 Infection in Primary Human Macrophages and CD4+ T Lymphocytes through Two Distinct Mechanisms.

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV-inhibitory effects of the MTOR-independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T cells through the downregulation of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages. Thus, we showed that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

In Vitro Evaluation of a Peptide-Mesoporous Silica Nanoparticle Drug Release System against HIV-1

It has been shown that the optimized VIR-576 derivative of the natural HIV-1 entry inhibitor targeting the viral gp41 fusion peptide is safe and effective in infected individuals. However, high doses of this peptide were required, and stability, as well as delivery, must be improved for clinical application. Here, we examined the loading and release of VIR-576 into/from mesoporous silica nanoparticles (MSNs) in vitro. We found that a moderately high peptide loading of 11.5 wt % could be achieved by adsorption from PBS buffer (pH 7.2), i.e., under mild, fully peptide-compatible conditions. The release rate of peptide into the same buffer was slow and the equilibrium concentration as indicated by the adsorption isotherm could not be reached even within 50 h at the particle concentrations studied. However, a faster release was observed at lower particle concentrations, indicating that partial particle dissolution had a positive influence on peptide release. To determine the antiviral activity of VIR-576-loaded MSNs, TZM-bl indicator cells were exposed to HIV-1 and the infection rates were followed as a function of time and VIR-576 concentration. The inhibitory activity observed for VIR-576 released from the MSNs was virtually identical to that of free VIR-576 at the 48 h time point, indicating that (a) VIR-576 was released in an active form from the MSNs, and (b) the release rate in the presence of serum proteins was clearly higher than that observed under protein-free conditions. These observations are discussed based on competitive peptide/protein adsorption, as well as potential influences of serum proteins on the dissolution-reprecipitation of silica under conditions where the total silica concentration is above the saturation level for dissolved silica. Our results highlight the need for studying drug release kinetics in the presence of serum proteins, in order to allow for a better extrapolation of in vitro data to in vivo conditions. Furthermore, due to the high peptide loadings that can be achieved using MSNs as carriers, such a formulation appears promising for local release applications. For systemic administration, however, peptides with a higher potency would be needed, due to their high molar masses limiting the drug loading in terms of moles per gram carrier.

Inactivation of HIV-1 Infection through Integrative Blocking with Amino Phenylboronic Acid Attributed Carbon Dots.

Current antiretroviral HIV therapies continue to have problems related to procedural complications, toxicity, and uncontrolled side effects. In this study, amino phenylboronic acid-modified carbon dots (APBA-CDs) were introduced as a new nanoparticle-based on gp120 targeting that inhibits HIV-1 entry processes. Prolonged by simple pyrolysis for preparing carbon dots, this report further explores attributing amino phenylboronic acid on carbon dots, which prove the formation of graphene-like structures on carbon dots and boronic acid sites, thereby enabling the enhancement of positive optical properties through photoluminescent detection. Aside from performing well in terms of biocompatibility and low cytotoxicity (the CC50 reach up to 11.2 mg/mL), APBA-CDs exhibited superior capabilities in terms of prohibiting HIV-1 entry onto targeted MOLT-4 cells recognized by the delimitations of syncitia formation and higher ATP signal rather than bare carbon dots. The modified carbon dots also promote dual-action on HIV-1 treatment by both intracellularly and extracellularly viral blocking by combining with the Duviral drug, along with compressing p24 antigen signals that are better than APBA-CDs and Duviral itself.

The Conformational States of the HIV-1 Envelope Glycoproteins.

During HIV-1 entry into target cells, binding of the virus to host receptors, CD4 and CCR5/CXCR4, triggers serial conformational changes in the envelope glycoprotein (Env) trimer that result in the fusion of the viral and cell membranes. Recent discoveries have refined our knowledge of Env conformational states, allowing characterization of the targets of small-molecule HIV-1 entry inhibitors and neutralizing antibodies, and identifying a novel off-pathway conformation (State 2A). Here, we provide an overview of the current understanding of these conformational states, focusing on (i) the events during HIV-1 entry; (ii) conformational preferences of HIV-1 Env ligands; (iii) evasion of the host antibody response; and (iv) potential implications for therapy and prevention of HIV-1 infection.

Development of a generative adversarial neural network for identification of potential HIV-1 inhibitors by deep learning methods

A generative adversarial autoencoder for the rational design of potential HIV-1 entry inhibitors able to block the region of the viral envelope protein gp120 critical for the virus binding to cellular receptor CD4 was developed using deep learning methods. The research were carried out to create the architecture of the neural network, to form virtual compound library of potential anti-HIV-1 agents for training the neural network, to make molecular docking of all compounds from this library with gp120, to calculate the values of binding free energy, to generate molecular fingerprints for chemical compounds from the training dataset. The training the neural network was implemented followed by estimation of the learning outcomes and work of the autoencoder. The validation of the neural network on a wide range of compounds from the ZINC database was carried out. The use of the neural network in combination with virtual screening of chemical databases was shown to form a productive platform for identifying the basic structures promising for the design of novel antiviral drugs that inhibit the early stages of HIV infection.

Composition and Orientation of the Core Region of Novel HIV-1 Entry Inhibitors Influences Metabolic Stability.

Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage therapies only. As such, the development of a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy (cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore, in this short communication, we assess the metabolic stabilities of five of our novel chemotype entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor with more than twice the metabolic stability of temsavir and demonstrated that the orientation of the core replacement is critical for this increase. This work further demonstrates the feasibility of our long-term goal—to design an entry inhibitor with improved drug-like qualities—and warrants expanded studies to achieve this.

Daphnane Diterpenoids from Trigonostemon lii and Inhibition Activities Against HIV-1

Natural products are the important source for the discovery of more potent anti-HIV agents. In this study, six daphnane diterpenoids including three unreported structures were isolated from Trigonostemon lii, which showed significant activities against HIV-1 strains replication in the nanomolar/picomolar range. Meanwhile, these diterpenoids significantly inhibited the fusion of H9/HIV-1 IIIB cells with uninfected C8166 cells, with the EC50s from 1.06 to 8.73 ng/mL, and did not show any inhibition activities against HIV-1 reverse transcriptase. Moreover, all of the diterpenoids shows significant inhibitions against T20-resistan HIV-1 strains, PNL4-3gp41(36G)V38E, N42S and pNL4-3gp41(36G)V38A, N42T. The results revealed that the six diterpenoids could be a new type of potential lead candidate as an HIV entry inhibitor, particularly for those infected by T20-resistant variants.Graphic

Modification and optimization of the inhibition of HIV-1 cell-to-cell transmission assay

HIV-1 infection is caused by cell-free and cell-associated viruses. Currently most of the assays used to screen potential HIV-1 entry inhibitors focus on the inhibition of cell-free viruses. One assay that is widely employed is the TZM-bl neutralization assay that uses pseudotyped viruses. However, a study by Abela et al. showed that many inhibitors that potently inhibit cell-free HIV-1 in this assay can be less effective against the cell-to-cell transmission of the virus. These researchers then designed a method to screen entry inhibitors for activity against cell-associated HIV-1, using pseudotyped viruses. The main limitation of this method, however, was that it can only be reliably employed against viruses that cannot infect target cells as cell-free virion in the absence of a polycation supplement such as DEAE (diethylaminoethyl). Thus, in the current study we provide modifications to this method that solves the problem and makes it possible to study entry inhibitors against cell-to-cell infection of both polycation depend and independent viruses. The main modification involves the introduction of the relative light unit (RLU) vs. virus producing 293-T cells / corresponding supernatants graph. This graph is used to select a virus input that only allows for the detection of cell-associated viruses infection.•The method is a modification of the cell-to-cell transmission assay published by Abela et al.•The method allows for the study of the inhibition of cell-to-cell transmission of both polycation dependent and independent HIV-1 pseudoviruses.

Characterization of resistance to a potent d-peptide HIV entry inhibitor

BackgroundPIE12-trimer is a highly potent d-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket.ResultsUsing deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data.ConclusionsThese data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.

Strain-Dependent Activation and Inhibition of Human Immunodeficiency Virus Entry by a Specific PF-68742 Stereoisomer.

Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.

The Discovery and Development of Oxalamide and Pyrrole Small Molecule Inhibitors of gp120 and HIV Entry - A Review.

Human immunodeficiency virus type-1 (HIV-1) is the causative agent responsible for the acquired immunodeficiency syndrome (AIDS) pandemic. More than 60 million infections and 25 million deaths have occurred since AIDS was first identified in the early 1980s. Advances in available therapeutics, in particular combination antiretroviral therapy, have significantly improved the treatment of HIV infection and have facilitated the shift from high mortality and morbidity to that of a manageable chronic disease. Unfortunately, none of the currently available drugs are curative of HIV. To deal with the rapid emergence of drug resistance, off-target effects, and the overall difficulty of eradicating the virus, an urgent need exists to develop new drugs, especially against targets critically important for the HIV-1 life cycle. Viral entry, which involves the interaction of the surface envelope glycoprotein, gp120, with the cellular receptor, CD4, is the first step of HIV-1 infection. Gp120 has been validated as an attractive target for anti-HIV-1 drug design or novel HIV detection tools. Several small molecule gp120 antagonists are currently under investigation as potential entry inhibitors. Pyrrole, piperazine, triazole, pyrazolinone, oxalamide, and piperidine derivatives, among others, have been investigated as gp120 antagonist candidates. Herein, we discuss the current state of research with respect to the design, synthesis and biological evaluation of oxalamide derivatives and five-membered heterocycles, namely, the pyrrole-containing small molecule as inhibitors of gp120 and HIV entry.

Recent Progress in the Development of HIV-1 Entry Inhibitors: From Small Molecules to Potent Anti-HIV Agents.

Viral entry, the first process in the reproduction of viruses, primarily involves attachment of the viral envelope proteins to membranes of the host cell. The crucial components that play an important role in viral entry include viral surface glycoprotein gp120, viral transmembrane glycoprotein gp41, host cell glycoprotein (CD4), and host cell chemokine receptors (CCR5 and CXCR4). Inhibition of the multiple molecular interactions of these components can restrain viruses, such as HIV-1, from fusion with the host cell, blocking them from reproducing. This review article specifically focuses on the recent progress in the development of small-molecule HIV-1 entry inhibitors and incorporates important aspects of their structural modification that lead to the discovery of new molecular scaffolds with more potency.

Development of an Effective Scalable Enantioselective Synthesis of the HIV-1 Entry Inhibitor BNM-III-170 as the Bis-Trifluoroacetate Salt.

We report here the development and optimization of a process synthesis for the HIV-1 entry inhibitor BNM-III-170 bis-TFA salt (1). The synthesis features a dynamic-kinetic resolution (DKR) to establish the initial stereogenicity. By taking advantage of significant sequence modifications of our first generation synthesis, inconjunction with the low solubility of late-stage intermediates, the overall efficiency of the synthesis has been significantly improved, now to proceed in an overall yield of 9.64% for the 16-steps, requiring only a single chromatographic separation.

A Novel CXCR4 Targeting Protein SDF-1/54 as an HIV-1 Entry Inhibitor.

CXC chemokine receptor 4 (CXCR4) is a co-receptor for HIV-1 entry into target cells. Its natural ligand, the chemokine SDF-1, inhibits viral entry mediated by this receptor. However, the broad expression pattern of CXCR4 and its critical roles in various physiological and pathological processes indicate that the direct application of SDF-1 as an entry inhibitor might have severe consequences. Previously, we constructed an effective SDF-1 mutant, SDF-1/54, by deleting the α-helix of the C-terminal functional region of SDF-1. Of note, SDF-1/54 shows remarkable decreased chemotoxic ability, but maintains a similar binding affinity to CXCR4, suggesting SDF-1/54 might better serve as a CXCR4 inhibitor. Here, we found that SDF-1/54 exhibited potent antiviral activity against various X4 HIV-1 strains, including the infectious clone HIV-1 NL4-3, laboratory-adapted strain HIV-1 IIIB, clinical isolates and even drug-resistant strains. By using time-of-addition assay, non-infectious and infectious cell–cell fusion assay and CXCR4 internalization assay, we demonstrated SDF-1/54 is an HIV-1 entry inhibitor. A combination of SDF-1/54 with several antiretroviral drugs exhibited potent synergistic anti-HIV-1 activity. Moreover, SDF-1/54 was stable and its anti-HIV-1 activity was not significantly affected by the presence of seminal fluid, vaginal fluid simulant and human serum albumin. SDF-1/54 showed limited in vitro cytotoxicity to lymphocytes and vaginal epithelial cells. Based on these findings, SDF-1/54 could have a therapeutic potential as an HIV-1 entry inhibitor.

The anti-parasitic drug suramin potently inhibits formation of seminal amyloid fibrils and their interaction with HIV-1

Seminal amyloid fibrils are made up of naturally occurring peptide fragments and are key targets for the development of combination microbicides or antiviral drugs. Previously, we reported that the polysulfonic compound ADS-J1 is a potential candidate microbicide that not only inhibits HIV-1 entry, but also seminal fibrils. However, the carcinogenic azo moieties in ADS-J1 preclude its clinical application. Here, we screened several ADS-J1-like analogs and found that the antiparasitic drug suramin most potently inhibited seminal amyloid fibrils. Using various biochemical methods, including Congo red staining, CD analysis, transmission EM, viral infection assays, surface plasmon resonance imaging, and molecular dynamics simulations, we investigated suramin's inhibitory effects and its putative mechanism of action. We found that by forming a multivalent interaction, suramin binds to proteolytic peptides and mature fibrils, thereby inhibiting seminal fibril formation and blocking fibril-mediated enhancement of viral infection. Of note, suramin exhibited potent anti-HIV activities, and combining suramin with several antiretroviral drugs produced synergistic effects against HIV-1 in semen. Suramin also displayed a good safety profile for vaginal application. Moreover, suramin inhibited the semen-derived enhancer of viral infection (SEVI)/semen-mediated enhancement of HIV-1 transcytosis through genital epithelial cells and the subsequent infection of target cells. Collectively, suramin has great potential for further development as a combination microbicide to reduce the spread of the AIDS pandemic by targeting both viral and host factors involved in HIV-1 sexual transmission.

In Silico Identification of Novel Aromatic Compounds as Potential HIV-1 Entry Inhibitors Mimicking Cellular Receptor CD4.

Despite recent progress in the development of novel potent HIV-1 entry/fusion inhibitors, there are currently no licensed antiviral drugs based on inhibiting the critical interactions of the HIV-1 envelope gp120 protein with cellular receptor CD4. In this connection, studies on the design of new small-molecule compounds able to block the gp120-CD4 binding are still of great value. In this work, in silico design of drug-like compounds containing the moieties that make the ligand active towards gp120 was performed within the concept of click chemistry. Complexes of the designed molecules bound to gp120 were then generated by molecular docking and optimized using semiempirical quantum chemical method PM7. Finally, the binding affinity analysis of these ligand/gp120 complexes was performed by molecular dynamic simulations and binding free energy calculations. As a result, five top-ranking compounds that mimic the key interactions of CD4 with gp120 and show the high binding affinity were identified as the most promising CD4-mimemic candidates. Taken together, the data obtained suggest that these compounds may serve as promising scaffolds for the development of novel, highly potent and broad anti-HIV-1 therapeutics.

N-Substituted Pyrrole Derivative 12m Inhibits HIV-1 Entry by Targeting Gp41 of HIV-1 Envelope Glycoprotein.

The combination of three or more antiviral agents that act on different targets is known as highly active antiretroviral therapy (HAART), which is widely used to control HIV infection. However, because drug resistance and adverse effects occur after long-term administration, an increasing number of HIV/AIDS patients do not tolerate HAART. It is necessary to continue developing novel anti-HIV drugs, particularly HIV entry/fusion inhibitors. Our group previously identified a small-molecule compound, NB-64, with weak anti-HIV activity. Here, we found that N-substituted pyrrole derivative 12m (NSPD-12m), which was derived from NB-64, had strong anti-HIV-1 activity, and NSPD-12m-treated cells showed good viability. The mechanism of action of NSPD-12m might be targeting the gp41 transmembrane subunit of the HIV envelope glycoprotein, thus inhibiting HIV entry. Site-directed mutagenesis confirmed that a positively charged lysine residue (K574) located in the gp41 pocket region is pivotal for the binding of NSPD-12m to gp41. These findings suggest that NSPD-12m can serve as a lead compound to develop novel virus entry inhibitors.