Activity Of HIV-1 Reverse Transcriptase Research Articles

Bioinspired Pyrano[2,3-f]chromen-8-ones: Ring C-Opened Analogues of Calanolide A: Synthesis and Anti-HIV-1 Evaluation.

We have designed and synthesized a series of bioinspired pyrano[2,3-f]coumarin-based Calanolide A analogs with anti-HIV activity. The design of these new calanolide analogs involved incorporating nitrogen heterocycles or aromatic groups in lieu of ring C, effectively mimicking and preserving their bioactive properties. Three directions for the synthesis were explored: reaction of 5-hydroxy-2,2-dimethyl-10-propyl-2H,8H-pyrano[2,3-f]chromen-8-one with (i) 1,2,4-triazines, (ii) sulfonylation followed by Suzuki cross-coupling with (het)aryl boronic acids, and (iii) aminomethylation by Mannich reaction. Antiviral assay of the synthesized compounds showed that compound 4 has moderate activity against HIV-1 on enzymes and poor activity on the cell model. A molecular docking study demonstrates a good correlation between in silico and in vitro HIV-1 reverse transcriptase (RT) activity of the compounds when docked to the nonnucleoside RT inhibitor binding site, and alternative binding modes of the considered analogs of Calanolide A were established.

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

Analysis of Biochemical and Antimicrobial Properties of Bioactive Molecules of Argemone mexicana.

This study identified phytochemicals in Argemone mexicana (A. mexicana) extracts that are responsible for its medicinal properties, and the best solvent for their extraction. The extracts of the stem, leaves, flowers, and fruits of A. mexicana were prepared at low (corresponding to room temperature) and high temperatures (corresponding to the boiling points) in various solvents, viz., hexane, ethyl acetate, methanol, and H2O. The UV-visible absorption spectra of various phytoconstituents in the isolated extracts were determined through spectrophotometry. Qualitative tests for the screening of phytoconstituents in the extracts were performed to identify various phytochemicals. We identified the presence of terpenoids, alkaloids, cardiac glycosides, and carbohydrates in the plant extracts. The antioxidant and anti-human immunodeficiency virus type 1 reverse transcriptase (anti-HIV-1RT) potential, as well as the antibacterial activity of various A. mexicana extracts were determined. These extracts showed strong antioxidant activities. The extracts exhibited antimicrobial activities against Salmonella typhi, Staphylococcus epidermis, Citrobacter, Neisseria gonorrhoeae, and Shigella flexineri. These extracts significantly inhibited HIV-1 reverse transcriptase activity. The aqueous leaf extract prepared at a temperature equivalent to the boiling point, i.e., 100 °C, was identified to be the most active against pathogenic bacteria and HIV-1 RT.

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.

Molecular Docking Study of HIV-1 Antiretroviral Candidate via Reverse Transcriptase Inhibitor from Zingiber officinale var. Roscoe

HIV-1 is a member of the Retrovirus and the virus causes AIDS in humans. AIDS affects the dynamics of the immune system leading to fatal opportunistic infections. Reverse transcriptase plays an important role as a functional enzyme in the viral replication process, the enzyme works to carry out the transcription process of ssRNA into cDNA and then initiates the viral integration process of the genome into the nucleus. Currently many use of HIV-1 NNRTIs, the nevirapine type, with a molecular mechanism that can bind to the active site of the HIV-1 reverse transcriptase enzyme to inhibit its activation. A new problem arises because the reverse transcriptase in HIV-1 undergoes a cross mutation and causes nevirapine resistance. Previous research using an in vitro approach showed the ability to inhibit the process of replication, attachment, and internalization of the virus shown by Zingiber officinale var. Roscoe, then another ability is that these herbal plants can trigger cell stimulation for interferon-β secretion. This study aims to screen the chemical compounds of Zingiber officinale var Roscoe for the discovery of new antiretrovirals through computational study. Zingiber officinale var. Roscoe is predicted to act as an antiretroviral agent through with a mechanism of HIV-1 reverse transcriptase activity inhibition at Pro95, Tyr181, Val179, Leu100, Tyr188, Val106, Leu234, Phe227, Tyr318, Asn103, Gly99 residues, β-sitosterol is predicted to act as a drug-like molecule, the antiretroviral potential of Zingiber officinale var. Roscoe must undergo further analysis to provide strong scientific evidence.

Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells.

Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity invitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.

Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations

ObjectiveMaintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations—a metabolite that indicates long-term PrEP adherence.SettingThe study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle.MethodsWe enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader—stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC–MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week).ResultsAmong 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC–MS/MS. RESTRICT fluorescence correlated with LC–MS/MS measurements (r = − 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity.ConclusionsThis novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

Retroviral RNase H: Structure, mechanism, and inhibition.

All retroviruses encode the enzyme, reverse transcriptase (RT), which is involved in the conversion of the single-stranded viral RNA genome into double-stranded DNA. RT is a multifunctional enzyme and exhibits DNA polymerase and ribonuclease H (RNH) activities, both of which are essential to the reverse-transcription process. Despite the successful development of polymerase-targeting antiviral drugs over the last three decades, no bona fide inhibitor against the RNH activity of HIV-1 RT has progressed to clinical evaluation. In this review article, we describe the retroviral RNH function and inhibition, with primary consideration of the structural aspects of inhibition.

Appraisal of a Simple and Effective RT-qPCR Assay for Evaluating the Reverse Transcriptase Activity in Blood Samples from HIV-1 Patients

Testing HIV-1 RNA in plasma by PCR is universally accepted as the ultimate standard to confirm diagnosis of HIV-1 infection and to monitor viral load in patients under treatment. However, in some cases, this assay could either underestimate or overestimate the replication capacity of a circulating or latent virus. In the present study, we performed the assessment of evaluating the HIV-1 reverse transcriptase (RT) activity by means of a new assay for the functional screening of the status of HIV-1 patients. To this purpose, we utilized, for the first time on blood samples, an adapted version of a real-time RT quantitative PCR assay, utilized to evaluate the HIV-1-RT inhibitory activity of compounds. The study analyzed blood samples from 28 HIV-1-infected patients, exhibiting a wide range of viremia and immunological values. Results demonstrated that plasma HIV-1 RT levels, expressed as cycle threshold values obtained with the assay under appraisal, were inversely and highly significantly correlated with the plasma HIV-1-RNA levels of the patients. Thus, an HIV-1 RT quantitative PCR assay was created which we describe in this study, and it may be considered as a promising basis for an additional tool capable of furnishing information on the functional virological status of HIV-1-infected patients.

Antibacterial and Anti-HIV-1 Reverse Transcriptase Activities of Selected Medicinal Plants and Their Synthesized Zinc Oxide Nanoparticles

Human Immunodeficiency Virus infection and Acquired Immune Deficiency Syndrome (HIV/AIDS) and associated opportunistic infections are still global health concerns. Traditional medicines have been used for managing these infections, with little about their biological activities being known. This study evaluated antimicrobial and anti-HIV-1 Reverse Transcriptase (RT) activities of medicinal plants; Harungana madagascariensis, Sapium ellipticum, Pseudospondias microcarpa, Capparis erythrocarpos, and Plectranthus barbatus; and activities of zinc oxide nanoparticles synthesized from aqueous extracts of H. madagascariensis. Results revealed that aqueous and ethyl acetate extracts of H. madagascariensis, S. ellipticum, P. microcarpa; and ethyl acetate extracts from P. barbatus exhibited minimum inhibitory concentrations ranging from 3.1 to 100 mg mL־1, while aqueous and ethyl acetate extracts from C. erythrocarpos showed no antibacterial activity. Furthermore, the study revealed that ethyl acetate extracts from P. barbatus, S. ellipticum, C. erythrocarpos and aqueous extract from H. madagascariensis have anti-HIV-1 RT inhibition greater than 50% at 10 mg mL־1. Aqueous crude extract of H. madagascariensis revealed higher anti-HIV-1-RT (IC50 = 0.9 mg mL־1) than all other extracts. On the other hand, zinc oxide nanoparticles synthesized from aqueous extract of H. madagascariensis exhibited antibacterial activity greater than all the tested extracts and anti-HIV-1 RT activities comparable to aqueous extract of H. madagascariensis. The results provide scientific information towards drug discovery from medicinal plants.&#x0D; Keywords: HIV/AIDS, Nanoparticles, Plant extracts, HIV-1 reverse transcriptase activity, Antibacterial activity

Polyether antibiotics K-41A and K-41Am inhibit HIV-1 replication via suppressing the activities of HIV-1 reverse transcriptase and integrase

IntroductionK-41A is a known compound with antibacterial activity against gram-positive bacteria and coccidia. In our screening of anti-HIV compounds, we found polyether antibiotic K-41A and its analogue K-41Am isolated from a marine-derived Streptomyces sp. SCSIO 01680 exhibited anti-HIV activity.Material and methodsIn this study, we characterised the anti-HIV activity of K-41A and K-41Am as well as the mechanism(s) involved. The dose-dependent inhibitory effects of K-41A and K-41Am on HIV-1 replication were observed in the TZM-bl-HIV-1IIIB system, MT-2-HIV-1IIIB system, and peripheral blood mononuclear cells (PBMCs)-HIV-1BaL system. The 50% inhibitory concentrations (IC50) of K-41A on HIV-1 replication in three systems were 0.75 µM, 0.09 µM, and 0.13 µM, respectively, and the selective indexes (SIs) were 5.81, 49.44, and 598.00, respectively. The IC50 of K-41Am in three systems were 5.57 µM, 0.24 µM, and 1.15 µM, respectively, and the SIs were &gt; 1.04, 80.71, and &gt; 5.03, respectively.ResultsMechanism research demonstrates that two compounds inhibited the activities of HIV-1 reverse transcriptase (RT) and integrase (IN) in a dose-dependent manner. Molecular docking shows that the docking scores were relatively high between the compound and HIV-1 RT or IN.ConclusionsK-41A and K-41Am possess anti-HIV-1 activity via a multi-target inhibition mechanism, which provides a novel molecular pattern for anti-HIV drug design and structural modification to improve the anti-HIV activity.

Isolation, identification, and evaluation of biological activities of Daldinia eschscholtzii, an endophytic fungus isolated from the leaves of Musa paradisiaca

Fungal endophytes of medicinal plants are gaining interest in the prospecting for biorelevant molecules due to their capacity to contribute novel compounds. This study was designed to identify and evaluate secondary metabolites of Daldinia eschscholtzii isolated from leaves of Musa paradisiaca for biorelevance. Plant leaves collection, fungal isolation, molecular identification, fermentation, and extraction of secondary metabolites were carried out following standard procedures. The crude extract was tested for antimicrobial, antioxidant, cytotoxic and antiviral properties; and further analyzed for their phyto-constituents using High performance liquid chromatography – diode array detection (HPLC-DAD). At 1000 µg/ml, D. eschscholtzii extract exhibited antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli producing IZD of 3 mm, 5 mm, and 7 mm respectively, but showed no antifungal activity. It showed antioxidant activity in the DPPH free radical scavenging assay producing an inhibition of 65.9% at a concentration of 500 µg/mL. At a concentration of 10 µg/mL, the D. eschscholtzii extract showed weak cytotoxic activity against L5178Y mouse lymphoma cells with growth inhibitions of 17.4% but showed a robust inhibition of HIV-1 reverse transcriptase activity of 97 % at 250 µg/mL. HPLC analysis of the extract/fraction revealed the presence of four bioactive compounds: aureonitol, pestalotiolactone A, cyclopenol, and palitantin. The detection of these metabolites further confirms the potentials of these endophytes as possible sources of bioactive molecules.

Targeting HIV-1 RNase H: N'-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants.

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N’-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.

Inhibition of HIV-1 RT activity by a new series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent potent anti-HIV agents targeting HIV-1 reverse transcriptase (RT), a crucial enzyme for the viral life cycle. We have previously identified a series of NNRTIs bearing a 2,3-diaryl-1,3-thiazolidin-4-one core and some compounds proved to be effective in inhibiting HIV-1 replication at micromolar concentration. As a continuation in this research work we report the design, the synthesis and the structure–activity relationship studies of a further series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives containing an arylthioacetamide group as pharmacophoric structural requirement for binding to the RT catalytic area. The new compounds proved to be effective to inhibit RT activity at micromolar concentrations. Finally, docking studies were carried out in order to rationalize the biological results of the new synthesized inhibitors.

Inhibition of HIV-1 Reverse Transcriptase Activity by the Extracts of Indian Plants

Inhibition of HIV-1 Reverse Transcriptase Activity by the Extracts of Indian Plants

Determinants of Active-Site Inhibitor Interaction with HIV-1 RNase H.

The ribonuclease H (RNH) activity of HIV-1 reverse transcriptase (RT) is essential for viral replication and can be a target for drug development. Yet, no RNH inhibitor to date has substantial antiviral activity to allow advancement into clinical development. Herein, we describe our characterization of the detailed binding mechanisms of RNH active-site inhibitors, YLC2-155 and ZW566, that bind to the RNH domain through divalent metal ions, using NMR, molecular docking, and quantum mechanical calculations. In the presence of Mg2+, NMR spectra of RNH exhibited split (two) resonances for some residues upon inhibitor binding, suggesting two binding modes, an observation consistent with the docking results. The relative populations of the two binding conformers were independent of inhibitor or Mg2+ concentration, with one conformation consistently more favored. In our docking study, one distinctive pose of ZW566 showed more interactions with surrounding residues of RNH compared to the analogous binding pose of YLC2-155. Inhibitor titration experiments revealed a lower dissociation constant for ZW566 compared to YLC2-155, in agreement with its higher inhibitory activity. Mg2+ titration data also indicated a stronger dependence on Mg2+ for the RNH interaction with ZW566 compared to YLC2-155. Combined docking and quantum mechanical calculation results suggest that stronger metal coordination as well as more protein-inhibitor interactions may account for the higher binding affinity of ZW566. These findings support the idea that strategies for the development of potent competitive active site RNH inhibitors should take into account not only metal-inhibitor coordination but also protein-inhibitor interaction and conformational selectivity.

In vitro inhibition of HIV-1 reverse transcriptase and anti-inflammatory activities of some herbal concoctions sold in the Limpopo Province

HIV-1 is the principal virus that causes acquired immunodeficiency syndrome (AIDS), a condition characterized by extreme immunosuppression and inflammation that leads to life-threatening opportunistic infections. The pitfalls of current anti-retroviral therapies are the lack of medical access in developing countries, adverse side effects and the rise of resistant strains. Natural products used in traditional medicine that can interfere with the replication of the HIV serve as good sources of anti-HIV drugs and anti-inflammation agents. The aim of this study was to validate the anti-HIV and anti-inflammatory properties of some herbal concoctions sold in the Limpopo Province, reported to treat HIV and inflammation related symptoms. The anti-HIV activity was evaluated using the HIV-1 reverse transcriptase (RT) inhibitory bioassay. Anti-inflammatory activity was determined using cyclooxygenase 1 and 2 inhibitory assays. Compared to the controls, Lamivudine, Zidovudine, Lopinavir and Ritonavir, the laboratory concoction showed the highest HIV-reverse transcriptase inhibition (IC50: 2.90 μg/mL), followed by herbal concoction 5 (IC50: 11.74 μg/mL). It is likely the herbal concoctions induced a conformational change on the HIV-RT by binding to its allosteric sites, thus inactivating it. Generally, the herbal concoctions achieved higher percentage inhibitions (≥70%) for COX-2 than for COX-1. The anti-inflammatory activity of the herbal concoctions thus may also assist to alleviate pain in the patients. This research validated the ethnopharmacological use of herbal concoctions towards the treatment of HIV infections. However, more studies are needed to further elucidate the pharmacokinetics and toxicological effects of these medicines.

Small molecule ONC201 inhibits HIV-1 replication in macrophages via FOXO3a and TRAIL

Despite the success of antiretroviral therapy (ART), eradication of HIV-1 from brain reservoirs remains elusive. HIV-1 brain reservoirs include perivascular macrophages that are behind the blood-brain barrier and difficult to access by ART. Macrophages express transcription factor FOXO3a and the TNF superfamily cytokine TRAIL, which are known to target HIV-1-infected macrophages for viral inhibition. ONC201 is a novel and potent FOXO3a activator capable of inducing TRAIL. It can cross the blood-brain barrier, and has shown antitumor effects in clinical trials. We hypothesized that activation of FOXO3a/TRAIL by ONC201 will inhibit HIV-1 replication in macrophages. Using primary human monocyte-derived macrophages, we demonstrated that ONC201 dose-dependently decreased replication levels of both HIV-1 laboratory strain and primary strains as determined by HIV-1 reverse transcriptase activity assay. Consistent with data on HIV-1 replication, ONC201 also reduced intracellular and extracellular p24, viral RNA, and integrated HIV-1 DNA in infected macrophages. Blocking TRAIL or knockdown of FOXO3a with siRNA reversed ONC201-mediated HIV-1 suppression, suggesting that ONC201 inhibits HIV-1 through FOXO3a and TRAIL. The anti-HIV-1 effect of ONC201 was further validated in vivo in NOD/scid-IL-2Rgcnull mice. After intracranial injection of HIV-1-infected macrophages into the basal ganglia, we treated the mice daily with ONC201 through intraperitoneal injection for six days. ONC201 significantly decreased p24 levels in both the macrophages and the brain tissues, suggesting that ONC201 suppresses HIV-1 in vivo. Therefore, ONC201 can be a promising drug candidate to combat persistent HIV-1 infection in the brain.

Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.

HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

Anti-HIV-1 activity and safety profile of a polyherbal gel formulation as a candidate microbicide

Microbicides represent one of the promising options for the prevention of sexually transmitted HIV infection. Medicinal plants have a long history of use to provide treatment against many diseases. In this regard, polyherbal aqueous gel formulation comprising 50% ethanolic extract prepared from the heart wood of Acacia catechu, leaves of Lagerstroemia speciosa, fruits of Aegle marmelos, Phyllanthus emblica and Terminalia chebula using carbopol 974 P NF polymer has been prepared. The gel formulation showed significant anti-HIV-1 activity against both CXCR4 tropic (HIV-1NL4.3) and CCR5 tropic (HIV-1NLAD8) viruses with IC50 values of 58.17 ± 4.4 and 63.54 ± 6.8 μg/ml respectively. It also showed significant inhibition of HIV-1 reverse transcriptase (p = 0.004), protease (p = 0.014), and integrase (p = 0.018) enzymes activity as compared to gel base. The gel formulation did not show any adverse effect on the viability of the vaginal lactobacilli, human vaginal keratinocytes (Vk2/E6E7), and the monolayer formed by Caco-2 cells. Furthermore, no significant changes in the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF from the gel formulation treated Vk2/E6E7 cells were observed as compared to cells treated with gal base or medium control. Gel formulation did not show any significant increase in mutagenic index using Salmonella typhimurium. Gel formulation stored at either 30 ± 2 °C or 40 ± 2 °C was stable for 6 months for anti-HIV-1 activity. In conclusion, the polyherbal gel formulation reported herein having potent anti-HIV-1 activity and with limited safety profile may be a promising candidate as an herbal microbicide.