Mutant HIV-1 Research Articles

Insights into the binding properties of calf thymus DNA with lopinavir from spectroscopic and computational studies

Lopinavir (LPV), an important antiviral drug, can inhibit effectively wild type and mutant HIV protease. In this work, molecular interaction mechanism between LPV and calf thymus deoxyribonucleic acid (ct-DNA) was characterized by spectroscopic, viscosity and molecular docking approaches to attain some important information about the LPV-ct-DNA interaction including binding affinity, binding mode, main driving-forces and structural change of ct-DNA. UV–vis titration data demonstrated that the LPV-ct-DNA complex was formed, and the binding affinity under the studied conditions was weak due to the binding constant at the level of 103 M−1. The negative values of ΔH0 (−26.63 kJ∙mol−1) and ΔS0 (−24.11 J∙mol−1∙K−1) revealed that hydrogen bonding and Van der Waals forces played an indispensable part in the LPV-ct-DNA complexation process. The outcomes from viscosity measurements excluded the possibility of intercalation mode between LPV and ct-DNA. The results from UV–vis titration experiments, displacement experiments and influence of ionic strength indicated that LPV bound to the minor groove of ct-DNA, which was further confirmed by docking studies. Besides, a minor change in the conformation of ct-DNA can be observed from CD spectral analysis. Our studies scientifically elucidated the binding behavior between LPV and ct-DNA for the first time and were beneficial to assessing the pharmacological properties of this antiviral drug.

Identification of novel potent HIV-1 inhibitors by exploiting the tolerant regions of the NNRTIs binding pocket.

With our previously identified potent NNRTIs 25a and HBS-11c as leads, series of novel thiophene[3,2-d]pyrimidine and thiophene[2,3-d]pyrimidine derivatives were designed via molecular hybridization strategy. All the target compounds were evaluated for their anti-HIV-1 activity and cytotoxicity in MT-4cells. Compounds 16a1 and 16b1 turned out to be the most potent inhibitors against WT and mutant HIV-1 strains (L100I, K103N, and E138K), with EC50 values ranging from 0.007μM to 0.043μM. Gratifyingly, 16b1 exhibited significantly reduced cytotoxicity (CC50>217.5μM) and improved water solubility (S=49.3μg/mL at pH 7.0) compared to the lead 25a (S<1μg/mL at pH 7.0, CC50=2.30μM). Moreover, molecular docking was also conducted to rationalize the structure-activity relationships of these novel derivatives and to understand their key interactions with the binding pocket.

First Assessment of Acquired HIV-1 Drug Resistance and Mutation Patterns in Suriname.

HIV drug resistance testing is fundamental in clinical patient management, but data on HIV-1 drug-resistant mutations (DRMs) is scarce in the Caribbean and in Suriname limited to one survey on transmitted resistance. The aim of this study was to address this gap, to gain insight in acquired HIV drug resistance (ADR) prevalence and mutation patterns, and to improve HIV-1 treatment outcome of people living with HIV (PLHIV) in Suriname. A prospective cross-sectional study was conducted from July 2018 through January 2019 among treatment-experienced PLHIV (n = 72), with either treatment failure or antiretroviral therapy restart. Genotypic drug resistance testing was performed and DRM impact on drug effectiveness was examined. Genotypic drug resistance testing revealed 97.2% HIV-1 subtype B, 2.8% B/D recombinants and a ADR prevalence of 63.2% in treatment failure patients, with a predominance of nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations. The most common DRMs were M184V (23.6%) and K103N (18.8%). A high level of non-DRM polymorphisms was observed in both the reverse transcriptase (RT) and protease (PR) gene. Interesting deviations from the existing mutation datasets were noted at position E248 and R83 of the RT gene and L63 and V77 in the PR gene. Full susceptibility to all examined drugs was 54.2%, while high-level drug resistance was estimated at 37.5%, which seems promising for treatment outcomes for PLHIV in Suriname, although cross-resistance to next-generation NNRTIs was already estimated for nearly a quarter of the patients. The meager 2.9% of PR DRMs rendered protease inhibitors as an effective rescue HIV-1 treatment.

Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus

ABSTRACT EK1 peptide is a membrane fusion inhibitor with broad-spectrum activity against human coronaviruses (CoVs). In the outbreak of COVID-19, we generated a lipopeptide EK1V1 by modifying EK1 with cholesterol, which exhibited significantly improved antiviral activity. In this study, we surprisingly found that EK1V1 also displayed potent cross-inhibitory activities against divergent HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. Consistently, the recently reported EK1 derivative EK1C4 and SARS-CoV-2 derived fusion inhibitor lipopeptides (IPB02 ∼ IPB09) also inhibited HIV-1 Env-mediated cell–cell fusion and infection efficiently. In the inhibition of a panel of HIV-1 mutants resistant to HIV-1 fusion inhibitors, EK1V1 and IPB02-based inhibitors exhibited significantly decreased or increased activities, suggesting the heptad repeat-1 region (HR1) of HIV-1 gp41 being their target. Furthermore, the sequence alignment and molecular docking analyses verified the target site and revealed the mechanism underlying the resistance. Combined, we conclude that this serendipitous discovery provides a proof-of-concept for a common mechanism of viral fusion and critical information for the development of broad-spectrum antivirals.

Spontaneous Mutations in HIV-1 Gag, Protease, RT p66 in the First Replication Cycle and How They Appear: Insights from an In Vitro Assay on Mutation Rates and Types.

While drug resistant mutations in HIV-1 are largely credited to its error prone HIV-1 RT, the time point in the infection cycle that these mutations can arise and if they appear spontaneously without selection pressures both remained enigmatic. Many HIV-1 RT mutational in vitro studies utilized reporter genes (LacZ) as a template to investigate these questions, thereby not accounting for the possible contribution of viral codon usage. To address this gap, we investigated HIV-1 RT mutation rates and biases on its own Gag, protease, and RT p66 genes in an in vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease, and RT p66 at 4.71 × 10−5, 6.03 × 10−5, and 7.09 × 10−5 mutations/bp, respectively. Gag and p66 genes showed a large number of ‘A to G’ mutations. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 × 10−4 mutations/bp) and that ‘A to G’ mutations occurred in regions reminiscent of ADAR neighbor sequence preferences. Mutational free energies of the ‘A to G’ mutations revealed an avoidance of destabilizing effects, with the natural p66 gene codon usage providing barriers to disruptive amino acid changes. Our study demonstrates the importance of studying mutation emergence in HIV genes in a RT-PCR in vitro selection pressure free system to understand how fast drug resistance can emerge, providing transferable applications to how new viral diseases and drug resistances can emerge.

Enhancement of HIV-1 Env-Specific CD8 T Cell Responses Using Interferon-Stimulated Gene 15 as an Immune Adjuvant.

Induction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens.IMPORTANCE Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime-MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines.

Advances and challenges in antiretroviral therapy for acquired immunodeficiency syndrome.

Advances and challenges in antiretroviral therapy for acquired immunodeficiency syndrome.

Absence of Integrase Inhibitor-Associated Resistance Among Antiretroviral Therapy-Naïve HIV-1-Infected Adults in Guangdong Province, China, in 2018.

BackgroundAntiretroviral therapy (ART) containing an integrase strand transfer inhibitor (INSTI) plus two nucleoside reverse-transcriptase inhibitors has been recommended as a first-line regimen for ART-naïve HIV-1-infected patients in the latest Chinese Guidelines for Diagnosis and Treatment of HIV/AIDS.ObjectiveTo determine the prevalence of INSTI-related mutations among ART-naïve HIV-1-infected adults in Guangdong, China, in 2018.MethodsThe entire integrase gene was amplified from blood plasma. Demographic and epidemiological information was collected. INSTI mutations and antiretroviral susceptibility were interpreted using the Stanford University HIV Drug Resistance Database HIVdb program.ResultsOf 927 samples, 827 integrase sequences were successfully obtained. Among them, no major resistance mutations to INSTIs were identified, and four accessory mutations, including T97A (0.12%, 1/827), A128T (0.24%, 2/827), E157Q (0.85%, 7/827), and G163R (0.24%, 2/827), were found in twelve individuals. Two patient samples contained the G163R mutation conferring low-level resistance to elvitegravir and raltegravir.ConclusionThe overall prevalence of INSTI mutations remains low. Drug resistance mutation testing for the detection of INSTI drug resistance mutations in HIV treatment-naïve patients should be considered due to the circulation of polymorphisms contributing to INSTI resistance and the expected increasing use of this class of drugs.

Elucidating the Basis for Permissivity of the MT-4 T-Cell Line to Replication of an HIV-1 Mutant Lacking the gp41 Cytoplasmic Tail

HIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virus particles and viral transmission. In most physiologically relevant cell types, the gp41 CT is required for HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study, we investigated the viral and cellular factors that contribute to the permissivity of MT-4 cells to gp41 CT truncation. We found that the kinetics of HIV-1 production and virus release are faster in MT-4 than in the other T-cell lines tested, but MT-4 cells express equivalent amounts of HIV-1 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 cells express higher levels of plasma-membrane-associated Env than nonpermissive cells, and Env internalization from the plasma membrane is less efficient than that from another T-cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4 cells, 2-fold less Env is incorporated into virus particles produced from MT-4 than SupT1 cells. Contact-dependent transmission between cocultured 293T and MT-4 cells is higher than in cocultures of 293T with most other T-cell lines tested, indicating that MT-4 cells are highly susceptible to cell-to-cell infection. These data help to clarify the long-standing question of how MT-4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in physiologically relevant cell lines.IMPORTANCE The HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into nascent particles and viral transmission in primary CD4+ T cells. The MT-4 T-cell line has been reported to support multiple rounds of infection of HIV-1 encoding a gp41 CT truncation. Uncovering the underlying mechanism of MT-4 T-cell line permissivity to gp41 CT truncation would provide key insights into the role of the gp41 CT in HIV-1 transmission. This study reveals that multiple factors contribute to the unique ability of a gp41 CT truncation mutant to spread in cultures of MT-4 cells. The lack of a requirement for the gp41 CT in MT-4 cells is associated with the combined effects of rapid HIV-1 protein production, high levels of cell-surface Env expression, and increased susceptibility to cell-to-cell transmission compared to nonpermissive cells.

Coordinating Cytoskeleton and Molecular Traffic in T Cell Migration, Activation, and Effector Functions.

Dynamic localization of receptors and signaling molecules at the plasma membrane and within intracellular vesicular compartments is crucial for T lymphocyte sensing environmental cues, triggering membrane receptors, recruiting signaling molecules, and fine-tuning of intracellular signals. The orchestrated action of actin and microtubule cytoskeleton and intracellular vesicle traffic plays a key role in all these events that together ensure important steps in T cell physiology. These include extravasation and migration through lymphoid and peripheral tissues, T cell interactions with antigen-presenting cells, T cell receptor (TCR) triggering by cognate antigen–major histocompatibility complex (MHC) complexes, immunological synapse formation, cell activation, and effector functions. Cytoskeletal and vesicle traffic dynamics and their interplay are coordinated by a variety of regulatory molecules. Among them, polarity regulators and membrane–cytoskeleton linkers are master controllers of this interplay. Here, we review the various ways the T cell plasma membrane, receptors, and their signaling machinery interplay with the actin and microtubule cytoskeleton and with intracellular vesicular compartments. We highlight the importance of this fine-tuned crosstalk in three key stages of T cell biology involving cell polarization: T cell migration in response to chemokines, immunological synapse formation in response to antigen cues, and effector functions. Finally, we discuss two examples of perturbation of this interplay in pathological settings, such as HIV-1 infection and mutation of the polarity regulator and tumor suppressor adenomatous polyposis coli (Apc) that leads to familial polyposis and colorectal cancer.

Structure-based linker optimization of 6-(2-cyclohexyl-1-alkyl)-2-(2-oxo-2-phenylethylsulfanyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

In continuation of our efforts toward the discovery of potent HIV-1 NNRTIs with diverse structures, a series of novel S-DACO analogues of 6-(2-cyclohexyl-1-alkyl)-2-(2-oxo-2-phenyl-ethylsulfanyl)pyrimidin-4(3H)-ones were designed, synthesized and evaluated for their antiviral activities in MT-4 cells. Most of these new compounds showed moderate to good activities against wild type HIV-1 with IC50 values ranging from 7.55 μmol/L to 0.018 μmol/L. Among them, compound 5c was identified as the most promising inhibitor against HIV-1 replication with an IC50 = 0.018 μmol/L, CC50 = 194 μmol/L, and SI = 12791, which was much more potent than the reference drugs NVP and DLV and comparable to AZT and EFV. In addition, 5c also exhibited improved activity against double mutant HIV-1 strain RES056 compared to that of the reference drugs NVP/DLV and DB02. The preliminary structure-activity relationship (SAR) and molecular modeling studies were also discussed, which provides some useful indications for guiding the further rational design of new S-DACO analogues.

Targeting dual tolerant regions of binding pocket: Discovery of novel morpholine-substituted diarylpyrimidines as potent HIV-1 NNRTIs with significantly improved water solubility.

To address the intractable issues of drug resistance and poor solubility, a novel series of morpholine-substituted diarylpyrimidines targeting the tolerant region I and tolerant region II of NNIBP were rationally designed by utilizing the available crystallography studies. The biological evaluation results showed that four most promising compounds (14e1, 14g1, 14g2 and 14j2) displayed excellent potency against WT HIV-1 strain with EC50 values ranging from 58 to 87nM, being far more potent than NVP and comparable to ETV. Besides, some derivatives exhibited moderate activity in inhibiting the mutant HIV-1 strains. More encouragingly, 14d2 (RF=0.4) possessed higher antiresistance profile than ETV (RF=6.3) and K-5a2 (RF=3.0) toward the double mutant strain F227L+V106A. The HIV-1 RT inhibition assay confirmed their binding target. The molecular docking studies were conducted and discussed in detail to rationalize the preliminary SARs. Further test indicated that morpholine could indeed promote the improvement of water solubility. Additionally, the in silico prediction of physicochemical properties and CYP enzymatic inhibitory ability were investigated to evaluate their drug-like features.

New indolylarylsulfone non-nucleoside reverse transcriptase inhibitors show low nanomolar inhibition of single and double HIV-1 mutant strains.

We designed and synthesized 21 new indolylarylsulfones (IASs) as new HIV-1 NNRTIs. Among these, IAS 12 exhibited a remarkable antiviral activity against single and double mutants (K103N EC50 = <0.7nM; Y181C EC50 = <0.7nM; Y188L EC50 = 21.3nM; K103N-Y181C EC50 = 6.2nM), resulting equally or more active than previuosly reported IAS 6 and some approved anti-HIV-1 drugs. Docking and molecular dynamics simulations of compound 12 in complex with WT, Y181C, Y188L, K103N and K103N-Y181C RTs clarified a general binding mode that was consistent with biological results. Kinetic experiments disclosed that derivative 12 preferentially binds WT and K103N-Y181C RTs to binary and ternary complexes, respectively.

Regulation of Cas9 by viral proteins Tat and Rev for HIV-1 inactivation

While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells.In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.

Detection of HIV-1 resistant to antiretroviral drugs among tomsk oblast population with newly diagnosed HIV-infection

The purpose of this paper is to evaluate the spreading of HIV-1 resistant to antiretroviral drugs among Tomsk Oblast population with newly diagnosed HIV-infection. Materials and methods. It was collected 122 clinical samples of peripheral blood of HIV-infected patients from Tomsk Oblast who did not take antiretroviral drugs. In HIV-1 isolated from clinical samples we studied nucleotide sequence of genome fragments encoding virus protease and reverse transcriptase. Complex analyses of epidemiologic data from patients and the presence in genome HIV-1 mutations associated with resistance development to protease inhibitors and virus reverse transcriptase were carried out. Results. Analysis of HIV-1 isolated from Tomsk Oblast naive HIV-infected population made it possible to detect HIV-1 mutations associated with a decrease of virus sensitivity to antiretroviral drugs in 9,8% of cases. Among described mutations 50% were associated with resistance to virus protease inhibitors; 33,3% were resistance mutations to nonnuclease inhibitors of reverse transcriptase, and 16.7% were resistance mutations to nucleoside inhibitors of virus reverse transcriptase. Out of 9,8% of resistant viruses 7,3% of cases included mutations associated with the development of potentially low level of reduction of HIV-1 sensitivity to drugs. Main HIV-1 resistance mutations of high and average levels were registered only in 2,5% of genotyped HIV-1 isolated from people who inject drugs. Conclusion. Current study detected considerably low sampling rate of HIV-1 carrying mutations associated with resistance to antiretroviral drugs among Tomsk Oblast naive HIV-infected population. It is believed to be caused by a relatively short period of extensive application of antiretroviral therapy in that territory. Analysis of epidemiologic data resulted in detection of factors negatively affecting prediction of further development of HIV-infection epidemic in the region including prevalence of risk behavior practice contributing to resistant HIV-1 transmission both among patients via heterosexual contacts and among people who inject drugs.

Neutralizing antibody VRC01 failed to select for HIV-1 mutations upon viral rebound.

Infusion of the broadly neutralizing antibody VRC01 has been evaluated in individuals chronically infected with HIV-1. Here, we studied how VRC01 infusions affected viral rebound after cessation of antiretroviral therapy (ART) in 18 acutely treated and durably suppressed individuals. Viral rebound occurred in all individuals, yet VRC01 infusions modestly delayed rebound and participants who showed a faster decay of VRC01 in serum rebounded more rapidly. Participants with strains most sensitive to VRC01 or with VRC01 epitope motifs similar to known VRC01-susceptible strains rebounded later. Upon rebound, HIV-1 sequences were indistinguishable from those sampled at diagnosis. Across the cohort, participant-derived Env showed different sensitivity to VRC01 neutralization (including 2 resistant viruses), yet neutralization sensitivity was similar at diagnosis and after rebound, indicating the lack of selection for VRC01 resistance during treatment interruption. Our results showed that viremia rebounded despite the absence of HIV-1 adaptation to VRC01 and an average VRC01 trough of 221 μg/mL. Although VRC01 levels were insufficient to prevent a resurgent infection, knowledge that they did not mediate Env mutations in acute-like viruses is relevant for antibody-based strategies in acute infection.

Novel radial distribution function approach in the study of point mutations: the HIV-1 protease case study.

Background: Mutations are one of the engines of evolution. Under constant stress pressure, mutations can lead to the emergence of unwanted, drug-resistant entities. Methodology: The radial distribution function weighted by the number of valence shell electrons is used to design quantitative structure-activity relationship (QSAR)model relating descriptors with the inhibition constant for a series of wild-type HIV-1 protease inhibitor complexes. The residuals of complexes with mutant HIV-1 protease were correlated with the energy of the highest occupied molecular orbitals of the residues introduced to enzyme via point mutations. Conclusion: Successful identification of residues Ile3, Asp25, Val32 and Ile50 as the one whose substitution influences the inhibition constant the most, demonstrates the potential of the proposed methodology for the study of the effects of point mutations.

Privileged scaffold inspired design of novel oxime-biphenyl-DAPYs in treatment of HIV-1.

Oxime is a key pharmacophore in drug development. The biphenyl diarylpyrimidines (DAPYs) have been developed by our group as novel non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, fourteen oxime-biphenyl-DAPYs were designed and synthesized through a privileged scaffold inspired design strategy. They exhibited promising activity toward wild type HIV-1 and single mutant strains. Compound 7d was found to be the most potent one against both wild type (EC50=12.1nM) and E138K mutant strains (EC50=0.0270µM). It also had a much lower cytotoxicity (CC50>292µM) and higher selective index (SI>24105) than those of the FDA-approved drugs efavirenz and etravirine. Molecular docking and dynamics simulation predicted and disclosed the binding mode of compound 7d with the RT, providing the explanation on the antiviral activity. These results were helpful for subsequent structural optimizations in anti-HIV-1 drug discovery.

Antiviral Activity of Tenofovir Alafenamide against HIV-1 with Thymidine Analog-Associated Mutations and M184V

Tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF) are prodrugs of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). In vivo, TAF achieves >4-fold-higher intracellular levels of TFV diphosphate (TFV-DP) compared to TDF. Since thymidine analog-associated mutations (TAMs) in HIV-1 confer reduced TFV susceptibility, patients with TAM-containing HIV-1 may benefit from higher TFV-DP levels delivered by TAF. Moreover, the presence of the M184V mutation increases TFV susceptibility during TDF- or TAF-based therapy. The susceptibilities to antiviral drugs of site-directed mutants (SDMs) and patient-derived mutants containing combinations of TAMs (M41L, D67N, K70R, L210W, T215Y, and K219Q) with or without the M184V mutation (TAMs±M184V) were evaluated using either 5-day multicycle (MC; n = 110) or 2-day single-cycle (SC; n = 96) HIV assays. The presence of M184V in TAM-containing HIV-1 SDMs (n = 48) significantly increased TAF sensitivity compared to SDMs without M184V (n = 48). The comparison of TAF and TDF resistance profiles was further assessed in viral breakthrough (VB) experiments mimicking clinically relevant drug concentrations. A total of 68 mutants were assayed at physiological concentration in VB experiments, with 15/68 mutants breaking through with TDF (TFV, the in vitro equivalent of TDF, was used in these experiments), and only 3 of 68 mutants breaking through under TAF treatment. Overall, in the VB assay mimicking the 4-fold-higher intracellular levels of TFV-DP observed clinically with TAF versus TDF, TAF inhibited viral breakthrough of most TAM-containing HIV-1, whereas TDF did not. These results indicate that TAF has a higher resistance threshold than TDF and suggest that higher resistance cutoffs should be applied for TAF compared to TDF in genotypic and phenotypic resistance algorithms.

In situ click chemistry-based rapid discovery of novel HIV-1 NNRTIs by exploiting the hydrophobic channel and tolerant regions of NNIBP

HIV-1 RT has been considered as one of the most important targets for the development of anti-HIV-1 drugs for their well-solved three-dimensional structure and well-known mechanism of action. In this study, with HIV-1 RT as target, we used miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening to discover novel potent HIV-1 NNRTIs. A 156 triazole-containing inhibitor library was assembled in microtiter plates and in millimolar scale. The enzyme inhibition screening results showed that 22 compounds exhibited improved inhibitory activity. Anti-HIV-1 activity results demonstrated that A3N19 effected the most potent activity against HIV-1 IIIB (EC50 = 3.28 nM) and mutant strain RES056 (EC50 = 481 nM). The molecular simulation analysis suggested that the hydrogen bonding interactions of A3N19 with the main chain of Lys101 and Lys104 was responsible for its potency. Overall, the results indicated the in situ click chemistry-based strategy was rational and might be amenable for the future discovery of more potent HIV-1 NNRTIs.