Wild-type Reverse Transcriptase Research Articles

Exploring novel HIV-1 reverse transcriptase inhibitors with drug-resistant mutants: A double mutant surprise.

HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.

Covalent and noncovalent strategies for targeting Lys102 in HIV-1 reverse transcriptase

Reverse transcriptase (RT) is one of three key proteins responsible for the replication cycle of HIV-1 in the host. Several classes of inhibitors have been developed to target the enzyme, with non-nucleoside reverse transcriptase inhibitors forming first-line treatment. Previously, covalent RT inhibitors have been identified and found to bind irreversibly to commonly mutated residues such as Y181C. In this work we aim to circumvent the issue of NNRTI resistance through targeting K102, which has not yet been identified to confer drug resistance. As reported here, 34 compounds were synthesized and characterized biochemically and structurally with wild-type (WT) HIV-1 RT. Two of these inhibitors demonstrate covalent inhibition as evidenced by protein crystallography, enzyme kinetics, mass spectrometry, and antiviral potency in HIV-1 infected human T-cell assays.

Correlation of tumor-associated macrophage infiltration in glioblastoma with magnetic resonance imaging characteristics: a retrospective cross-sectional study.

Glioblastoma (Gb) is the most common primary malignant tumor of brain with poor prognosis. Immune cells are the main factors affecting the prognosis of Gb, tumor-associated macrophages (TAMs) are the predominant infiltrating immune cell population in the immune microenvironment of Gb. Analyzing the relationship between magnetic resonance imaging (MRI) features and TAMs of Gb, and using imaging features to characterize the infiltration level of TAMs in tumor tissue may provide indicators for clinical decision-making and prognosis evaluation of Gb. Data from 140 in patients with isocitrate dehydrogenase (IDH) wild-type Gb diagnosed via histopathology and molecular diagnosis in the Second Hospital of Lanzhou University from January 2018 to April 2022 were collected in this retrospective, cross-sectional study. MRI images were reviewed for lesion location, cyst, necrosis, hemorrhage, contrast-enhanced T1-weighted MRI signal intensity, average apparent diffusion coefficient (ADCmean), and minimum apparent diffusion coefficient (ADCmin). Immunohistochemical staining with anti-CD163 and anti-CD68 antibodies was employed for macrophage detection. The positive cell percentage was estimated in 9 microscopic fields at 400× magnification per whole-slide image with ImageJ software (National Institutes of Health). Additionally, the relationship between MRI features, molecular, states and the positive CD68 and CD163 expression was analyzed. Our study discovered that the mean or median values of CD68+ and CD163+ TAMs were 7.39% and 14.98%, respectively. There was an obvious correlation between CD163+ TAMs and CD68+ TAMs (r=0.497; P=0.000). CD68+ and CD163+ macrophage infiltration correlated with age at diagnosis in patients with Gb (CD68+: r=0.230, P=0.006; CD163+: r=0.172, P=0.042). The levels of Gb TAM infiltration in different tumor locations varied, with the temporal lobe having the highest CD163+ macrophage and CD68+ macrophage infiltration (18.58% and 9.46%, respectively). CD163+ macrophage infiltration was positively correlated with ADCmean (r=0.208; P=0.014). The infiltration of CD68+ macrophages differed significantly between groups with varying degrees of tumor enhancement (H =4.228; P=0.017). There was a significant difference in CD68+ TAMs and CD163+ TAMs between the wild-type and mutant-type telomerase reverse transcriptase (TERT) types (P=0.004 and P=0.031, respectively). Age, location of the tumor, degree of tumor enhancement, ADC value, and TERT mutation status were associated with macrophage infiltration. These findings may serve as an effective tool for characterizing the tumor microenvironment in patients with Gb.

Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.

Structure-directed expansion of biphenyl-pyridone derivatives as potent non-nucleoside reverse transcriptase inhibitors with significantly improved potency and safety

Following our previous work on human immunodeficiency virus-1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs), a series of novel biphenyl-pyridone derivatives were synthesized and evaluated for their anti-HIV-1 activity to expand their structure–activity relationship. Some of them exhibited low nanomolar activity toward wild-type HIV-1 and clinically relevant single/double mutant strains. The most active compound B1 was 231-fold more potent (EC50 = 17 nmol/L) than the lead compound 2 (EC50 = 3.93 µmol/L) against wild-type (WT) HIV-1. This compound was approximately 3.5-fold less cytotoxic (CC50 = 100.58 µmol/L) than compound 2 (CC50 = 28.24 µmol/L), presenting a higher selectivity index (SI) value of 5923. Compared with 2, the antiviral potency of B1 was significantly increased against five single mutant strains (L100I, K103N, E138K, Y181C and Y188L) and two double mutant strains (F227L+V106A and K103N+Y181C). Especially, K103N, Y181C and K103N+Y181C were more sensitive to B1 than both 2 and doravirine. Besides, the enzymatic inhibitory activity of B1 against wild-type HIV-1 reverse transcriptase was approximately 32-fold higher (IC50 = 100 nmol/L) than 2 (IC50 = 3.21 µmol/L). Molecular docking studies and dynamic simulations were conducted to explain their potent activity. Taken together, this research represents an important step toward the discovery of novel biphenyl-pyridone drug candidates for HIV therapy.

Development of novel S-N3-DABO derivatives as potent non-nucleoside reverse transcriptase inhibitors with improved potency and selectivity

Following on our initial discovery of S-CN-DABOs as non-nucleoside reverse transcriptase inhibitors (NNRTIs), a series of novel S-N3-DABO derivatives F1-F31 were developed by substituting the cyano group of S-CN-DABOs with azide group. Some of these compounds were conferred significantly increased potency against wild-type HIV-1 and clinically observed mutant strains. Remarkably, the best compound F10 exerted a 7-fold improvement in potency (EC50 = 0.053 μM) and 12.5-fold higher selectivity (SI = 6818) in MT-4 cells infected with wild-type HIV-1, compared to that of the parent compound B1 (EC50 = 370 nM, SI = 547). The anti-HIV-1 activity of F10 against the tested mutant strains was prominently enhanced. For wild-type reverse transcriptase, it was approximately 19-fold more potent (IC50 = 0.080 μM) than B1 (IC50 = 1.51 μM). It was not found that this analog had significant inhibition of hERG, CYP, and acute toxicity after a single dose of F10 (1.0 g/kg).

Identification of Novel Diarylpyrimidines as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors by Exploring the Primer Grip Region.

HIV-1 reverse transcriptase (RT) plays a crucial role in the viral replication cycle, and RT inhibitors can represent a promising pathway in treating AIDS. To explore the primer grip region of HIV-1 RT, using -CH2O- as a linker, substituted benzene or pyridine rings were introduced into the left wing of diarylpyrimidines (DAPYs). A total of 17 compounds with new structures were synthesized. It showed that all compounds exhibited anti-HIV-1 (wild-type) activity values ranging from 7.6-199.0 nM. Among them, TF2 (EC50 = 7.6 nM) showed the most potent activity, which was better than that of NVP (EC50 = 122.6 nM). Notably, compared with RPV (CC50 = 3.98 μM), TF2 (CC50 > 279,329.6 nM) showed low cytotoxicity. For HIV-1 mutant strains K103N and E138K, most compounds showed effective activities. Especially for K103N, TF2 (EC50 = 28.1 nM), TF12 (EC50 = 34.7 nM) and TF13 (EC50 = 28.0 nM) exhibited outstanding activity, being superior to that of NVP (EC50 = 7495.1 nM) and EFV (EC50 = 95.1 nM). Additionally, TF2 also showed the most potent activity against E138K (EC50 = 44.0 nM) and Y181C mutant strains (EC50 = 139.3 nM). In addition, all the compounds showed strong enzyme inhibition (IC50 = 0.036-0.483 μM), which demonstrated that their target was HIV-1 RT. Moreover, molecular dynamics simulation studies were implemented to predict the binding mode of TF2 in the binding pocket of wild-type and K103N HIV-1 RT.

Linker optimization of HEPT derivatives as potent non-nucleoside HIV-1 reverse transcriptase inhibitors: From S=O to CHOR

A novel series of CHOR-HEPT non-nucleoside HIV-1 reverse transcriptase inhibitors were developed by means of structure-based design strategy based on compound 6 reported previously by our group. Most of these compounds showed moderate to good activity toward wild-type HIV-1 strain with EC50 values in the range of 0.18–51.88 µmol/L and SI values in the range of 4–907. The compound 14aj with a CHOH linker and compound 13i with a CHOTMS linker in this series exhibited improved anti-HIV-1 activity (EC50 = 0.18 µmol/L, and 0.20 µmol/L) with higher selectivity (SI = 907, and 665) as comparison with the lead compound 6 (EC50 = 0.59 µmol/L, SI = 9). These two compounds 14aj and 13i were more sensitive than 6 toward clinically relevant mutant L100I, K103N and E138K viruses, which were further evaluated for their activity against wild-type reverse transcriptase and displayed a good correlation with the cell-based activity. Preliminary molecular modeling investigations provided insight for further structural optimization of HEPT.

Structure-Based design of [(2-Hydroxyethoxy)methyl]-6-(phenylthio)-thymine derivatives as nonnucleoside HIV-1 reverse transcriptase Inhibitors: From HEPTs to Sulfinyl-substituted HEPTs

The [(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs were reported to be a kind of promising lead compounds as nonnucleoside HIV-1 reverse transcriptase inhibitors. In this work, a series of novel sulfinyl-substituted analogs were designed by structure-based design strategy with the purpose of improving the activity of HEPT, followed by evaluating their anti-HIV-1 activity in MT-4 cells. Most of the final compounds had moderate to strong activity against wild-type HIV-1 strain (IIIB) with EC50 values in the range of 0.21–1.91 μM, which were around 4 ∼ 32-fold better than the reference compound HEPT. Some of them showed higher sensitivity toward clinically relevant mutant L100I and E138K viruses than NVP. Selected compounds were further evaluated for their activity against wild-type reverse transcriptase (RT), and most of them exhibited nanomolar activity, suggesting a good correlation with the cell-based activity. The compounds 11h, 11l, and 11ab displayed the best anti-HIV-1 activity against wild-type HIV-1 strain (EC50 = 0.280, 0.209, and 0.290 μM) and nanomolar activity against mutant strains (L100I and E138K), superior to HEPT and NVP. Molecular modeling studies were also performed to elucidate the biological activity, providing a structural insight for follow-up research on HEPT optimization.

CHARACTERISATION AND STUDY OF 1- [2- (2-BENZOYLPHENOXY) ETHYL] -6-METHYLURACIL MECHANISM OF ACTION

The aim of the study is to identify 1-[2-(2-benzoylphenoxy) ethyl]-6-methyluracil using various methods of analysis, as well as to study its action mechanism against wild-type and mutant forms of HIV-1 reverse transcriptase (RT).Materials and methods. To characterize the structure of the test substance, a few kinds of analysis (X-ray diffraction, elemental, thermal) as well as a few kinds of spectroscopy (UV, IR, and NMR) have been used. The study of the action mechanism of the compound as a potential drug was carried out by evaluating the inhibitory activity against HIV-1 RT wild-type and its mutant forms corresponding to drug-resistant viral strains.Results. The studies have been carried out to confirm the structure of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil. The UV spectrum has a pronounced absorption maximum when measuring a solution of the substance in tetrahydrofuran at the concentration of 0.10 mg / ml. In the IR spectrum, there are specific bands in the range of 4000-370 cm–1. These factors make it possible to use UV and IR spectra to identify the test compound in the substance. It has also been established that the number and mutual arrangement of functional groups, the integrated intensity of signals in the 1H-NMR spectrum, as well as the structure of the carbon skeleton, correspond to the structure of 1-[2-(2-benzoylphenoxy) ethyl]-6-methyluracil. The results of studying the action mechanism showed that the test compound is an effective inhibitor of wild-type HIV-1 RT with an inhibition constant of 0.2 µM, as well as an enzyme inhibitor (mutation G190A) with an inhibition constant of 8 µM; enzyme (mutation Y181C) with an inhibition constant of 10 µM, as well as a reverse transcriptase (RT) inhibitor (mutation L100I, K103N, V106A) and a double mutant K103N / Y181C with an inhibition constant of more than 20 µM.Conclusion. As a result of the performed X-ray structural, elemental, 1H-NMR and 13C-NMR analyzes, the structure of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil has been confirmed. The possibility of using UV, IR and NMR spectroscopy, as well as thermal analyzes to confirm the authenticity during the verification of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil, has been shown. The developed methods can be used in the quality control and included in the draft of practice guidelines for the investigated substance. The studies of the action mechanism of the compound of HIV-1 RT reverse transcriptase have shown that this compound belongs to the group of non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1.

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction.

HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342–351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of β-sheets 17 and 18 and their connecting loop (residues 342–350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.

Small Conformational Changes Underlie Evolution of Resistance to NNRTI in HIV Reverse Transcriptase

Despite achieving considerable success in reducing the number of fatalities due to acquired immunodeficiency syndrome, emergence of resistance against the reverse transcriptase (RT) inhibitor drugs remains one of the biggest challenges of the human immunodeficiency virus antiretroviral therapy (ART). Non-nucleoside reverse transcriptase inhibitors (NNRTIs) form a large class of drugs and a crucial component of ART. In NNRTIs, even a single resistance mutation is known to make the drugs completely ineffective. Additionally, several inhibitor-bound RTs with single resistance mutations do not exhibit any significant variations in their three-dimensional structures compared with the inhibitor-bound RT but completely nullify their inhibitory functions. This makes understanding the structural mechanism of these resistance mutations crucial for drug development. Here, we study several single resistance mutations in the allosteric inhibitor (nevirapine)-bound RT to analyze the mechanism of small structural changes leading to these large functional effects. In this study, we have shown that in absence of significant conformational variations in the inhibitor-bound wild-type RT and RT with single resistance mutations, the protein contact network analysis of their static structures, along with molecular dynamics simulations, can be a useful approach to understand the functional effect of small local conformational variations. The simple network analysis exposes the localized contact changes that lead to global rearrangement in the communication pattern within RT. Furthermore, these conformational changes have implications on the overall dynamics of RT. Using various measures, we show that a single resistance mutation can change the network structure and dynamics of RT to behave more like unbound RT, even in the presence of the inhibitor. This combined coarse-grained contact network and molecular dynamics approach promises to be a useful tool to analyze structure-function studies of proteins that show large functional changes with negligible variations in their overall conformation.

Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV agents targeting reverse transcriptase enzyme

Infection by human immunodeficiency virus still represents a continuous serious concern and a global threat to human health. Due to appearance of multi-resistant virus strains and the serious adverse side effects of the antiretroviral therapy administered, there is an urgent need for the development of new treatment agents, more active, less toxic and with increased tolerability to mutations. Quinoxaline derivatives are an emergent class of heterocyclic compounds with a wide spectrum of biological activities and therapeutic applications. These types of compounds have also shown high potency in the inhibition of HIV reverse transcriptase and HIV replication in cell culture. For these reasons we propose, in this work, the design, synthesis and biological evaluation of quinoxaline derivatives targeting HIV reverse transcriptase enzyme. For this, we first carried out a structure-based development of target-specific compound virtual chemical library of quinoxaline derivatives. The rational construction of the virtual chemical library was based on previously assigned pharmacophore features. This library was processed by a virtual screening protocol employing molecular docking and 3D-QSAR. Twenty-five quinoxaline compounds were selected for synthesis in the basis of their docking and 3D-QSAR scores and chemical synthetic simplicity. They were evaluated as inhibitors of the recombinant wild-type reverse transcriptase enzyme. Finally, the anti-HIV activity and cytotoxicity of the synthesized quinoxaline compounds with highest reverse transcriptase inhibitory capabilities was evaluated. This simple screening strategy led to the discovery of two selective and potent quinoxaline reverse transcriptase inhibitors with high selectivity index.

DNA polymerase activity on synthetic N3'→P5' phosphoramidate DNA templates.

Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3′→P5′ phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.

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.

Aryl Substituted Benzimidazolones as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors.

Since the discovery of HIV as the etiological agent of AIDS, the virus has infected millions of people each year. Fortunately, with the use of HAART, viremia can be suppressed to below detectable levels in the infected individuals, which significantly improves their quality of life and prevents the onset of AIDS. However, HAART is not curative and issues relating to adherence and drug resistance may lead to the re-emergence of viremia, the development of AIDS, and ultimately death. To address a pressing need for the development of new and efficacious antiretroviral agents with activity against viruses bearing prevalent resistant mutations, we have designed two generations of benzimidazolone derivatives as HIV non-nucleoside reverse transcriptase inhibitors. The first generation benzimidazolone inhibitors were found to be potent inhibitors of wild-type HIV reverse transcriptase but were ineffective in the presence of common resistance mutations such as K103N and Y181C. A second generation benzimidazolone inhibitor (compound 42) not only showed inhibitory activity against wild-type HIV but also remained active against HIV containing the K103N, Y181C, and K103N/Y181C drug resistance mutations.

Discovery of the Novel Entecavir-Resistant Hepatitis B Virus Reverse Transcriptase A181C Substitution From an Integrated Genotypic Analysis.

Entecavir (ETV) is a first‐line therapy for chronic hepatitis B virus (HBV), demonstrating potent suppression of HBV DNA and a high barrier to viral resistance. Previous studies revealed that ETV‐resistant (ETVr) HBV DNA resulted from substitutions in the HBV reverse transcriptase (RT) at positions rtT184, rtS202, or rtM250 in combination with lamivudine resistance (LVDr) substitutions rtM204I/V±rtL180M. In vitro, viral variants exhibit varying degrees of ETV susceptibility and replication capacity depending on specific resistance substitutions. To explore the potential for additional pathways to ETVr, HBV RT sequences from 982 evaluable patients enrolled in 17 ETV clinical studies were analyzed. Thirty novel emergent substitutions at amino acid positions not previously associated with HBV nucleos(t)ide drug resistance were observed in at least 2 patients and were identified in patient‐derived HBV with a wild‐type, LVDr, or ETVr RT sequence. Phenotypic analysis of these substitutions indicated that they had no effect on ETV susceptibility. Phenotypic analysis was also performed on patient‐derived HBV RT sequences from 10 LVD‐naive and 13 LVD‐experienced patients with virologic breakthrough and emergent novel substitutions while on ETV treatment. One LVD‐experienced patient‐derived HBV RT harboring LVDr substitutions rtL180M+rtM204V with rtA181C displayed reduced ETV susceptibility (122‐fold greater than wild‐type HBV) and remained susceptible to adefovir and tenofovir. HBV harboring the rtA181C substitution without LVDr substitutions rtL180M+rtM204V remained susceptible to inhibition by ETV, adefovir, and tenofovir, although cross‐resistance to LVD and telbivudine was observed. Conclusion: An integrated genotypic analysis of HBV RT sequences from patients with chronic HBV treated with ETV led to the discovery of the novel ETVr substitution rtA181C. This substitution was always detected in combination with LVDr substitutions rtL180M+rtM204V in ETV‐treated patients.

Structural basis for potent and broad inhibition of HIV-1 RT by thiophene[3,2-d]pyrimidine non-nucleoside inhibitors.

Rapid generation of drug-resistant mutations in HIV-1 reverse transcriptase (RT), a prime target for anti-HIV therapy, poses a major impediment to effective anti-HIV treatment. Our previous efforts have led to the development of two novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) with piperidine-substituted thiophene[3,2-d]pyrimidine scaffolds, compounds K-5a2 and 25a, which demonstrate highly potent anti-HIV-1 activities and improved resistance profiles compared with etravirine and rilpivirine, respectively. Here, we have determined the crystal structures of HIV-1 wild-type (WT) RT and seven RT variants bearing prevalent drug-resistant mutations in complex with K-5a2 or 25a at ~2 Å resolution. These high-resolution structures illustrate the molecular details of the extensive hydrophobic interactions and the network of main chain hydrogen bonds formed between the NNRTIs and the RT inhibitor-binding pocket, and provide valuable insights into the favorable structural features that can be employed for designing NNRTIs that are broadly active against drug-resistant HIV-1 variants.

A Machine Learning Approach for Predicting HIV Reverse Transcriptase Mutation Susceptibility of Biologically Active Compounds.

HIV resistance emerging against antiretroviral drugs represents a great threat to the continued prolongation of the lifespans of HIV-infected patients. Therefore, methods capable of predicting resistance susceptibility in the development of compounds are in great need. By targeting the major reverse transcription residues Y181, K103, and L100, we used the biological activities of compounds against these enzymes and the wild-type reverse transcriptase to create Naïve Bayes Networks. Through this machine learning approach, we could predict, with high accuracy, whether a compound would be susceptible to a loss of potency due to resistance. Also, we could perfectly predict retrospectively whether compounds would be susceptible to both a K103 mutant RT and a Y181 mutant RT. In the study presented here, our method outperformed a traditional molecular mechanics approach. This method should be of broad interest beyond drug discovery efforts, and serves to expand the utility of machine learning for the prediction of physical, chemical, or biological properties using the vast information available in the literature.

Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid (“surrogate challenge”). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199–220 and aa 528–543. Drug-resistance mutations disrupted the epitope at aa 205–220, while the CTL epitope at aa 202–210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.