HIV-1 Reverse Transcriptase Inhibitors Research Articles

Design and synthesis of a novel series of non-nucleoside HIV-1 inhibitors bearing pyrimidine and N-substituted aromatic piperazine

A novel series of substituted piperazine-1-yl-pyrimidine derivatives were designed and synthesized as a new type of HIV-1 non-nucleoside inhibitors. Various N-substituted aromatic groups were incorporated into the piperazine ring through a simple and practical route to investigate the biological activity of these target compounds against wild-type and resistant strains of HIV-1. All of the target compounds were also evaluated as HIV-1 reverse transcriptase inhibitors in MT-4 cell cultures. The biological results showed that six of these compounds displayed inhibitory activities against the wild-type strain, among of which 7q and 7t were found to be the two most active analogues possessing EC50 values of 31.50 μM and 3.36 μM, respectively. Molecular modeling studies of 7q provide valuable information for developing new anti-HIV-1 inhibitors.

Phytochemical analysis of Canna indica L. roots and rhizomes extract

Canna indica L. (Cannaceae) roots and rhizomes were reported to possess various biological properties like antimicrobial, anthelmintic potential and HIV-1 reverse transcriptase inhibition. In our previous studies, they showed antidiabetic activity on normal rats and rats co-addicted with caffeine and nicotine. In the pursuit of the phytochemical/s responsible for these biological activities, present study was aimed at phytochemical evaluation of hydroalcoholic extract (HAE) of C. indica L. roots and rhizomes; including preliminary screening, thin layer chromatography, H1-NMR and HR-LC/MS-MS analysis. After preliminary detection of flavonoids, tannins and sterols, HAE was tested for presence of β-sitosterol using TLC. H1-NMR spectrum of HAE revealed the presence of around 761 deshielded protons corresponding to different polar compounds. HR-LC/MS-MS analysis carried out at both positive and negative ion mode, indicated the presence of more than 90 compounds including short fragment of peptide. As per METLIN database, predicted major phytochemicals were 3′-hydroxytrimethoprim, 3,7-epoxycaryophyllan-6-one, swietenine, typhasterol, hexacosanedioic acid and 3β, 6α,7α-trihydroxy-5β-cholan-24-oic acid few of which, are biologically active.

Hydrophilic and amphiphilic water-soluble dendrimer prodrugs suitable for parenteral administration of a non-soluble non-nucleoside HIV-1 reverse transcriptase inhibitor thiocarbamate derivative

Drugs delivered by proper carriers enter into the cells much more rapidly and carry out their action much more promptly than in the free forms. A high drug concentration can be sustained for longer periods of time at the target site in the cell. In in vivo conditions, this would translate into a reduction of systemic toxicity, dosage and frequency of dosing. Dendritic polymers significantly affect drug delivery in terms of reaching the target site, modifying the bio-distribution of the drug, and enhancing the efficacy of different drugs including anticancer compounds. 2-({[2-({[(2-tolyl)amino]carbonothioyl}oxy)ethyl]amino}carbo-nyl)benzoic acid 1 is a thiocarbamate derivative belonging to an already reported class of non-nucleoside HIV-1 reverse transcriptase inhibitors. In in vitro assay it showed no cytotoxic effects but was endowed with very low solubility and poor activity against wild-type HIV-1 (EC50 = 27 μM). With the aim at improving its water solubility, 1 has been successfully incorporated inside non-toxic amino acids-modified core-shell hetero dendrimers. IR, NMR, zeta potential, mean size of particles, buffer capacity and in vitro release profile of prepared materials were reported. All dendriplexes were evaluated in cell-based assays to assess their cytotoxic profile. The obtained complexes, which harmonize a peripheral polycationic character and a buffer capacity which presuppose efficient cells penetration and increased residence time with a not PAMAM structured biodegradable scaffold, were well water-soluble and could rationally appear as a promising set of prodrugs for safe in vivo administrations.

Design, synthesis, docking studies and biological screening of 2-thiazolyl substituted -2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazines as potent HIV-1 reverse transcriptase inhibitors

1,3-oxazine nucleus and thiazolyl group features prominently in many biologically important natural products as well as bioactive molecules. A series of novel 2-thiazolyl substituted-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine derivatives were designed and synthesized based on their structure-activity relationships (SARs) from 2-naphthol, substituted thiazolyl amines and formalin through ring closure by one-pot three component reaction. These derivatives were first evaluated for their inhibitory effect on HIV-1 Reverse Transcriptase (RT) enzyme activity. Out of 14 compounds, 4 showed potent inhibition of HIV-1 RT activity at significantly low concentration. Docking studies of these molecules revealed their high affinity binding to several amino acids of HIV-1 RT which are less sensitive to point mutations. Furthermore, anti-HIV activity of these molecules was analysed in a CD4+ T cell-line, which indicates that Therapeutic Index (TI) of some of these compounds is better than Zidovudine and Efavirenz, known HIV-1 RT inhibitors. Taken together, our studies report for the first time some novel naphthoxazine derivatives with significant TI, which is through inhibition of HIV-1 RT activity.

Uvaria angolensis as a promising source of inhibitors of HIV-1 Reverse Transcriptase associated RNA-dependent DNA polymerase and RNase H functions

Uvaria angolensis as a promising source of inhibitors of HIV-1 Reverse Transcriptase associated RNA-dependent DNA polymerase and RNase H functions

Docking-based 3D-QSAR and pharmacophore studies on diarylpyrimidines as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Diarylpyrimidines (DAPYs), a type of effective HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), have been considered as one of the most successful agents for treating AIDS. A number of structurally diverse DAPYs have been designed and synthesized in the past decade, and most of them exhibited potent anti-HIV-1 activities; however, the structure-activity relationships of recently reported DAPYs and their pharmacophore features that interacted with HIV-1 reverse transcriptase (RT) remain to be studied. In the present study, molecular docking studies were first performed on three novel classes of DAPYs to study their binding pattern in the HIV-1 RT. Based on the docking conformations of these DAPYs, 3D-QSAR models were constructed using CoMSIA and Topomer CoMFA methods, and pharmacophore models were also built using distance comparison technique. All selected DAPYs presented preferred U- or L-shaped conformations while being docked into the non-nucleoside inhibitor-binding pocket of the HIV-1 RT. The best CoMSIA model exhibited powerful predictivity, with satisfactory statistical parameters such as a q2 of 0.572, an r2 of 0.952, and an [Formula: see text] of 0.728. Contour maps of the best CoMSIA model were in accordance with those of the Topomer CoMFA model, giving the insight into the feature requirements of DAPYs for the anti-HIV-1 activity. Three potential pharmacophore models were constructed, and each of them was consisted of five hypothesis features. All results suggested that the aromatic ring on the left wing of DAPYs and the central pyrimidine ring contained key pharmacophore features for the anti-HIV-1 activity, and also indicated that the right wing of DAPYs had potential for further structural modification to improve activity. Eight novel DAPY molecules with potential anti-HIV-1 activities were designed on the basis of the obtained results. The findings in this study might provide important information for further design and development of novel HIV-1 NNRTIs.

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.

HIV antiretroviral therapy drugs induce premature senescence and altered physiology in HUVECs

Developments in medicine have led to a significant increase in the average human lifespan. This increase in aging is most readily apparent in the case of HIV where antiretroviral therapy has shifted infection from a terminal to a chronic but manageable disease. Despite this advance, patients suffer from co-morbidities best described as an accelerated aging phenotype. A potential contributor is cellular senescence, an aging-associated growth arrest, which has already been linked to other HIV co-morbidities such as lipodystrophies and osteoporosis in response to antiretroviral drugs. We have previously demonstrated that astrocytes senescence in response to antiretroviral drugs. As endothelial cells play a critical role regulating the blood brain barrier (BBB) and senescence could severely impact barrier permeability, we investigate the role of a commonly used combination of HIV reverse transcriptase inhibitors on the senescence program of human umbilical vein endothelial cells (HUVECs). Our studies indicate that HUVECs underwent premature senescence associated with inflammation, oxidative stress and altered eNOS activation. Treated cells had detrimental paracrine effects on astrocytes including paracrine senescence, suggesting that senescent HUVECs could influence astrocytes, which line the other side of the BBB. These results may have implications for HIV-associated neurocognitive disorders (HAND), a set of neurological deficits.

In Silico SAR Studies of HIV-1 Inhibitors.

Quantitative Structure Activity Relationships (QSAR or SAR) have helped scientists to establish mathematical relationships between molecular structures and their biological activities. In the present article, SAR studies have been carried out on 89 tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepine (TIBO) derivatives using different classifiers, such as support vector machines, artificial neural networks, random forests, and decision trees. The goal is to propose classification models that will be able to classify TIBO compounds into two groups: high and low inhibitors of HIV-1 reverse transcriptase. Each molecular structure was encoded by 10 descriptors. To check the validity of the established models, all of them were subjected to various validation tests: internal validation, Y-randomization, and external validation. The established classification models have been successful. The correct classification rates reached 100% and 90% in the learning and test sets, respectively. Finally, molecular docking analysis was carried out to understand the interactions between reverse transcriptase enzyme and the TIBO compounds studied. Hydrophobic and hydrogen bond interactions led to the identification of active binding sites. The established models could help scientists to predict the inhibition activity of untested compounds or of novel molecules prior to their synthesis. Therefore, they could reduce the trial and error process in the design of human immunodeficiency virus (HIV) inhibitors.

Binding property of HIV p24 and Reverse transcriptase by chalcones from Pongamia pinnata seeds.

HIV remains a challenging life threatening viral agent for humans despite available anti HIV drugs. The known effective drug named HAART clears the circulating viruses but not the intracellular viruses. Therefore, it is of interest to identify molecules with improved anti-HIV activity from natural plant sources. Hence, we studied the anti-HIV potency of an Indian medicinal plant named Pongamia pinnata. Aqueous extracts were made from leaf, seed and roots of Pongmia pinnata and screened for anti HIV-1 activity using HIV-1 p24 and reverse transcriptase (RT) inhibition assays. Further, the active chalcone derivatives namely, P24 protein and RT enzymes showed promising binding score against Glabarachalcone and Karanijin. Among these extracts, P. pinnta aqueous seed extracts have shown HIV-1 p24 inhibition at 66.9 ± 4.4 percentage. However, RT inhibition assay showed only 36.8%. Hence, the HIV-1 p24 inhibition infers either the prevention of virus entry or inhibits other enzymes and or interferes with virion assembly.

Application of Substituent Electronic Descriptors QSAR Model of 2-amino-6-arylsulfonylbenzonitriles as HIV-1 Reverse Transcriptase Inhibitors based on the MOLMAP Approach

The HIV-1 reverse transcriptase (RT) is a major target for drug development. Inhibition of this enzyme has been one of the primary therapeutic strategies in suppressing the replication of HIV-1. A series of 2-amino-6-arylsulfonylbenzonitrile derivatives were subjected to quantitative structure-activity relationship (QSAR) analysis. Very recently, we proposed the use of substituent electronic descriptors (SED) instead of the electronic descriptors of whole molecules as new and expedite source of electronic descriptors. In this study, we used SED parameters in QSAR modeling of anti HIV-1 activity of 6-arylsulfonylbenzonitrile derivatives. In SED methodology produces a vector of electronic descriptors for each substituent and thus a matrix of SED is generated for each molecule. Consequently, a three-dimensional array is obtained by staking the data matrices of different molecules beside each other. As a novel multiway data analysis method, molecular maps of atom-level properties (MOLMAP) approach was also used to transfer a three-dimensional array of SED descriptors into new two-dimensional parameters using Kohonen network, following by genetic algorithm-based partial least square(GA-PLS) to connect a quantitative relationship between the Kohonen scores and biological activity.In unfolding data, HOMO1, HOMOB1, SOFB1 and EPHA4 represent the most important indices on QSAR equation derived by PLS analysis. Accurate QSAR models were obtained by both approaches. The resulted GA-PLS model of MOLMAP approach possessed high statistical quality r2= 0.83 and q2=0.70. It could explain and predict about 70% of variances in the anti-HIV1 inhibitory activity of the studied molecules. However, the superiority of three-way analysis of SED parameters based on MOLMAP approach with respect to simple unfolding was obtained.

A transgenic mouse model for HLA-B*57:01-linked abacavir drug tolerance and reactivity.

Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

QSAR studies of TIBO derivatives as HIV-1 reverse transcriptase inhibitors using HQSAR, CoMFA and CoMSIA

The study deals with CoMFA, CoMSIA and HQSAR to explore the important features of tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepinone (TIBO) derivatives for exerting potent HIV-1 reverse transcriptase (HIV-1 RT) inhibitors activity. The cross-validated q2 value of CoMFA model is 0.641 and the non-cross-validated r2 value is 0.847. The best cross-validated q2 value of CoMSIA Model is 0.706 and the non-cross-vaildated r2 value is 0.939. The most effective HQSAR model was obtained that the cross-validation q2 value of 0.839, the non-cross-validated r2 value of 0.942, the standard error of prediction SDCV value of 0.604, and the best hologram length value of 307 using atoms and bonds as fragment distinctions. The statistical parameters from models indicate that the data are well fitted and have high predictive ability. Furthermore, Molecular docking was employed to explore the binding requirements between the ligands and the receptor protein which included several hydrogen bonds between the TIBO inhibitors and active site residues. Observations derived from these QSAR modeling study may be utilized further in designing promising HIV-1 reverse transcriptase inhibitors.

Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067.

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.

Inhibition of HIV-1 Reverse Transcriptase of Selected Indonesia Medicinal Plants and Isolation of the Inhibitor from Erythrina variegata L. Leaves

Objective: This research was conducted to screen inhibition of HIV-1 reverse transcriptase activity of selected Indonesia medicinal plants and to isolate HIV-1 reverse transcriptase inhibitor from Erythrina variegata leaves. Method: Screening inhibition of HIV-1 RT activity of selected Indonesia medicinal plants and isolated compounds were performed using HIV-1 RT colorimetric assay. The isolation of HIV-1 reverse transcriptase inhibitor was conducted using chromatography technique. The isolated compound was determined based on the data of UV, IR spectrophotometry, MS, 1D and 2D NMR spectroscopy. Results: Beside that, in vitro study of the leaves methanolic extract exhibited inhibition against HIV-1 RT activity with percent inhibition of 97.64% at concentration of 5 mg/mL. Ethyl acetate fraction from the extract showed the strongest HIV-1 RT inbitory activity with IC50 of 429.28 μg/mL. Isolation the HIV-1 RT inhibitor from the fraction give compound 1. Conclusion: Erythryna variegata leave extract exhibited potent inhibition on HIV-1 RT activity. The isolated compound from the leaves was determined as apigenin-7-O-β-D-glucopyranoside and demonstrated HIV-1 RT inhibitory activity with the IC50 value of 100.59 μg/mL. Key words: Erythrina variegata, Flavonoid, HIV-1 RT, in vitro.

MALDI Mass Spectrometry Imaging Reveals Heterogenous Distribution of Tenofovir and Tenofovir‐Diphosphate in Human Colorectal Tissue

Tenofovir (TFV) disoproxil fumarate is currently used orally in combination with other antiretroviral drugs for HIV treatment and pre‐exposure prophylaxis. Tenofovir disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV, which upon phosphorylation to the nucleotide triphosphate analog TFV‐diphosphate (TFV‐DP) competitively inhibits HIV reverse transcriptase. In addition, TFV is under investigation as a topical microbicide for application to mucosal tissues that are routes of sexual transmission of HIV, such as colorectal tissue, in order to reduce the likelihood of infection upon exposure to HIV. In order to be efficacious as a microbicide it is necessary for a drug to be present in sufficient concentrations in the relevant tissues and over intervals of time; however, the disposition of TFV in mucosal tissues is not fully understood. With this in mind, we developed a Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) approach in order to determine the spatial distribution of TFV and TFV‐DP in colorectal tissue. For this study, colorectal biopsies were obtained from healthy volunteers (n=4) who received TFV‐containing enemas, and these biopsies were embedded with OCT medium and sectioned into 20 micron slices. Using α‐cyano‐4‐hydroxycinnamic acid as the matrix, both TFV and TFV‐DP were detected in these samples as their protonated molecular ions, [M+H]+ ions at m/z 288.0861 ± 5 ppm and 448.0188 ± 5 ppm, respectively, in positive ion mode. Confirmation of the detection of TFV and TFV‐DP in the colorectal biopsies was performed by comparing the ionization patterns to their authentic standards that yielded [M+H]+ ions at m/z 288.0851 and 448.0169, respectively. Interestingly, the MALDI MS ion images of TFV and TFV‐DP revealed heterogeneity in the distribution of both TFV and the pharmacologically active metabolite, TFVDP. In addition, a time dependence in the abundance of TFV and TFV‐DP was evident, as the intensities of both the TFV and TFV‐DP ions were greater in biopsies collected 6 hours following treatment as compared to those collected at 24 hours. In order to confirm that the patterns of heterogeneity that we observed in the spatial distributions of TFV and TFV‐DP were specific to these molecules, we simultaneously imaged the spatial localization of three different lipid species, namely PC (34:1), PC (16:0/OH) and PC (36:2) at m/z 760.5856 ± 5 ppm, 496.3403 ± 5 ppm and 786.6012 ± 5 ppm, respectively. The homogeneous distribution of these phospholipids suggested that there is specificity in the observed TFV and TFV‐DP distribution in colorectal tissue. In conclusion, the above results demonstrate the heterogeneity in the spatial distribution of both TFV and TFV‐DP in colorectal tissue. Further, our results provide important insights into the disposition of topical TFV in colorectal tissue.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of agarolytic enzymes of Arthrobacter spp. AG-1 for the whole cell conversion of agar into 3,6-anhydro-α- l -galactose in one pot

Abstract 3,6-anhydro-α- l -galactose ( l -AHG) is a commercially unavailable monosaccharide with high therapeutic value, in particular, anti-viral. l -AHG is a selective inhibitor of HIV reverse transcriptase. Till date, the synthesis of l -AHG remains an intricate process and there is a need for a simpler approach producing l -AHG industrially. Herein, we report an agar-hydrolyzing Arthrobacter spp. AG-1 isolated from Gulf of Mannar, Tamil Nadu which completely converts agar to l -AHG with their extracellular agarase in one pot. The optimal temperature and pH for the maximal activity of Arthrobacter spp. strain AG-1 were 45 °C and pH 7 respectively. We purified and characterized the 44 kDa agarase involved in the glycoside hydrolysis. The Vmax, Km, and Kcat value of the agarase were determined as 1.7848 × 10−2 U/mg, 0.01825 ± 0.001 M and 0.1748 ± 0.006 S−1 respectively. Presence of l -AHG was confirmed by TLC. Detailed ESI–MS revealed the presence of a relatively higher abundance of l -AHG and chemical fingerprinting analysis by FT-IR, GC–MS and CD confirmed that the end product as l -AHG. Additionally, l -AHG exhibited notable anti-inflammatory and antibacterial activity. The whole cell biocatalytic system is predicted as a starting point for a more detailed study on Arthrobacter spp. extracellular agarase.

How to Control HTLV-1-Associated Diseases: Preventing de Novo Cellular Infection Using Antiviral Therapy.

Five to ten million individuals are infected by Human T-cell Leukemia Virus type 1 (HTLV-1). HTLV-1 is transmitted through prolonged breast-feeding, by sexual contacts and by transmission of infected T lymphocytes through blood transfusion. One to ten percent of infected carriers will develop a severe HTLV-1-associated disease: Adult-T-cell leukemia/lymphoma (ATLL), or a neurological disorder named Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). In vivo, HTLV-1 is mostly detected in CD4+ T-cells, and to a lesser extent in CD8+ T cells and dendritic cells. There is a strong correlation between HTLV-1 proviral load (PVL) and clinical status of infected individuals. Thus, reducing PVL could be part of a strategy to prevent or treat HTLV-1-associated diseases among carriers. Treatment of ATLL patients using conventional chemotherapy has very limited benefit. Some chronic and acute ATLL patients are, however, efficiently treated with a combination of interferon α and zidovudine (IFN-α/AZT), to which arsenic trioxide is added in some cases. On the other hand, no efficient treatment for TSP/HAM patients has been described yet. It is therefore crucial to develop therapies that could either prevent the occurrence of HTLV-1-associated diseases or at least block the evolution of the disease in the early stages. In vivo, reverse transcriptase (RT) activity is low in infected cells, which is correlated with a clonal mode of viral replication. This renders infected cells resistant to nucleoside RT inhibitors such as AZT. However, histone deacetylase inhibitors (HDACi) associated to AZT efficiently induces viral expression and prevent de novo cellular infection. In asymptomatic STLV-1 infected non-human primates, HDACi/AZT combination allows a strong decrease in the PVL. Unfortunately, rebound in the PVL occurs when the treatment is stopped, highlighting the need for better antiviral compounds. Here, we review previously used strategies targeting HTLV-1 replication. We also tested a series of HIV-1 RT inhibitors in an in vitro anti-HTLV-1 screen, and report that bis-POM-PMEA (adefovir dipivoxil) and bis-POC-PMPA (tenofovir disoproxil) are much more efficient compared to AZT to decrease HTLV-1 cell-to-cell transmission in vitro. Our results suggest that revisiting already established antiviral drugs is an interesting approach to discover new anti-HTLV-1 drugs.

Genetic Variation of the Kinases That Phosphorylate Tenofovir and Emtricitabine in Peripheral Blood Mononuclear Cells.

Tenofovir (TFV) disoproxil fumarate and emtricitabine (FTC) are used in combination for HIV treatment and pre-exposure prophylaxis (PrEP). TFV disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV. FTC and TFV are nucleoside/nucleotide reverse transcriptase inhibitors that upon phosphorylation to nucleotide triphosphate analogs competitively inhibit HIV reverse transcriptase. We previously demonstrated that adenylate kinase 2, pyruvate kinase, muscle and pyruvate kinase, liver and red blood cell phosphorylate TFV in peripheral blood mononuclear cells (PBMC). To identify the kinases that phosphorylate FTC in PBMC, siRNAs targeted toward kinases that phosphorylate compounds structurally similar to FTC were delivered to PBMC, followed by incubation with FTC and the application of a matrix-assisted laser desorption ionization-mass spectrometry method and ultra high performance liquid chromatography-UV to detect the formation of FTC phosphates. Knockdown of deoxycytidine kinase decreased the formation of FTC-monophosphate, while siRNA targeted toward thymidine kinase 1 decreased the abundance of FTC-diphosphate. Knockdown of either cytidine monophosphate kinase 1 or phosphoglycerate kinase 1 decreased the abundance of FTC-triphosphate. Next-generation sequencing of genomic DNA isolated from 498 HIV-uninfected participants in the HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 clinical study, revealed 17 previously unreported genetic variants of TFV or FTC phosphorylating kinases. Of note, four individuals were identified as simultaneous carriers of variants of both TFV and FTC activating kinases. These results identify the specific kinases that activate FTC in PBMC, while also providing further insight into the potential for genetic variation to impact TFV and FTC activation.

Pharmacophore requirements for HIV-1 reverse transcriptase inhibitors that selectively “Freeze” the pre-translocated complex during the polymerization catalytic cycle

Reverse transcriptase (RT) is responsible for replicating the HIV-1 genome and is a validated therapeutic target for the treatment of HIV infections. During each cycle of the RT-catalyzed DNA polymerization process, inorganic pyrophosphate is released as the by-product of nucleotide incorporation. Small molecules were identified that act as bioisosteres of pyrophosphate and can selectively freeze the catalytic cycle of HIV-1 RT at the pre-translocated stage of the DNA- or RNA-template-primer-enzyme complex.