HIV-1 Integrase Strand Transfer Research Articles

Inhibition of lipid synthesis by the HIV integrase strand transfer inhibitor elvitegravir in primary rat oligodendrocyte cultures.

Combined antiretroviral therapy (cART) has greatly decreased mortality and morbidity among persons with HIV; however, neurologic impairments remain prevalent, in particular HIV-associated neurocognitive disorders (HANDs). White matter damage persists in cART-treated persons with HIV and may contribute to neurocognitive dysfunction as the lipid-rich myelin membrane of oligodendrocytes is essential for efficient nerve conduction. Because of the importance of lipids to proper myelination, we examined the regulation of lipid synthesis in oligodendrocyte cultures exposed to the integrase strand transfer inhibitor elvitegravir (EVG), which is administered to persons with HIV as part of their initial regimen. We show that protein levels of genes involved in the fatty acid pathway were reduced, which correlated with greatly diminished de novo levels of fatty acid synthesis. In addition, major regulators of cellular lipid metabolism, the sterol regulatory element-binding proteins (SREBP) 1 and 2, were strikingly altered following exposure to EVG. Impaired oligodendrocyte differentiation manifested as a marked reduction in mature oligodendrocytes. Interestingly, most of these deleterious effects could be prevented by adding serum albumin, a clinically approved neuroprotectant. These new findings, together with our previous study, strengthen the possibility that antiretroviral therapy, at least partially through lipid dysregulation, may contribute to the persistence of white matter changes observed in persons with HIV and that some antiretrovirals may be preferable as life-long therapy.

RP-HPLC hyphenated with ESI -Q-TOF –MS for the detection of potential degradants in dolutegravir

Dolutegravir is an antiviral agent, a second-generation HIV integrase strand transfer inhibitor (INSTI). A precise liquid chromatography-tandem mass spectrometry method has been developed and validated for the detection of potential degradants in Dolutegravir. The goal of this study was to detect major and minor potential degradants that can develop during the various stages of the drug molecule, start from acquiring of raw material for the synthesis, manufacturing, formulation and storage of the final product. With this objective in mind preparative scale HPLC was used to detect possible oxidative degradants in Dolutegravir. The oxidative degradation sample was isolated using Prominence preparative high performance liquid chromatograph equipped with a reverse phase C18 Inertsil ODS (300mm×19mm ×7µ) column. Mobile phase was 0.1% trifluoro acetic acid in water (A) and acetonitrile (B) (50:50). Ethyl acetate was used for the extraction of aqueous layer and the same was distilled and the collected fractions were evaporated to dryness for LCMS studies. To detect the degradation products mass spectra of the drug was established by direct infusion of degradant into mass spectrometry systems. The LC-MS spectrum of Dolutegravir was recorded by Shimadzu LCMS spectrometer in the electron spray ionization mode. The outcome was intense protonated molecular ion peak at m/z 304.27 and 249.06 in the positive ionization mode, scan range 120–560 amu and the other possible degradant structures with m/z 320.32, m/z 207.18, m/z 185.17, m/z 385.41, m/z 155.14. The information obtained from degradation behaviour of dolutegravir by stress degradation studies, under peroxide oxidation and the degradation products formed were identified by HPLC, LCMS analytical techniques are useful information regarding manufacturing, formulation and storage of the drug.

Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism

Dolutegravir is a highly potent HIV integrase strand transfer inhibitor that is recommended for first-line anti-retroviral treatment in all major treatment guidelines. A recent study has shown that people taking this class of anti-retroviral treatment have a substantially higher risk of early-onset cardiovascular disease, a condition shown previously to be associated with increased platelet reactivity. To date, few studies have explored the effects of dolutegravir on platelet activation. Accordingly, the current study was undertaken with the primary objective of investigating the effects of dolutegravir on the reactivity of human platelets in vitro. Platelet-rich plasma, isolated platelets, or buffy coat cell suspensions prepared from the blood of healthy adults were treated with dolutegravir (2.5–10 µg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin, or a thromboxane A2 receptor agonist U46619. Expression of platelet CD62P (P-selectin), formation of heterotypic neutrophil:platelet aggregates, and calcium (Ca2+) fluxes were measured using flow cytometry and fluorescence spectrometry, respectively. Dolutegravir caused dose-related potentiation of ADP-, thrombin- and U46619-activated expression of CD62P by platelets, as well as a significant increases in formation of neutrophil:platelet aggregates. These effects were paralleled by a spontaneous, receptor-independent elevation in cytosolic Ca2+ that appears to underpin the mechanism by which the antiretroviral agent augments the responsiveness of these cells to ADP, thrombin and U46619. The most likely mechanism of dolutegravir-mediated increases in platelet cytosolic Ca2+ relates to a combination of lipophilicity and divalent/trivalent metal-binding and/or chelating properties of the anti-retroviral agent. These properties are likely to confer ionophore-type activities on dolutegravir that would promote movement of Ca2+ across the plasma membrane, delivering the cation to the cytosol where it would augment Ca2+-dependent intracellular signaling mechanisms. These effects of dolutegravir may lead to hyper-activation of platelets which, if operative in vivo, may contribute to an increased risk for cardiometabolic co-morbidities.

Development of Synthetic Approaches Towards HIV Integrase Strand Transfer Inhibitors (INSTIs)

AbstractA clear and precise report on synthetic methods for INSTIs and their intermediates are provided in the present work. HIV Integrase strand transfer inhibitors (INSTIs), a class of antiretroviral agents employed to treat HIV infected patients are placed as the second option of treatment after nucleosides. As INSTIs are employed as first line therapy to newly infected HIV positive individuals, thus synthetic routes must be easily accessible to the medicinal chemists towards lead optimization or process development. The current perspective elaborates synthetic routes of first to next generation INSTIs investigated in two decades of research and their development efforts. Further, drug resistance occurred by mutations against usage of INSTI drugs with respect to their generation also discussed. Later, discussion about synthetic development and investigation of next generation INSTIs against drug resistance HIV infections also covers to fill the gap between first/second generation INSTIs. The current perspective will draw attention of medicinal chemists and multi‐disciplinary researchers in HIV drug discovery.

Advances in the development of HIV integrase strand transfer inhibitors

HIV-1 integrase (IN) is a key enzyme in viral replication that catalyzes the covalent integration of viral cDNA into the host genome. Currently, five HIV-1 IN strand transfer inhibitors (INSTIs) are approved for clinical use. These drugs represent an important addition to the armamentarium for antiretroviral therapy. This review briefly illustrates the development history of INSTIs. The characteristics of the currently approved INSTIs, as well as their future perspectives, are critically discussed.

Pharmacokinetics of vaginally applied integrase inhibitors in macaques.

We conducted a detailed pharmacokinetic assessment in macaques treated with vaginal gels formulated with HIV integrase strand transfer inhibitors (INSTIs) to better understand drug distribution and identify INSTI concentrations associated with previously demonstrated in vivo protection against vaginal simian HIV challenge. Six macaques received vaginal gel containing 1% raltegravir (30 mg) once-weekly over 6 weeks. Following a washout period, five macaques received once-weekly gel containing 0.23% L-870,812 (7 mg). Drug concentrations were measured in plasma, mucosal fluids and vaginal tissues at baseline and 2, 5 and 24 h post-dosing. The median maximum concentration (Cmax) for raltegravir and L-870,812 in plasma was below the limit of quantification and 41.1 ng/mL, respectively. The Cmax in vaginal fluids (1441 and 1250 μg/mL) and tissues (266.7 and 368.4 μg/g) was achieved 2-5 h after dosing, respectively. A similar half-life was observed for raltegravir and L-870,812 in vaginal fluids (8-10 h) and remained 3-4 orders of magnitude above the protein-adjusted IC95 (0.016 and 0.106 μg/mL, respectively) at 24 h. Drug concentrations in vaginal fluids correlated well with those in vaginal tissues (Pearson r ≥ 0.788). Both drugs were consistently detected in rectal fluids 2 h after vaginal dosing, albeit at much lower levels (31-92-fold) than those in vaginal fluids. To the best of our knowledge, this study provides the first data on INSTI levels in vaginal tissues associated with in vivo protection and demonstrates rectal drug distribution of INSTIs after vaginal dosing. These findings may inform dose selection for topical products with INSTIs for HIV prevention.

Safety and Effectiveness Analysis of Dolutegravir in Patients with HIV-1: Interim Report of Post-Marketing Surveillance in Japan.

IntroductionDolutegravir (DTG), a novel HIV-integrase strand transfer inhibitor (INSTI), is usually used with multiple antiretrovirals (ARVs) for treatment of HIV. DTG is now approved as Tivicay tablets in over 120 countries and Triumeq combination tablets (DTG/abacavir [ABC]/lamivudine [3TC]) in over 90 countries. In Japan, these formulations have been marketed since 2014 and 2015. The post-marketing prospective surveillance has been conducted as part of the HIV-Related Drug (HRD) cooperative survey aimed to collect actual drug use information in all of these DTG-treated patients in accordance with conditions for initial approvals.MethodsThe survey has been conducted to evaluate long-term safety and effectiveness of DTG since 2014, for approximately 6 years. The safety was evaluated by incidence of adverse drug reactions (ADRs) and change in body weight. The effectiveness was evaluated by plasma HIV RNA copies/mL and peripheral CD4+ cell counts.ResultsOf 2292 patients in 30 Japanese sites, 565 (24.65%) reported ADRs. The most common ADR was blood creatinine increased (4.28%). Incidence of ADRs was statistically significantly higher in patients with severe symptoms (Centers for Disease Control and Prevention [CDC] categories B and C) than those with category A, and in patients with comorbidities than those without comorbidities. Whereas incidence of ADRs was statistically significantly lower in antiretroviral therapy (ART)-experienced patients than that in ART-naïve patients. Incidence of ADRs related to suicide or self-injurious behavior was statistically significantly higher in patients with comorbidities of psychiatric disorders than those without comorbidities. The body weight tended to increase over time and those changes and percentage changes from baseline were greater in ART-naïve patients compared with ART-experienced patients. HIV RNA copies/mL and CD4+ cell counts showed favorable shifts from baseline in both ART-naïve and ART-experienced patients.ConclusionThe results of the survey identified no new safety and effectiveness risks in Japanese patients with HIV/AIDS treated with DTG.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12325-021-01842-3.

RETRACTED: A low-cost in-house HIV integrase strand transfer inhibitor drug resistance test for resource-limited settings

At the request of the authors, the article titled 'A low-cost in-house HIV integrase strand transfer inhibitor drug resistance test for resource-limited settings' ([version 1; peer review: awaiting peer review]. F1000Research 2021, 10:260, https://doi.org/10.12688/f1000research.28404.1) has been retracted from F1000Research. Since publication, it has come to the attention of the authors that the primers described in Table 1 were incorrect. As this article contains information which should not be publicly available the content of the article has been removed. The authors apologise for this honest error, and intend to republish the article with the correct primer information. Unfortunately, Dr Graeme B. Jacobs has passed away since publication of version 1 of this article.

Design, Synthesis and In Vitro Evaluation of 4-Oxo-6-Substituted Phenyl- 2-Thioxo1,2,3,4-Tetrahydropyrimidine-5-Carbonitrile Derivatives as HIV Integrase Strand Transfer Inhibitors

Aim:: To design, synthesis and in vitro evaluation of 4-oxo-6-substituted phenyl-2- thioxo1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives as HIV integrase strand transfer inhibitors. Background:: Human immunodeficiency virus-1 (HIV-1), a member of retroviridae family, is the primary causative agent of acquired immunodeficiency syndrome (AIDS). Three enzymes viz: integrase (IN), reverse transcriptase (RT) and protease play important role in its replication cycle. HIV-1 integrase is responsible for the incorporation of viral DNA into human chromosomal DNA by catalyzing two independent reactions, 3′-processing (3′-P) and strand transfer (ST), which are observed as the “point of no-return” in HIV infection. Objective:: To develop inhibitors against HIV integrase strand transfer step. Methods:: Our previous results indicated that tetrahydro pyrimidine-5-carboxamide derivatives are potent HIV-1 IN inhibitors (unpublished results from our laboratory). Taking clue from above studies and our own experience, we hypothesized 4-oxo-6-substituted phenyl-2-thioxo1,2,3,4- tetrahydropyrimidine-5-carbonitrile analogues (14a to 14n) as inhibitors of HIV-1 Integrase strand transfer. Prototype compound 14 can be viewed as hybrid structure having characteristics of dihydropyrimidine derivatives 10-12 and tyrphostin 13. Result:: A total of fourteen derivatives of 4-oxo-6-substituted phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine- 5-carbonitrile (14a-14n) were synthesized and evaluated using HIV-1 Integrase Assay Kit (Xpressbio Life Science Products, USA). The percentage inhibition of all compounds was investigated at 10 μM concentration and IC50 value of few highly active compounds was studied. The obtained results were validated by in silico molecular docking study using Glide (maestro version 9.3, Schrödinger suite) in extra precision (XP) mode. Conclusion:: Fourteen 4-oxo-6-substituted phenyl-2-thioxo 1,2,3,4-tetrahydropyrimidine-5-carbonitrile analogues were synthesized and evaluated for HIV-1 IN inhibitory activity. Three compounds 14a, 14e, and 14h exhibited significant percentage inhibition of HIV-1 IN. There was good in vitro - in silico correlation. However, none of the derivative was active against HIV-1 and HIV-2 below their cytotoxic concentration. It needs to be seen whether these compounds can be explored further for their anti-HIV or cytotoxic potential.

A low-cost in-house HIV integrase strand transfer inhibitor drug resistance test for resource-limited settings

Background: HIV-1 drug resistance testing (DRT) is vital for monitoring of individual patient treatment outcomes and for public health surveillance. Access to HIV-1 DRT is limited in resource-poor settings, including Kenya due to its costly nature. Recent inclusion of integrase strand transfer inhibitors (INSTIs) in first-line treatment for all people living with HIV-1 (PLHIV-1) underscores the need for an INSTI DRT. This study aims to validate a cost-effective in-house DRT method to detect HIV-1 Integrase resistance-associated mutations (RAMs) using HIV positive plasma derived samples Methods: Thirty-six plasma derived samples were used to assess the performance characteristics including accuracy, precision, reproducibility and amplification sensitivity of an in-house method in comparison with a reference assay. Cost estimation per test followed an incremental ingredient costing approach. Clinical application of the in-house test was evaluated on a plasma sample from a patient failing an INSTI-based regimen. Results: Comparison of the in-house and reference assay gave mean nucleotide and amino acid sequence identity of 99.49 %, CI [99.21- 99.77] and 99%, CI [98.58, 99.42] respectively. Complete concordance was observed by both assays in detection of the T97TA INSTI RAM. Precision and reproducibility assessment revealed mean nucleotide sequence identities of 100% and 99.14% respectively. The amplification sensitivity was 100% for samples with VL> 1000 copies/mL (n=8) and 50% for samples with VL<1000 copies/mL. Two major (G118R and E188K) and two accessory INSTI mutations (G149A and E157Q) were detected from the clinical sample of a patient failing an INSTI-based therapy. Cost analysis estimated the cost per test at $50.31. Conclusion: The developed HIV-Integrase assay met the validation acceptance criteria and demonstrates the ability to detect clinically relevant INSTI-resistance-associated mutations, highlighting its potential use as an alternative to commercial INSTI tests in resource-limited settings.

Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

Long Dissociation of Bictegravir from HIV-1 Integrase-DNA Complexes.

The HIV integrase (IN) strand transfer inhibitor (INSTI) bictegravir (BIC) has a long dissociation half-life (t1/2) from wild-type IN-DNA complexes: BIC 163 hr > dolutegravir (DTG) 96 hr > raltegravir (RAL) 10 hr > elvitegravir (EVG) 3.3 hr. In cells, BIC had more durable antiviral activity against wild-type HIV after drug washout than RAL or EVG. BIC also had a longer t1/2 and maintained longer antiviral activity after drug washout than DTG with the clinically relevant resistance IN mutant G140S+Q148H. Structural analyses indicate that BIC makes more contacts with the IN-DNA complex than DTG mainly via its bicyclic ring system which may contribute to more prolonged residence time and resilience against many resistance mutations.

Elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide discontinuation and return to normal weight

HIV integrase strand transfer inhibitors (INSTI) are considered well tolerated with few treatment-limiting adverse effects. However, emerging data from clinical trials has identified excessive weight gain possibly due to INSTI alone or with tenofovir alafenamide as a new and possible long-term complication of combination antiretroviral therapy (cART). Identifying who is at greatest risk and whether the unintended weight gain is reversible remain unanswered questions. We report a return to baseline weight after switching back to tenofovir disoproxil/emtricitabine/efavirenz (Atripla®) in a woman who had profound weight gain due to tenofovir alafenamide/emtricitabine/cobicistat/elvitegravir (Genvoya®).

Design, Synthesis and In Vitro Evaluation of 2-Oxo-N-substituted Phenyl- 2H-chromene-3-carboxamide Derivatives as HIV Integrase Strand Transfer Inhibitors

Background: A series of eighteen 2-Oxo-N-substituted phenyl- 2H-chromene-3- carboxamide analogues has been evaluated for HIV-1 integrase (IN) inhibition. Methods: The derivatives were synthesized via a two-step pathway commencing with 2- hydroxybenzaldehyde and diethyl malonate followed by hydrolysis of ester and coupling with various aromatic amines. The HIV-1 IN inhibitory potential of these compounds has been studied relative to dolutegravir, a known HIV-1 IN inhibitor using a standard available kit. Results: Six molecules (compounds 13h, 13i, 13l, 13p to 13r) showed significant inhibition of HIV- 1 integrase 3′-strand transfer with IC50 values less than 1.7 μM. The presence of chromene-3- carboxamide motif was shown to be crucial for the enzymatic activity. In addition, molecular modelling studies were also done to justify the IN inhibition and in vitro-in silico correlation was drawn. Conclusion: However, these compounds did not show HIV-1 and HIV-2 inhibition below their cytotoxic concentration indicating that these compounds cannot be taken further for anti-HIV activity as such and require structural modification.

Strengths and weaknesses of an HIV drug

Structural Biology Retroviruses replicate by inserting a copy of their RNA, which has been reverse transcribed into DNA, into the host genome. This process involves the intasome, a nucleoprotein complex comprising copies of the viral integrase bound at the ends of the viral DNA. HIV integrase strand-transfer inhibitors (INSTIs) stop HIV from replicating by blocking the viral integrase and are widely used in HIV treatment. Cook et al. describe structures of second-generation inhibitors bound to the simian immunodeficiency virus (SIV) intasome and to an intasome with integrase mutations known to cause drug resistance. Passos et al. describe the structures of the HIV intasome bound to a second-generation inhibitor and to developmental compounds that are promising drug leads. These structures show how mutations can cause subtle changes in the active site that affect drug binding, show the basis for the higher activity of later-generation inhibitors, and may guide development of better drugs. Science , this issue p. [806][1], p. [810][2] [1]: /lookup/doi/10.1126/science.aay4919 [2]: /lookup/doi/10.1126/science.aay8015

Cutting into the Substrate Dominance: Pharmacophore and Structure-Based Approaches toward Inhibiting Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) contains two distinct functional domains: a DNA polymerase (pol) domain and a ribonuclease H (RNase H) domain, both of which are required for viral genome replication. Over the last 3 decades, RT has been at the forefront of HIV drug discovery efforts with numerous nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) approved by the FDA. However, all these RT inhibitors target only the pol function, and inhibitors of RT-associated RNase H have yet to enter the development pipeline, which in itself manifests both the opportunity and challenges of targeting RNase H: if developed, RT RNase H inhibitors would represent a mechanistically novel class of HIV drugs that can be particularly valuable in treating HIV strains resistant to current drugs. The challenges include (1) the difficulty in selectively targeting RT RNase H over RT pol due to their close interplay both spatially and temporally and over HIV-1 integrase strand transfer (INST) activity because of their active site similarities; (2) to a larger extent, the inability of active site inhibitors to confer significant antiviral effect, presumably due to a steep substrate barrier by which the pre-existing substrate prevents access of small molecules to the active site. As a result, previously reported RT RNase H inhibitors typically lacked target specificity and significant antiviral potency. Achieving meaningful antiviral activity via active site targeting likely entails selective and ultrapotent RNase H inhibition to allow small molecules to cut into the dominance of substrates. Based on a pharmacophore model informed by prior work, we designed and redesigned a few metal-chelating chemotypes, such as 2-hydroxyisoquinolinedione (HID), hydroxypyridonecarboxylic acid (HPCA), 3-hydroxypyrimidine-2,4-dione (HPD), and N-hydroxythienopyrimidine-2,4-dione (HTPD). Analogues of these chemotypes generally exhibited improved potency and selectivity inhibiting RT RNase H over the best previous compounds and further validated the pharmacophore model. Extended structure-activity relationship (SAR) on the HPD inhibitor type by mainly altering the linkage generated a few subtypes showing exceptional potency (single-digit nanomolar) and excellent selectivity over the inhibition of RT pol and INST. In parallel, a structure-based approach also allowed us to design a unique double-winged HPD subtype to potently and selectively inhibit RT RNase H and effectively compete against the RNA/DNA substrate. Significantly, all potent HPD subtypes consistently inhibited HIV-1 in the cell culture, suggesting that carefully designed active site RNase H inhibitors with ultrapotency could partially overcome the barrier to antiviral phenotype. Overall, in addition to identifying our own inhibitor types, our medicinal chemistry efforts demonstrated the value of pharmacophore and structure-based approaches in designing active side-directed RNase H inhibitors and could provide a viable path to validating RNase H as a novel antiviral target.

Synthesis, Molecular Modelling and Biological Studies of 3-hydroxypyrane- 4-one and 3-hydroxy-pyridine-4-one Derivatives as HIV-1 Integrase Inhibitors.

Despite the progress in the discovery of antiretroviral compounds for treating HIV-1 infection by targeting HIV integrase (IN), a promising and well-known drug target against HIV-1, there is a growing need to increase the armamentarium against HIV, for avoiding the drug resistance issue. To develop novel HIV-1 IN inhibitors, a series of 3-hydroxy-pyrane-4-one (HP) and 3- hydroxy-pyridine-4-one (HPO) derivatives have been rationally designed and synthesized. To provide a significant characterization of the novel compounds, in-depth computational analysis was performed using a novel HIV-1 IN/DNA binary 3D-model for investigating the binding mode of the newly conceived molecules in complex with IN. The 3D-model was generated using the proto-type foamy virus (PFV) DNA as a structural template, positioning the viral polydesoxyribonucleic chain into the HIV-1 IN homology model. Moreover, a series of in vitro tests were performed including HIV-1 activity inhibition, HIV-1 IN activity inhibition, HIV-1 IN strand transfer activity inhibition and cellular toxicity. Bioassay results indicated that most of HP analogues including HPa, HPb, HPc, HPd, HPe and HPg, showed favorable inhibitory activities against HIV-1-IN in the low micromolar range. Particularly halogenated derivatives (HPb and HPd) offered the best biological activities in terms of reduced toxicity and optimum inhibitory activities against HIV-1 IN and HIV-1 in cell culture. Halogenated derivatives, HPb and HPd, displayed the most promising anti-HIV profile, paving the way to the optimization of the presented scaffolds for developing new effective antiviral agents.

Synthesis and Evaluation of 3-(1,3-dioxoisoindolin-2-yl)-N-substituted Phenyl Benzamide Analogues as HIV Integrase Strand Transfer Inhibitors

<P>Background: A series of novel 3-(1,3-dioxoisoindolin-2-yl)-N-substituted phenyl benzamide derivatives was synthesized and tested in vitro against human immunodeficiency virus type-1 Integrase (HIV-1 IN). Methods: Out of the 18 analogues, six (compounds 16c, 16h, 16i, 16m, 16n and 16r) showed significant inhibition of strand transfer by HIV-1 integrase. For these six compounds. IC50 was below 5.0 µM. In silico docking studies revealed that the presence of 2-phenyl isoindoline-1,3-dione motif was essential as it was found to interact with active site magnesium. Results: To further confirm the results, cell-based HIV-1 and HIV-2 inhibitory assay was carried out. Conclusion: These compounds possess structural features not seen in previously reported HIV-1 integrase inhibitors and thus can help further optimization of anti-HIV-1 integrase activity.</P>

Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase

Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes. The atomic structures of five different retrovirus IN-DNA complexes, termed intasomes, have revealed varying IN subunit compositions ranging from tetramers to octamers, dodecamers, and hexadecamers. Intasomes containing two IN-associated viral DNA ends capable of concerted integration are termed stable synaptic complexes (SSC), and those formed with a viral/target DNA substrate representing the product of strand-transfer reactions are strand-transfer complexes (STC). Here, we investigated the mechanisms associated with the assembly of the Rous sarcoma virus SSC and STC. C-terminal truncations of WT IN (286 residues) indicated a role of the last 18 residues ("tail" region) in assembly of the tetrameric and octameric SSC, physically stabilized by HIV-1 IN strand-transfer inhibitors. Fine mapping through C-terminal truncations and site-directed mutagenesis suggested that at least three residues (Asp-268-Thr-270) past the last β-strand in the C-terminal domain (CTD) are necessary for assembly of the octameric SSC. In contrast, the assembly of the octameric STC was independent of the last 18 residues of IN. Single-site substitutions in the CTD affected the assembly of the SSC, but not necessarily of the STC, suggesting that STC assembly may depend less on specific interactions of the CTD with viral DNA. Additionally, we demonstrate that trans-communication between IN dimer-DNA complexes facilitates the association of native long-terminal repeat (LTR) ends with partially defective LTR ends to produce a hybrid octameric SSC. The differential assembly of the tetrameric and octameric SSC improves our understanding of intasomes.

Placental transfer of elvitegravir and cobicistat in an ex-vivo human cotyledon double perfusion model.

To determine the transplacental pharmacokinetics of the HIV integrase strand transfer inhibitor elvitegravir and of cobicistat, a cytochrome P450 inhibitor used as a pharmacoenhancer in antiretroviral therapy. Maternal-to-fetal transfer across the term human placenta was investigated with the ex-vivo dually perfused cotyledon model, in seven open-circuit experiments and 10 closed-circuit (recirculating) experiments. Elvitegravir and cobicistat were added to a maternal perfusate containing 2 g/l of human serum albumin and antipyrine, as a marker to validate the cotyledon's viability. Elvitegravir and cobicistat concentrations were measured using ultraperformance liquid chromatography coupled with tandem mass spectrometry. For elvitegravir, in open-circuit experiments the mean (±SD) fetal transfer rate (FTR) (fetal/maternal concentration at steady state from 30 to 90 min) was 19 ± 13% and the mean clearance index was 0.46 ± 0.21; in the closed-circuit model, after 3 h of perfusion the FTR was 20 ± 10% and the mean accumulation index was 12.28 ± 5.57. For cobicistat, in the open perfusions the FTR was 23 ± 13% and the mean clearance index was 0.63 ± 0.34; in the closed perfusions after 3 h the fetal-to-maternal ratio of cobicistat was 21 ± 11%. The mean accumulation index was 3.46 ± 2.19 CONCLUSION:: The two models concurred to show moderate placental transfer of elvitegravir and cobicistat across the placenta as well as elvitegravir accumulation in the placenta tissue. Whether this may lead to toxicities and modifications in fetal or placental metabolism requires clinical studies.