Effects of integrase inhibitor-based antiretroviral therapy on brain outcomes according to time since acquisition of HIV-1 infection.

Integrase strand transfer inhibitors (INSTI) are a main component of the current antiretroviral regimens recommended for treatment of HIV infection. However, little is known about the impact of INSTI on neurocognition and neuroimaging. We developed a prospective observational trial to evaluate the effects of INSTI-based antiretroviral therapy on comprehensive brain outcomes (cognitive, functional, and imaging) according to the time since HIV-1 acquisition. We recruited men living with HIV who initiated antiretroviral therapy with INSTI < 3 months since the estimated date of HIV-1 acquisition (n = 12) and > 6 months since estimated date of HIV-1 acquisition (n = 15). We also recruited a group of matched seronegative individuals (n = 15). Assessments were performed at baseline (before initiation of therapy in HIV arms) and at weeks 4 and 48. Baseline cognitive functioning was comparable between the arms. At week 48, we did not find cognitive differences between starting therapy with INSTI earlier than 3 months or later than 6 months after acquisition of HIV-1 infection. Functional status was poorer in individuals diagnosed earlier. This effect recovered 48 weeks after initiation of therapy. Regarding brain imaging, we found that men living with HIV initiating antiretroviral therapy later experienced a greater decrease in medial orbitofrontal cortex over time, with expected negative repercussions for decision-making tasks.

Body Fat Distribution and Metabolic Changes in a Cohort of Adolescents Living With HIV Switched to an Antiretroviral Regimen Containing Dolutegravir.

Use of antiretrovirals is associated to body fat accumulation. We measured body composition in adolescents living with HIV switched to a dolutegravir-containing regimen. Trunk fat and trunk/body fat ratio markedly increased after 12 months. Total and low density lipoprotein cholesterol decreased after 3 months. Increase in trunk fat may put at risk of future cardiovascular problems, despite improvement in the lipid profile.

Optimized binding of substituted quinoline ALLINIs within the HIV-1 integrase oligomer.

During the integration step, human immunodeficiency virus type 1 integrase (IN) interacts with viral DNA and the cellular cofactor LEDGF/p75 to effectively integrate the reverse transcript into the host chromatin. Allosteric human immunodeficiency virus type 1 integrase inhibitors (ALLINIs) are a new class of antiviral agents that bind at the dimer interface of the IN catalytic core domain and occupy the binding site of LEDGF/p75. While originally designed to block IN-LEDGF/p75 interactions during viral integration, several of these compounds have been shown to also severely impact viral maturation through an IN multimerization mechanism. In this study, we tested the hypothesis that these dual properties of ALLINIs could be decoupled toward late stage viral replication effects by generating additional contact points between the bound ALLINI and a third subunit of IN. By sequential derivatization at position 7 of a quinoline-based ALLINI scaffold, we show that IN multimerization properties are enhanced by optimizing hydrophobic interactions between the compound and the C-terminal domain of the third IN subunit. These features not only improve the overall antiviral potencies of these compounds but also significantly shift the ALLINIs selectivity toward the viral maturation stage. Thus, we demonstrate that to fully maximize the potency of ALLINIs, the interactions between the inhibitor and all three IN subunits need to be simultaneously optimized.

Structural Comparison of Diverse HIV-1 Subtypes using Molecular Modelling and Docking Analyses of Integrase Inhibitors.

The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance.

Molecular dynamic simulations to investigate the structural impact of known drug resistance mutations on HIV-1C Integrase-Dolutegravir binding.

Resistance associated mutations (RAMs) threaten the long-term success of combination antiretroviral therapy (cART) outcomes for HIV-1 treatment. HIV-1 Integrase (IN) strand transfer inhibitors (INSTIs) have proven to be a viable option for highly specific HIV-1 therapy. The INSTI, Dolutegravir is recommended by the World Health Organization for use as first-line cART. This study aims to understand how RAMs affect the stability of IN, as well as the binding of the drug Dolutegravir to the catalytic pocket of the protein. A homology model of HIV-1 subtype C IN was successfully constructed and validated. The site directed mutator webserver was used to predict destabilizing and/or stabilizing effects of known RAMs while FoldX confirmed any changes in protein energy upon introduction of mutation. Also, interaction analysis was performed between neighbouring residues. Three mutations known to be associated with Raltegravir, Elvitegravir and Dolutegravir resistance were selected; E92Q, G140S and Y143R, for molecular dynamics simulations. The structural quality assessment indicated high reliability of the HIV-1C IN tetrameric structure, with more than 90% confidence in modelled regions. Change in free energy for the three mutants indicated different effects, while simulation analysis showed G140S to have the largest affect on protein stability and flexibility. This was further supported by weaker non-bonded pairwise interaction energy and binding free energy values between the drug DTG and E92Q, Y143R and G140S mutants suggesting reduced binding affinity, as indicated by interaction analysis in comparison to the WT. Our findings suggest the G140S mutant has the strongest effect on the HIV-1C IN protein structure and Dolutegravir binding. To the best of our knowledge, this is the first study that uses the consensus wild type HIV-1C IN sequence to build an accurate 3D model to understand the effect of three known mutations on DTG drug binding in a South Africa context.

Synthesis and biological evaluation of bis-N2,N2'-(4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccinodihydrazides.

A series of N2,N2'-bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccino-hydrazides, containing 4-hydroxycoumarin, hydrazine and tartaric acid moieties, have been prepared and examined for possible biological activity. Several of these compounds exhibit promising HIV-1 integrase inhibition (IC50 = 3.5 µM), and anti-T. brucei (32% viability) and anti-mycobacterial (Visual MIC90 = 15.63 µM) activity.

Heterocycle amide isosteres: An approach to overcoming resistance for HIV-1 integrase strand transfer inhibitors.

A series of heterocyclic pyrimidinedione-based HIV-1 integrase inhibitors was prepared and screened for activity against purified integrase enzyme and/or viruses modified with the following mutations within integrase: Q148R, Q148H/G140S and N155H. These are mutations that result in resistance to the first generation integrase inhibitors raltegravir and elvitegravir. Based on consideration of drug-target interactions, an approach was undertaken to replace the amide moiety of the first generation pyrimidinedione inhibitor with azole heterocycles that could retain potency against these key resistance mutations. An imidazole moiety was found to be the optimal amide substitute and the observed activity was rationalized with the use of calculated properties and modeling. Rat pharmacokinetic (PK) studies of the lead imidazole compounds demonstrated moderate clearance and moderate exposure.

Different kinetics of viral replication and DNA integration in the main HIV-1 cellular reservoirs in the presence and absence of integrase inhibitors.

To compare the kinetics of integration, p24 production and equilibrium of the different HIV-DNA forms in human primary cells in the presence/absence of integrase-inhibitors (INIs) in vitro. Monocyte-derived-macrophages (MDMs), CD4+ T-cells and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 in the presence/absence of raltegravir and dolutegravir. HIV-DNA levels and p24 production were measured by qPCR and ELISA assays, respectively. In the absence of INIs, levels of HIV-DNA forms were initially very low, with an increase in the integration process starting at 3 dpi. HIV-DNA increased more slowly in MDMs than it did in CD4+ T-cells and PMBCs peaking at 21 dpi with a mean of 1580 (±890) and 615 (±37) copies/103 cells for proviral and unintegrated HIV-DNA, and 455,972 (±213,255) pg/mL of p24 at the same time point. In CD4+ T-cells the proviral HIV-DNA increased together with unintegrated HIV-DNA peaking at 7 dpi (583 ±â€¯261 and 338 ±â€¯254 copies/103 cells) when the p24 was 218,000 (±75,600) pg/mL. A similar trend was observed in PBMCs (494 ±â€¯361 and 350 ±â€¯123 copies/103 cells for proviral and unintegrated HIV-DNA, and p24 production of 149,400 ±â€¯131,800 pg/mL). Both INIs inhibited viral replication and integration in all the cell types that were tested, especially starting at 3 dpi. However, a small but measurable amount of HIV-DNA (<5 copies/103 cells) was still observed in treated-MDMs up to 30 dpi. In conclusion, our study showed differences in HIV-DNA kinetic integration between CD4+ T-cells and MDMs, which could explain the divergent kinetics of viral-replication. Both INIs inhibited HIV-1 integration and replication with no difference found between CD4+ T-cells and MDMs. However, residual HIV-DNA remained detectable up to 30 dpi in INI-treated MDMs although complete inhibition of HIV replication was achieved. The clinical significance of this minor DNA persistence deserves further investigation considering the role of macrophages as reservoirs.

CYP1A1 and 1B1-mediated metabolic pathways of dolutegravir, an HIV integrase inhibitor.

Dolutegravir (DTG), a potent integrase inhibitor, is part of a recommended initial regimen for the treatment of human immunodeficiency virus (HIV). Prior reports demonstrated that the clearance of DTG was higher in current smokers than non-smokers, but the mechanism remains unclear. Using a metabolomic approach, M4 (an aldehyde) was identified as a novel metabolite of DTG. In addition, the formation of M4 was found to be mediated by cytochrome P450 (CYP) 1A1 and 1B1, the enzymes that can be highly induced by cigarette smoking. CYP1A1 and 1B1 were also identified as the major enzymes contributing to the formation of M1 (an N-dealkylated metabolite of DTG) and M5 (an aldehyde). Furthermore, the production of M1 and M4 was significantly increased in the lung of mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an inducer of CYP1A1 and 1B1. In summary, the current study uncovered the CYP1A1 and 1B1-mediated metabolic pathways of DTG. These data suggest that persons with HIV infection receiving DTG should be cautious to cigarettes, and drugs, or exposure to environmental chemicals that induce CYP1A1 and 1B1.

In Vitro Study on the Effect of Maraviroc or Dolutegravir on Bilirubin to Albumin Binding.

We performed an in vitro evaluation of the effect of maraviroc or dolutegravir on bilirubin to albumin binding. At typical treatment and low albumin concentrations, maraviroc had no impact, while dolutegravir affected bilirubin to albumin binding to an equivalent extent as sulfisoxazole. However in vivo, neither is likely to significantly impact bilirubin to albumin binding because of their low concentrations relative to albumin.

1,2,3,4-Tetrahydroisoquinolines as inhibitors of HIV-1 integrase and human LEDGF/p75 interaction.

Alkaloids are a class of organic compounds with a wide range of biological properties, including anti-HIV activity. The 1,2,3,4-tetrahydroisoquinoline is a ubiquitous structural motif of many alkaloids. Using a short and an efficient route for synthesis, a series of 1,2,3,4-tetrahydroisoquinolines/isoquinolines was developed. These compounds have been analysed for their ability to inhibit an important interaction between HIV-1 integrase enzyme (IN) and human LEDGF/p75 protein (p75) which assists in the viral integration into the active genes. A lead compound 6d is found to inhibit the LEDGF/p75-IN interaction in vitro with an IC50 of ~10 µm. Molecular docking analysis of the isoquinoline 6d reveals its interactions with the LEDGF/p75-binding residues of IN. Based on an order of addition experiment, the binding of 6d or LEDGF/p75 to IN is shown to be mutually exclusive. Also, the activity of 6d in vitro is found to be unaffected by the presence of a non-specific DNA. As reported earlier for the inhibitors of LEDGF/p75-IN interaction, 6d exhibits a potent inhibition of both the early and late stages of HIV-1 replication. Compound 6d differing from the known inhibitors in the chemical moieties and interactions with CCD could potentially be explored further for developing small molecule inhibitors of LEDGF/p75-IN interaction having a higher potency.

Application of the Morita-Baylis-Hillman reaction in the synthesis of 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones as potential HIV-1 integrase inhibitors.

A practicable six-step synthetic pathway has been developed to access a library of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones using Morita-Baylis-Hillman methodology. These compounds and their 3-[(N-cycloalkylamino)methyl]-2-quinolone precursors have been screened as potential HIV-1 integrase (IN) inhibitors. A concomitant survey of their activity against HIV-1 protease and reverse-transcriptase reveals selective inhibition of HIV-1 IN.

Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence.

FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) efficiently inhibit HIV-1 replication. Here, we present fluorescence properties of these inhibitors. Dolutegravir displays an excitation mode particularly dependent on Mg2+ chelation, allowing to directly probe its Mg2+-dependent binding to the prototype foamy virus (PFV) integrase. Dolutegravir-binding studied by both its fluorescence anisotropy and subsequent emission enhancement, strictly requires a preformed integrase/DNA complex, the ten terminal base pairs from the 3'-end of the DNA reactive strand being crucial to optimize dolutegravir-binding in the context of the ternary complex. From the protein side, mutation of any catalytic residue fully abolishes dolutegravir-binding. We also compared dolutegravir-binding to PFV F190Y, G187R and S217K mutants, corresponding to HIV-1 F121Y, G118R and G140S/Q148K mutations that confer low-to-high resistance levels against raltegravir/dolutegravir. The dolutegravir-binding properties derived from fluorescence-based binding assays and drug susceptibilities in terms of catalytic activity, are well correlated. Indeed, dolutegravir-binding to wild-type and F190Y integrases are comparable while strongly compromised with G187R and S217K. Accordingly, the two latter mutants are highly resistant to dolutegravir while F190Y shows only moderate or no resistance. Intrinsic fluorescence properties of dolutegravir are thus particularly suitable for a thorough characterization of both DNA-binding properties of integrase and resistance mutations.

Interactions between integrase inhibitors and human arginase 1.

The neuro-pathogenic mechanism(s) underlying HIV-associated neurocognitive disorders are mostly unknown. HIV-infected macrophages and microglial cells play a crucial role and the metabolic fate of l-arginine may be highly relevant to microglia activation. In this context, arginase (ARG), which uses l-arginine as substrate, can be on the same time a target and source of oxidative stress and inflammation. In this study, we investigated whether integrase strand transfer inhibitors share with the other antiretroviral drugs the ability to inhibit ARG activity. We used the previously validated cell model, namely the human microglia cell line, as well as the computational chemistry approach. Furthermore, here we characterized the activity of purified human ARG in a cell-free in vitro system, and investigated the effects of integrase strand transfer inhibitors in this newly validated model. Overall evidence shows that Dolutegravir, Raltegravir and Elvitegravir inhibit ARG activity.

New insights into the interaction between pyrrolyl diketoacids and HIV-1 integrase active site and comparison with RNase H.

HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg2+ cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding. In this study, we investigated the binding mode of four pyrrolyl DKAs to the HIV-1 IN active site by molecular modeling coupled with site-directed mutagenesis studies showing that the DKA pyrrolyl scaffold primarily interacts with the IN amino residues P145, Q146 and Q148. Importantly, the tested DKAs demonstrated good effectiveness against HIV-1 Raltegravir resistant Y143A and N155H INs, thus showing an interaction pattern with relevant differences if compared with the first generation IN inhibitors. These data provide precious insights for the design of new HIV inhibitors active on clinically selected Raltegravir resistant variants. Furthermore, this study provides new structural information to modulate IN and RNase H inhibitory activities for development of dual-acting anti-HIV agents.

Bidirectional Transfer of Raltegravir in an Ex Vivo Human Cotyledon Perfusion Model.

Placental transfer of the HIV integrase inhibitor raltegravir (RLT) was investigated in term human cotyledons in the maternal-to-fetal (n = 3) and fetal-to-maternal (n = 6) directions. In the maternal-to-fetal direction, the mean ± standard deviation (SD) fetal transfer rate (FTR) was 9.1% ± 1.4%, and the mean ± SD clearance index (IC), i.e., RLT FTR/antipyrine FTR, was 0.28 ± 0.05. In the fetal-to-maternal direction, the mean ± SD CI was 0.31 ± 0.09. Placental transfer of RLT was high in both directions.

Anti-HIV-1 integrase effect of compounds from Aglaia andamanica leaves and molecular docking study with acute toxicity test in mice.

CONTEXT: Acquired immunodeficiency syndrome (AIDS) is a serious health problem worldwide. It has been reported that Aglaia andamanica Hiern (Meliaceae) leaves possessed an antiviral effect. Therefore, a search of anti-HIV-1 integrase (HIV-1 IN) agents from A. andamanica is a promising target. OBJECTIVE: The objective of this study is to evaluate anti-HIV-1 IN activity of isolated compounds from A. andamanica using an in vitro assay and molecular docking study as well as testing acute toxicity in mice using the up and down method. MATERIALS AND METHODS: The leaves and compounds (3-100 µg/mL) from A. andamanica were determined for the anti-HIV-1 IN effect using the multiplate integration assay (MIA) by detection the absorbance of the final product, p-nitrophenol, at 405 nm. The molecular docking with the HIV-1 IN of the active compound N-methyl-trans-4-hydroxy-l-proline (10) was also studied. The Swiss albino mice were used for an acute toxicity test. RESULTS AND DISCUSSION: Among the isolated compounds, 10 showed marked anti-HIV-1 IN effect with an IC50 value of 11.8 µg/mL, whereas other compounds were inactive (IC50 value > 100 µg/mL). The molecular docking of compound 10 with an HIV-1 IN enzyme was also studied. The result revealed that this compound formed the hydrogen bonding with the Thr66, Asn155, and Lys159 of the HIV-1 IN binding site. The acute toxicity of the A. andamanica extract was not observed at the dose 2000 mg/kg mice. This is the first report of A. andamanica for anti-HIV-1 IN activity.

Kuwanon-L as a New Allosteric HIV-1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation.

HIV-1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand-transfer drug-resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking-based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon-L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon-L is able to inhibit the HIV-1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon-L also inhibited HIV-1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon-L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents.

Correlation between UGT1A1 polymorphisms and raltegravir plasma trough concentrations in Japanese HIV-1-infected patients.

Raltegravir (RAL), an HIV integrase inhibitor, is metabolized mainly by UDP-glucuronosyltransferase 1A1 (UGT1A1). Polymorphisms in UGT1A1 may cause alterations in the pharmacodynamics of RAL, which is taken twice daily with no dietary restrictions. We compared the effect of two polymorphic alleles in this gene, UGT1A1*6 and UGT1A1*28 on plasma RAL concentrations in Japanese HIV-1-infected patients. Of 114 Japanese HIV-1-infected patients who received RAL, the frequencies of UGT1A1*6 and UGT1A1*28 were 18% and 13%, respectively. The percentage of homozygotes for UGT1A1*6 and UGT1A1*28 was 6% and 4%, respectively, the percentage of compound heterozygotes for UGT1A1*6 and UGT1A1*28 was 2%, and that of heterozygotes for UGT1A1*6 and UGT1A1*28 was 22% and 17%, respectively. RAL plasma trough concentrations were compared for each polymorphism. Significantly higher levels of RAL were observed with patients who were homozygous for UGT1A1*6 (median: 1.0 µg/mL) than for the normal allele (median: 0.11 µg/mL; p = 0.021). Multivariate logistic regression analysis showed that the presence of one or two alleles of UGT1A1*6 or two alleles of UGT1A1*28 were independent factors associated with high RAL plasma trough concentrations (≥ 0.17 µg/mL). These results indicated that UGT1A1*6 and UGT1A1*28 are both factors influencing the RAL plasma trough concentrations in Japanese HIV-1-infected patients.

Structural Studies of the HIV-1 Integrase Protein: Compound Screening and Characterization of a DNA-Binding Inhibitor.

Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance.