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1.  Resistance Mutations outside the Integrase Coding Region Have an Effect on Human Immunodeficiency Virus Replicative Fitness but Do Not Affect Its Susceptibility to Integrase Strand Transfer Inhibitors 
PLoS ONE  2013;8(6):e65631.
Most studies describing phenotypic resistance to integrase strand transfer inhibitors have analyzed viruses carrying only patient-derived HIV-1 integrase genes (INT-recombinant viruses). However, to date, many of the patients on INSTI-based treatment regimes, such as raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) are infected with multidrug-resistant HIV-1 strains. Here we analyzed the effect of drug resistance mutations in Gag (p2/NCp7/p1/p6), protease (PR), reverse transcriptase (RT), and integrase (IN) coding regions on susceptibility to INSTIs and viral replicative fitness using a novel HIV-1 phenotyping assay. Initial characterization based on site-directed mutant INSTI-resistant viruses confirmed the effect of a series of INSTI mutations on reduced susceptibility to EVG and RAL and viral replicative fitness (0.6% to 99% relative to the HIV-1NL4-3 control). Two sets of recombinant viruses containing a 3,428-bp gag-p2/NCp7/p1/p6/pol-PR/RT/IN (p2-INT) or a 1,088 bp integrase (INT) patient-derived fragment were constructed from plasma samples obtained from 27 virologic failure patients participating in a 48-week dose-ranging study of elvitegravir, GS-US-183-0105. A strong correlation was observed when susceptibility to EVG and RAL was assayed using p2-INT- vs. INT-recombinant viruses (Pearson coefficient correlation 0.869 and 0.918, P<0.0001 for EVG and RAL, respectively), demonstrating that mutations in the protease and RT have limited effect on susceptibility to these INSTIs. On the other hand, the replicative fitness of viruses harboring drug resistance mutations in PR, RT, and IN was generally impaired compared to viruses carrying only INSTI-resistance mutations. Thus, in the absence of drug pressure, drug resistance mutations in the PR and RT contribute to decrease the replicative fitness of the virus already impaired by mutations in the integrase. The use of recombinant viruses containing most or all HIV-1 regions targeted by antiretroviral drugs might be essential to understand the collective effect of epistatic interactions in multidrug-resistant viruses.
doi:10.1371/journal.pone.0065631
PMCID: PMC3679210  PMID: 23776513
2.  The Combination of the R263K and T66I Resistance Substitutions in HIV-1 Integrase Is Incompatible with High-Level Viral Replication and the Development of High-Level Drug Resistance 
Journal of Virology  2015;89(22):11269-11274.
ABSTRACT
The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and has been reported for several treatment-experienced individuals receiving DTG as part of salvage therapy. The R263K substitution seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG-treated individuals. Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand transfer and 3′ processing activities were measured biochemically. The combination of the R263K and T66I substitutions decreased HIV-1 infectivity, replicative capacity, and strand transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL. These findings suggest that the presence of the T66I substitution will not compromise the activity of DTG and may also help to prevent the additional generation of the R263K mutation. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution.
IMPORTANCE The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus type 1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution, whereas R263K is a signature resistance substitution against dolutegravir. In order to determine how different combinations of integrase resistance mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir therapy who harbor the T66I resistance substitution.
doi:10.1128/JVI.01881-15
PMCID: PMC4645644  PMID: 26311878
3.  Effect of HIV-1 Integrase Resistance Mutations When Introduced into SIVmac239 on Susceptibility to Integrase Strand Transfer Inhibitors 
Journal of Virology  2014;88(17):9683-9692.
ABSTRACT
Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity. Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared to wild-type virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36- and 62-fold, respectively), whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R, and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred high-level resistance to both RAL and EVG (>300- and 286-fold, respectively). DTG remained fully effective against all site-directed mutants except G118R and R263K. Thus, HIV INSTI mutations, when inserted into SIV, resulted in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful nonhuman primate model for studies of HIV resistance to INSTIs.
IMPORTANCE The goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. An SIV-primate model remains an invaluable tool for investigating questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.
doi:10.1128/JVI.00947-14
PMCID: PMC4136349  PMID: 24920794
4.  HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M 
Journal of the International AIDS Society  2014;17(4Suppl 3):19738.
Introduction
HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations.
Viruses and Methods
We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M (subtype B, HIV-M/B) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs.
Results
HIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC) is easier than substitution of alanine to tyrosine (153AGGC→153YTAC), with only a transversion needed instead of one transition plus one transversion.
Conclusions
This is the first report of susceptibility and resistance in vitro to INSTIs for HIV-O. Our study confirmed the impact of HIV-O polymorphism, on susceptibility to INSTIs and the emergence of resistance mutations.
doi:10.7448/IAS.17.4.19738
PMCID: PMC4225329  PMID: 25397483
5.  Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors▿  
The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.
doi:10.1128/AAC.01720-09
PMCID: PMC2935022  PMID: 20479206
6.  Biochemical Analysis of the Role of G118R-Linked Dolutegravir Drug Resistance Substitutions in HIV-1 Integrase 
Antimicrobial Agents and Chemotherapy  2013;57(12):6223-6235.
Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3′ processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility.
doi:10.1128/AAC.01835-13
PMCID: PMC3837891  PMID: 24080645
7.  Natural Polymorphisms of Human Immunodeficiency Virus Type 1 Integrase and Inherent Susceptibilities to a Panel of Integrase Inhibitors▿  
Antimicrobial Agents and Chemotherapy  2009;53(10):4275-4282.
We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drug-resistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance. A phenotypic analysis was done on 10 recombinant viruses derived from nine patients against a panel of six diverse InSTI. Few of the polymorphisms associated with in vitro InSTI resistance were identified in the samples from newly infected individuals or those patients with MDR HIV-1. We identified polymorphisms V72I, L74I, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N. V72I was the most common, seen in 63 (56.3%) of the AHI samples. E157Q was the only naturally occurring mutation thought to contribute to resistance to elvitegravir, raltegravir, and L-870,810. None of the patient-derived viruses demonstrated any significant decrease in susceptibility to the drugs tested. In summary, the integrase coding region contains as much natural variation as that seen in protease, but mutations associated with high-level resistance to existing InSTI are rarely, if ever, present in integrase naïve patients, especially those being used clinically. Most of the highly prevalent polymorphisms have little effect on InSTI susceptibility in the absence of specific primary mutations. Baseline testing for integrase susceptibility in InSTI-naïve patients is not currently warranted.
doi:10.1128/AAC.00397-09
PMCID: PMC2764199  PMID: 19651917
8.  Therapy-Emergent Drug Resistance to Integrase Strand Transfer Inhibitors in HIV-1 Patients: A Subgroup Meta-Analysis of Clinical Trials 
PLoS ONE  2016;11(8):e0160087.
Background
Integrase strand transfer inhibitors (INSTIs) are a novel class of anti-HIV agents that show high activity in inhibiting HIV-1 replication. Currently, licensed INSTIs include raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG); these drugs have played a critical role in AIDS therapy, serving as additional weapons in the arsenal for treating patients infected with HIV-1. To date, long-term data regarding clinical experience with INSTI use and the emergence of resistance remain scarce. However, the literature is likely now sufficiently comprehensive to warrant a meta-analysis of resistance to INSTIs.
Methods
Our team implemented a manuscript retrieval protocol using Medical Subject Headings (MeSH) via the Web of Science, MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials databases. We screened the literature based on inclusion and exclusion criteria and then performed a quality analysis and evaluation using RevMan software, Stata software, and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). We also performed a subgroup analysis. Finally, we calculated resistance rates and risk ratios (RRs) for the three types of drugs.
Results
We identified 26 references via the database search. A meta-analysis of the RAL data revealed that the resistance rate was 3.9% (95% CI = 2.9%-4.9%) for the selected randomized controlled trials (RCTs). However, the RAL resistance rate reached 40.9% (95% CI = 8.8%-72.9%) for the selected observational studies (OBSs). The rates of resistance to RAL that were associated with HIV subtypes A, B, and C as well as with more complex subtypes were 0.1% (95% CI = -0.7%-0.9%), 2.5% (95% CI = 0.5%-4.5%), 4.6% (95% CI = 2.7%-6.6%) and 2.2% (95% CI = 0.7%-3.7%), respectively. The rates of resistance to EVG and DTG were 1.2% (95% CI = 0.2%-2.2%) and 0.1% (95% CI = -0.2%-0.5%), respectively. Furthermore, we found that the RRs for antiviral resistance were 0.414 (95% CI = 0.210–0.816) between DTG and RAL and 0.499 (95% CI = 0.255–0.977) between EVG and RAL. When RAL was separately co-administered with nuclear nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs), the rates of resistance to RAL were 0.2% (95% CI = -0.1%-0.5%) and 0.2% (95% CI = -0.2%-0.6%), respectively. The ten major integrase mutations (including N155H, Y143C/R, Q148H/R, Y143Y/H, L74L/M, E92Q, E138E/A, Y143C, Q148Q and Y143S) can reduce the sensitivity of RAL and EVG. The resistance of DTG is mainly shown in 13 integrase mutations (including T97T/A, E138E/D, V151V/I, N155H, Q148, Y143C/H/R, T66A and E92Q).
Conclusions
Our results reveal that the DTG resistance rate was lower than the RAL resistance rate in a head-to-head comparison. Moreover, we confirmed that the EVG resistance rate was lower than the RAL resistance rate. In addition, our results revealed that the resistance rate of RAL was lower than that of efavirenz. The rates of resistance to RAL, EVG and DTG were specifically 3.9%, 1.2% and 0.1%, respectively. Compared with other types of antiviral drugs, the rates of resistance to INSTIs are generally lower. Unfortunately, the EVG and DTG resistance rates could not be compared because of a lack of data.
doi:10.1371/journal.pone.0160087
PMCID: PMC4988762  PMID: 27532886
9.  Dolutegravir, the Second-Generation of Integrase Strand Transfer Inhibitors (INSTIs) for the Treatment of HIV 
The integrase strand transfer inhibitors (INSTIs) are the newest antiretroviral class in the HIV treatment armamentarium. Dolutegravir (DTG) is the only second-generation INSTI with FDA approval (2013). It has potential advantages in comparison to first-generation INSTI’s, including unboosted daily dosing, limited cross resistance with raltegravir and elvitegravir, and a high barrier to resistance. Clinical trials have evaluated DTG as a 50-mg daily dose in both treatment-naïve and treatment-experienced, INSTI-naïve participants. In those treatment-naïve participants with baseline viral load <100,000 copies/mL, DTG combined with abacavir and lamivudine was non-inferior and superior to fixed-dose combination emtricitabine/tenofovir/efavirenz. DTG was also superior to the protease inhibitor regimen darunavir/ritonavir in treatment-naïve participants regardless of baseline viral load. Among treatment-experienced patients naïve to INSTI, DTG (50 mg daily) demonstrated both non-inferiority and superiority when compared to the first-generation INSTI raltegravir (400 mg twice daily) regardless of the background regimen. No phenotypically significant DTG resistance has been demonstrated in INSTI-naïve participant trials. The VIKING trials evaluated DTG’s ability to treat persons with HIV with prior INSTI exposure. VIKING demonstrated twice-daily DTG was more efficacious than daily dosing when treating participants receiving and failing first-generation INSTI regimens. DTG maintained potency against single mutations from any of the three major INSTI pathways (Y143, H155, Q148); however, the Q148 mutation with two or more additional mutations significantly reduced its potency. The long-acting formulation of DTG, GSK1265744LA, is the next innovation in this second-generation INSTI class, holding promise for the future of HIV prevention and treatment.
Electronic supplementary material
The online version of this article (doi:10.1007/s40121-014-0029-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s40121-014-0029-7
PMCID: PMC4269626  PMID: 25134686
Antiretroviral therapy (ART); Dolutegravir (DTG); GSK1265744LA; HIV; Integrase strand transfer inhibitor (INSTI); Nanoparticle formulation
10.  Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Integrase Strand Transfer Inhibitor with an Improved Resistance Profile 
Antimicrobial Agents and Chemotherapy  2016;60(12):7086-7097.
Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.
doi:10.1128/AAC.01474-16
PMCID: PMC5118987  PMID: 27645238
11.  Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239 
Journal of Virology  2015;89(23):12002-12013.
ABSTRACT
We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3′-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km′ and Vmax′/Km′ for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV.
IMPORTANCE Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.
doi:10.1128/JVI.02131-15
PMCID: PMC4645305  PMID: 26378179
12.  Comparative biochemical analysis of HIV-1 subtype B and C integrase enzymes 
Retrovirology  2009;6:103.
Background
Integrase inhibitors are currently being incorporated into highly active antiretroviral therapy (HAART). Due to high HIV variability, integrase inhibitor efficacy must be evaluated against a range of integrase enzymes from different subtypes.
Methods
This study compares the enzymatic activities of HIV-1 integrase from subtypes B and C as well as susceptibility to various integrase inhibitors in vitro. The catalytic activities of both enzymes were analyzed in regard to each of 3' processing and strand transfer activities both in the presence and absence of the integrase inhibitors raltegravir (RAL), elvitegravir (EVG), and MK-2048.
Results
Our results show that integrase function is similar with enzymes of either subtype and that the various integrase strand transfer inhibitors (INSTIs) that were employed possessed similar inhibitory activity against both enzymes.
Conclusion
This suggests that the use of integrase inhibitors against HIV-1 subtype C will result in comparable outcomes to those obtained against subtype B infections.
doi:10.1186/1742-4690-6-103
PMCID: PMC2779801  PMID: 19906306
13.  Altered Viral Fitness and Drug Susceptibility in HIV-1 Carrying Mutations That Confer Resistance to Nonnucleoside Reverse Transcriptase and Integrase Strand Transfer Inhibitors 
Journal of Virology  2014;88(16):9268-9276.
ABSTRACT
Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) and integrase (IN) strand transfer inhibitors (INSTI) are key components of antiretroviral regimens. To explore potential interactions between NNRTI and INSTI resistance mutations, we investigated the combined effects of these mutations on drug susceptibility and fitness of human immunodeficiency virus type 1 (HIV-1). In the absence of drug, single-mutant viruses were less fit than the wild type; viruses carrying multiple mutations were less fit than single-mutant viruses. These findings were explained in part by the observation that mutant viruses carrying NNRTI plus INSTI resistance mutations had reduced amounts of virion-associated RT and/or IN protein. In the presence of efavirenz (EFV), a virus carrying RT-K103N together with IN-G140S and IN-Q148H (here termed IN-G140S/Q148H) mutations was fitter than a virus with a RT-K103N mutation alone. Similarly, in the presence of EFV, the RT-E138K plus IN-G140S/Q148H mutant virus was fitter than one with the RT-E138K mutation alone. No effect of INSTI resistance mutations on the fitness of RT-Y181C mutant viruses was observed. Conversely, RT-E138K and -Y181C mutations improved the fitness of the IN-G140S/Q148H mutant virus in the presence of raltegravir (RAL); the RT-K103N mutation had no effect. The NNRTI resistance mutations had no effect on RAL susceptibility. Likewise, the IN-G140S/Q148H mutations had no effect on EFV or RPV susceptibility. However, both the RT-K103N plus IN-G140S/Q148H and the RT-E138K plus IN-G140S/Q148H mutant viruses had significantly greater fold increases in 50% inhibitory concentration (IC50) of EFV than viruses carrying a single NNRTI mutation. Likewise, the RT-E138K plus IN-G140S/Q148H mutant virus had significantly greater fold increases in RAL IC50 than that of the IN-G140S/Q148H mutant virus. These results suggest that interactions between RT and IN mutations are important for NNRTI and INSTI resistance and viral fitness.
IMPORTANCE Nonnucleoside reverse transcriptase inhibitors and integrase inhibitors are used to treat infection with HIV-1. Mutations that confer resistance to these drugs reduce the ability of HIV-1 to reproduce (that is, they decrease viral fitness). It is known that reverse transcriptase and integrase interact and that some mutations can disrupt their interaction, which is necessary for proper functioning of these two enzymes. To determine whether resistance mutations in these enzymes interact, we investigated their effects on drug sensitivity and viral fitness. Although individual drug resistance mutations usually reduced viral fitness, certain combinations of mutations increased fitness. When present in certain combinations, some integrase inhibitor resistance mutations increased resistance to nonnucleoside reverse transcriptase inhibitors and vice versa. Because these drugs are sometimes used together in the treatment of HIV-1 infection, these interactions could make viruses more resistant to both drugs, further limiting their clinical benefit.
doi:10.1128/JVI.00695-14
PMCID: PMC4136249  PMID: 24899199
14.  HIV-1 Group O Integrase Displays Lower Enzymatic Efficiency and Higher Susceptibility to Raltegravir than HIV-1 Group M Subtype B Integrase 
Antimicrobial Agents and Chemotherapy  2014;58(12):7141-7150.
HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3′ processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3′ processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.
doi:10.1128/AAC.03819-14
PMCID: PMC4249541  PMID: 25224008
15.  Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor ▿  
Journal of Virology  2010;84(18):9210-9216.
MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.
doi:10.1128/JVI.01164-10
PMCID: PMC2937597  PMID: 20610719
16.  Impact of Primary Elvitegravir Resistance-Associated Mutations in HIV-1 Integrase on Drug Susceptibility and Viral Replication Fitness 
Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.
doi:10.1128/AAC.02568-12
PMCID: PMC3716146  PMID: 23529738
17.  In-Silico docking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate 
Retrovirology  2007;4:21.
Background
HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain.
Methods
Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD).
Results
Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC50s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N-Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those displayed by fungal molecules from Fusarium sp., shown in the 1990s to inhibit HIV-1 integration.
Conclusion
The 3D model presented here supports the idea that INSTIs dock at the putative acceptor DNA-binding site in a IN/viral DNA complex. This mechanism of enzyme inhibition, likely to be exploited by some natural products, might disclose future strategies for inhibition of nucleic acid-manipulating enzymes.
doi:10.1186/1742-4690-4-21
PMCID: PMC1847836  PMID: 17374162
18.  In vitro activity of dolutegravir against wild-type and integrase inhibitor-resistant HIV-2 
Retrovirology  2015;12:10.
Background
Dolutegravir recently became the third integrase strand transfer inhibitor (INSTI) approved for use in HIV-1–infected individuals. In contrast to the extensive dataset for HIV-1, in vitro studies and clinical reports of dolutegravir for HIV-2 are limited. To evaluate the potential role of dolutegravir in HIV-2 treatment, we compared the susceptibilities of wild-type and INSTI-resistant HIV-1 and HIV-2 strains to the drug using single-cycle assays, spreading infections of immortalized T cells, and site-directed mutagenesis.
Findings
HIV-2 group A, HIV-2 group B, and HIV-1 isolates from INSTI-naïve individuals were comparably sensitive to dolutegravir in the single-cycle assay (mean EC50 values = 1.9, 2.6, and 1.3 nM, respectively). Integrase substitutions E92Q, Y143C, E92Q + Y143C, and Q148R conferred relatively low levels of resistance to dolutegravir in HIV-2ROD9 (2- to 6-fold), but Q148K, E92Q + N155H, T97A + N155H and G140S + Q148R resulted in moderate resistance (10- to 46-fold), and the combination of T97A + Y143C in HIV-2ROD9 conferred high-level resistance (>5000-fold). In contrast, HIV-1NL4-3 mutants E92Q + N155H, G140S + Q148R, and T97A + Y143C showed 2-fold, 4-fold, and no increase in EC50, respectively, relative to the parental strain. The resistance phenotypes for E92Q + N155H, and G140S + Q148R HIV-2ROD9 were also confirmed in spreading infections of CEM-ss cells.
Conclusions
Our data support the use of dolutegravir in INSTI-naïve HIV-2 patients but suggest that, relative to HIV-1, a broader array of replacements in HIV-2 integrase may enable cross-resistance between dolutegravir and other INSTI. Clinical studies are needed to evaluate the efficacy of dolutegravir in HIV-2–infected individuals, including patients previously treated with raltegravir or elvitegravir.
doi:10.1186/s12977-015-0146-8
PMCID: PMC4328052  PMID: 25808007
19.  Selectivity for strand-transfer over 3′-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme–DNA interactions in the active site 
Nucleic Acids Research  2016;44(14):6896-6906.
Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3′-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and −1 bases, which flank the 3′-processing site, play a critical role for 3′-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3′-processing activity, becomes more active and more resistant to inhibition of 3′-processing by RAL and DTG in the absence of the −1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3′-processing and ST reactions. The potency of integrase inhibitors against 3′-processing and their ability to overcome resistance is discussed.
doi:10.1093/nar/gkw592
PMCID: PMC5001616  PMID: 27369381
20.  Prevalence of Integrase Strand Transfer Inhibitors (INSTI) Resistance Mutations in Taiwan 
Scientific Reports  2016;6:35779.
Antiretroviral therapy containing an integrase strand transfer inhibitor (INSTI) plus two NRTIs has become the recommended treatment for antiretroviral-naive HIV-1-infected patients in the updated guidelines. We aimed to determine the prevalence of INSTI-related mutations in Taiwan. Genotypic resistance assays were performed on plasma from ARV-naïve patients (N = 948), ARV-experienced but INSTI-naive patients (N = 359), and raltegravir-experienced patients (N = 63) from 2006 to 2015. Major INSTI mutations were defined according to the IAS-USA list and other substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were defined as minor mutations. Of 1307 HIV-1 samples from patients never exposed to INSTIs, the overall prevalence of major resistance mutations to INSTIs was 0.9% (n = 12), with an increase to 1.2% in 2013. Of these 12 sequences, 11 harboured Q148H/K/R, one Y143R, and none N155H. Of 30 sequences (47.6%) with INSTI-resistant mutations from raltegravir-experienced patients, 17 harboured Q148H/K/R, 8 N155H, and 6 Y143C/R. Other than these major mutations, the prevalence of minor mutations were 5.3% and 38.1%, respectively, in ARV-naive and raltegravir-experienced patients. The overall prevalence of INSTI mutations remains low in Taiwan. Surveillance of INSTI resistance is warranted due to circulation of polymorphisms contributing to INSTI resistance and expected increasing use of INSTIs.
doi:10.1038/srep35779
PMCID: PMC5078839  PMID: 27779200
21.  The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness 
Retrovirology  2014;11:7.
Background
First-generation integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG), have been clinically proven to be effective antiretrovirals for the treatment of HIV-positive patients. However, their relatively low genetic barrier for resistance makes them susceptible to the emergence of drug resistance mutations. In contrast, dolutegravir (DTG) is a newer INSTI that appears to have a high genetic barrier to resistance in vivo. However, the emergence of the resistance mutation R263K followed by the polymorphic substitution M50I has been observed in cell culture. The M50I polymorphism is also observed in 10-25% of INSTI-naïve patients and has been reported in combination with R263K in a patient failing treatment with RAL.
Results
Using biochemical cell-free strand-transfer assays and resistance assays in TZM-bl cells, we demonstrate that the M50I polymorphism in combination with R263K increases resistance to DTG in tissue culture and in biochemical assays but does not restore the viral fitness cost associated with the R263K mutation.
Conclusions
Since the combination of the R263K mutation and the M50I polymorphism results in a virus with decreased viral fitness and limited cross-resistance, the R263K resistance pathway may represent an evolutionary dead-end. Although this hypothesis has not yet been proven, it may be more advantageous to treat HIV-positive individuals with DTG in first-line than in second or third-line therapy.
doi:10.1186/1742-4690-11-7
PMCID: PMC3898230  PMID: 24433497
HIV integrase; Subtype B; Antiretrovirals; R263K; Resistance mutation; M50I; Polymorphism; INSTI-naïve
22.  Clinical Pharmacokinetic, Pharmacodynamic and Drug-Interaction Profile of the Integrase Inhibitor Dolutegravir 
Clinical pharmacokinetics  2013;52(11):981-994.
Dolutegravir is a second generation integrase strand transfer inhibitor (INSTI) currently under review by the US FDA for marketing approval. Dolutegravir’s in vitro, protein adjusted 90% inhibitory concentration (IC90) for wild-type virus is 0.064 μg/ml, and it retains in vitro anti-HIV 1 activity across a broad range of viral phenotypes known to confer resistance to the currently marketed INSTIs, raltegravir and elvitegravir. Dolutegravir has a half-life (t½) of 13 to 14 hours and maintains concentrations over the in vitro, protein adjusted IC90 for more than 30 hours following a single dose. Additionally, dolutegravir has comparatively low intersubject variability compared to raltegravir and elvitegravir. A plasma exposure-response relationship has been well described, with antiviral activity strongly correlating to trough concentration (Ctrough) values. Phase III trials have assessed the antiviral activity of dolutegravir compared with efavirenz and raltegravir in antiretroviral (ARV)-naïve patients and found dolutegravir to achieve more rapid and sustained virologic suppression in both instances. Additionally, studies of dolutegravir activity in patients with known INSTI-resistant mutations have been favorable, indicating that dolutegravir retains activity in a variety of INSTI resistant phenotypes. Much like currently marketed INSTIs, dolutegravir is very well tolerated. Because dolutegravir inhibits the renal transporter, organic cation transporter (OCT) 2, reduced tubular secretion of creatinine leads to non-progressive increases in serum creatinine. These serum creatinine increases have not been associated with decreased glomerular filtration rate or progressive renal impairment. Dolutegravir’s major and minor metabolic pathways are UDP glucuronosyltransferase (UGT)1A1 and cytochrome (CYP)3A4, respectively, and it neither induces nor inhibits CYP isozymes. Thus dolutegravir has a modest drug interaction profile. However, antacids significantly decrease dolutegravir plasma exposure and should be separated by 2 hours before, or 6 hours after, a dolutegravir dose. In summary, dolutegravir is the first of the second generation INSTIs, which exhibits a predictable pharmacokinetic profile and a well-defined exposure-response relationship. Dolutegravir retains activity despite the presence of some class resistant mutations and achieves rapid and sustained virologic suppression in ARV-naïve and -experienced patients. Clinically dolutegravir is poised to become a commonly used component of antiretroviral regimens.
doi:10.1007/s40262-013-0093-2
PMCID: PMC3805712  PMID: 23824675
23.  Lack of resistance to integrase inhibitors among antiretroviral-naive subjects with primary HIV-1 infection, 2007–2013 
Antiviral therapy  2014;20(1):77-80.
Background
U.S. guidelines recommend genotyping for persons newly diagnosed with HIV infection to identify transmitted drug resistance mutations associated with decreased susceptibility to NRTIs, NNRTIs, and PIs. To date, testing for integrase strand transfer inhibitor (INSTI) mutations has not been routinely recommended. We aimed to evaluate the prevalence of transmitted INSTI mutations among persons with primary HIV-1 infection in Seattle, WA.
Methods
Persons with primary HIV-1 infection have enrolled in an observational cohort at the University of Washington Primary Infection Clinic since 1992. We performed a retrospective analysis of plasma specimens collected prospectively from the 82 antiretroviral-naive subjects who were enrolled from 2007–13, after FDA-approval of the first INSTI. Resistance testing was performed by consensus sequencing.
Results
Specimens for analysis had been obtained a median of 24 (lQR 18–41, range 8–108) days after the estimated date of HIV-1 infection. All subjects were infected with HIV-1 subtype B except for one subject infected with subtype C. Consensus sequencing identified no subjects with major INSTI mutations (T66I, E92Q, G140S, Y143C/H/R, S147G, Q148H/K/R, N155H). Using exact binomial confidence intervals, the upper bound of the 95% CI was 4.4%.
Conclusions
Although our sample size was small, this study does not support the need at this time to evaluate integrase mutations as part of routine consensus sequencing among persons newly diagnosed with HIV-1 infection. However, it is likely that the prevalence of transmitted INSTI mutations may increase with the recent commercial introduction of additional INSTIs and presumably greater INST1 use among persons living with HIV-1.
doi:10.3851/IMP2780
PMCID: PMC4312242  PMID: 24831260
24.  Bicyclic 1-Hydroxy-2-oxo-1,2-dihydropyridine-3-carboxamide-Containing HIV-1 Integrase Inhibitors Having High Antiviral Potency against Cells Harboring Raltegravir-Resistant Integrase Mutants 
Journal of Medicinal Chemistry  2014;57(4):1573-1582.
Integrase (IN) inhibitors are the newest class of antiretroviral agents developed for the treatment of HIV-1 infections. Merck’s Raltegravir (RAL) (October 2007) and Gilead’s Elvitegravir (EVG) (August 2012), which act as IN strand transfer inhibitors (INSTIs), were the first anti-IN drugs to be approved by the FDA. However, the virus develops resistance to both RAL and EVG, and there is extensive cross-resistance to these two drugs. New “2nd-generation” INSTIs are needed that will have greater efficacy against RAL- and EVG-resistant strains of IN. The FDA has recently approved the first second generation INSTI, GSK’s Dolutegravir (DTG) (August 2013). Our current article describes the design, synthesis, and evaluation of a series of 1,8-dihydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides, 1,4-dihydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides, and 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides. This resulted in the identification of noncytotoxic inhibitors that exhibited single digit nanomolar EC50 values against HIV-1 vectors harboring wild-type IN in cell-based assays. Importantly, some of these new inhibitors retain greater antiviral efficacy compared to that of RAL when tested against a panel of IN mutants that included Y143R, N155H, G140S/Q148H, G118R, and E138K/Q148K.
doi:10.1021/jm401902n
PMCID: PMC3983366  PMID: 24471816
25.  Antiviral Characteristics of GSK1265744, an HIV Integrase Inhibitor Dosed Orally or by Long-Acting Injection 
GSK1265744 is a new HIV integrase strand transfer inhibitor (INSTI) engineered to deliver efficient antiviral activity with a once-daily, low-milligram dose that does not require a pharmacokinetic booster. The in vitro antiviral profile and mechanism of action of GSK1265744 were established through integrase enzyme assays, resistance passage experiments, and cellular assays with site-directed molecular (SDM) HIV clones resistant to other classes of anti-HIV-1 agents and earlier INSTIs. GSK1265744 inhibited HIV replication with low or subnanomolar efficacy and with a selectivity index of at least 22,000 under the same culture conditions. The protein-adjusted half-maximal inhibitory concentration (PA-EC50) extrapolated to 100% human serum was 102 nM. When the virus was passaged in the presence of GSK1265744, highly resistant mutants with more than a 10-fold change (FC) in EC50 relative to that of the wild-type were not observed for up to 112 days of culture. GSK1265744 demonstrated activity against SDM clones containing the raltegravir (RAL)-resistant Y143R, Q148K, N155H, and G140S/Q148H signature variants (FC less than 6.1), while these mutants had a high FC in the EC50 for RAL (11 to >130). Either additive or synergistic effects were observed when GSK1265744 was tested in combination with representative anti-HIV agents, and no antagonistic effects were seen. These findings demonstrate that, similar to dolutegravir, GSK1265744 is differentiated as a new INSTI, having a markedly distinct resistance profile compared with earlier INSTIs, RAL, and elvitegravir (EVG). The collective data set supports further clinical development of GSK1265744.
doi:10.1128/AAC.03909-14
PMCID: PMC4291378  PMID: 25367908

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