Non-nucleoside Reverse Transcriptase Inhibitor Binding Research Articles

Expanding the Solvent/Protein Region Occupation of the Non-Nucleoside Reverse Transcriptase Inhibitor Binding Pocket for Improved Broad-Spectrum Anti-HIV-1 Efficacy: from Rigid Phenyl-Diarylpyrimidines to Flexible Hydrophilic Piperidine-Diarylpyrimidines.

Considering the nonideal antiresistance efficacy of our previously reported non-nucleoside reverse transcriptase inhibitor 7, a series of novel piperidine-diarylpyrimidine derivatives were designed through expanding solvent/protein region occupation. The representative compound 15f proved to be exceptionally potent against Y188L (EC50 = 23 nM), F227L + V106A (EC50 = 15 nM) and RES056 (EC50 = 45 nM), significantly better than 7. This analog exerted strong inhibition against wild-type HIV-1 (EC50 = 3 nM) and single mutant strains (L100I, K103N, Y181C, E138 K). Notably, its cytotoxicity and selectivity (CC50 = 18.23 μM, SI = 6537) were 4-fold better than etravirine and rilpivirine. Additionally, it exhibited minimal suppression of CYP isoenzymes and hERG, indicating low potential for drug-drug interactions and cardiotoxicity. No significant acute toxicity and tissue damage at a dose of 2 g/kg were revealed. These findings lay the groundwork for the advancement of 15f as a highly potent, safe, and broad-spectrum NNRTI for HIV therapy.

Identification of novel diarylpyrimidine derivatives as potent HIV-1 non-nucleoside reverse transcriptase inhibitors against wild-type and K103N mutant viruses

HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) play a crucial role in combination antiretroviral therapy (cART). To further enhance their antiviral activity and anti-resistance properties, we developed a series of novel NNRTIs, by specifically targeting tolerant region I of the NNRTI binding pocket. Among them, compound 9t-2 displayed excellent anti-HIV-1 potency against wild-type and prevalent mutant strains with EC50 values between 0.0019 and 0.012 μM. This outperformed the positive drugs ETR, NVP and RPV. Aslo, ELISA results confirmed that these compounds can effectively inhibit the activity of HIV-1 RT. Molecular dynamics (MD) simulation studies indicated that the thiomorpholine-1,1-dioxide moiety of 9t-2 is capable of establishing additional interactions with residues P225, F227 and P236 in the tolerant region I, which contributed to its enhanced activity. Compound 9t-2 possessed negligible inhibitory effect on the five main CYP isoenzymes (IC50 > 10 μM), indicating a low potential for inducing CYP-mediated drug-drug interactions. In conclusion, compound 9t-2, with its enhanced anti-resistance properties, stands out as a promising lead compound for further optimization towards discovering the new generation of anti-HIV agents.

Phylogenetic position and genetic features of HIV-1 in CNS

Background. Due to the wide coverage with antiretroviral therapy, the life expectancy of HIV infected people has significantly increased. Against the background of a decrease in mortality from HIV infection, HIV-associated neurocognitive disorders, which develop even during effective treatment, are of high importance. The overall prevalence of this pathology among HIV-infected people reaches 42.6%.
 The objective of the study was to research the genetic features and phylogenetic position of HIV-1 persisting in the central nervous system.
 Materials and methods. The clinical study group consisted of 38 patients with severe neurocognitive disorders against the background of HIV infection in stage 4B. The viral load of HIV-1 in blood plasma and cerebrospinal fluid (CSF) was measured using the "AmpliSens HIV Monitor-FRT" reagents kit. Sanger sequencing was performed using the AmpliSens HIV-Resist-Seq assay kit on an Applied Biosystems 3500 analyzer. Phylogenetic analysis of the pol gene fragments of HIV-1 strains (the site encoding the viral protease and part of the reverse transcriptase) was carried out using maximum likelihood method with the GTR+G nucleotide substitution model. Comparisons of the tertiary structure of viral proteins were performed according to three-dimensional models of the protease and p51 and p66 reverse transcriptase subunits obtained by homologous reconstruction using the SWISS-MODEL tools.
 Results. The viral load in the sample of patients with severe CNS lesions in blood plasma was 6.27 times higher than in CSF and amounted to 4.67 and 3.87 lg copies/ml respectively by median (p = 0,004).
 Phylogenetic analysis with the use of all available HIV-1 genomes from GenBank, which differed from the studied ones by less than 5% showed close genetic relations of viruses circulating in Chelyabinsk region, apart from strains circulating in Russian Federation, with viruses circulating in neighboring countries, in most abundance — from Ukraine and Kyrgyzstan, slightly less — from Belarus, Tajikistan, Kazakhstan and Armenia and also with strains from certain foreign countries: Poland and Germany. Phylogenetic analysis of 38 HIV-1 genomes revealed significant genetic distances between HIV isolates from blood plasma and CSF in 5 patients, 4 of whom were PWID, which may indicate an event of superinfection.
 The amount of independent amino acid substitutions in protease in isolates from blood plasma ranged from 1 to 3, in isolates from CSF — from 1 to 2. An amount of such substitutions in a fragment of reverse transcriptase in isolates from blood plasma ranged from 1 to 6, while in isolates from CSF, it ranged from 1 to 7. HIV isolates from blood plasma and CSF from 5 patients had differences in the tertiary structure of HIV-1 reverse transcriptase p51 subunit in amino acid positions 16–20 and 210–235. Isolates from 3 other patients differed in the tertiary structure only in amino acid positons 210–235. Isolates from 3 patients differed in the structure of HIV-1 RT p66 subunit in a non-nucleoside reverse transcriptase inhibitor binding pocket (NNRTI) region. Fixed differences in the tertiary structure of p51 subunit required at minimum only 1 amino acid substitution to emerge. Alterations in the tertiary structure of p66 subunit required at least 3 amino acid substitutions.
 Conclusion. Microevolution of HIV-1 proceeds in parallel within the same patient, in different compartments, which is reflected in the accumulation of amino acid substitutions different from another compartment in the conserved pol gene. There is a weak correlation between the viral load level in plasma and in CSF. The genetic heterogeneity of HIV strains from patients of the Chelyabinsk region indicates a high frequency of reintroduction of HIV infection in the region from other countries. Differences in the tertiary structure of HIV-1 reverse transcriptase between blood plasma and CSF isolates are regularly fixed in certain domens, which also confirms the presence of parallel HIV microevolution during virus persistence in tissues separated by the blood-brain barrier which allows a better understanding of the fixation trends of individual amino acid substitutions during HIV-induced damage to central nervous system.

Structure-Based Optimization of 2,4,5-Trisubstituted Pyrimidines as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: Exploiting the Tolerant Regions of the Non-Nucleoside Reverse Transcriptase Inhibitors' Binding Pocket.

Although non-nucleoside reverse transcriptase inhibitors (NNRTIs) exhibit potent anti-HIV-1 activity and play an important role in the active antiretroviral therapy of AIDS, the emergence of drug-resistant strains has seriously reduced their clinical efficacy. Here, we report a series of 2,4,5-trisubstituted pyrimidines as potent HIV-1 NNRTIs by exploiting the tolerant regions of the NNRTI binding pocket. Compounds 16b and 16c were demonstrated to have excellent activity (EC50 = 3.14-22.1 nM) against wild-type and a panel of mutant HIV-1 strains, being much superior to that of etravirine (EC50 = 3.53-52.2 nM). Molecular modeling studies were performed to illustrate the detailed interactions between RT and 16b, which shed light on the improvement of the drug resistance profiles. Moreover, 16b possessed favorable pharmacokinetic (T1/2 = 1.33 h, F = 31.8%) and safety profiles (LD50 > 2000 mg/kg), making it a promising anti-HIV-1 drug candidate for further development.

Novel indolylarylsulfone derivatives as covalent HIV-1 reverse transcriptase inhibitors specifically targeting the drug-resistant mutant Y181C

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are widely used in combination therapies against HIV-1. However, emergent and transmitted drug resistance compromise their efficacy in the clinical setting. Y181C is selected in patients receiving nevirapine, etravirine and rilpivirine, and together with K103N is the most prevalent NNRTI-associated mutation in HIV-infected patients. Herein, we report on the design, synthesis and biological evaluation of a novel series of indolylarylsulfones bearing acrylamide or ethylene sulfonamide reactive groups as warheads to inactivate Cys181-containing HIV-1 RT via a Michael addition reaction. Compounds I-7 and I-9 demonstrated higher selectivity towards the Y181C mutant than against the wild-type RT, in nucleotide incorporation inhibition assays. The larger size of the NNRTI binding pocket in the mutant enzyme facilitates a better fit for the active compounds, while stacking interactions with Phe227 and Pro236 contribute to inhibitor binding. Mass spectrometry data were consistent with the covalent modification of the RT, although off-target reactivity constitutes a major limitation for further development of the described inhibitors.

Exploiting the tolerant region I of the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding pocket. Part 2: Discovery of diarylpyrimidine derivatives as potent HIV-1 NNRTIs with high Fsp3 values and favorable drug-like properties.

To yield potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with favorable drug-like properties, a series of novel diarylpyrimidine derivatives targeting the tolerant region I of the NNRTI binding pocket were designed, synthesized and biologically evaluated. The most active inhibitor 10c exhibited outstanding antiviral activity against most of the viral panel, being about 2-fold (wild-type, EC50=0.0021μM), 1.7-fold (K103N, EC50=0.0019μM), and slightly more potent (E138K, EC50=0.0075μM) than the NNRTI drug etravirine (ETR). Additionally, 10c was endowed with relatively low cytotoxicity (CC50=18.52μM). More importantly, 10c possessed improved drug-like properties compared to those of ETR with an increased Fsp3 (Fraction of sp3 carbon atoms) value. Furthermore, the molecular dynamics simulation and molecular docking studies were implemented to reveal the binding mode of 10c in the binding pocket. Taken together, 10c is a promising lead compound that is worth further investigation.

Structure-based non-nucleoside inhibitor design: Developing inhibitors that are effective against resistant mutants.

Non‐nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit reverse transcription and block the replication of HIV‐1. Currently, NNRTIs are usually used as part of a three‐drug combination given to patients as antiretroviral therapy. These combinations involve other classes of anti‐HIV‐1 drugs, commonly nucleoside reverse transcriptase inhibitors (NRTIs). However, attempts are being made to develop two‐drug maintenance therapies, some of which involve an NNRTI and an integrase strand transfer inhibitor. This has led to a renewed interest in developing novel NNRTIs, with a major emphasis on designing compounds that can effectively inhibit the known NNRTI‐resistant mutants. We have generated and tested novel rilpivirine (RPV) analogs. The new compounds were designed to exploit a small opening in the upper right periphery of the NNRTI‐binding pocket. The best of the new compounds, 12, was a more potent inhibitor of the NNRTI‐resistant mutants we tested than either doravirine or efavirenz but was inferior to RPV. We describe the limitations on the modifications that can be appended to the “upper right side” of the RPV core and the effects of substituting other cores for the central pyrimidine core of RPV and make suggestions about how this information can be used in NNRTI design.

Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase.

Substitutions at residue Y181 in HIV-1 reverse transcriptase (RT), in particular, Y181C, Y181I, and Y181V, are associated with nonnucleoside RT inhibitor (NNRTI) cross-resistance. In this study, we used kinetic and thermodynamic approaches, in addition to molecular modeling, to gain insight into the mechanisms by which these substitutions confer resistance to nevirapine (NVP), efavirenz (EFV), and rilpivirine (RPV). Using pre-steady-state kinetics, we found that the dissociation constant (Kd ) values for inhibitor binding to the Y181C and Y181I RT-template/primer (T/P) complexes were significantly reduced. In the presence of saturating concentrations of inhibitor, the Y181C RT-T/P complex incorporated the next correct deoxynucleoside triphosphate (dNTP) more efficiently than the wild-type (WT) complex, and this phenotype correlated with decreased mobility of the RT on the T/P substrate. Interestingly, we found that the Y181F substitution in RT-which represents a transitional mutation between Y181 and Y181I/V, or a partial revertant-conferred hypersusceptibility to EFV and RPV at both the virus and enzyme levels. EFV and RPV bound more tightly to Y181F RT-T/P. Furthermore, inhibitor-bound Y181F RT-T/P was less efficient than the WT complex in incorporating the next correct dNTP, and this could be attributed to increased mobility of Y181F RT on the T/P substrate. Collectively, our data highlight the key role that Y181 in RT plays in NNRTI binding.

Discovery of piperidine-substituted thiazolo[5,4-d]pyrimidine derivatives as potent and orally bioavailable HIV-1 non-nucleoside reverse transcriptase inhibitors

HIV-1 reverse transcriptase offers a key target for antiviral therapy. However, the rapid emergence of drug-resistant mutations in reverse transcriptase as well as the poor pharmacokinetic properties of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) limits their clinical use. Starting from a previous piperidine-substituted thiophene[3,2-d]pyrimidine compound (K-5a2), here we explore the chemical space around the thiophene ring located in the solvent-exposed regions of the NNRTI binding pocket in detail. Bioisosterism-based structural modification leads to the discovery of a number of compounds as potent in vitro reverse transcriptase inhibitors, providing improved drug resistance profiles compared to the listed drug Etravirine. Furthermore, 14a and 19a are identified as lead compounds with good solubility, appropriate ligand efficiency, and lower cytochrome P450 liability. Compound 19a exhibits useful in vivo pharmacokinetic properties in rat and safety in mice, suggesting that it may have the potential to be an effective drug candidate for treating AIDS.

Predicting binding modes and affinities for non-nucleoside inhibitors to HIV-1 reverse transcriptase using molecular docking

The HIV/AIDS epidemic has become one of the most dangerous causes leading to millions of deaths around the world a year. To date, there have not had effective anti-HIV drugs in the treatment of HIV/AIDS because of emerging drug-resistant HIV mutants. In this work, potential non-nucleoside reverse transcriptase inhibitors (NNRTIs) were studied by means of molecular docking. The Diversity “drug-like” database from the National Cancer Institute, is composed of 1.420 compounds, was performed docking into the NNRTI binding pocket of HIV-1 reverse transcriptase crystal structure (1fk9) by using Autodock version 4.2.6. Pharmacokinetic properties (absorption, distribution, metabolism and excretion (ADME)) and toxicity of potential inhibitors within the body were predicted by the PreADMET version 2.0. The obtained results point out that the compound, coded 2518, was discovered as a potential inhibitor that has good human intestinal absorption, weakly bound to plasma proteins as well as is negative to mutagenicity and carcinogenicity. This rational inhibitor would be further studied in order to contribute informations finding new anti-HIV drugs.

Exploiting the Components Leading to Mutational Resistance in Rigid and Flexible Non‐nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

Non-nucleoside reverse transcriptase Inhibitors (NNRTIs) are antiretroviral drugs that bind to an allosteric site in HIV reverse transcriptase (RT). NNRTIs are often associated with treatment failure due to emergence of resistance mutations. Nevirapine is a first generation, rigid NNRTI that binds only one conformation in the binding site. Rilpivirine was designed as a flexible diarylpyrimidine that can adapt to multiple mutations in the NNRTI binding site. We have established a computational, structure-based method to predict resistance mutations to NNRTIs and similar compounds in development. The objective of the current study is to examine this approach for 2 types of NNRTIs: rigid and flexible inhibitors. We selected nevirapine and rilpivirine for the analysis and resistance prediction. In our approach, we employed molecular docking and residue scanning to predict resistance to both NNRTIs. Results from residue scanning predicted that the K101P mutation conferred high level resistance to rilpivirine and intermediate-level resistance to nevirapine. These predicted results were further validated through structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results indicate that resistance may be caused by the loss of a salt bridge required for the stability and affinity of the NNRTI. As this is a proof of concept, we believe that our computational and structure-based approach may be used to predict resistance to diverse inhibitors in development. Support or Funding Information This study was supported and funded by my advisor Dr. Kathleen Frey. Superposition of RT (WT) crystal structure 2ZD1 (green) with RT (K101P) model (orange) generated by residue scans in complex with Rilpivirine (gray). Superposition of RT (WT) crystal structure 1VRT (green) with RT (K101P) model (orange) generated by residue scans in complex with Nevirapine (yellow). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the entrance channel of NNIBP: Discovery of diarylnicotinamide 1,4-disubstituted 1,2,3-triazoles as novel HIV-1 NNRTIs with high potency against wild-type and E138K mutant virus

Inspired by our previous efforts on the modifications of diarylpyrimidines as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) and reported crystallography study, novel diarylnicotinamide derivatives were designed with a “triazole tail” occupying the entrance channel in the NNRTI binding pocket of the reverse transcriptase to afford additional interactions. The newly designed compounds were then synthesized and evaluated for their anti-HIV activities in MT-4 cells. All the compounds showed excellent to good activity against wild-type HIV-1 strain with EC50 of 0.02–1.77 μM. Evaluations of selected compounds against more drug-resistant strains showed these compounds had advantage of inhibiting E138K mutant virus which is a key drug-resistant mutant to the new generation of NNRTIs. Among this series, propionitrile (3b2, EC50(IIIB) = 0.020 μM, EC50(E138K) = 0.015 μM, CC50 = 40.15 μM), pyrrolidin-1-ylmethanone (3b8, EC50(IIIB) = 0.020 μM, EC50(E138K) = 0.014 μM, CC50 = 58.09 μM) and morpholinomethanone (3b9, EC50(IIIB) = 0.020 μM, EC50(E138K) = 0.027 μM, CC50 = 180.90 μM) derivatives are the three most promising compounds which are equally potent to the marketed drug Etravirine against E138K mutant strain but with much lower cytotoxicity. Furthermore, detailed SAR, inhibitory activity against RT and docking study of the representative compounds are also discussed.

Mechanistic Study of Common Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Mutations with K103N and Y181C Substitutions.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromatic π–π stacking plays a crucial role in the binding of NNRTIs to RT.

A Triazinone Derivative Inhibits HIV‐1 Replication by Interfering with Reverse Transcriptase Activity

A novel HIV-1 inhibitor, 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (compound 1), was identified from a compound library screened for the ability to inhibit HIV-1 replication. EC50 values of compound 1 were found to range from 107.9 to 145.4 nm against primary HIV-1 clinical isolates. In in vitro assays, HIV-1 reverse transcriptase (RT) activity was inhibited by compound 1 with an EC50 of 4.3 μm. An assay for resistance to compound 1 selected a variant of HIV-1 with a RT mutation (RTL100I ); this frequently identified mutation confers mild resistance to non-nucleoside RT inhibitors (NNRTIs). A recombinant HIV-1 bearing RTL100I exhibited a 41-fold greater resistance to compound 1 than the wild-type virus. Compound 1 was also effective against HIV-1 with RTK103N , one of the major mutations that confers substantial resistance to NNRTIs. Computer-assisted docking simulations indicated that compound 1 binds to the RT NNRTI binding pocket in a manner similar to that of efavirenz; however, the putative compound 1 binding site is located further from RTK103 than that of efavirenz. Compound 1 is a novel NNRTI with a unique drug-resistance profile.

Rilpivirine and Doravirine Have Complementary Efficacies Against NNRTI-Resistant HIV-1 Mutants.

Rilpivirine (RPV) is the latest non-nucleoside reverse transcriptase inhibitor (NNRTI) to be approved by Food and Drug Administration to combat HIV-1 infections. NNRTIs inhibit the chemical step in viral DNA synthesis by binding to an allosteric site located about 10 Å from the polymerase active site of reverse transcriptase (RT). Although NNRTIs potently inhibit the replication of wild-type HIV-1, the binding site is not conserved, and mutations arise in the binding pocket. Doravirine (DOR) is a new NNRTI in phase III clinical trials. Using a single round HIV-1 infection assay, we tested RPV and DOR against a broad panel of NNRTI-resistant mutants to determine their respective activities. We also used molecular modeling to determine if the susceptibility profile of each compound was related to how they bind RT. Several mutants displayed decreased susceptibility to DOR. However, with the exception of E138K, our data suggest that the mutations that reduce the potency of DOR and RPV are non-overlapping. Thus, these 2 NNRTIs have the potential to be used together in combination therapy. We also show that the location at which DOR and RPV bind with the NNRTI binding pocket of RT correlates with the differences in their respective susceptibility to the panel of NNRTI-resistance mutations. This shows that (1) DOR is susceptible to a number of well-known NNRTI resistance mutations and (2) an understanding of the mutational susceptibilities and binding interactions of NNRTIs with RT could be used to develop pairs of compounds with non-overlapping mutational susceptibilities.

Arylazolyl(azinyl)thioacetanilides. Part 20: Discovery of novel purinylthioacetanilides derivatives as potent HIV-1 NNRTIs via a structure-based bioisosterism approach

By means of structure-based bioisosterism approach, a series of novel purinylthioacetanilide derivatives were designed, synthesized and evaluated as potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Some of the tested compounds were found to be active against wild-type (WT) HIV-1(IIIB) with EC50 in the range of 0.78–4.46μM. Among them, LAD-8 displayed the most potent anti-HIV activity (EC50=0.78μM, SI=24). In addition, LBD-6 showed moderate activity against L100I mutant (EC50=5.64μM) and double mutant strain RES056 (EC50=22.24μM). Preliminary structure–activity relationships (SARs) were discussed in detail. Molecular modeling study was used to predict the optimal conformation in the NNRTI binding site, which may play a guiding role in further rational optimization.

Rilpivirine analogs potently inhibit drug-resistant HIV-1 mutants.

BackgroundNonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of antiretroviral compounds that bind in an allosteric binding pocket in HIV-1 RT, located about 10 Å from the polymerase active site. Binding of an NNRTI causes structural changes that perturb the alignment of the primer terminus and polymerase active site, preventing viral DNA synthesis. Rilpivirine (RPV) is the most recent NNRTI approved by the FDA, but like all other HIV-1 drugs, suboptimal treatment can lead to the development of resistance. To generate better compounds that could be added to the current HIV-1 drug armamentarium, we have developed several RPV analogs to combat viral variants that are resistant to the available NNRTIs.ResultsUsing a single-round infection assay, we identified several RPV analogs that potently inhibited a broad panel of NNRTI resistant mutants. Additionally, we determined that several resistant mutants selected by either RPV or Doravirine (DOR) caused only a small increase in susceptibility to the most promising RPV analogs.ConclusionsThe antiviral data suggested that there are RPV analogs that could be candidates for further development as NNRTIs, and one of the most promising compounds was modeled in the NNRTI binding pocket. This model can be used to explain why this compound is broadly effective against the panel of NNRTI resistance mutants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0244-2) contains supplementary material, which is available to authorized users.

Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have been developed for the treatment of acquired immunodeficiency syndrome. HIV-1 RT binding to NNRTIs has been characterized by various biophysical techniques. However, these techniques are often hampered by the low water solubility of the inhibitors, such as the current promising diarylpyrimidine-based inhibitors rilpivirine and etravirine. Hence, a conventional and rapid method that requires small sample amounts is desirable for studying NNRTIs with low water solubility. Here we successfully applied a recently developed mass spectrometric technique under non-denaturing conditions to characterize the interactions between the heterodimeric HIV-1 RT enzyme and NNRTIs with different inhibitory activities. Our data demonstrate that mass spectrometry serves as a semi-quantitative indicator of NNRTI binding affinity for HIV-1 RT using low and small amounts of samples, offering a new high-throughput screening tool for identifying novel RT inhibitors as anti-HIV drugs.

Design, synthesis, biochemical, and antiviral evaluations of C6 benzyl and C6 biarylmethyl substituted 2-hydroxylisoquinoline-1,3-diones: dual inhibition against HIV reverse transcriptase-associated RNase H and polymerase with antiviral activities.

Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). Although numerous chemotypes have been reported to inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV. We report herein the design, synthesis, and biological evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety. The synthesis involved a recently reported metal-free direct benzylation between tosylhydrazone and boronic acid, which allowed the generation of structural diversity for the hydrophobic aromatic region. Biochemical studies showed that the C-6 benzyl and biarylmethyl HID analogues, previously unknown chemotypes, consistently inhibited HIV RT-associated RNase H and polymerase with IC50s in low to submicromolar range. The observed dual inhibitory activity remained uncompromised against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs), suggesting the involvement of binding site(s) other than the NNRTI binding pocket. Intriguingly, these same compounds inhibited the polymerase, but not the RNase H function of Moloney Murine Leukemia Virus (MoMLV) RT and also inhibited Escherichia coli RNase H. Additional biochemical testing revealed a substantially reduced level of inhibition against HIV integrase. Molecular docking corroborates favorable binding of these analogues to the active site of HIV RNase H. Finally, a number of these analogues also demonstrated antiviral activity at low micromolar concentrations.

Design, Synthesis, and Anti‐HIV Evaluation of Novel Triazine Derivatives Targeting the Entrance Channel of the NNRTI Binding Pocket

A novel series of triazine derivatives targeting the entrance channel of the HIV-1 non-nucleoside reverse transcriptase inhibitor binding pocket (NNIBP) were designed and synthesized on the basis of our previous work. The results of a cell-based antiviral screening assay indicated that most compounds showed good-to-moderate activity against wild-type HIV-1 with EC50 values within the concentration range of 0.0078-0.16μm (compound DCS-a4, EC50 =7.8nm). Some compounds displayed submicromolar activity against the K103N/Y181C resistant mutant strain (such as compound DCS-a4, EC50 =0.65μm). Molecular modeling studies confirmed that the new compounds could bind into the NNIBP similarly as the lead compound, and the newly introduced flexible heterocycles could occupy the entrance channel effectively. In addition, the preliminary structure-activity relationship and the RT inhibitory assay are presented in this study.