HIV-1 Reverse Transcriptase Research Articles

Nucleocapsid Protein Precursors NCp9 and NCp15 Suppress ATP-Mediated Rescue of AZT-Terminated Primers by HIV-1 Reverse Transcriptase.

In HIV-1, development of resistance to AZT (3'-azido-3'-deoxythymidine) is mediated by the acquisition of thymidine analogue resistance mutations (TAMs) (i.e., M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q) in the viral reverse transcriptase (RT). Clinically relevant combinations of TAMs, such as M41L/T215Y or D67N/K70R/T215F/K219Q, enhance the ATP-mediated excision of AZT monophosphate (AZTMP) from the 3' end of the primer, allowing DNA synthesis to continue. Additionally, during HIV-1 maturation, the Gag polyprotein is cleaved to release a mature nucleocapsid protein (NCp7) and two intermediate precursors (NCp9 and NCp15). NC proteins interact with the viral genome and facilitate the reverse transcription process. Using wild-type and TAM-containing RTs, we showed that both NCp9 and NCp15 inhibited ATP-mediated rescue of AZTMP-terminated primers annealed to RNA templates but not DNA templates, while NCp7 had no effect on rescue activity. RNase H inactivation by introducing the active-site mutation E478Q led to the loss of the inhibitory effect shown by NCp9. NCp15 had a stimulatory effect on the RT's RNase H activity not observed with NCp7 and NCp9. However, analysis of RNase H cleavage patterns revealed that in the presence of NCp9, RNA/DNA complexes containing duplexes of 12 bp had reduced stability in comparison with those obtained in the absence of NC or with NCp7 or NCp15. These effects are expected to have a strong influence on the inhibitory action of NCp9 and NCp15 by affecting the efficiency of RNA-dependent DNA polymerization after unblocking DNA primers terminated with AZTMP and other nucleotide analogues.

HIV-1 transmitted drug resistance surveillance: shifting trends in study design and prevalence estimates.

IntroductionHIV‐1 transmitted drug resistance (TDR) prevalence increased during the initial years of the antiretroviral therapy (ART) global scale‐up. Few studies have examined recent trends in TDR prevalence using published genetic sequences and described the characteristics of ART‐naïve persons from whom these published sequences have been obtained.MethodsWe identified 125 studies published between 2014 and 2019 for which HIV‐1 reverse transcriptase (RT) with or without protease from ≥50 ART‐naïve adult persons were submitted to the GenBank sequence database. The population characteristics and TDR prevalence were compared to those in 122 studies published in the preceding five years between 2009 and 2013. TDR prevalence was analysed using median study‐level and person‐level data.Results and discussionThe 2009 to 2013 and 2014 to 2019 studies reported sequence data from 32,866 and 41,724 ART‐naïve persons respectively. Studies from the low‐ and middle‐income country (LMIC) regions in sub‐Saharan Africa, South/Southeast Asia and Latin America/Caribbean accounted for approximately two‐thirds of the studies during each period. Between the two periods, the proportion of studies from sub‐Saharan Africa and from South/Southeast Asia countries other than China decreased from 43% to 32% and the proportion of studies performed at sentinel sites for recent HIV‐1 infection decreased from 33% to 22%. Between 2014 and 2019, median study‐level TDR prevalence was 4.1% in South/Southeast Asia, 6.0% in sub‐Saharan Africa, 9.1% in Latin America/Caribbean, 8.5% in Europe and 14.2% in North America. In the person‐level analysis, there was an increase in overall, NNRTI and two‐class NRTI/NNRTI resistance in sub‐Saharan Africa; an increase in NNRTI resistance in Latin America/Caribbean, and an increase in overall, NNRTI and PI resistance in North America.ConclusionsOverall, NNRTI and dual NRTI/NNRTI‐associated TDR prevalence was significantly higher in sub‐Saharan Africa studies published between 2014 and 2019 compared with those published between 2009 and 2013. The decreasing proportion of studies from the hardest hit LMIC regions and the shift away from sentinel sites for recent infection suggests that global TDR surveillance efforts and publication of findings require renewed emphasis.

Computational Approaches to Identify a Hidden Pharmacological Potential in Large Chemical Libraries

To improve the discovery of more effective and less toxic pharmaceutical agents, large virtual repositories of synthesizable molecules have been generated to increase the explored chemical-pharmacological space diversity. Such libraries include billions of structural formulae of drug-like molecules associated with data on synthetic schemes, required building blocks, estimated physical-chemical parameters, etc. Clearly, such repositories are “Big Data”. Thus, to identify the most promising compounds with the required pharmacological properties (hits) among billions of available opportunities, special computational methods are necessary. We have proposed using a combined computational approach, which combines structural similarity assessment, machine learning, and molecular modeling. Our approach has been validated in a project aimed at finding new pharmaceutical agents against HIV/AIDS and associated comorbidities from the Synthetically Accessible Virtual Inventory (SAVI), a 1.75 billion compound database. Potential inhibitors of HIV-1 protease and reverse transcriptase and agonists of toll-like receptors and STING, affecting innate immunity, were computationally identified. The activity of the three synthesized compounds has been confirmed in a cell-based assay. These compounds belong to the chemical classes, in which the agonistic effect on TLR 7/8 had not been previously shown. Synthesis and biological testing of several dozens of compounds with predicted antiretroviral activity are currently taking place at the NCI/NIH. We also carried out virtual screening among one billion substances to find compounds potentially possessing anti-SARS-CoV-2 activity. The selected hits' information has been accepted by the European Initiative “JEDI Grand Challenge against COVID-19” for synthesis and further biological evaluation. The possibilities and limitations of the approach are discussed.

Study Design Characteristics and Pharmacological Mechanisms in International Clinical Trials Registry Platform: Registered Clinical Trials on Antiviral Drugs for COVID-19.

ObjectiveThis study aimed to evaluate the pharmacological mechanisms of antiviral drugs against the novel coronavirus disease (COVID-19) and the study designs in clinical trials registered with the International Clinical Trials Registry Platform (ICTRP).MethodsClinical trials involving antiviral drugs for treating COVID-19 were retrieved from the ICTRP database. For each trial, the study design, number of participants, primary endpoints, source register, antiviral mechanism, and results were evaluated.ResultsOn June 10, 2020, 145 eligible clinical trials were retrieved from the ICTRP, of which 99 (68.3%) were randomized trials, 109 (75.2%) were parallel assignment trials, 38 (26.2%) were double or single blinded, 130 (89.7%) involved two groups, and 75 (51.6%) included more than 100 participants; and clinical improvement or recovery and virus-negative conversion were the two most common endpoints, accounting for 40.7% and 18.6%, respectively. The drugs were divided according to the antiviral mechanism into HIV reverse transcriptase inhibitors, RNA-dependent RNA polymerase inhibitors, HIV protease inhibitors (PIs), hepatitis C virus NS3 PIs, and anti-influenza drugs.ConclusionThe design characteristics of clinical trials of antiviral drugs for treating COVID-19 as well as the mechanism of action and antiviral efficacy of the drugs were evaluated in this study. The results of these trials could constitute a reference for future clinical trials to be executed on COVID-19 treatment and prevention.

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives.

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18-mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.

Novel peptides with HIV-1 reverse transcriptase inhibitory activity derived from the fruits of Quercus infectoria.

The HIV-1 reverse transcriptase (HIV-1 RT), which is responsible for transcription of viral RNA genomes into DNA genomes, has become an important target for the treatment of patients with HIV infection. Hydrolyzed peptides from plants are considered a new source of potential drugs. In order to develop new effective inhibitors, peptides extracted from 111 Asian medicinal plants were screened against the HIV-1 RT. The crude hydrolyzed peptides from the fruit peel of Quercus infectoria were selected for purification and peptide sequence determination by HPLC and LC-MS. Two peptides of interest were synthesized, and an IC50 test was performed to determine their ability to inhibit the HIV-1 RT. The IC50 values of the peptides AIHIILI and LIAVSTNIIFIVV were determined to be 274±5.10nm and 236.4±7.07nm, respectively. This indicated that these peptides could be further developed as potential HIV-1 RT inhibitors.

Elucidating the Inhibitory Effect of Resveratrol and Its Structural Analogs on Selected Nucleotide-Related Enzymes.

Resveratrol, the most widely studied natural phytochemical, has been shown to interact with different target proteins. Previous studies show that resveratrol binds and inhibits DNA polymerases and some other enzymes; however, the binding and functioning mechanisms remain unknown. The elucidated knowledge of inhibitory mechanisms of resveratrol will assist us in new drug discovery. We utilized molecular docking and molecular dynamics (MD) simulation to reveal how resveratrol and structurally similar compounds bind to various nucleotide-dependent enzymes, specifically, DNA polymerases, HIV-1 reverse transcriptase, and ribonucleotide reductase. The results show that resveratrol and its analogs exert their inhibitory effects by competing with the substrate dNTPs in these enzymes and blocking elongation of chain polymerization. In addition, the results imply that resveratrol binds to a variety of other ATP-/NTP-binding proteins.

Polyether antibiotics K-41A and K-41Am inhibit HIV-1 replication via suppressing the activities of HIV-1 reverse transcriptase and integrase

IntroductionK-41A is a known compound with antibacterial activity against gram-positive bacteria and coccidia. In our screening of anti-HIV compounds, we found polyether antibiotic K-41A and its analogue K-41Am isolated from a marine-derived Streptomyces sp. SCSIO 01680 exhibited anti-HIV activity.Material and methodsIn this study, we characterised the anti-HIV activity of K-41A and K-41Am as well as the mechanism(s) involved. The dose-dependent inhibitory effects of K-41A and K-41Am on HIV-1 replication were observed in the TZM-bl-HIV-1IIIB system, MT-2-HIV-1IIIB system, and peripheral blood mononuclear cells (PBMCs)-HIV-1BaL system. The 50% inhibitory concentrations (IC50) of K-41A on HIV-1 replication in three systems were 0.75 µM, 0.09 µM, and 0.13 µM, respectively, and the selective indexes (SIs) were 5.81, 49.44, and 598.00, respectively. The IC50 of K-41Am in three systems were 5.57 µM, 0.24 µM, and 1.15 µM, respectively, and the SIs were > 1.04, 80.71, and > 5.03, respectively.ResultsMechanism research demonstrates that two compounds inhibited the activities of HIV-1 reverse transcriptase (RT) and integrase (IN) in a dose-dependent manner. Molecular docking shows that the docking scores were relatively high between the compound and HIV-1 RT or IN.ConclusionsK-41A and K-41Am possess anti-HIV-1 activity via a multi-target inhibition mechanism, which provides a novel molecular pattern for anti-HIV drug design and structural modification to improve the anti-HIV activity.

Design, synthesis, and biological evaluation of piperidinyl‐substituted [1,2,4]triazolo[1,5‐a]pyrimidine derivatives as potential anti‐HIV‐1 agents with reduced cytotoxicity

Taking the previously reported compound BH-7d as the lead, we designed and synthesized a series of piperidinyl-substituted [1,2,4]triazolo[1,5-a]pyrimidines, and their anti-HIV activities as well as cytotoxicities were evaluated. Several compounds exhibited moderate anti-HIV (IIIB) potency, among which 2b was the most active one (EC50 =4.29μM). Structure-activity relationships derived from the antiretroviral results were analyzed. Additionally, most compounds demonstrated reduced cytotoxicity (CC50 >200μM) compared with those of BH-7d and etravirine. Molecular docking study further revealed the binding conformation of 2b in the binding pocket of HIV-1 reverse transcriptase.

Isolation, identification, and evaluation of biological activities of Daldinia eschscholtzii, an endophytic fungus isolated from the leaves of Musa paradisiaca

Fungal endophytes of medicinal plants are gaining interest in the prospecting for biorelevant molecules due to their capacity to contribute novel compounds. This study was designed to identify and evaluate secondary metabolites of Daldinia eschscholtzii isolated from leaves of Musa paradisiaca for biorelevance. Plant leaves collection, fungal isolation, molecular identification, fermentation, and extraction of secondary metabolites were carried out following standard procedures. The crude extract was tested for antimicrobial, antioxidant, cytotoxic and antiviral properties; and further analyzed for their phyto-constituents using High performance liquid chromatography – diode array detection (HPLC-DAD). At 1000 µg/ml, D. eschscholtzii extract exhibited antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli producing IZD of 3 mm, 5 mm, and 7 mm respectively, but showed no antifungal activity. It showed antioxidant activity in the DPPH free radical scavenging assay producing an inhibition of 65.9% at a concentration of 500 µg/mL. At a concentration of 10 µg/mL, the D. eschscholtzii extract showed weak cytotoxic activity against L5178Y mouse lymphoma cells with growth inhibitions of 17.4% but showed a robust inhibition of HIV-1 reverse transcriptase activity of 97 % at 250 µg/mL. HPLC analysis of the extract/fraction revealed the presence of four bioactive compounds: aureonitol, pestalotiolactone A, cyclopenol, and palitantin. The detection of these metabolites further confirms the potentials of these endophytes as possible sources of bioactive molecules.

2'-fluoro-modified pyrimidines enhance affinity of RNA oligonucleotides to HIV-1 reverse transcriptase.

Nucleic acid aptamers can be chemically modified to enhance function, but modifying previously selected aptamers can have nontrivial structural and functional consequences. We present a reselection strategy to evaluate the impact of several modifications on preexisting aptamer pools. RNA aptamer libraries with affinity to HIV-1 reverse transcriptase (RT) were retranscribed with 2′-F, 2′-OMe, or 2′-NH2 pyrimidines and subjected to three additional selection cycles. RT inhibition was observed for representative aptamers from several structural families identified by high-throughput sequencing when transcribed with their corresponding modifications. Thus, reselection identified specialized subsets of aptamers that tolerated chemical modifications from unmodified preenriched libraries. Inhibition was the strongest with the 2′-F-pyrimidine (2′-FY) RNAs, as compared to inhibition by the 2′-OMeY and 2′-NH2Y RNAs. Unexpectedly, a diverse panel of retroviral RTs were strongly inhibited by all 2′-FY-modified transcripts, including sequences that do not inhibit those RTs as unmodified RNA. The magnitude of promiscuous RT inhibition was proportional to mole fraction 2′-FY in the transcript. RT binding affinity by 2′-FY transcripts was more sensitive to salt concentration than binding by unmodified transcripts, indicating that interaction with retroviral RTs is more ionic in character for 2′-FY RNA than for unmodified 2′-OH RNA. These surprising features of 2′-FY-modified RNA may have general implications for applied aptamer technologies.

Anti-HIV/SIV activity of icariin and its metabolite anhydroicaritin mainly involve reverse transcriptase

AIDS, a serious fatal disease caused by the human immunodeficiency virus (HIV), is an epidemic disease for which no effective vaccine has been established. The current therapeutic interventions for AIDS have limited efficacy because they are unable to clear HIV infections and the continuous occurrence of resistant HIV strains. Therefore, the exploitation of new drugs to prevent the spread of AIDS remains a high priority. In this study, the effects of icariin and its metabolite anhydroicaritin on SIV/HIV replication were investigated. In CEM × 174 cells and PBMC cells, both icariin and anhydroicaritin can significantly inhibit HIV-1 or SIVmac251 replication. Furthermore, molecular docking studies revealed that icariin and anhydroicaritin can act on both HIV reverse transcriptase and protease but could not bind to integrase. Reverse transcriptase and protease inhibition biological assays showed that both icariin and anhydroicaritin could significantly inhibit only HIV reverse transcriptase. In summary, the two compounds can significantly inhibit HIV/SIV in vitro and their targets may be mainly involved with HIV reverse transcriptase.

Uracil-Containing Heterodimers of a New Type: Synthesis and Study of Their Anti-Viral Properties.

Widespread latent herpes viral infections within a population can lead to the development of co-infections in HIV-infected patients. These infections are not particularly dangerous for healthy individuals and often occur with minimal symptoms, but for those who are immunocompromised, these infections can accelerate the acute phase of HIV infection and AIDS. Thus, the idea of designing compounds that could combine activity against HIV and co-infections would seem promising. In that regard, eleven compounds were synthesized that represent conjugates of non-nucleoside HIV reverse transcriptase inhibitors and nucleoside inhibitors of the herpes family viruses with the hope that these novel heterodimers will result in dual activity against HIV and concomitant herpes virus infections.

Synergistic Efficacy of Aframomum Melegueta [Roscoe] K. Schum and Spondias mombin (Linn), a Predictive Treatment of SARS-CoV-2 (COVID-19) Infection

SARS-CoV-2 is closely related to the original SARS-CoV. It is thought to have animal based origin from Bats(Betacoronavirus) in subgenus (Sarbecoronavirus) together with two bat-derived strains The virus is mainly spread between people during physical contact, often through small droplets produced during coughing, sneezing, even talking to patient or infected people. It is primarily spread during close contact and via small droplets produced when infected people cough, sneeze or talk with close contact being within 1–3 m (3 ft 3 in–9 ft 10 in). Those infected with the virus may be asymptomatic (Appearance of No symptoms) or develop flu-like symptom which include fever, cough, fatigue, and shortness of breath. The exposure time to initial symptom is day five (5 days). Majority of cases result in mild symptoms, some progress to viral pneumonia and multiple organ failure. Two medicinal plants may be used for the treatment of SARS-CoV-2, the two medicinal plants are Aframomum melegueta and Spondis mombin (Linn). Both contains arrays of natural product that may be used for drug formulation to combat SARS-CoV-2, Aframomum melegueta medicinal plant belong to the family Zingiberaceae which can be used to detoxify liver and bronchitis during lungs infection. Aframomum melegueta contains 6-Gingerol, 8-Gingerol, Methyl-6-Gingerol, 6-Shogaol, 6-Rac-6-Dihydro paradol,6-Gingeredione, 2-(5 butylfuran-2-yl) ethyl}-2-methoxyphenol and 6-Paradol. Secondary metabolite(Phytochemical)investigations of Aframomum melegueta extracts revealed the presence of (-)-buplerol, (-)-arctigenin, (E)- 14-hydroxy-15-norlabda-8(17), 12-dien- 16-al, labda-8(17), 12-dien-15, 16- dial, 16-oxo-8(17), 12(E)-labdadien-15-oic acid, 5-hydroxy-7-methoxy flavone and apigenin, The combination of all this novel compound can be used for the treatment of SARS-CoV-2. Spondias mombin belongs to the family of Anacardiaceae medicinal plants and contains Catechin and Epigalocatechin, Epicatechin, Stigmasterol and Phytosterol, a very important flavonoid that can be used in drug formulation because of its extensive therapeutic properties which may inhibit specific enzymes, blocking the angiotensin-converting enzyme (ACE) that raises blood pressure of infected patient during elevated fever clinical symptoms of SARS-CoV-2 (COVID-19). Spondias monbin extract may act by blocking the “suicide” enzyme cyclooxygenase that breaks down prostaglandins, they also prevent platelet aggregation. Cancer-battling antioxidants, flavonoids, have been found in relative abundance in Aframomum melegueta and Spondias mombin. Epicatechin have been proven to have diverse benefits to human health. It reduces the risks of diabetes mellitus and cardiovascular diseases, EGC acts as a strong inhibitor of HIV replication in cultured peripheral blood cells and inhibition of HIV-1 reverse transcriptase in vitro. EGC binds directly to CD4 molecules with consequent inhibition of Gp 120 binding and inactivate viruses in vitro by deformation of phospholipids, Epicatechin and other polyphenols decreases the susceptibility of low density lipoprotein to oxidation which prevents the initiation of artherosclerosis, HIV protein (Tat) and gp120.In conclusion. SARS-CoV-2(COVID-19) pandemic infection should not be a death sentence, it should be an advancement into the scientific world especially the naturally occurring product that can be used in the formulation of new antimicrobial agents.

Probing the Interaction between HIV-1 Protease and the Homodimeric p66/p66' Reverse Transcriptase Precursor by Double Electron-Electron Resonance EPR Spectroscopy.

Following excision from the Gag-Pol polyprotein, HIV-1 reverse transcriptase is released as an asymmetric homodimer comprising two p66 subunits that are structurally dissimilar but identical in amino acid sequence. Subsequent cleavage of the RNase H domain from only one of the subunits, denoted p66', results in the formation of the mature p66/p51 enzyme in which catalytic activity resides in the p66 subunit, and the p51 subunit (derived from p66') provides a supporting structural scaffold. Here, we probe the interaction of the p66/p66' asymmetric reverse transcriptase precursor with HIV-1 protease by pulsed Q-band double electron-electron resonance EPR spectroscopy to measure distances between nitroxide labels introduced at surface-engineered cysteine residues. The data suggest that the flexible, exposed linker between the RNaseH and connection domains in the open state of the p66' subunit binds to the active site of protease in a configuration that is similar to that of extended peptide substrates.

Targeting HIV-1 RNase H: N'-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants.

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N’-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.

Bioisosterism-based design and enantiomeric profiling of chiral hydroxyl-substituted biphenyl-diarylpyrimidine nonnucleoside HIV-1 reverse transcriptase inhibitors

The single enantiomers of seven hydroxyl-substituted biphenyl-diarylpyrimidines were designed and synthesized by a bioisosterism strategy as novel HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). The cellular and enzymatic assays indicated that the novel obtained compounds had significant activities and relatively low cytotoxicity. The supercritical fluid chromatography (SFC) enantioseparations of the racemic compounds and the enantiomeric profiling resulted that the (S) forms were generally more potent than the (R) counterparts. Among all the chiral derivatives, (S)-(−)-12a showed the best potency with the antiviral activities against wild-type (WT) and single mutant strains (L100I, K103 N, Y181C, E138K; especially Y188L), and RT enzyme in the low nanomolar concentration range. Toward double mutant virus strains (F227L + V106A, RES056), (S)-(−)-12a possessed submicromolar antiviral activities. In addition, (S)-(−)-12a showed a high cell-based selectivity index (SI WT = 5822) and no apparent toxicity was observed in the in vivo acute toxicity assay and electrocardiogram. The molecular docking studies predicted the binding modes of the compounds with RT and explained the activity differences for the enantiomers. Although the rat pharmacokinetic assay indicated a poor oral metabolism of the hydroxyl compound, the promising antiviral activity of the chiral hydroxyl-substituted biphenyl-diarylpyrimidines provided valuable lead compounds for further anti-HIV drug design.

Effect of Lysyl-tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase.

In human immunodeficiency virus-1 (HIV-1), reverse transcriptase (RT) is encoded as a 66 kDa protein, p66, in the Gag-Pol polyprotein. This protein is proteolytically cleaved by HIV-1 protease (PR) to finally generate a mature RT that is a heterodimer, composed of a p66 subunit and a p66-derived 51 kDa subunit, p51. In our prior work, we demonstrated that tRNALys3 binding to p66/p66 facilitates efficient cleavage of p66 to p51 by PR. However, tRNALys3 is known to be recruited to the virus by forming a complex with lysyl–tRNA synthetase (LysRS). Herein, we tested whether LysRS can have an effect on RT maturation in vitro. Importantly, our data show no significant differences in RT maturation in the presence of LysRS. Furthermore, no apparent p66/66 interaction with LysRS was observed. Although PR cleaved LysRS, it did not immediately release tRNALys3 from LysRS. Thus, we conclude that a free fraction of tRNALys3, which is in equilibrium with a LysRS-bound form, interacts with p66/p66 without any additional mechanism involving release of tRNALys3 from LysRS. Given that only transient tRNALys3–p66/p66 interaction is needed for efficient RT maturation, a small amount of free tRNA may be sufficient for this process. These studies reveal molecular level insights into RT maturation and will be useful for the design of cellular/viral experiments to better understand the role of tRNA in HIV-1 replication.

Insights into Biophysical Methods to Study Interactions Between HIV-1 Reverse Transcriptase and Non-nucleoside Reverse Transcriptase Inhibitors

Background:Reverse Transcriptase (RT) of immunodeficiency virus type-1 (HIV-1) remains an essential target for new antiretroviral therapies. Non-nucleoside reverse transcriptase inhibitors (or NNRTIs) constitute a major class of RT inhibitors whose characterization is essential.Introduction:Several biochemical, biological, and biophysical methods have been previously used to analyze the biological effects of NNRTIs. We explored here the use of surface plasmonic resonance to characterize the affinity of RT towards selected NNRTIs and compared the results with those obtained with in vitro and in cellulo assays.Methods:The solubility and stability in buffers of the tested NNRTIs were assessed by spectrophotometry and fluorescence. Surface plasmonic resonance experiments to study direct NNRTIs binding to immobilized RT and intramolecular quenching of RT tryptophan fluorescence were used to determine the KA association constants (= 1/KD) between RT and the inhibitors. The in vitro inhibition constants of RT were determined using kinetics and the effects on three other potential targets (proteasome, HIV-1 integrase, and HIV-1 protease) were analyzed.Results:The results obtained with two typical molecules belonging to our previous N-hydroxyureido acylnucleoside derivatives series using the above biophysical assays matched those obtained in in vitro and previous in cellulo assays.Conclusion:Surface plasmonic resonance provides reliable thermodynamic information on the interaction of RT with NNRTIs and appears as a useful method for understanding their inhibitory mechanism.

Application of Molecular Docking for the Development of Improved HIV-1 Reverse Transcriptase Inhibitors

Inhibition of the reverse transcriptase (RT) enzyme of the human immunodeficiency virus (HIV) by low molecular weight inhibitors is still an active area of research. Here, protein-ligand interactions and possible binding modes of novel compounds with the HIV-1 RT binding pocket (the wild-type as well as Y181C and K103N mutants) were obtained and discussed. A molecular fragment-based approach using FDA-approved drugs were followed to design novel chemical derivatives using delavirdine, efavirenz, etravirine and rilpivirine as the scaffolds. The drug-likeliness of the derivatives was evaluated using Swiss-ADME. The parent molecule and derivatives were then docked into the binding pocket of related crystal structures (PDB ID: 4G1Q, 1IKW, 1KLM and 3MEC). Genetic Optimization for Ligand Docking (GOLD) Suite 5.2.2 software was used for docking and the results analyzed in the Discovery Studio Visualizer 4. A derivative was chosen for further analysis, if it passed drug-likeliness and the docked energy was more favorable than that of its parent molecule. Out of the fifty-seven derivatives, forty-eight failed in drug-likeness screening by Swiss-ADME or at the docking stage. The final results showed that the selected compounds had higher predicted binding affinities than their parent scaffolds in both wild-type and the mutants. Binding energy improvement was higher for the structures designed based on second-generation NNRTIs (etravirine and rilpivirine) than the first-generation NNRTIs (delavirdine and efavirenz). For example, while the docked energy for rilpivirine was -51 KJ/mol, it was improved for its derivatives RPV01 and RPV15 up to - 58.3 and -54.5 KJ/mol, respectively. In this study, we have identified and proposed some novel molecules with improved binding capacity for HIV RT using a fragment-based approach.