Islatravir Is Not Expected to Be a Victim or Perpetrator of Drug-Drug Interactions via Major Drug-Metabolizing Enzymes or Transporters.

Islatravir (MK-8591) is a nucleoside reverse transcriptase translocation inhibitor in development for the treatment and prevention of HIV-1. The potential for islatravir to interact with commonly co-prescribed medications was studied in vitro. Elimination of islatravir is expected to be balanced between adenosine deaminase-mediated metabolism and renal excretion. Islatravir did not inhibit uridine diphosphate glucuronosyltransferase 1A1 or cytochrome p450 (CYP) enzymes CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4, nor did it induce CYP1A2, 2B6, or 3A4. Islatravir did not inhibit hepatic transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 1, bile salt export pump (BSEP), multidrug resistance-associated protein (MRP) 2, MRP3, or MRP4. Islatravir was neither a substrate nor a significant inhibitor of renal transporters organic anion transporter (OAT) 1, OAT3, OCT2, multidrug and toxin extrusion protein (MATE) 1, or MATE2K. Islatravir did not significantly inhibit P-glycoprotein and breast cancer resistance protein (BCRP); however, it was a substrate of BCRP, which is not expected to be of clinical significance. These findings suggest islatravir is unlikely to be the victim or perpetrator of drug-drug interactions with commonly co-prescribed medications, including statins, diuretics, anti-diabetic drugs, proton pump inhibitors, anticoagulants, benzodiazepines, and selective serotonin reuptake inhibitors.

Biochemical and Structural Properties of Entecavir-Resistant Hepatitis B Virus Polymerase with L180M/M204V Mutations.

Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations. IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.

Layer-by-Layer Assembled Nanostructured Lipid Carriers for CD-44 Receptor-Based Targeting in HIV-Infected Macrophages for Efficient HIV-1 Inhibition.

Macrophages act as a cellular reservoir in HIV infection. Elimination of HIV from macrophages has been an unfulfilled dream due to the failure of drugs to reach them. To address this, we developed CD44 receptor-targeted, novel hyaluronic acid (HA)-coated nanostructured lipid carriers (NLCs) of efavirenz via washless layer-by-layer (LbL) assembly of HA and polyallylamine hydrochloride (PAH). NLCs were subjected to TEM analysis, size and zeta potential, in vitro release and encapsulation efficiency studies. The uptake of NLCs in THP-1 cells was studied using fluorescence microscopy and flow cytometry. The anti-HIV efficacy was evaluated using p24 antigen inhibition assay. NLCs were found to be spherical in shape with anionic zeta potential (-23.66 ± 0.87 mV) and 241.83 ± 5.38 nm particle size. NLCs exhibited prolonged release of efavirenz during in vitro drug release studies. Flow cytometry revealed 1.73-fold higher uptake of HA-coated NLCs in THP-1 cells. Cytotoxicity studies showed no significant change in cell viability in presence of NLCs as compared with the control. HA-coated NLCs distributed throughout the cell including cytoplasm, plasma membrane and nucleus, as observed during fluorescence microscopy. HA-coated NLCs demonstrated consistent and significantly higher inhibition (81.26 ± 1.70%) of p24 antigen which was 2.08-fold higher than plain NLCs. The obtained results suggested preferential uptake of HA-coated NLCs via CD44-mediated uptake. The present finding demonstrates that HA-based CD44 receptor targeting in HIV infection is an attractive strategy for maximising the drug delivery to macrophages and achieve effective viral inhibition.

Effects of islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) on renal tubular cells and islatravir's interactions with organic anion transporters.

Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) is a novel reverse transcriptase translocation inhibitor and has a unique structure and high antiviral activity against wild-type and multidrug resistant HIV strains. In this study, we investigated whether islatravir (ISL) can cause kidney damage compared to tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). We also investigated interactions of these drugs with organic anion transporters (OATs). There is a large gap in ISL concentration between the pharmacological dose to proximal tubular cells and the clinical dose. ISL is unlikely to be taken up via OAT1 or OAT3; therefore, OAT1 and OAT3 may not be involved in the injury to tubular cells. Present data strongly suggests that ISL is not toxic to proximal tubules because blood levels of ISL are not high enough to cause kidney damage in the clinical setting.

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 ± 5.10 nm and 236.4 ± 7.07 nm, respectively. This indicated that these peptides could be further developed as potential HIV-1 RT inhibitors.

Current status of antivirals and druggable targets of SARS CoV-2 and other human pathogenic coronaviruses.

Coronaviridae is a peculiar viral family, with a very large RNA genome and characteristic appearance, endowed with remarkable tendency to transfer from animals to humans. Since the beginning of the 21st century, three highly transmissible and pathogenic coronaviruses have crossed the species barrier and caused deadly pneumonia, inflicting severe outbreaks and causing human health emergencies of inconceivable magnitude. Indeed, in the past two decades, two human coronaviruses emerged causing serious respiratory illness: severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), causing more than 10,000 cumulative cases, with mortality rates of 10 % for SARS-CoV-1 and 34.4 % for MERS-CoV. More recently, the severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) has emerged in China and has been identified as the etiological agent of the recent COVID-19 pandemic outbreak. It has rapidly spread throughout the world, causing nearly 22 million cases and ∼ 770,000 deaths worldwide, with an estimated mortality rate of ∼3.6 %, hence posing serious challenges for adequate and effective prevention and treatment. Currently, with the exception of the nucleotide analogue prodrug remdesivir, and despite several efforts, there is no known specific, proven, pharmacological treatment capable of efficiently and rapidly inducing viral containment and clearance of SARS-CoV-2 infection as well as no broad-spectrum drug for other human pathogenic coronaviruses. Another confounding factor is the paucity of molecular information regarding the tendency of coronaviruses to acquire drug resistance, a gap that should be filled in order to optimize the efficacy of antiviral drugs. In this light, the present review provides a systematic update on the current knowledge of the marked global efforts towards the development of antiviral strategies aimed at coping with the infection sustained by SARS-CoV-2 and other human pathogenic coronaviruses, displaying drug resistance profiles. The attention has been focused on antiviral drugs mainly targeting viral protease, RNA polymerase and spike glycoprotein, that have been tested in vitro and/or in clinical trials as well as on promising compounds proven to be active against coronaviruses by an in silico drug repurposing approach. In this respect, novel insights on compounds, identified by structure-based virtual screening on the DrugBank database endowed by multi-targeting profile, are also reported. We specifically identified 14 promising compounds characterized by a good in silico binding affinity towards, at least, two of the four studied targets (viral and host proteins). Among which, ceftolozane and NADH showed the best multi-targeting profile, thus potentially reducing the emergence of resistant virus strains. We also focused on potentially novel pharmacological targets for the development of compounds with anti-pan coronavirus activity. Through the analysis of a large set of viral genomic sequences, the current review provides a comprehensive and specific map of conserved regions across human coronavirus proteins which are essential for virus replication and thus with no or very limited tendency to mutate. Hence, these represent key druggable targets for novel compounds against this virus family. In this respect, the identification of highly effective and innovative pharmacological strategies is of paramount importance for the treatment and/or prophylaxis of the current pandemic but potentially also for future and unavoidable outbreaks of human pathogenic coronaviruses.

New indolylarylsulfone non-nucleoside reverse transcriptase inhibitors show low nanomolar inhibition of single and double HIV-1 mutant strains.

We designed and synthesized 21 new indolylarylsulfones (IASs) as new HIV-1 NNRTIs. Among these, IAS 12 exhibited a remarkable antiviral activity against single and double mutants (K103N EC50 = <0.7 nM; Y181C EC50 = <0.7 nM; Y188L EC50 = 21.3 nM; K103N-Y181C EC50 = 6.2 nM), resulting equally or more active than previuosly reported IAS 6 and some approved anti-HIV-1 drugs. Docking and molecular dynamics simulations of compound 12 in complex with WT, Y181C, Y188L, K103N and K103N-Y181C RTs clarified a general binding mode that was consistent with biological results. Kinetic experiments disclosed that derivative 12 preferentially binds WT and K103N-Y181C RTs to binary and ternary complexes, respectively.

Structure-Based Bioisosterism Yields HIV-1 NNRTIs with Improved Drug-Resistance Profiles and Favorable Pharmacokinetic Properties.

The development of efficacious NNRTIs for AIDS therapy commonly encountered the rapid generation of drug-resistant mutations, which becomes a major impediment to effective anti-HIV treatment. Using a structure-based bioisosterism strategy, a series of piperidine-substituted thiophene[2,3-d]pyrimidine derivatives were designed and synthesized. Compound 9a yielded the greatest potency, exhibiting significantly better anti-HIV-1 activity than ETR against all of the tested NNRTI-resistant HIV-1 strains. In addition, the phenotypic (cross)resistance of 9a and other NRTIs to the different selected HIV-1 strains was evaluated. As expected, no phenotypic cross-resistance against the NRTIs (AZT and PMPA) was observed with the mutant 9ares strain. Furthermore, 9a was identified with improved solubility, lower CYP liability, and hERG inhibition. Remarkably, 9a exhibited optimal pharmacokinetic properties in rats (F = 37.06%) and safety in mice (LD50 > 2000 mg/kg), which highlights 9a as a promising anti-HIV-1 drug candidate.

Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine.

Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent non-nucleoside reverse transcriptase inhibitors of wild-type and mutant HIV-1 strains.

A novel series of dihydroquinazolin-2-amine derivatives were synthesized and evaluated for their anti-HIV-1 activity in MT-4 cell cultures. All of the molecules were active against wild-type HIV-1 with EC50 values ranging from 0.61 µM to 0.84 nM. The most potent inhibitor, compound 4b, had an EC50 value of 0.84 nM against HIV-1 strain IIIB, and thus was more active than the reference drugs efavirenz and etravirine. Moreover, most of the compounds maintained high activity (low-micromolar EC50 values) against strains bearing the reverse transcriptase (RT) E138K mutation. Compound 4b had EC50 values of 3.5 nM and 66 nM against non-nucleoside reverse transcriptase inhibitor-resistant strains bearing the RT E138K and RES056 mutations. In enzyme activity assays, compound 4b exhibited an IC50 value of 10 nM against HIV-1 RT. Preliminary SARs and molecular docking studies provide valuable insights for further optimization.

First discovery of a potential carbonate prodrug of NNRTI drug candidate RDEA427 with submicromolar inhibitory activity against HIV-1 K103N/Y181C double mutant strain.

In the present work, we described the synthesis, antiviral profiles and metabolic stability in human plasma of compound 6, a potential carbonate prodrug of HIV-1 NNRTI drug candidate RDEA427. Compound 6 was found to inhibit the wild-type (WT) and K103N/Y181C double mutant HIV-1 strains at nano- and submicromolar concentrations, respectively. Moreover, it displayed potent HIV-1 reverse transcriptase inhibitory activity (IC50 = 0.264 µM). Further stability test in human plasma showed that 6 could release its active form RDEA427 in a linearly time-independent manner, possibly acting as a potential prodrug.

Deaminase-Dead Mouse APOBEC3 Is an In Vivo Retroviral Restriction Factor.

The apolipoprotein B editing complex 3 (APOBEC3) proteins are potent retroviral restriction factors that are under strong positive selection, both in terms of gene copy number and sequence diversity. A common feature of all the members of the APOBEC3 family is the presence of one or two cytidine deamination domains, essential for cytidine deamination of retroviral reverse transcripts as well as packaging into virions. Several studies have indicated that human and mouse APOBEC3 proteins restrict retrovirus infection via cytidine deaminase (CD)-dependent and -independent means. To understand the relative contribution of CD-independent restriction in vivo, we created strains of transgenic mice on an APOBEC3 knockout background that express a deaminase-dead mouse APOBEC3 due to point mutations in both CD domains (E73Q/E253Q). Here, we show that the CD-dead APOBEC3 can restrict murine retroviruses in vivo Moreover, unlike the wild-type protein, the mutant APOBEC3 is not packaged into virions but acts only as a cell-intrinsic restriction factor that blocks reverse transcription by incoming viruses. Finally, we show that wild-type and CD-dead mouse APOBEC3 can bind to murine leukemia virus (MLV) reverse transcriptase. Our findings suggest that the mouse APOBEC3 cytidine deaminase activity is not required for retrovirus restriction.IMPORTANCE APOBEC3 proteins are important host cellular restriction factors essential for restricting retrovirus infection by causing mutations in the virus genome and by blocking reverse transcription. While both methods of restriction function in vitro, little is known about their role during in vivo infection. By developing transgenic mice with mutations in the cytidine deamination domains needed for enzymatic activity and interaction with viral RNA, we show that APOBEC3 proteins can still restrict in vivo infection by interacting with reverse transcriptase and blocking its activity. These studies demonstrate that APOBEC3 proteins have evolved multiple means for blocking retrovirus infection and that all of these means function in vivo.

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption.

Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir.

Exploring the mechanism of inhibition of human telomerase by cysteine-reactive compounds.

Telomerase is an almost universal cancer target that consists minimally of a core protein human telomerase reverse transcriptase (hTERT) and a RNA component human telomerase RNA (hTR). Some inhibitors of this enzyme are thought to function by the covalent binding to one or several cysteine residues; however, this inhibition mechanism has never been investigated because of the difficulty in producing telomerase. In this study, we use a recent method to produce recombinant hTERT to analyze the effect of cysteine-reactive inhibitors on telomerase. Using mass spectrometry and mutagenesis analysis, we identify several targeted residues in separated domains of the hTERT protein and show that cysteine-reactive reagents abolish the interaction with the CR4/5 region of hTR.

Concentrations of Pro-Inflammatory Cytokines Are Not Associated with Senescence Marker p16INK4a or Predictive of Intracellular Emtricitabine/Tenofovir Metabolite and Endogenous Nucleotide Exposures in Adults with HIV Infection.

OBJECTIVES: As the HIV-infected population ages, the role of cellular senescence and inflammation on co-morbid conditions and pharmacotherapy is increasingly of interest. p16INK4a expression, a marker for aging and senescence in T-cells, is associated with lower intracellular concentrations of endogenous nucleotides (EN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs). This study expands on these findings by determining whether inflammation is contributing to the association of p16INK4a expression with intracellular metabolite (IM) exposure and endogenous nucleotide concentrations. METHODS: Samples from 73 HIV-infected adults receiving daily tenofovir/emtricitabine (TFV/FTC) with either efavirenz (EFV) or atazanavir/ritonavir (ATV/r) were tested for p16INK4a expression, and plasma cytokine and intracellular drug concentrations. Associations between p16INK4a expression and cytokine concentrations were assessed using maximum likelihood methods, and elastic net regression was applied to assess whether cytokines were predictive of intracellular metabolite/endogenous nucleotide exposures. RESULTS: Enrolled participants had a median age of 48 years (range 23-73). There were no significant associations between p16INK4a expression and cytokines. Results of the elastic net regression showed weak relationships between IL-1Ra and FTC-triphosphate and deoxyadenosine triphosphate exposures, and MIP-1ß, age and TFV-diphosphate exposures. CONCLUSIONS: In this clinical evaluation, we found no relationships between p16INK4a expression and cytokines, or cytokines and intracellular nucleotide concentrations. While inflammation is known to play a role in this population, it is not a major contributor to the p16INK4a association with decreased IM/EN exposures in these HIV-infected participants.

Synthesis and polymerase incorporation of ß,γ-modified α-l-threofuranosyl thymine triphosphate mimics.

Three ß,γ-modified α-l-threofuranosyl nucleoside triphosphates were synthesized. The ß,γ-modified tTTPs undergo a single incorporation event with HIV RT but undergo multiple incorporations to form full-length product with engineered thermophilic polymerases.

Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol.

Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase γ (mtDNA Pol γ) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPol-mediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol γ that may manifest in toxicity.

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent.

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H: quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16-F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16-F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ~80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16-F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells.

Structure and conformational analysis of the anti-HIV reverse transcriptase inhibitor AZT using MP2 and DFT methods. Differences with the natural nucleoside thymidine. Simulation of the 1st phosphorylation step with ATP.

A comprehensive quantum-chemical investigation of the conformational landscape of the HIV-1 reverse transcriptase inhibitor AZT (3'-azido-3'-deoxythymidine) nucleoside analogue was carried out. The whole conformational parameters (χ, γ, ß, δ, ϕ, P, νmax) were analysed as well as the NBO charges. The search located at least 55 stable structures, 9 of which were by MP2 within a 1 kcal mol(-1) electronic energy range of the global minimum. Most conformers were anti or high-anti around the glycoside bond and with North sugar ring puckering angles. The distribution of all the conformers according to the ranges of stability of the characteristic torsional angles was established. The results obtained were in accordance with those found in related anti-HIV nucleoside analogues. The best conformer in the anti form corresponded to the calculated values by MP2 of χ = -126.9°, ß = 176.4° and γ = 49.1°. An analysis of the lowest vibrations in conformer C1 was carried out. The first hydration shell was simulated and the structural differences with the natural nucleoside deoxythymidine (dT) were determined. The first phosphorylation step was simulated by interacting ATP with the best hydrated clusters of AZT and dT. The Na cations act as a bridge between the phosphate moieties of ATP making it easy for -P3O3 to receive the H5' proton from AZT or dT. A proton-transfer mechanism is proposed through the water molecules. When the number of the water molecules surrounding AZT is lower than 8, the first phosphorylation step of AZT can be carried out. However, the appropriate orientation of the O5'-H in dT avoids this limitation and it can be performed with large numbers of water molecules.