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.

Evaluation of the effect of synthetic compounds derived from azidothymidine on MDA-MB-231 type breast cancer cells.

The present study aimed to investigate the effect of AZT derivates containing tellurium (Te) on human breast cancer cell lines and the mechanisms underlying cell death. The inhibitory effect of AZT and its derivatives (7m and 7r) was determined by the MTT assay (6.25, 12.5, 25, 50 and 100 µM in 24 and 48 h time points), meanwhile the induction of apoptosis and the cell cycle phases was investigated by flow cytometry. The MTT assay showed that AZT derivatives decreased the rate of cell proliferation at concentrations of 12.5 µM, while commercial AZT showed low antitumor potential. In flow cytometric analysis, we demonstrate that the AZT derivatives do not induce apoptosis at the concentration tested and promote the cell cycle arrest in the S phase. Besides, predicted absorption, distribution, metabolization, excretion and toxicity analysis suggest that the compounds possess a good pharmacokinetic profile and possibly less toxicity when compared to conventional AZT. These compounds containing tellurium in their formulation are potential therapeutic agents for breast cancer.

A Clinical Metabolite of Azidothymidine Inhibits Experimental Choroidal Neovascularization and Retinal Pigmented Epithelium Degeneration.

Purpose: Azidothymidine (AZT), a nucleoside reverse transcriptase inhibitor, possesses anti-inflammatory and anti-angiogenic activity independent of its ability to inhibit reverse transcriptase. The aim of this study was to evaluate the efficacy of 5'-glucuronyl azidothymidine (GAZT), an antiretrovirally inert hepatic clinical metabolite of AZT, in mouse models of retinal pigment epithelium (RPE) degeneration and choroidal neovascularization (CNV), hallmark features of dry and wet age-related macular degeneration (AMD), respectively. Methods: RPE degeneration was induced in wild-type (WT) C57BL/6J mice by subretinal injection of Alu RNA. RPE degeneration was assessed by fundus photography and confocal microscopy of zonula occludens-1-stained RPE flat mounts. Choroidal neovascularization was induced by laser injury in WT mice, and CNV volume was measured by confocal microscopy. AZT and GAZT were delivered by intravitreous injections. Inflammasome activation was monitored by western blotting for caspase-1 and by ELISA for IL-1ß in Alu RNA-treated bone marrow-derived macrophages (BMDMs). Results: GAZT inhibited Alu RNA-induced RPE degeneration and laser-induced CNV. GAZT also reduced Alu RNA-induced caspase-1 activation and IL-1ß release in BMDMs. Conclusions: GAZT possesses dual anti-inflammatory and anti-angiogenic properties and could be a viable treatment option for both forms of AMD.

Synthesis and evaluation of the anti-hepatitis B virus activity of 4'-Azido-thymidine analogs and 4'-Azido-2'-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent.

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4'-Azido-thymidine/4'-Azido-2'-deoxy-5-methylcytidine derivatives (6, 10-15) were synthesized, and their anti-HBV activities evaluated. Compounds 10-15 were synthesized via an SNAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11-15 showed no antiviral activity. However, 4'-Azido thymidine (6) and 4'-Azido-2'-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC50 = 0.63 and 5.99 µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100 µM.

New phosphazine and phosphazide derivatives as multifunctional ligands targeting acetylcholinesterase and ß-Amyloid aggregation for treatment of Alzheimer's disease.

Phosphazine and phosphazide derivatives are described herein as a new class of selective and potent acetylcholinesterase (AChE) inhibitors and ß-amyloid aggregation inhibitors. Phosphazines (5-7) were synthesized smoothly via a redox-condensation reaction of 1,2-bis(diphenylphosphino)ethane with different amines derivatives in the presence of dialkyl azodicarboxylate (Staudinger reaction) while phosphazides (8) via electrophilic attack of azido derivatives. Structures of the synthesized compounds were justified on the basis of compatible elementary and spectroscopic analyses. All the compounds were evaluated for their acetylcholinesterase inhibitory activity. The most three potent compounds (5b-c and 8b) showing AChE IC50 values (29.85-34.96 nM) comparable to that of donepezil (34.42 nM) were subjected to further investigation by testing their butyrylcholinesterase, MMP-2 and self-induced Aß aggregation inhibition activity. Especially, the coumarin phosphazide derivative (8b) presented the best AChE inhibition selectivity index (IC50 = 34.96 nM, AChE/BuChE; 3.81) together with good inhibition ability against MMP-2 (IC50 = 441.33 nM) and self-induced Aß1-42 aggregation (IC50 = 337.77 nM). In addition, the inhibition of metal-induced Aß aggregation by 8b was confirmed by thioflavine T fluorescence. The most potent effect of 8b was observed on the Zn2+-induced Aß42 aggregation. Kinetic study of compound 8b suggested it to be a competitive AChE inhibitor. Also, it specifically chelates metal and is predicted to be permeable to BBB. It also possesses low toxicity on SH-SY5Y neuroblastoma cells with a safety index of 15.37. In addition, it was demonstrated that compound 8b can improve the cognitive impairment of scopolamine-induced model in mice with % alternations and transfer latency time comparable to that of donepezil. Also, a docking study was carried out and it was in accordance with the in vitro results. These promising in vitro and in vivo findings highlight compound 8b as a possible drug candidate in searching for new multifunctional AD drugs.

[The experience in the application of nikavir in the schemes of perinatal chemoprophylactics of HIV infection: evaluation of the immunological and virusological effectiveness.]

OBJECTIVES: The goal of this work was to study the immunological and virological efficacy of the domestic antiretroviral drug nicavir (at the optimal dose, as proven by previous clinical studies) with lamivudine, in comparison with other drugs of the group of nucleoside reverse transcriptase inhibitors in combination with kaletra in perinatal HIV chemoprophylaxis regimens. METHODS: 658 pregnant women aged 16-39 years and children born to them were examined. The first group (281 people) and the third group (66 people) received the nicavir (manufactured by AZT PHARMA KB LLC) with lamivudine in combination with calyx; the second (281 people) and the fourth (30 people) of the comparison group, stag and zidovudine, respectively, with lamivudine in combination with calyx. The effectiveness of CP was assessed from the increase in the number of CD4 lymphocytes, reduction of the viral load, and the number of children born without HIV DNA in the blood. RESULTS: Against the backdrop of the therapy, the viral load below the detectable level and the positive dynamics of CD4 lymphocytes were registered in all examined women prior to childbirth. When applying the scheme of niacavir + lamivudine + kaletra, a more rapid decrease in the level of BH, most pronounced by week 4 of therapy, was found, as compared with the rate of decline of the same index in pregnant comparison groups. CONCLUSIONS: The obtained results allow us to consider ART with the inclusion of nicavir effective and recommend its priority use in perinatal prevention of mother-to-child transmission of HIV.

Chalcogenozidovudine Derivatives With Antitumor Activity: Comparative Toxicities in Cultured Human Mononuclear Cells.

Considering a novel series of zidovudine (AZT) derivatives encompassing selenoaryl moieties promising candidates as therapeutics, we examined the toxicities elicited by AZT and derivatives 5'-(4-Chlorophenylseleno)zidovudine (SZ1); 5'-(Phenylseleno)zidovudine (SZ2); and 5'-(4-Methylphenylseleno)zidovudine (SZ3) in healthy cells and in mice. Resting and stimulated cultured human peripheral blood mononuclear cells (PBMCs) were treated with the compounds at concentrations ranging from 10 to 200 µM for 24 and/or 72 h. Adult mice received a single injection of compounds (100 µmol/kg, s.c.) and 72 h after administration, hepatic/renal biomarkers were analyzed. Resting and stimulated PBMCs exposed to SZ1 displayed loss of viability, increased reactive species production, disruption in cell cycle, apoptosis and increased transcript levels and production of pro-inflammatory cytokines. In a mild way, most of these effects were also induced by SZ2. AZT and SZ3 did not cause significant toxicity towards resting PBMCs. Differently, both compounds elicited apoptosis and S phase arrest in stimulated cells. AZT and derivatives administration did not change the body weight and plasma biochemical markers in mice. However, the absolute weight and organ-to-body weight ratio of liver, kidneys and spleen were altered in AZT, SZ1-, and SZ2-treated mice. Our results highlighted the involvement of derivatives SZ1 and SZ2 in redox and immunological dyshomeostasis leading to activation of apoptotic signaling pathways in healthy cells under different division phases. On the other hand, the derivative SZ3 emerged as a promising candidate for further viral infection/antitumor studies as a new effective therapy with low toxicity for immune cells and after acute in vivo treatment.

Dual inhibitors of the human blood-brain barrier drug efflux transporters P-glycoprotein and ABCG2 based on the antiviral azidothymidine.

The brain provides a sanctuary site for HIV due, in part, to poor penetration of antiretroviral agents at the blood-brain barrier. This lack of penetration is partially attributed to drug efflux transporters such as P-glycoprotein (P-gp) and ABCG2. Inhibition of both ABCG2 and P-gp is critical for enhancing drug accumulation into the brain. In this work, we have developed a class of homodimers based on the HIV reverse transcriptase inhibitor azidothymidine (AZT) that effectively inhibits P-gp and ABCG2. These agents block transporter mediated efflux of the P-gp substrate calcein-AM and the ABCG2 substrate mitoxantrone. The homodimers function by interacting with the transporter drug binding sites as demonstrated by competition studies with the photo-affinity agent and P-gp/ABCG2 substrate [125I]iodoarylazidoprazosin. As such, these dual inhibitors of both efflux transporters provide a model for the future development of delivery vehicles for antiretroviral agents to the brain.

Recent progress in prodrug design strategies based on generally applicable modifications.

The development of prodrugs has progressed with the aim of improving drug bioavailability by overcoming various barriers that reduce drug benefits in clinical use, such as stability, duration, water solubility, side effect profile, and taste. Many conventional drugs act as the precursors of an active agent in vivo; for example, the anti-HIV agent azidothymidine (AZT) is converted into its corresponding active triphosphate ester in the body, meaning that AZT is a prodrug in the broadest sense. However prodrug design is generally difficult owing to the lack of general versatility. Thus, these prodrugs, broadly defined, are often discovered by chance or trial-and-error. Recently, many prodrugs that could release the corresponding parent drugs with or without enzymatic action under physiological conditions have been reported. These prodrugs can be easily designed and synthesized because of their generally applicable modifications. This digest paper provides an overview of recent development in prodrug strategies for drugs with a carboxylic acid or hydroxyl/amino group on the basis of a generally applicable modification strategy, such as esterification, amidation, or benzylation.

Phosphazide (nikavir) is a highly effective drug for the treatment of HIV/AIDS infection.

Federation Convincing evidence for high therapeutic activity and tolerability of Phosphazide in the treatment of HIV/AIDS-infection is given. Phosphazide is currently used in various regimens of highly active antiretroviral therapy, as well as in the HIV therapy in patients with simultaneously acquired chronic hepatitis C or tuberculosis. Therapeutic possibilities of Phosphazide were clearly manifested in the prevention of HIV transmission from mother to child. There is every reason to use Phosphazide in first-line antiretroviral therapy.

Self-assemblies of 5'-cholesteryl-ethyl-phosphoryl zidovudine.

Anti-HIV prodrugs are recently focused on due to their ability of self-assembly, macrophage targeting, and enhanced antiviral effects. Here, an amphiphilic prodrug of zidovudine, an anti-HIV nucleoside analogue, 5'-cholesteryl-ethyl-phosphoryl zidovudine (CEPZ) was synthesized. CEPZ showed some unique physicochemical properties. The solubility of CEPZ in the noncompetitive solvents chloroform and tetrahydrofuran (THF) was very high based on the hydrogen bonds between zidovudine groups, though CEPZ was sparing soluble in alcohols and almost insoluble in water. The typical amphiphilic property of CEPZ was demonstrated according to the Langmuir monolayers at the air/water interface. The LogP of CEPZ was high to 13.78, indicating the high hydrophobicity of amphiphilic CEPZ similar to phospholipids. Homogenous and stable self-assemblies were formed with the mean size of 128.7nm and the zeta potential of -35.4mV after injecting the CEPZ-in-THF solution into water. Hydrophobic interaction between the cholesteryl moieties of CEPZ could drive molecular self-assembly and lead to the formation of spherical vesicles. CEPZ self-assemblies showed strong stability even under high temperature and gravity probably due to the high surface charge. CEPZ was very slowly degraded in neutral solutions (e.g., pH 7.4), but fast in acid solutions (e.g., pH 5.0) and some tissue homogenates. CEPZ was quickly eliminated from the circulation and distributed into the mononuclear phagocyte system (MPS) including the liver, spleen and lung after bolus intravenous administration of CEPZ self-assemblies to mice. The MPS targeting effect of CEPZ self-assemblies makes them become a promising self-assembled drug delivery system to eradicate the HIV hidden in the macrophages.

Immobilization of Zidovudine Derivatives on the SBA-15 Mesoporous Silica and Evaluation of Their Cytotoxic Activity.

Novel zidovudine derivatives, able to be covalently conjugated to silica surface, have been obtained and grafted to SBA-15 mesoporous silica. Cytotoxic activity of the hybrid organic-inorganic (zidovudine derivatives-silica) systems against HeLa and KB cell lines has been analyzed. Addition of folic acid had a positive influence on the cytotoxicity. Up to 69% of HeLa and 65% of KB tumor cells growth inhibition has been achieved at low silica concentration used (10 µg/mL).

5'-Silylated 3'-1,2,3-triazolyl Thymidine Analogues as Inhibitors of West Nile Virus and Dengue Virus.

West Nile virus (WNV) and Dengue virus (DENV) are important human pathogens for which there are presently no vaccine or specific antivirals. We report herein a 5'-silylated nucleoside scaffold derived from 3'-azidothymidine (AZT) consistently and selectively inhibiting WNV and DENV at low micromolar concentrations. Further synthesis of various triazole bioisosteres demonstrated clear structure-activity relationships (SARs) in which the antiviral activity against WNV and DENV hinges largely on both the 5'-silyl group and the substituent of 3'-triazole or its bioisosteres. Particularly interesting is the 5' silyl group which turns on the antiviral activity against WNV and DENV while abrogating the previously reported antiviral potency against human immunodeficiency virus (HIV-1). The antiviral activity was confirmed through a plaque assay where viral titer reduction was observed in the presence of selected compounds. Molecular modeling and competitive S-adenosyl-l-methionine (SAM) binding assay suggest that these compounds likely confer antiviral activity via binding to methyltransferase (MTase).

Efficient syntheses of [¹¹C]zidovudine and its analogs by convenient one-pot palladium(0)-copper(I) co-mediated rapid C-[¹¹C]methylation.

The nucleosides zidovudine (AZT), stavudine (d4T), and telbivudine (LdT) are approved for use in the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections. To promote positron emission tomography (PET) imaging studies on their pharmacokinetics, pharmacodynamics, and applications in cancer diagnosis, a convenient one-pot method for Pd(0)-Cu(I) co-mediated rapid C-C coupling of [(11)C]methyl iodide with stannyl precursor was successfully established and applied to synthesize the PET tracers [(11)C]zidovudine, [(11)C]stavudine, and [(11)C]telbivudine. After HPLC purification and radiopharmaceutical formulation, the desired PET tracers were obtained with high radioactivity (6.4-7.0 GBq) and specific radioactivity (74-147 GBq/µmol) and with high chemical (>99%) and radiochemical (>99.5%) purities. This one-pot Pd(0)-Cu(I) co-mediated rapid C-[(11)C]methylation also worked well for syntheses of [methyl-(11)C]thymidine and [methyl-(11)C]4'-thiothymidine, resulting twice the radioactivity of those prepared by a previous two-pot method. The mechanism of one-pot Pd(0)-Cu(I) co-mediated rapid C-[(11)C]methylation was also discussed.

Drug-induced nanocarrier assembly as a strategy for the cellular delivery of nucleotides and nucleotide analogues.

The natural nucleotide adenosine triphosphate (ATP) and nucleotide analogues such as azidothymidine triphosphate (AZT-TP) display important pharmacological activities for the treatment of ischemia and HIV infections, respectively. Their clinical use is, however, limited mostly due to their hydrophilicity, which highly restricts their diffusion into the target cells. Few nanocarriers have been proposed to address the challenge of ATP/AZT-TP cellular delivery, but the loading efficiency, preparation complexity, and efficient cellular delivery remain important barriers to their development. In this study, we propose an original, straightforward and versatile design of nucleotide and nucleotide analogue nanocarriers based on the natural polysaccharide chitosan (CS). We show that the drugs ATP and AZT-TP can induce ionotropic gelation of CS, leading to CS/ATP and CS/AZT-TP nanoparticles with high drug entrapment efficiency and loading rate-up to 44%. Such nanocarriers release ATP and AZT-TP in physiological media and allow an efficient in vitro cellular delivery of these molecules down to the cell cytoplasm.

A pyrophosphatase activity associated with purified HIV-1 particles.

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.

A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.

Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3'-deoxy-3'-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position -7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the -7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the -7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.

HIV-1 polymerase inhibition by nucleoside analogs: cellular- and kinetic parameters of efficacy, susceptibility and resistance selection.

Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.

Structures of reverse transcriptase pre- and post-excision complexes shed new light on HIV-1 AZT resistance.

HIV-1 resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) results from mutations in reverse transcriptase that increase the ability of the enzyme to excise AZT-monophosphate after it has been incorporated. Crystal structures of complexes of wild type and mutant reverse transcriptase with double-stranded DNA with or without the excision product, AZT adenosine dinucleoside tetraphosphate (AZTppppA), have recently been reported. The excision-enhancing mutations dramatically change the way the enzyme interacts with the excision product.

A structural frame for understanding the role of thymidine analogue resistance mutations in resistance to zidovudine and other nucleoside analogues.

The acquisition of resistance to nucleoside reverse transcriptase inhibitors (NRTIs) can be mediated by amino acid changes at the dNTP binding site that affect the catalytic efficiency of nucleotide analogue incorporation, or by mutations that, in the presence of a pyrophosphate donor, facilitate excision of 3'-terminal chain-terminating inhibitors from blocked primers. These mutations, known as thymidine analogue resistance mutations (TAMs) are M41L, D67N, K70R, L210W, T215F/Y and K219E/Q. Recently published crystal structures of wild-type and TAM-containing HIV-1 reverse transcriptases bound to double-stranded DNA and the excision product, azidothymidine adenosine dinucleoside tetraphosphate have shed light into the molecular mechanism of excision.