RESUMO
Gemcitabine (dFdC) and emtricitabine (FTC) are first-line drugs that are used for the treatment of pancreatic cancer and human immunodeficiency virus, respectively. The above drugs must undergo sequential phosphorylation to become pharmacologically active. Interindividual variability associated with the responses of the above drugs has been reported. The molecular mechanisms underlying the observed variability are yet to be elucidated. Although this could be multifactorial, nucleotidases may be involved in the dephosphorylation of drug metabolites due to their structural similarity to endogenous nucleosides. With these in mind, we performed in vitro assays using recombinant nucleotidases to assess their enzymatic activities toward the metabolites of dFdC and FTC. From the above in vitro experiments, we noticed the dephosphorylation of dFdC-monophosphate in the presence of two 5'-nucleotidases (5'-NTs), cytosolic 5'-nucleotidase IA (NT5C1A) and cytosolic 5'-nucleotidase III (NT5C3), individually. Interestingly, FTC monophosphate was dephosphorylated only in the presence of NT5C3 enzyme. Additionally, nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) exhibited enzymatic activity toward both triphosphate metabolites of dFdC and FTC. Enzyme kinetic analysis further revealed Michaelis-Menten kinetics for both NT5C3-mediated dephosphorylation of monophosphate metabolites, as well as NTPDase 1-mediated dephosphorylation of triphosphate metabolites. Immunoblotting results confirmed the presence of NT5C3 and NTPDase 1 in both pancreatic and colorectal tissue that are target sites for dFdC and FTC treatment, respectively. Furthermore, sex-specific expression patterns of NT5C3 and NTPDase 1 were determined using mass spectrometry-based proteomics approach. Based on the above results, NT5C3 and NTPDase 1 may function in the control of the levels of dFdC and FTC metabolites. SIGNIFICANCE STATEMENT: Emtricitabine and gemcitabine are commonly used drugs for the treatment of human immunodeficiency virus and pancreatic cancer. To become pharmacologically active, both the above drugs must be phosphorylated. The variability in the responses of the above drugs can lead to poor clinical outcomes. Although the sources of drug metabolite concentration variability are multifactorial, it is vital to understand the role of nucleotidases in the tissue disposition of the above drug metabolites due to their structural similarities to endogenous nucleosides.
Assuntos
Gencitabina , Neoplasias Pancreáticas , Polifosfatos , Feminino , Humanos , Masculino , 5'-Nucleotidase/metabolismo , Desoxicitidina , Emtricitabina/química , Emtricitabina/metabolismo , Cinética , Nucleotidases/metabolismo , Nucleotídeos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismoRESUMO
In shallow, open-water engineered wetlands, design parameters select for a photosynthetic microbial biomat capable of robust pharmaceutical biotransformation, yet the contributions of specific microbial processes remain unclear. Here, we combined genome-resolved metatranscriptomics and oxygen profiling of a field-scale biomat to inform laboratory inhibition microcosms amended with a suite of pharmaceuticals. Our analyses revealed a dynamic surficial layer harboring oxic-anoxic cycling and simultaneous photosynthetic, nitrifying, and denitrifying microbial transcription spanning nine bacterial phyla, with unbinned eukaryotic scaffolds suggesting a dominance of diatoms. In the laboratory, photosynthesis, nitrification, and denitrification were broadly decoupled by incubating oxic and anoxic microcosms in the presence and absence of light and nitrogen cycling enzyme inhibitors. Through combining microcosm inhibition data with field-scale metagenomics, we inferred microbial clades responsible for biotransformation associated with membrane-bound nitrate reductase activity (emtricitabine, trimethoprim, and atenolol), nitrous oxide reduction (trimethoprim), ammonium oxidation (trimethoprim and emtricitabine), and photosynthesis (metoprolol). Monitoring of transformation products of atenolol and emtricitabine confirmed that inhibition was specific to biotransformation and highlighted the value of oscillating redox environments for the further transformation of atenolol acid. Our findings shed light on microbial processes contributing to pharmaceutical biotransformation in open-water wetlands with implications for similar nature-based treatment systems.
Assuntos
Compostos de Amônio , Áreas Alagadas , Atenolol , Biotransformação , Desnitrificação , Emtricitabina/metabolismo , Metoprolol , Nitrato Redutases/metabolismo , Nitrificação , Nitrogênio/metabolismo , Óxido Nitroso , Oxigênio , Preparações Farmacêuticas , Fotossíntese , Trimetoprima , ÁguaRESUMO
The increasing global prevalence of human immunodeficiency virus (HIV) is estimated at 36.7 million people currently infected. Lifelong antiretroviral (ARV) drug combination dosing allows management as a chronic condition by suppressing circulating viral load to allow for a near-normal life; however, the daily burden of oral administration may lead to non-adherence and drug resistance development. Long-acting (LA) depot injections of nanomilled poorly water-soluble ARVs have shown highly promising clinical results with drug exposure largely maintained over months after a single injection. ARV oral combinations rely on water-soluble backbone drugs which are not compatible with nanomilling. Here, we evaluate a unique prodrug/nanoparticle formation strategy to facilitate semi-solid prodrug nanoparticles (SSPNs) of the highly water-soluble nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (FTC), and injectable aqueous nanodispersions; in vitro to in vivo extrapolation (IVIVE) modelling predicts sustained prodrug release, with activation in relevant biological environments, representing a first step towards complete injectable LA regimens containing NRTIs.
Assuntos
Antirretrovirais/uso terapêutico , Sistemas de Liberação de Medicamentos , Infecções por HIV/tratamento farmacológico , Nanopartículas/uso terapêutico , Pró-Fármacos/uso terapêutico , Água/química , Antirretrovirais/química , Antirretrovirais/farmacocinética , Simulação por Computador , Quimioterapia Combinada , Emtricitabina/sangue , Emtricitabina/química , Emtricitabina/metabolismo , Emtricitabina/farmacocinética , Emulsões , Humanos , Pró-Fármacos/química , SolubilidadeRESUMO
Tenofovir (TFV) disoproxil fumarate and emtricitabine (FTC) are used in combination for HIV treatment and pre-exposure prophylaxis (PrEP). TFV disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV. FTC and TFV are nucleoside/nucleotide reverse transcriptase inhibitors that upon phosphorylation to nucleotide triphosphate analogs competitively inhibit HIV reverse transcriptase. We previously demonstrated that adenylate kinase 2, pyruvate kinase, muscle and pyruvate kinase, liver and red blood cell phosphorylate TFV in peripheral blood mononuclear cells (PBMC). To identify the kinases that phosphorylate FTC in PBMC, siRNAs targeted toward kinases that phosphorylate compounds structurally similar to FTC were delivered to PBMC, followed by incubation with FTC and the application of a matrix-assisted laser desorption ionization-mass spectrometry method and ultra high performance liquid chromatography-UV to detect the formation of FTC phosphates. Knockdown of deoxycytidine kinase decreased the formation of FTC-monophosphate, while siRNA targeted toward thymidine kinase 1 decreased the abundance of FTC-diphosphate. Knockdown of either cytidine monophosphate kinase 1 or phosphoglycerate kinase 1 decreased the abundance of FTC-triphosphate. Next-generation sequencing of genomic DNA isolated from 498 HIV-uninfected participants in the HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 clinical study, revealed 17 previously unreported genetic variants of TFV or FTC phosphorylating kinases. Of note, four individuals were identified as simultaneous carriers of variants of both TFV and FTC activating kinases. These results identify the specific kinases that activate FTC in PBMC, while also providing further insight into the potential for genetic variation to impact TFV and FTC activation.
Assuntos
Antivirais/metabolismo , Emtricitabina/metabolismo , Variação Genética , Leucócitos Mononucleares/enzimologia , Fosfotransferases/metabolismo , Tenofovir/metabolismo , Biotransformação , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfotransferases/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase ß (hPolß), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolß. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolß, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolß follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolß discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolß to complete thumb domain closure.
Assuntos
DNA Polimerase beta/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , DNA Polimerase beta/química , DNA Polimerase beta/genética , Nucleotídeos de Desoxicitosina/metabolismo , Emtricitabina/química , Emtricitabina/metabolismo , Humanos , Cinética , Lamivudina/química , Lamivudina/metabolismo , Modelos Moleculares , Conformação Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
1. Emtricitabine is a nucleoside reverse transcriptase inhibitor used in combination antiretroviral therapy of HIV (cART). Although active transport mechanisms are believed to mediate tubular secretion of the drug into urine, the responsible transporter and its potential to cause pharmacokinetic drug--drug interactions (DDI) has not been identified so far. The aim of this study was to investigate whether drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), OCT1 (SLC22A1), OCT2 (SLC22A2) or MATE1 (SLC47A1) can mediate active transcellular transfer of emtricitabine. 2. We employed transport assays in polarized monolayers of MDCK cells stably expressing P-gp, BCRP, MRP2, OCT1, OCT2 and/or MATE1. Among the transporters studied only MATE1 accelerated basal-to-apical transport of emtricitabine over a wide range of concentrations (6 nM to 1 mM). The transport was enhanced by an oppositely directed pH gradient and significantly reduced (p < 0.001) at low temperature in MDCK-MATE1, MDCK-OCT1/MATE1 and MDCK-OCT2/MATE1 cells. Co-administration of cimetidine or ritonavir decreased MATE1-mediated transport of emtricitabine by up to 42 and 39%, respectively (p < 0.01) and augmented intracellular accumulation of emtricitabine (p < 0.05). 3. We demonstrate emtricitabine as a substrate of MATE1 and suggest that MATE1 might cause DDI between emtricitabine and other co-administrated drugs including antiretrovirals.
Assuntos
Emtricitabina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Cães , Células Madin Darby de Rim CaninoRESUMO
DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.1-3 The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PPi) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events. Here, by using the triphosphates of chain-terminating antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP), we structurally reveal the correct sequence of post-chemistry steps during nucleotide incorporation by human DNA polymerase ß (hPolß) and provide a structural basis for PPi release. These post-catalytic structures reveal hPolß in an open conformation with PPi bound in the active site, thereby strongly suggesting that the reverse conformational changes occur prior to PPi release. The results also help to refine the role of the newly discovered third divalent metal ion for DNA polymerase-catalyzed nucleotide incorporation. Furthermore, a post-chemistry structure of hPolß in the open conformation, following incorporation of (-)3TC-MP, with a second (-)3TC-TP molecule bound to the active site in the absence of PPi, suggests that nucleotide binding stimulates PPi dissociation and occurs before polymerase translocation. Our structural characterization defines the order of the elusive post-chemistry steps in the canonical mechanism of a DNA polymerase.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos/metabolismo , Antivirais/química , Antivirais/metabolismo , Biocatálise , DNA Polimerase Dirigida por DNA/química , Emtricitabina/química , Emtricitabina/metabolismo , Humanos , Lamivudina/química , Lamivudina/metabolismo , Nucleotídeos/química , Conformação ProteicaRESUMO
Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).
Assuntos
Coenzimas/metabolismo , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Enzimas Multifuncionais/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Antivirais/metabolismo , Antivirais/uso terapêutico , Arabinofuranosilcitosina Trifosfato/metabolismo , Arabinofuranosilcitosina Trifosfato/uso terapêutico , Cátions Bivalentes/metabolismo , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/metabolismo , Citidina Trifosfato/uso terapêutico , Desoxirribonucleotídeos/metabolismo , Didesoxinucleotídeos/metabolismo , Didesoxinucleotídeos/uso terapêutico , Emtricitabina/análogos & derivados , Emtricitabina/metabolismo , Emtricitabina/uso terapêutico , Humanos , Cinética , Lamivudina/análogos & derivados , Lamivudina/metabolismo , Lamivudina/uso terapêuticoRESUMO
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.
Assuntos
Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Citocinas/metabolismo , Emtricitabina/metabolismo , Infecções por HIV/metabolismo , Inflamação/metabolismo , Nucleotídeos/metabolismo , Tenofovir/metabolismo , Adulto , Idoso , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/uso terapêutico , Biomarcadores/metabolismo , Senescência Celular/efeitos dos fármacos , Emtricitabina/uso terapêutico , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/uso terapêutico , Ritonavir/uso terapêutico , Tenofovir/uso terapêutico , Adulto JovemRESUMO
The coformulation of the nucleos(t)ide analogs (NA) tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC) is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP). Such complexities manifest in nonlinear intracellular pharmacokinetics (PK). In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP) at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD) study among forty subjects receiving daily TDF/FTC (300 mg/200 mg) from the first-dose to pharmacological intracellular steady-state (30 days). TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC) were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM). Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV)) of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) production were 1020 fmol/106 cells (130%) and 44.4 pmol/106 cells (82.5%), resulting in (90% prediction interval) 11% (0.45%, 53%) and 14% (2.6%, 35%) reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP). Simulation-based intracellular operational multiple dosing half-lives of TFV-DP and FTC-TP were 6.7 days and 33 hours. This model described the formation of intracellular TFV-DP/FTC-TP and the interaction with dNTPs, and can be used to simulate analog:dNTP time course for various dosing strategies.