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Critical Reviews in Analytical Chemistry 2022Human beings are in dire need of developing an efficient treatment against fierce viruses like hepatitis C virus (HCV) and Coronavirus (COVID-19). These viruses have... (Review)
Review
Human beings are in dire need of developing an efficient treatment against fierce viruses like hepatitis C virus (HCV) and Coronavirus (COVID-19). These viruses have already caused the death of over two million people all over the world. Therefore, over the last years, many direct-acting antiviral drugs (DAADs) were developed targeting nonstructural proteins of these two viruses. Among these DAADs, several drugs were found more effective and safer than the others as sofosbuvir, ledipasvir, grazoprevir, glecaprevir, voxilaprevir, velpatasvir, elbasvir, pibrentasvir and remdesivir. The last one is indicated for COVID-19, while the rest are indicated for HCV treatment. Due to the valuable impact of these DAADs, larger number of analytical methods were required to meet the needs of the clinical studies. Therefore, this review will highlight the current approaches, published in the period between 2017 to present, dealing with the determination of these drugs in two different matrices: pharmaceuticals and biological fluids with the challenges of analyzing these drugs either alone, with other drugs, in presence of interferences (pharmaceutical excipients or endogenous plasma components) or in presence of matrix impurities, degradation products and metabolites. These approaches include spectroscopic, chromatographic, capillary electrophoretic, voltametric and nuclear magnetic resonance methods that have been reported during this period. Moreover, the analytical instrumentation and methods used in determination of these DAADs will be illustrated in tabulated forms.
Topics: Humans; Antiviral Agents; Hepatitis C, Chronic; Sofosbuvir; Hepacivirus; COVID-19 Drug Treatment
PubMed: 34138669
DOI: 10.1080/10408347.2021.1923456 -
Proceedings of the National Academy of... Apr 2016Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine...
Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct.
Topics: Arabidopsis; Cell Line; DNA Damage; DNA Repair; DNA, Plant; Deoxyribodipyrimidine Photo-Lyase; Dose-Response Relationship, Radiation; Pyrimidine Dimers; Pyrimidinones; Radiation Dosage; Ultraviolet Rays
PubMed: 27071131
DOI: 10.1073/pnas.1604097113 -
Clinical Pharmacokinetics Nov 2018Ledipasvir/sofosbuvir (Harvoni), a fixed-dose combination tablet of an NS5A inhibitor ledipasvir and an NS5B polymerase inhibitor sofosbuvir, is approved for the... (Review)
Review
Ledipasvir/sofosbuvir (Harvoni), a fixed-dose combination tablet of an NS5A inhibitor ledipasvir and an NS5B polymerase inhibitor sofosbuvir, is approved for the treatment of chronic hepatitis C virus infection. Ledipasvir/sofosbuvir exhibits a favorable drug-drug interaction profile and can be administered with various medications that may be used by hepatitis C virus-infected patients, including patients with comorbidities, such as co-infection with human immunodeficiency virus or immunosuppression following liver transplantation. Ledipasvir/sofosbuvir is not expected to act as a victim or perpetrator of cytochrome P450- or UDP-glucuronosyltransferase 1A1-mediated drug-drug interactions. With the exception of strong inducers of P-glycoprotein, such as rifampin, ledipasvir/sofosbuvir is not expected to act as a victim of clinically relevant drug-drug interactions. As a perpetrator of pharmacokinetic drug-drug interactions via P-glycoprotein/BCRP, ledipasvir/sofosbuvir should not be used with rosuvastatin and elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate, whereas its co-administration with amiodarone is not recommended because of a pharmacodynamic interaction. This review summarizes a number of drug interaction studies conducted in support of the clinical development of ledipasvir/sofosbuvir.
Topics: Benzimidazoles; Drug Interactions; Fluorenes; Humans; Sofosbuvir; Uridine Monophosphate
PubMed: 29644537
DOI: 10.1007/s40262-018-0654-5 -
Journal of Photochemistry and... Jan 2018The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage...
The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage.
Topics: Base Sequence; DNA Damage; DNA Repair; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 29149689
DOI: 10.1016/j.jphotobiol.2017.10.034 -
Nutrients Jul 2019Medicines containing citicoline (cytidine-diphosphocholine) as an active principle have been marketed since the 1970s as nootropic and psychostimulant drugs available on...
Medicines containing citicoline (cytidine-diphosphocholine) as an active principle have been marketed since the 1970s as nootropic and psychostimulant drugs available on prescription. Recently, the inner salt variant of this substance was pronounced a food ingredient in the major world markets. However, in the EU no nutrition or health claim has been authorized for use in commercial communications concerning its properties. Citicoline is considered a dietetic source of choline and cytidine. Cytidine does not have any health claim authorized either, but there are claims authorized for choline, concerning its contribution to normal lipid metabolism, maintenance of normal liver function, and normal homocysteine metabolism. The applicability of these claims to citicoline is discussed, leading to the conclusion that the issue is not a trivial one. Intriguing data, showing that on a molar mass basis citicoline is significantly less toxic than choline, are also analyzed. It is hypothesized that, compared to choline moiety in other dietary sources such as phosphatidylcholine, choline in citicoline is less prone to conversion to trimethylamine (TMA) and its putative atherogenic N-oxide (TMAO). Epidemiological studies have suggested that choline supplementation may improve cognitive performance, and for this application citicoline may be safer and more efficacious.
Topics: Cytidine Diphosphate Choline; Dietary Supplements; Dose-Response Relationship, Drug; European Union; Food Analysis; Humans; Methylamines
PubMed: 31336819
DOI: 10.3390/nu11071569 -
The Journal of Neuroscience : the... Feb 2016Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders....
Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Cells, Cultured; Colon; Female; Ganglia, Spinal; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; NAV1.9 Voltage-Gated Sodium Channel; Nociceptors; Purine Nucleotides; Pyrimidine Nucleotides; Receptors, Purinergic P2Y; Species Specificity
PubMed: 26911685
DOI: 10.1523/JNEUROSCI.3369-15.2016 -
Biochemistry Nov 2021The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5'-monophosphate decarboxylase...
The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5'-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5'-monophosphate () is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of . The D37 and T100' side chains of OMPDC interact, respectively, with the C-3' and C-2' hydroxyl groups of enzyme-bound . D37G and T100'A substitutions result in 1.4 kcal/mol increases in the activation barrier Δ for catalysis of decarboxylation of the phosphodianion-truncated substrate 1-(β-d-erythrofuranosyl)orotic acid () but result in larger 2.1-2.9 kcal/mol increases in Δ for decarboxylation of and for phosphite dianion-activated decarboxylation of . This shows that these substitutions reduce transition-state stabilization by the Q215, Y217, and R235 side chains at the dianion binding site. The D37G and T100'A substitutions result in <1.0 kcal/mol increases in Δ for activation of OMPDC-catalyzed decarboxylation of the phosphoribofuranosyl-truncated substrate by phosphite dianions. Experiments to probe the effect of D37 and T100' substitutions on the kinetic parameters for d-glycerol 3-phosphate and d-erythritol 4-phosphate activators of OMPDC-catalyzed decarboxylation of show that Δ for sugar phosphate-activated reactions is increased by 2.5 kcal/mol for each -OH interaction eliminated by D37G or T100'A substitutions. We conclude that the interactions between the D37 and T100' side chains and ribosyl or ribosyl-like hydroxyl groups are utilized to activate OMPDC for catalysis of decarboxylation of , , and .
Topics: Binding Sites; Biophysical Phenomena; Catalysis; Cell Communication; Erythritol; Hydroxides; Kinetics; Orotic Acid; Orotidine-5'-Phosphate Decarboxylase; Phagocytosis; Phosphites; Protein Domains; Ribose; Sugar Phosphates; Uridine Monophosphate
PubMed: 34726391
DOI: 10.1021/acs.biochem.1c00589 -
ELife Nov 2023As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly...
As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K-D-K-E [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP--acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.
Topics: Receptors, Purinergic; Nucleotides; Uridine Diphosphate Glucose; Sugars; Receptors, Purinergic P2Y
PubMed: 37955640
DOI: 10.7554/eLife.85449 -
Biomolecules Apr 2022Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP.... (Review)
Review
Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
Topics: Adenosine Triphosphate; Allosteric Regulation; Carbon-Nitrogen Ligases; Cryoelectron Microscopy; Cytidine Triphosphate; Glutaminase; Glutamine; Guanosine Triphosphate; Nitric Oxide Synthase; Uridine Triphosphate
PubMed: 35625575
DOI: 10.3390/biom12050647 -
Xenobiotica; the Fate of Foreign... Dec 2022Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds,...
Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest (1203 pmol·minmg·protein). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.
Topics: Animals; Uridine Diphosphate Glucuronic Acid; Turtles; Magnesium; Uridine Diphosphate; Glucuronosyltransferase; Microsomes, Liver; Alamethicin
PubMed: 36594659
DOI: 10.1080/00498254.2022.2164750