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Journal of Pharmaceutical and... Mar 2022In this study, a sensitive and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous...
Development and application of a rapid and sensitive liquid chromatography-mass spectrometry method for simultaneous analysis of cytarabine, cytarabine monophosphate, cytarabine diphosphate and cytarabine triphosphate in the cytosol and nucleus.
In this study, a sensitive and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous analysis of cytarabine (ara-C), cytarabine monophosphate (ara-CMP), cytarabine diphosphate (ara-CDP) and cytarabine triphosphate (ara-CTP) in the cytosol and nucleus. The separation of analytes and endogenous interferents was achieved in 8 min on a hypercarb column (2.1 mm × 100 mm, 3 µm) by using a gradient elution with 95% acetonitrile and aqueous 5 mM hexylamine with 0.4% (v/v) diethylamine adjusted to pH 10. The analytes were detected with both negative and positive electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve demonstrated good linearity ranging from 5 to 750 nM for ara-C, 50-7500 nM for ara-CMP, 20-3000 nM for ara-CDP and 1-150 nM for ara-CTP in the cytosol. In the nucleus, good linearity was achieved over a concentration range of 1-100 nM for ara-C, 5-500 nM for ara-CMP, 2.5-250 nM for ara-CDP and 0.5-50 nM for ara-CTP. Intra- and interbatch accuracies and precisions met the standards of validation. The matrix effect, recovery and stability were also within acceptable ranges. After incubation with 10 μM ara-C for 3 h, the levels of ara-C, ara-CMP, ara-CDP and ara-CTP in the cytosol and nucleus of HL-60 cells and HL-60/ara-C cells were determined. Most of the metabolites were found within the quantitation range. The results showed that the nuclear ara-CTP level was significantly different than the intracellular ara-CTP level between HL-60 and HL-60/ara-C cells.
Topics: Arabinofuranosylcytosine Triphosphate; Chromatography, High Pressure Liquid; Cytarabine; Cytosol; Diphosphates; Humans; Tandem Mass Spectrometry
PubMed: 35101802
DOI: 10.1016/j.jpba.2022.114582 -
Cell Nov 2021Cyclic pyrimidines cCMP and cUMP were known to be present in a variety of organisms and cell types, but their biological roles remained mysterious. Tal et al. show that...
Cyclic pyrimidines cCMP and cUMP were known to be present in a variety of organisms and cell types, but their biological roles remained mysterious. Tal et al. show that bacteria use cCMP and cUMP as second messengers that function in anti-phage defense.
Topics: Bacteriophages; Cyclic CMP; Nucleotides, Cyclic; Pyrimidines; Second Messenger Systems
PubMed: 34767773
DOI: 10.1016/j.cell.2021.10.012 -
Database : the Journal of Biological... Nov 2021Protein domains are functional and structural units of proteins. They are responsible for a particular function that contributes to protein's overall role. Because of...
Protein domains are functional and structural units of proteins. They are responsible for a particular function that contributes to protein's overall role. Because of this essential role, the majority of the genetic variants occur in the domains. In this study, the somatic mutations across 21 cancer types were mapped to the individual protein domains. To map the mutations to the domains, we employed the whole human proteome to predict the domains in each protein sequence and recognized about 149 668 domains. A novel Perl-API program was developed to convert the protein domain positions into genomic positions, and users can freely access them through GitHub. We determined the distribution of protein domains across 23 chromosomes with the help of these genomic positions. Interestingly, chromosome 19 has more number of protein domains in comparison with other chromosomes. Then, we mapped the cancer mutations to all the protein domains. Around 46-65% of mutations were mapped to their corresponding protein domains, and significantly mutated domains for all the cancer types were determined using the local false discovery ratio (locfdr). The chromosome positions for all the protein domains can be verified using the cross-reference ensemble database. Database URL: https://dcmp.vit.ac.in/.
Topics: Deoxycytidine Monophosphate; Humans; Mutant Proteins; Neoplasms; Protein Domains; Proteome
PubMed: 34791106
DOI: 10.1093/database/baab066 -
Pharmacology & Therapeutics Aug 2023Drug-drug interactions (DDIs) arising from the perturbation of drug metabolising enzyme activities represent both a clinical problem and a potential economic loss for... (Review)
Review
Drug-drug interactions that alter the exposure of glucuronidated drugs: Scope, UDP-glucuronosyltransferase (UGT) enzyme selectivity, mechanisms (inhibition and induction), and clinical significance.
Drug-drug interactions (DDIs) arising from the perturbation of drug metabolising enzyme activities represent both a clinical problem and a potential economic loss for the pharmaceutical industry. DDIs involving glucuronidated drugs have historically attracted little attention and there is a perception that interactions are of minor clinical relevance. This review critically examines the scope and aetiology of DDIs that result in altered exposure of glucuronidated drugs. Interaction mechanisms, namely inhibition and induction of UDP-glucuronosyltransferase (UGT) enzymes and the potential interplay with drug transporters, are reviewed in detail, as is the clinical significance of known DDIs. Altered victim drug exposure arising from modulation of UGT enzyme activities is relatively common and, notably, the incidence and importance of UGT induction as a DDI mechanism is greater than generally believed. Numerous DDIs are clinically relevant, resulting in either loss of efficacy or an increased risk of adverse effects, necessitating dose individualisation. Several generalisations relating to the likelihood of DDIs can be drawn from the known substrate and inhibitor selectivities of UGT enzymes, highlighting the importance of comprehensive reaction phenotyping studies at an early stage of drug development. Further, rigorous assessment of the DDI liability of new chemical entities that undergo glucuronidation to a significant extent has been recommended recently by regulatory guidance. Although evidence-based approaches exist for the in vitro characterisation of UGT enzyme inhibition and induction, the availability of drugs considered appropriate for use as 'probe' substrates in clinical DDI studies is limited and this should be a research priority.
Topics: Humans; Glucuronosyltransferase; Clinical Relevance; Drug Interactions; Uridine Diphosphate
PubMed: 37263383
DOI: 10.1016/j.pharmthera.2023.108459 -
Handbook of Experimental Pharmacology 2017After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems,... (Review)
Review
After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems, also the noncanonical congeners cyclic cytidine-3',5'-monophosphate (cCMP) and cyclic uridine-3',5'-monophosphate (cUMP) gained more and more interest. Until the late 1980s, only a small number of cCMP and cUMP analogs with sometimes undefined purities had been described. Moreover, most of these compounds had been rather synthesized as precursors of antitumor and antiviral nucleoside-5'-monophosphates and hence had not been tested for any second messenger activity. Along with the recurring interest in cCMP- and cUMP-related signaling in the early 2000s, it became evident that well-characterized small molecule analogs with reliable purities would serve as highly valuable tools for the evaluation of a putative second messenger role of cyclic pyrimidine nucleotides. Meanwhile, for this purpose new cCMP and cUMP derivatives have been developed, and already known analogs have been resynthesized and highly purified. This chapter summarizes early medicinal chemistry work on cCMP and cUMP and analogs thereof, followed by a description of recent synthetic developments and an outlook on potential future directions.
Topics: Animals; Crystallization; Cyclic CMP; Humans; Molecular Structure; Nucleotides, Cyclic; Permeability; Prodrugs; Uridine Monophosphate
PubMed: 27896476
DOI: 10.1007/164_2015_41 -
The FEBS Journal Jan 2020Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch...
Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch repair function of S. pneumoniae has been assigned to the hexA-hexB gene products, an enzyme capable of the direct elimination of noncanonical nucleotides from the cytoplasm has not been described for this bacterium. Our results show that Spr1057, a protein with previously unknown function, is involved in the inactivation of mutagenic pyrimidine nucleotides and was accordingly designated PynA (pyrimidine nucleotidase A). Biochemical assays confirmed the phosphatase activity of the recombinant enzyme and revealed its metal ion dependence for optimal enzyme activity. We demonstrated that PynA forms a homodimer with higher in vitro activity towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate than towards canonical thymidine monophosphate. Furthermore, we showed via in vivo assays that PynA protects cells against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine and prevents the incorporation of the potentially mutagenic 5-bromo-2'-deoxyuridine (5-BrdU) into DNA. Fluctuation analysis performed under S. pneumoniae exposure to 5-BrdU revealed that the pynA null strain accumulates random mutations with high frequency, resulting in a 30-fold increase in the mutation rate. The data support a model in which PynA, a protein conserved in other Gram-positive bacteria, functions as a house-cleaning enzyme by selectively eliminating noncanonical nucleotides and maintaining the purity of dNTP pools, similar to the YjjG protein described for Escherichia coli.
Topics: 5'-Nucleotidase; Bacterial Proteins; Cations; Deoxyuridine; Mutation Rate; Streptococcus pneumoniae; Substrate Specificity; Thymidine Monophosphate
PubMed: 31437335
DOI: 10.1111/febs.15049 -
Handbook of Experimental Pharmacology 2017cCMP and cUMP are pyrimidine cyclic nucleotides which are present in several types of cells. These molecules could exert diverse cellular functions and might act as... (Review)
Review
cCMP and cUMP are pyrimidine cyclic nucleotides which are present in several types of cells. These molecules could exert diverse cellular functions and might act as second messengers. In the last years, diverse approaches were performed to analyze possible cellular substrates and signaling pathways of cCMP and cUMP. In this review these approaches are summarized, and probable cross talk of these signaling molecules is described. These analyses might lead to the (patho)physiological and pharmacological relevance of these noncanonical cyclic nucleotides.
Topics: Animals; Biological Assay; Cells, Cultured; Cyclic CMP; Humans; Nucleotides, Cyclic; Second Messenger Systems; Uridine Monophosphate
PubMed: 26721673
DOI: 10.1007/164_2015_38 -
Annals of the New York Academy of... Nov 2015The nucleotide analog NS5B polymerase inhibitor sofosbuvir was approved by the U.S. Food and Drug Administration (FDA) in December 2013 for the treatment of chronic... (Review)
Review
The nucleotide analog NS5B polymerase inhibitor sofosbuvir was approved by the U.S. Food and Drug Administration (FDA) in December 2013 for the treatment of chronic hepatitis C virus (HCV) infection in combination with ribavirin or peginterferon and ribavirin. Sofosbuvir was developed to meet an urgent medical need for shorter, safer, simplified, more effective HCV treatment regimens and to reduce or eliminate the need for peginterferon. New treatment regimens were especially required for patient populations with limited treatment options, including patients who had failed prior HCV therapy, those with compensated and decompensated cirrhosis, and those who were either intolerant of or had contraindications to interferon. Sofosbuvir plus ribavirin for patients with genotype 2 or 3 HCV infection was the first approved all-oral treatment option. Sofosbuvir is also the backbone of the first regimen available for patients awaiting liver transplantation to prevent HCV recurrence, as well as the first oral interferon-free regimen for patients coinfected with HCV and HIV. This paper describes the development of sofosbuvir up to its original FDA approval.
Topics: Antiviral Agents; Drug Discovery; Hepacivirus; Hepatitis C; Humans; Sofosbuvir
PubMed: 26235748
DOI: 10.1111/nyas.12832 -
Digestive and Liver Disease : Official... Dec 2014Sofosbuvir is the first-in-class NS5B nucleotide analogues to be launched for hepatitis C virus (HCV) treatment. Its viral potency, pangenotypic activity and high... (Review)
Review
Sofosbuvir is the first-in-class NS5B nucleotide analogues to be launched for hepatitis C virus (HCV) treatment. Its viral potency, pangenotypic activity and high barrier to resistance make it the ideal candidate to become a backbone for several IFN-free regimens. Recent data demonstrated that sofosbuvir either with ribavirin alone or in combination with other direct-acting antivirals (DAAs) as daclatasvir, ledipasvir or simeprevir are able to cure HCV in at least 90% or over of patients. Treatment experienced genotype 3 population may remain the most difficult to treat population, but ongoing DAA combination studies will help to fill this gap. Safety profile of sofosbuvir or combination with other DAAs is good. Resistance to sofosbuvir did not appear as a significant issue. The rationale for using this class of drug and the available clinical data are reviewed.
Topics: Antiviral Agents; Drug Therapy, Combination; Hepacivirus; Hepatitis C; Hepatitis D, Chronic; Humans; Interferons; Sofosbuvir; Treatment Outcome; Uridine Monophosphate
PubMed: 25453869
DOI: 10.1016/j.dld.2014.09.024 -
Biochemistry Nov 2022Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive...
Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the of human UCK2 without altering its , these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.
Topics: Humans; Pyrimidine Nucleotides; Uridine; Uridine Kinase
PubMed: 36190114
DOI: 10.1021/acs.biochem.2c00451