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Molecules (Basel, Switzerland) May 2024DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is...
DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.
Topics: 8-Hydroxy-2'-Deoxyguanosine; DNA; Deoxycytidine; Oxazines; Deoxyguanosine; DNA Damage; Nucleotides; Polyphosphates
PubMed: 38792131
DOI: 10.3390/molecules29102270 -
Spectrochimica Acta. Part A, Molecular... Oct 2024X-ray diffraction is a commonly used technique in the pharmaceutical industry for the determination of the atomic and molecular structure of crystals. However, it is...
X-ray diffraction is a commonly used technique in the pharmaceutical industry for the determination of the atomic and molecular structure of crystals. However, it is costly, sometimes time-consuming, and it requires a considerable degree of expertise. Vibrational circular dichroism (VCD) spectroscopy resolves these limitations, while also exhibiting substantial sensitivity to subtle modifications in the conformation and molecular packaging in the solid state. This study showcases VCD's ability to differentiate between various crystal structures of the same molecule (polymorphs, cocrystals). We examined the most effective approach for producing high-quality spectra and unveiled the intricate link between structure and spectrum via quantum-chemical computations. We rigorously assessed, using alanine as a model compound, multiple experimental conditions on the resulting VCD spectra, with the aim of proposing an optimal and efficient procedure. The proposed approach, which yields reliable, reproducible, and artifact-free results with maximal signal-to-noise ratio, was then validated using a set comprising of three amino acids (serine, alanine, tyrosine), one hydroxy acid (tartaric acid), and a monosaccharide (ribose) to mimic active pharmaceutical components. Finally, the optimized approach was applied to distinguish three polymorphs of the antiviral drug sofosbuvir and its cocrystal with piperazine. Our results indicate that solid-state VCD is a prompt, cost-effective, and easy-to-use technique to identify crystal structures, demonstrating potential for application in pharmaceuticals. We also adapted the cluster and transfer approach to calculate the spectral properties of molecules in a periodic crystal environment. Our findings demonstrate that this approach reliably produces solid-state VCD spectra of model compounds. Although for large molecules with many atoms per unit cell, such as sofosbuvir, this approach has to be simplified and provides only a qualitative match, spectral calculations, and energy analysis helped us to decipher the observed differences in the experimental spectra of sofosbuvir.
Topics: Circular Dichroism; Sofosbuvir; Crystallization; Vibration; Models, Molecular; Antiviral Agents
PubMed: 38788502
DOI: 10.1016/j.saa.2024.124478 -
Science Advances May 2024The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to...
The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.
Topics: Base Pairing; Catalytic Domain; Humans; DNA Polymerase gamma; Models, Molecular; Mutation; Deoxycytosine Nucleotides; Crystallography, X-Ray; Protein Binding
PubMed: 38787958
DOI: 10.1126/sciadv.adl3214 -
Scientific Reports May 2024The treatment of HCV and its sequelae are used to be predominantly based on Interferon (IFN). However, this was associated with significant adverse events as a result of... (Comparative Study)
Comparative Study
The treatment of HCV and its sequelae are used to be predominantly based on Interferon (IFN). However, this was associated with significant adverse events as a result of its immunostimulant capabilities. Since their introduction, the directly acting antiviral drugs (DAAs), have become the standard of care to treat of HCV and its complications including mixed cryoglobulinemic vasculitis (MCV). In spite of achieving sustained viral response (SVR), there appeared many reports describing unwelcome complications such as hepatocellular and hematological malignancies as well as relapses. Prolonged inflammation induced by a multitude of factors, can lead to DNA damage and affects BAFF and APRIL, which serve as markers of B-cell proliferation. We compared, head-to-head, three antiviral protocols for HCV-MCV treatment As regards the treatment response and relapse, levels of BAFF and APRIL among pegylated interferon α-based and free regimens (Sofosbuvir + Ribavirin; SOF-RIBA, Sofosbuvir + Daclatasvir; SOF-DACLA). Regarding clinical response HCV-MCV and SVR; no significant differences could be identified among the 3 different treatment protocols, and this was also independent form using IFN. We found no significant differences between IFN-based and free regimens DNA damage, markers of DNA repair, or levels of BAFF and APRIL. However, individualized drug-to-drug comparisons showed many differences. Those who were treated with IFN-based protocol showed decreased levels of DNA damage, while the other two IFN-free groups showed increased DNA damage, being the worst in SOF-DACLA group. There were increased levels of BAFF through follow-up periods in the 3 protocols being the best in SOF-DACLA group (decreased at 24 weeks). In SOF-RIBA, CGs relapsed significantly during the follow-up period. None of our patients who were treated with IFN-based protocol had significant clinico-laboratory relapse. Those who received IFN-free DAAs showed a statistically significant relapse of constitutional manifestations. Our findings suggest that IFN-based protocols are effective in treating HCV-MCV similar to IFN-free protocols. They showed lower levels of DNA damage and repair. We believe that our findings may offer an explanation for the process of lymphoproliferation, occurrence of malignancies, and relapses by shedding light on such possible mechanisms.
Topics: Humans; Cryoglobulinemia; Antiviral Agents; Male; Vasculitis; Middle Aged; Female; Aged; Hepacivirus; Ribavirin; Sofosbuvir; Imidazoles; Valine; Pyrrolidines; B-Cell Activating Factor; Interferon-alpha; Drug Therapy, Combination; Hepatitis C; Treatment Outcome; Hepatitis C, Chronic; Carbamates
PubMed: 38782988
DOI: 10.1038/s41598-024-60490-z -
Biochemistry Jun 2024The catalytic function of DNA polymerase β (pol β) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide...
The catalytic function of DNA polymerase β (pol β) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide into a gapped DNA substrate resulting from the removal of damaged DNA bases. Most importantly, pol β can select the correct nucleotide from a pool of similarly structured nucleotides to incorporate into DNA in order to prevent the accumulation of mutations in the genome. Pol β is likely to employ various mechanisms for substrate selection. Here, we use dCTP analogues that have been modified at the β,γ-bridging group of the triphosphate moiety to monitor the effect of leaving group basicity of the incoming nucleotide on precatalytic conformational changes, which are important for catalysis and selectivity. It has been previously shown that there is a linear free energy relationship between leaving group p and the chemical transition state. Our results indicate that there is a similar relationship with the rate of a precatalytic conformational change, specifically, the closing of the fingers subdomain of pol β. In addition, by utilizing analogue β,γ-CHX stereoisomers, we identified that the orientation of the β,γ-bridging group relative to R183 is important for the rate of fingers closing, which directly influences chemistry.
Topics: DNA Polymerase beta; Protein Conformation; Humans; Deoxycytosine Nucleotides; Substrate Specificity; Models, Molecular; Kinetics; DNA; DNA Repair
PubMed: 38780930
DOI: 10.1021/acs.biochem.4c00065 -
International Journal of Pharmaceutics Jun 2024This study aimed to develop, optimize, and evaluate hot-melt-extruded ophthalmic inserts capable of sustained release of diquafosol tetrasodium (DQS) via a design of...
This study aimed to develop, optimize, and evaluate hot-melt-extruded ophthalmic inserts capable of sustained release of diquafosol tetrasodium (DQS) via a design of experiments approach. DQS, a tear stimulant for dry eye management, faces challenges of frequent administration and low bioavailability. The developed insert uses biodegradable polymers in varied proportions to achieve sustained release. Optimized through mixture design, the insert completely dissolved within 24 h and maintained a stable drug content, thickness, and surface pH over three months at room temperature. In vitro corneal permeation studies on excised rabbit corneas demonstrated increased bioavailability, suggesting a reduced dosing frequency compared with conventional eye drops. Therefore, this insert has potential to enhance treatment outcomes by improving patient compliance and providing sustained drug effects.
Topics: Rabbits; Animals; Polyphosphates; Uracil Nucleotides; Cornea; Delayed-Action Preparations; Ophthalmic Solutions; Biological Availability; Drug Liberation; Administration, Ophthalmic; Drug Compounding; Drug Implants; Hot Temperature; Chemistry, Pharmaceutical
PubMed: 38772496
DOI: 10.1016/j.ijpharm.2024.124249 -
Trials May 2024The SARS CoV-2 pandemic has resulted in more than 1.1 million deaths in the USA alone. Therapeutic options for critically ill patients with COVID-19 are limited. Prior...
SCARLET (Supplemental Citicoline Administration to Reduce Lung injury Efficacy Trial): study protocol for a single-site, double-blinded, placebo-controlled, and randomized Phase 1/2 trial of i.v. citicoline (CDP-choline) in hospitalized SARS CoV-2-infected patients with hypoxemic acute respiratory...
BACKGROUND
The SARS CoV-2 pandemic has resulted in more than 1.1 million deaths in the USA alone. Therapeutic options for critically ill patients with COVID-19 are limited. Prior studies showed that post-infection treatment of influenza A virus-infected mice with the liponucleotide CDP-choline, which is an essential precursor for de novo phosphatidylcholine synthesis, improved gas exchange and reduced pulmonary inflammation without altering viral replication. In unpublished studies, we found that treatment of SARS CoV-2-infected K18-hACE2-transgenic mice with CDP-choline prevented development of hypoxemia. We hypothesize that administration of citicoline (the pharmaceutical form of CDP-choline) will be safe in hospitalized SARS CoV-2-infected patients with hypoxemic acute respiratory failure (HARF) and that we will obtain preliminary evidence of clinical benefit to support a larger Phase 3 trial using one or more citicoline doses.
METHODS
We will conduct a single-site, double-blinded, placebo-controlled, and randomized Phase 1/2 dose-ranging and safety study of Somazina® citicoline solution for injection in consented adults of any sex, gender, age, or ethnicity hospitalized for SARS CoV-2-associated HARF. The trial is named "SCARLET" (Supplemental Citicoline Administration to Reduce Lung injury Efficacy Trial). We hypothesize that SCARLET will show that i.v. citicoline is safe at one or more of three doses (0.5, 2.5, or 5 mg/kg, every 12 h for 5 days) in hospitalized SARS CoV-2-infected patients with HARF (20 per dose) and provide preliminary evidence that i.v. citicoline improves pulmonary outcomes in this population. The primary efficacy outcome will be the SO:FO ratio on study day 3. Exploratory outcomes include Sequential Organ Failure Assessment (SOFA) scores, dead space ventilation index, and lung compliance. Citicoline effects on a panel of COVID-relevant lung and blood biomarkers will also be determined.
DISCUSSION
Citicoline has many characteristics that would be advantageous to any candidate COVID-19 therapeutic, including safety, low-cost, favorable chemical characteristics, and potentially pathogen-agnostic efficacy. Successful demonstration that citicoline is beneficial in severely ill patients with SARS CoV-2-induced HARF could transform management of severely ill COVID patients.
TRIAL REGISTRATION
The trial was registered at www.
CLINICALTRIALS
gov on 5/31/2023 (NCT05881135).
TRIAL STATUS
Currently enrolling.
Topics: Humans; Cytidine Diphosphate Choline; Double-Blind Method; SARS-CoV-2; COVID-19; Randomized Controlled Trials as Topic; COVID-19 Drug Treatment; Clinical Trials, Phase II as Topic; Pneumonia, Viral; Treatment Outcome; Hypoxia; Male; Pandemics; Coronavirus Infections; Hospitalization; Female; Betacoronavirus; Clinical Trials, Phase I as Topic; Respiratory Insufficiency; Administration, Intravenous; Adult
PubMed: 38760804
DOI: 10.1186/s13063-024-08155-0 -
Nucleosides, Nucleotides & Nucleic Acids May 2024Glutamine amidotransferases (GATs) catalyze the synthesis of nucleotides, amino acids, glycoproteins and an enzyme cofactor, thus serving as key metabolic enzymes for...
Glutamine amidotransferases (GATs) catalyze the synthesis of nucleotides, amino acids, glycoproteins and an enzyme cofactor, thus serving as key metabolic enzymes for cell proliferation. arbamoyl-phosphate synthetase, spartate transcarbamoylase, and ihydroorotase (CAD) is a multifunctional enzyme of the GAT family and catalyzes the first three steps of the pyrimidine synthesis. Following our findings that cellular GATs are involved in immune evasion during herpesvirus infection, we discovered that CAD reprograms cellular metabolism to fuel aerobic glycolysis and nucleotide synthesis deamidating RelA. Deamidated RelA activates the expression of key glycolytic enzymes, rather than that of the inflammatory NF-κB-responsive genes. As such, cancer cells prime RelA for deamidation up-regulating CAD activity or accumulating RelA mutations. Interestingly, the recently emerged SARS-CoV-2 also activates CAD to couple evasion of inflammatory response to activated nucleotide synthesis. A small molecule inhibitor of CAD depletes nucleotide supply and boosts antiviral inflammatory response, thus greatly reducing SARS-CoV-2 replication. Additionally, we also found that CTP synthase 1 (CTPS1) deamidates interferon (IFN) regulatory factor 3 (IRF3) to mute IFN induction. Our previous studies have implicated phosphoribosyl formylglycinamidine synthase (PFAS) and phosphoribosyl pyrophosphate amidotransferase (PPAT) in deamidating retinoic acid-inducible gene I (RIG-I) and evading dsRNA-induced innate immune defense in herpesvirus infection. Overall, these studies have uncovered an unconventional enzymatic activity of cellular GATs in metabolism and immune defense, offering a molecular link intimately coupling these fundamental biological processes.
PubMed: 38743960
DOI: 10.1080/15257770.2024.2351135 -
PloS One 2024More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular...
More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.
Topics: Humans; Hepatocytes; Hepacivirus; Antiviral Agents; Sofosbuvir; Cell Line; Virus Replication; Interferon-alpha; Hepatitis C; Apoptosis; Mesenchymal Stem Cells
PubMed: 38739590
DOI: 10.1371/journal.pone.0303265 -
International Journal of Molecular... Apr 2024Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone...
Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y and P2Y receptors and K channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K (K) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K currents, and this effect was abolished with flutamide (an androgen receptor antagonist). K channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of K1.2 and K1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y signaling and K channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.
Topics: Animals; Uridine Triphosphate; Guinea Pigs; Muscle Relaxation; Male; Adenosine Triphosphate; Trachea; Testosterone; Adenylyl Cyclases; Muscle, Smooth; Potassium Channels, Voltage-Gated; Signal Transduction; Receptors, Purinergic P2
PubMed: 38731872
DOI: 10.3390/ijms25094652