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Chemical Research in Toxicology Apr 2023Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a...
Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant () in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.
Topics: Humans; Antineoplastic Agents; Doxorubicin; Deoxyguanine Nucleotides; Guanosine Triphosphate; Adenosine Triphosphate
PubMed: 37000908
DOI: 10.1021/acs.chemrestox.2c00367 -
Proceedings of the National Academy of... Jan 2018The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that...
The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the s of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively. DNA synthesis from dNDPs is about 17 times slower than from dNTPs, and DNA phosphorolysis about 200 times less efficient than pyrophosphorolysis. Such parameters allow DNA replication without requiring coupled metabolism to sequester the phosphate products, which consequently do not pose a threat to genome stability. This mechanism contrasts with DNA synthesis from dNTPs, which yield high-energy pyrophosphates that have to be hydrolyzed to phosphates to prevent the reverse reaction. Because the last common ancestor was likely a thermophile, dNDPs are plausible substrates for genome replication on early Earth and may represent metabolic intermediates later replaced by the higher-energy triphosphates.
Topics: Bacterial Proteins; DNA Replication; DNA, Bacterial; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Kinetics; Substrate Specificity; Taq Polymerase
PubMed: 29339523
DOI: 10.1073/pnas.1712193115 -
MSystems Apr 2023Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are...
Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are classified into three classes (I, II, and III) depending on their overall structure and metal cofactors. Pseudomonas aeruginosa is an opportunistic pathogen which harbors all three RNR classes, increasing its metabolic versatility. During an infection, P. aeruginosa can form a biofilm to be protected from host immune defenses, such as the production of reactive oxygen species by macrophages. One of the essential transcription factors needed to regulate biofilm growth and other important metabolic pathways is AlgR. AlgR is part of a two-component system with FimS, a kinase that catalyzes its phosphorylation in response to external signals. Additionally, AlgR is part of the regulatory network of cell RNR regulation. In this study, we investigated the regulation of RNRs through AlgR under oxidative stress conditions. We determined that the nonphosphorylated form of AlgR is responsible for class I and II RNR induction after an HO addition in planktonic culture and during flow biofilm growth. We observed similar RNR induction patterns upon comparing the P. aeruginosa laboratory strain PAO1 with different P. aeruginosa clinical isolates. Finally, we showed that during Galleria mellonella infection, when oxidative stress is high, AlgR is crucial for transcriptional induction of a class II RNR gene (). Therefore, we show that the nonphosphorylated form of AlgR, in addition to being crucial for infection chronicity, regulates the RNR network in response to oxidative stress during infection and biofilm formation. The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA. RNRs are classified into three classes (I, II, and III), and P. aeruginosa harbors all three of these classes, increasing its metabolic versatility. Transcription factors, such as AlgR, regulate the expression of RNRs. AlgR is involved in the RNR regulation network and regulates biofilm growth and other metabolic pathways. We determined that AlgR induces class I and II RNRs after an HO addition in planktonic culture and biofilm growth. Additionally, we showed that a class II RNR is essential during Galleria mellonella infection and that AlgR regulates its induction. Class II RNRs could be considered excellent antibacterial targets to be explored to combat P. aeruginosa infections.
Topics: Pseudomonas aeruginosa; Hydrogen Peroxide; Oxidative Stress; Reactive Oxygen Species; Deoxyribonucleotides
PubMed: 36794960
DOI: 10.1128/msystems.01005-22 -
Journal of the American Chemical Society Jul 2019Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base...
Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.
Topics: Base Pairing; Deoxyribonucleotides; Genetic Code; Nucleotides; Protein Biosynthesis; Synthetic Biology; Transcription, Genetic
PubMed: 31241334
DOI: 10.1021/jacs.9b02075 -
Nucleic Acids Research Apr 2019For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro...
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.
Topics: Acetylgalactosamine; Animals; Deoxyribonucleotides; Female; Fluorine; Humans; Male; RNA, Small Interfering; Rats; Rats, Sprague-Dawley
PubMed: 30820542
DOI: 10.1093/nar/gkz140 -
Biomedicine & Pharmacotherapy =... Apr 2023Elevated myocardial intracellular sodium ([Na]) was shown to decrease mitochondrial calcium ([Ca]) via mitochondrial sodium/calcium exchanger (NCX), resulting in...
BACKGROUND
Elevated myocardial intracellular sodium ([Na]) was shown to decrease mitochondrial calcium ([Ca]) via mitochondrial sodium/calcium exchanger (NCX), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na] in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na].
METHODS
Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCX inhibitor. Myocardial [Na] was measured by Na NMR spectroscopy.
RESULTS
HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na] was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na] to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts.
CONCLUSIONS
Elevated myocardial [Na] contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na] and/or NCX may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na].
Topics: Mice; Male; Animals; Diabetic Cardiomyopathies; Sodium-Glucose Transporter 2 Inhibitors; Sodium; Calcium; Deoxycytidine Monophosphate; Myocardial Contraction; Mice, Inbred C57BL; Myocardium; Adenosine Triphosphate; Diabetes Mellitus
PubMed: 36731341
DOI: 10.1016/j.biopha.2023.114310 -
ACS Chemical Biology Sep 2021While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers,...
While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNupp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of Rel complexed with pppGpp, we show that as an HD substrate mimic, the analogue serves as a orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.
Topics: Adenine Nucleotides; Allosteric Site; Bacillus subtilis; Bacterial Proteins; Deoxyribonucleotides; Escherichia coli; Gene Expression Regulation, Bacterial; Ligases; Protein Binding; Protein Conformation; Pyrophosphatases
PubMed: 34477366
DOI: 10.1021/acschembio.1c00398 -
Metabolism: Clinical and Experimental Nov 2022Few studies have explored the association of visit-to-visit variation in blood pressure (BP) and glycemic factors with cardiovascular disease (CVD) morbidity and...
OBJECTIVE
Few studies have explored the association of visit-to-visit variation in blood pressure (BP) and glycemic factors with cardiovascular disease (CVD) morbidity and mortality. This study aimed to examine the independent and joint effect of visit-to-visit BP and glycemic variation on CVD morbidity and mortality in persons with T2DM.
METHODS
The present study consisted of two retrospective cohort studies. The Taiwan Diabetes Study was based on a database of the National Diabetes Care Management Program (DCMP) and linked with cardiovascular morbidity incidence. The Taichung Diabetes Study was based on the DCMP database of a medical center, which can be linked with the National Death Registry dataset. The outcomes were analyzed by using Cox's proportional hazard models.
RESULTS
A total of 13,280 and 10,894 persons with T2DM in Taiwan and Taichung Diabetes Study, respectively, were included. SBP-CV, FPG-CV, and HbA1c-CV were significant predictors of stroke, CVD event or death, all-cause mortality, and expanded CVD mortality, whereas DBP-CV was a significant predictor of all-cause mortality and expanded and non-expanded CVD mortality. The joint effect of SBP, FPG, and HbA1c predicted the incidence of stroke and CVD event or death with increased risks of 16 %-35 %. In addition, the joint effect of SBP, DBP, FPG, and HbA1c was associated with all-cause and expanded CVD mortality with increased risks of 29 %-81 %.
CONCLUSIONS
The joint effect of BP and glucose variation improved the prediction of cardiovascular morbidity and mortality. Moreover, simultaneous measurement of visit-to-visit BP and glycemic variation may stratify persons with cardiovascular risks and may be regarded as important therapeutic goals in the care of T2DM.
Topics: Blood Glucose; Blood Pressure; Cardiovascular Diseases; Deoxycytidine Monophosphate; Diabetes Mellitus, Type 2; Glycated Hemoglobin; Humans; Retrospective Studies; Risk Factors; Stroke
PubMed: 36058287
DOI: 10.1016/j.metabol.2022.155308 -
Science Advances Jun 2021Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template...
Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2'-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.
Topics: Animals; DNA; DNA Repair; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Humans; Mammals; RNA; Ribonucleotides
PubMed: 34117057
DOI: 10.1126/sciadv.abf1771 -
Nucleic Acids Research Sep 2019A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read...
A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
Topics: DNA-Directed DNA Polymerase; Deoxyribonucleosides; Deoxyribonucleotides; High-Throughput Nucleotide Sequencing; Limit of Detection; Microscopy, Fluorescence; Oligodeoxyribonucleotides; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 31318971
DOI: 10.1093/nar/gkz611