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1.
J Transl Med ; 22(1): 449, 2024 May 13.
Article En | MEDLINE | ID: mdl-38741129

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.


DNA, Mitochondrial , Fibroblasts , Lysosomes , Mitochondria , Mitochondrial Encephalomyopathies , Nucleosides , Thymidine Phosphorylase , Humans , Lysosomes/metabolism , Thymidine Phosphorylase/metabolism , Thymidine Phosphorylase/deficiency , Thymidine Phosphorylase/genetics , Mitochondrial Encephalomyopathies/metabolism , Mitochondrial Encephalomyopathies/pathology , Mitochondrial Encephalomyopathies/genetics , Fibroblasts/metabolism , Fibroblasts/pathology , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Mitochondria/metabolism , Nucleosides/metabolism , Intestinal Pseudo-Obstruction/metabolism , Intestinal Pseudo-Obstruction/pathology , Intestinal Pseudo-Obstruction/enzymology , Intestinal Pseudo-Obstruction/genetics , Ophthalmoplegia/metabolism , Ophthalmoplegia/pathology , Ophthalmoplegia/congenital , Muscular Dystrophy, Oculopharyngeal/metabolism , Muscular Dystrophy, Oculopharyngeal/pathology , Male , Female , Skin/pathology , Skin/metabolism , Lysosomal-Associated Membrane Protein 2/metabolism
2.
DNA Repair (Amst) ; 137: 103668, 2024 May.
Article En | MEDLINE | ID: mdl-38460389

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.


Dideoxynucleosides , Nucleosides , Nucleosides/pharmacology , Nucleosides/genetics , Nucleosides/metabolism , DNA Replication , BRCA1 Protein/metabolism , DNA
3.
Int J Mol Sci ; 25(4)2024 Feb 17.
Article En | MEDLINE | ID: mdl-38397036

Nicotinamide (NA) derivatives play crucial roles in various biological processes, such as inflammation, regulation of the cell cycle, and DNA repair. Recently, we proposed that 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), an unusual derivative of NA, could be classified as an oncometabolite in bladder, breast, and lung cancer. In this study, we investigated the relations between NA metabolism and the progression, recurrence, metastasis, and survival of patients diagnosed with different histological subtypes of renal cell carcinoma (RCC). We identified alterations in plasma NA metabolism, particularly in the clear cell RCC (ccRCC) subtype, compared to papillary RCC, chromophobe RCC, and oncocytoma. Patients with ccRCC also exhibited larger tumor sizes and elevated levels of diagnostic serum biomarkers, such as hsCRP concentration and ALP activity, which were positively correlated with the plasma 4PYR. Notably, 4PYR levels were elevated in advanced stages of ccRCC cancer and were associated with a highly aggressive phenotype of ccRCC. Additionally, elevated concentrations of 4PYR were related to a higher likelihood of mortality, recurrence, and particularly metastasis in ccRCC. These findings are consistent with other studies, suggesting that NA metabolism is accelerated in RCC, leading to abnormal concentrations of 4PYR. This supports the concept of 4PYR as an oncometabolite and a potential prognostic factor in the ccRCC subtype.


Carcinoma, Renal Cell , Kidney Neoplasms , Pyridones , Ribonucleosides , Humans , Nucleosides/metabolism , Niacinamide
4.
Trends Endocrinol Metab ; 35(4): 290-299, 2024 Apr.
Article En | MEDLINE | ID: mdl-38423899

From our daily nutrition and synthesis within cells, nucleosides enter the bloodstream and circulate throughout the body and tissues. Nucleosides and nucleotides are classically viewed as precursors of nucleic acids, but recently they have emerged as a novel energy source for central carbon metabolism. Through catabolism by nucleoside phosphorylases, the ribose sugar group is released and can provide substrates for lower steps in glycolysis. In environments with limited glucose, such as at sites of infection or in the tumor microenvironment (TME), cells can use, and may even require, this alternative energy source. Here, we discuss the implications of these new findings in health and disease and speculate on the potential new roles of nucleosides and nucleic acids in energy metabolism.


Nucleic Acids , Nucleosides , Humans , Nucleosides/metabolism , Carbon/metabolism , Nucleotides/metabolism
5.
Mol Biochem Parasitol ; 258: 111616, 2024 Jun.
Article En | MEDLINE | ID: mdl-38401850

Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (Ki) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG0. Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens.


Guanine , Hypoxanthine , Trypanosoma cruzi , Trypanosoma cruzi/metabolism , Trypanosoma cruzi/genetics , Trypanosoma cruzi/chemistry , Guanine/metabolism , Hypoxanthine/metabolism , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/chemistry , Nucleobase Transport Proteins/metabolism , Nucleobase Transport Proteins/genetics , Nucleobase Transport Proteins/chemistry , Biological Transport , Substrate Specificity , Protein Binding , Nucleosides/metabolism
6.
Chem Res Toxicol ; 37(2): 248-258, 2024 02 19.
Article En | MEDLINE | ID: mdl-38198686

Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD+) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD+, the key redox cofactor in metabolism and bioenergetics maintenance.


NAD , Nucleotides , Humans , NAD/metabolism , Nucleotides/metabolism , Nucleosides/metabolism , Energy Metabolism , Pyridones
7.
Chem Commun (Camb) ; 60(12): 1607-1610, 2024 Feb 06.
Article En | MEDLINE | ID: mdl-38230513

Extensive research has focused on genetic code reprogramming using flexizymes (Fxs), ribozymes enabling diverse tRNA acylation. Here we describe a nucleoside-modification strategy for the preparation of flexizyme variants derived from 2'-OMe, 2'-F, and 2'-MOE modifications with unique and versatile activities, enabling the charging of tRNAs with a broad range of substrates. This innovative strategy holds promise for synthetic biology applications, offering a robust pathway to expand the genetic code for diverse substrate incorporation.


RNA, Catalytic , Transfer RNA Aminoacylation , Nucleosides/metabolism , RNA, Transfer/metabolism , Genetic Code , RNA, Catalytic/metabolism
8.
Curr Protein Pept Sci ; 25(2): 120-136, 2024.
Article En | MEDLINE | ID: mdl-37670708

Membrane protein human concentrative nucleoside transporter 3 (hCNT3) can not only transport extracellular nucleosides into the cell but also transport various nucleoside-derived anticancer drugs to the focus of infection for therapeutic effects. Typical nucleoside anticancer drugs, including fludarabine, cladabine, decitabine, and clofarabine, are recognized by hCNT3 and then delivered to the lesion site for their therapeutic effects. hCNT3 is highly conserved during the evolution from lower to higher vertebrates, which contains scaffold and transport domains in structure and delivers substrates by coupling with Na+ and H+ ions in function. In the process of substrate delivery, the transport domain rises from the lower side of transmembrane 9 (TM9) in the inward conformation to the upper side of the outward conformation, accompanied by the collaborative motion of TM7b/ TM4b and hairpin 1b (HP1b)/ HP2b. With the report of a series of three-dimensional structures of homologous CNTs, the structural characteristics and biological functions of hCNT3 have attracted increasing attention from pharmacists and biologists. Our research group has also recently designed an anticancer lead compound with high hCNT3 transport potential based on the structure of 5-fluorouracil. In this work, the sequence evolution, conservation, molecular structure, cationic chelation, substrate recognition, elevator motion pattern and nucleoside derivative drugs of hCNT3 were reviewed, and the differences in hCNT3 transport mode and nucleoside anticancer drug modification were summarized, aiming to provide theoretical guidance for the subsequent molecular design of novel anticancer drugs targeting hCNT3.


Antineoplastic Agents , Nucleosides , Animals , Humans , Nucleosides/pharmacology , Nucleosides/chemistry , Nucleosides/metabolism , Antineoplastic Agents/pharmacology , Biological Transport
9.
Plant J ; 117(5): 1432-1452, 2024 Mar.
Article En | MEDLINE | ID: mdl-38044809

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as ß-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.


Arabidopsis , Nucleosides , Nucleosides/metabolism , Nitrogen/metabolism , Plant Breeding , Plants/metabolism , Uridine/metabolism , Arabidopsis/genetics
10.
Am J Physiol Renal Physiol ; 326(1): F30-F38, 2024 01 01.
Article En | MEDLINE | ID: mdl-37916286

Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.


Diabetes Mellitus, Type 2 , Diabetic Nephropathies , Humans , Mice , Animals , Diabetic Nephropathies/metabolism , Pseudouridine/metabolism , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/metabolism , Nucleosides/metabolism , Renal Elimination , Kidney/metabolism , Guanosine/metabolism
11.
RNA Biol ; 21(1): 1-10, 2024 Jan.
Article En | MEDLINE | ID: mdl-38117089

Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5'-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5'-monophosphate (ΨMP) into uridine and ribose 5'-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5'-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.


Arabidopsis , Pseudouridine , Pseudouridine/metabolism , Kinetics , Ribose/metabolism , Escherichia coli/metabolism , Nucleosides/metabolism , Phosphates , Catalysis , Substrate Specificity , Crystallography, X-Ray
12.
mSphere ; 9(1): e0036323, 2024 Jan 30.
Article En | MEDLINE | ID: mdl-38126788

Nucleoside analogs have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono- and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1-related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.IMPORTANCEThere is substantial interest in developing nucleoside analogs as anti-parasitic agents. We used genome-scale genetic screening and discovered two proteins linked to purine analog resistance in African trypanosomes. Our screens also identified two nucleoside kinases required for pro-drug activation, further validating the approach. The top novel hit, HD82, is related to SAMHD1, a mammalian nuclear viral restriction factor. We validated HD82 and localized the protein to the trypanosome nucleus. HD82 appears to sensitize trypanosomes to nucleoside analogs by reducing native pools of nucleotides, providing insights into both nucleoside/nucleotide metabolism and nucleoside analog resistance in trypanosomatids.


Nucleosides , Trypanosoma , Animals , Nucleosides/metabolism , SAM Domain and HD Domain-Containing Protein 1 , Trypanosoma/metabolism , Purines/metabolism , Nucleotides/metabolism , Ganciclovir/metabolism , Mammals
13.
Int J Mol Sci ; 24(19)2023 Sep 26.
Article En | MEDLINE | ID: mdl-37834034

Neutrophils are innate immune cells that play a key role in pathogen clearance. They contribute to inflammatory diseases, including diabetes, by releasing pro-inflammatory cytokines, reactive oxygen species, and extracellular traps (NETs). NETs contain a DNA backbone and catalytically active myeloperoxidase (MPO), which produces hypochlorous acid (HOCl). Chlorination of the DNA nucleoside 8-chloro-deoxyguanosine has been reported as an early marker of inflammation in diabetes. In this study, we examined the reactivity of different chlorinated nucleosides, including 5-chloro-(deoxy)cytidine (5ClC, 5CldC), 8-chloro-(deoxy)adenosine (8ClA, 8CldA) and 8-chloro-(deoxy)guanosine (8ClG, 8CldG), with the INS-1E ß-cell line. Exposure of INS-1E cells to 5CldC, 8CldA, 8ClA, and 8CldG decreased metabolic activity and intracellular ATP, and, together with 8ClG, induced apoptotic cell death. Exposure to 8ClA, but not the other nucleosides, resulted in sustained endoplasmic reticulum stress, activation of the unfolded protein response, and increased expression of thioredoxin-interacting protein (TXNIP) and heme oxygenase 1 (HO-1). Exposure of INS-1E cells to 5CldC also increased TXNIP and NAD(P)H dehydrogenase quinone 1 (NQO1) expression. In addition, a significant increase in the mRNA expression of NQO1 and GPx4 was seen in INS-1E cells exposed to 8ClG and 8CldA, respectively. However, a significant decrease in intracellular thiols was only observed in INS-1E cells exposed to 8ClG and 8CldG. Finally, a significant decrease in the insulin stimulation index was observed in experiments with all the chlorinated nucleosides, except for 8ClA and 8ClG. Together, these results suggest that increased formation of chlorinated nucleosides during inflammation in diabetes could influence ß-cell function and may contribute to disease progression.


Diabetes Mellitus , Insulin-Secreting Cells , Humans , Nucleosides/pharmacology , Nucleosides/metabolism , Inflammation/metabolism , DNA/metabolism , Insulin/metabolism , Diabetes Mellitus/metabolism , Insulin-Secreting Cells/metabolism
14.
Reprod Toxicol ; 121: 108475, 2023 10.
Article En | MEDLINE | ID: mdl-37748715

Molnupiravir is a nucleoside analog antiviral that is authorized for use in the treatment of COVID-19. For its therapeutic action, molnupiravir is converted after ingestion to the active metabolite N4-hydroxycytidine, which is incorporated into the viral genome to cause lethal mutagenesis. Molnupiravir is not recommended for use during pregnancy, because preclinical animal studies suggest that it is hazardous to developing embryos. However, the mechanisms underlying the embryotoxicity of molnupiravir are currently unknown. To gain mechanistic insights into its embryotoxic action, the effects of molnupiravir and N4-hydroxycytidine were examined on the in vitro development of mouse preimplantation embryos. Molnupiravir did not prevent blastocyst formation even at concentrations that were much higher than the therapeutic plasma levels. By contrast, N4-hyroxycytidine exhibited potent toxicity, as it interfered with blastocyst formation and caused extensive cell death at concentrations below the therapeutic plasma levels. The adverse effects of N4-hydroxycytidine were dependent on the timing of exposure, such that treatment after the 8-cell stage, but not before it, caused embryotoxicity. Transcriptomic analysis of N4-hydroxycytidine-exposed embryos, together with the examination of eIF-2a protein phosphorylation level, suggested that N4-hydroxycytidine induced the integrated stress response. The adverse effects of N4-hydroxycytidine were significantly alleviated by the co-treatment with S-(4-nitrobenzyl)-6-thioinosine, suggesting that the embryotoxic potential of N4-hydroxycytidine requires the activity of nucleoside transporters. These findings show that the active metabolite of molnupiravir impairs preimplantation development at clinically relevant concentrations, providing mechanistic foundation for further studies on the embryotoxic potential of molnupiravir and other related nucleoside antivirals.


COVID-19 , Nucleosides , Pregnancy , Female , Mice , Animals , Nucleosides/metabolism , Nucleosides/pharmacology , Blastocyst , Hydroxylamines/metabolism , Hydroxylamines/pharmacology , Antiviral Agents/toxicity
15.
Chembiochem ; 24(22): e202300094, 2023 11 16.
Article En | MEDLINE | ID: mdl-37548117

We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l-methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.


Methyltransferases , Nucleosides , Nucleosides/metabolism , Methyltransferases/metabolism , Adenosine , DNA Methylation , Methionine , DNA/chemistry , S-Adenosylmethionine/metabolism
16.
Nat Commun ; 14(1): 4776, 2023 08 08.
Article En | MEDLINE | ID: mdl-37553334

Chitin is one of the most abundant natural biopolymers and serves as a critical structural component of extracellular matrices, including fungal cell walls and insect exoskeletons. As a linear polymer of ß-(1,4)-linked N-acetylglucosamine, chitin is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. In this study, we determine seven different cryo-electron microscopy structures of a Saccharomyces cerevisiae chitin synthase in the absence and presence of glycosyl donor, acceptor, product, or peptidyl nucleoside inhibitors. Combined with functional analyses, these structures show how the donor and acceptor substrates bind in the active site, how substrate hydrolysis drives self-priming, how a chitin-conducting transmembrane channel opens, and how peptidyl nucleoside inhibitors inhibit chitin synthase. Our work provides a structural basis for understanding the function and inhibition of chitin synthase.


Chitin Synthase , Chitin , Chitin Synthase/chemistry , Chitin Synthase/metabolism , Chitin/metabolism , Cryoelectron Microscopy , Nucleosides/metabolism , Saccharomyces cerevisiae/metabolism , Catalysis
17.
Nat Commun ; 14(1): 3175, 2023 06 01.
Article En | MEDLINE | ID: mdl-37264059

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.


Nucleosides , Pyrimidine Nucleosides , Humans , Mice , Animals , Nucleosides/metabolism , Membrane Transport Proteins/metabolism , Renal Elimination , Carrier Proteins/metabolism , Antimetabolites , Nucleoside Transport Proteins/metabolism , Kidney/metabolism
18.
Methods Mol Biol ; 2660: 95-121, 2023.
Article En | MEDLINE | ID: mdl-37191793

Nucleoside analogs (NAs) are an established class of anticancer agents being used clinically for the treatment of diverse cancers, either as monotherapy or in combination with other established anticancer or pharmacological agents. To date, nearly a dozen anticancer NAs are approved by the FDA, and several novel NAs are being tested in preclinical and clinical trials for future applications. However, improper delivery of NAs into tumor cells because of alterations in expression of one or more drug carrier proteins (e.g., solute carrier (SLC) transporters) within tumor cells or cells surrounding the tumor microenvironment stands as one of the primary reasons for therapeutic drug resistance. The combination of tissue microarray (TMA) and multiplexed immunohistochemistry (IHC) is an advanced, high-throughput approach over conventional IHC that enables researchers to effectively investigate alterations to numerous such chemosensitivity determinants simultaneously in hundreds of tumor tissues derived from patients. In this chapter, taking an example of a TMA from pancreatic cancer patients treated with gemcitabine (a NA chemotherapeutic agent), we describe the step-by-step procedure of performing multiplexed IHC, imaging of TMA slides, and quantification of expression of some relevant markers in these tissue sections as optimized in our laboratory and discuss considerations while designing and carrying out this experiment.


Antineoplastic Agents , Biological Transport , Drug Resistance, Neoplasm , Gemcitabine , Immunohistochemistry , Nucleosides , Tissue Array Analysis , Humans , Antibodies , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Fluorescence , Gemcitabine/metabolism , Gemcitabine/therapeutic use , Immunohistochemistry/methods , Nucleosides/analogs & derivatives , Nucleosides/metabolism , Nucleosides/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Paraffin Embedding , Tissue Array Analysis/methods , Tissue Fixation
19.
Int J Mol Sci ; 24(9)2023 Apr 26.
Article En | MEDLINE | ID: mdl-37175571

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global health concern. Three years since its origin, despite the approval of vaccines and specific treatments against this new coronavirus, there are still high rates of infection, hospitalization, and mortality in some countries. COVID-19 is characterised by a high inflammatory state and coagulation disturbances that may be linked to purinergic signalling molecules such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine (ADO), and purinergic receptors (P1 and P2). These nucleotides/nucleosides play important roles in cellular processes, such as immunomodulation, blood clot formation, and vasodilation, which are affected during SARS-CoV-2 infection. Therefore, drugs targeting this purinergic pathway, currently used for other pathologies, are being evaluated in preclinical and clinical trials for COVID-19. In this review, we focus on the potential of these drugs to control the release, degradation, and reuptake of these extracellular nucleotides and nucleosides to treat COVID-19. Drugs targeting the P1 receptors could have therapeutic efficacy due to their capacity to modulate the cytokine storm and the immune response. Those acting in P2X7, which is linked to NLRP3 inflammasome activation, are also valuable candidates as they can reduce the release of pro-inflammatory cytokines. However, according to the available preclinical and clinical data, the most promising medications to be used for COVID-19 treatment are those that modulate platelets behaviour and blood coagulation factors, mainly through the P2Y12 receptor.


COVID-19 , Nucleosides , Humans , Nucleosides/metabolism , COVID-19 Drug Treatment , SARS-CoV-2/metabolism , Adenosine Triphosphate/metabolism , Adenosine Diphosphate/metabolism , Receptors, Purinergic/metabolism
20.
ACS Chem Biol ; 18(4): 794-802, 2023 04 21.
Article En | MEDLINE | ID: mdl-37005433

Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5'-aminopseudouridine and formamidinylated, N-hydroxylated Gly-Gln dipeptide moieties to allow Watson-Crick base pairing and to mimic protein-ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (KM = 34 µM) over uridine (KM = 901 µM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5'-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5'-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (KM = 181 µM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.


Nucleosides , Pseudouridine , Biosynthetic Pathways , DNA-Directed RNA Polymerases/metabolism , Nucleosides/metabolism , Pseudouridine/biosynthesis , Pseudouridine/metabolism , Pyridoxal Phosphate/chemistry , Streptomyces/chemistry , Streptomyces/metabolism
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