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18 pages, 6183 KiB

Open AccessArticle

Functional Characterization of the Saccharomyces cerevisiae Equilibrative Nucleoside Transporter 1 (ScENT1)

by Rebba C. Boswell-Casteel, Jennifer M. Johnson and Franklin A. Hays

Molecules 2018, 23(4), 732; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules23040732 - 22 Mar 2018

Cited by 3 | Viewed by 3957

Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases—to a lesser extent—and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, [...] Read more.

Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases—to a lesser extent—and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, a detailed understanding of ENT function at the molecular level has remained elusive. ScENT1 (formerly known as Function Unknown Now 26 or FUN26) is the only known ENT ortholog endogenously expressed in S. cerevisiae, and a proteoliposome assay system was used to study homogenously overexpressed and purified ScENT1 (wildtype relative to L390A and F249I mutants). L390 and F249 are highly conserved residues and were found to alter transporter function. L390A produced a reduction of mean transport activity while F249I increased mean substrate translocation relative to wildtype protein. However, both mutations resulted in transport of UTP—a novel gain of function for any ENT. These residues were then mapped onto an ab initio model of FUN26 which suggests they function in substrate translocation (L390) or cytoplasmic gating (F249). Furthermore, wildtype, L390A, and F249I were found to be sensitive to the presence of alcohols. Ethanol attenuated ScENT1-mediated transport of uridine by ~50%. These findings further demonstrate functional similarities between ScENT1 and human ENT isoforms and support identification of FUN26 as ScENT1, the first ENT isoform in S. cerevisiae. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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2745 KiB

Open AccessArticle

Altering Residue 134 Confers an Increased Substrate Range of Alkylated Nucleosides to the E. coli OGT Protein

by Nadia M. Schoonhoven, Derek K. O’Flaherty, Francis P. McManus, Lauralicia Sacre, Anne M. Noronha, M. Judith Kornblatt and Christopher J. Wilds

Molecules 2017, 22(11), 1948; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22111948 - 11 Nov 2017

Cited by 3 | Viewed by 3478

Abstract

O⁶-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O⁶-atom of guanine and O⁴-atom of thymine. In the current study we set out to understand the role of the Ser134 residue [...] Read more.

O⁶-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O⁶-atom of guanine and O⁴-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O⁶-adducts of guanine and O⁴-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O⁶-atoms of guanine in a DNA duplex. When compared to the human AGT protein (hAGT), the S134P OGT variant displayed reduced activity towards O⁶-alkylation but a much broader substrate range for O⁴-alkylation damage reversal. The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O⁶-position of guanine. Finally, a method to generate a covalent conjugate between hAGT and a model nucleoside using a single-stranded oligonucleotide substrate is demonstrated. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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2227 KiB

Open AccessArticle

Synthesis of Disaccharide Nucleosides Utilizing the Temporary Protection of the 2′,3′-cis-Diol of Ribonucleosides by a Boronic Ester

by Hidehisa Someya, Taiki Itoh and Shin Aoki

Molecules 2017, 22(10), 1650; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22101650 - 01 Oct 2017

Cited by 9 | Viewed by 7277

Abstract

Disaccharide nucleosides are an important class of natural compounds that have a variety of biological activities. In this study, we report on the synthesis of disaccharide nucleosides utilizing the temporary protection of the 2′,3′-cis-diol of ribonucleosides, such as adenosine, guanosine, uridine, [...] Read more.

Disaccharide nucleosides are an important class of natural compounds that have a variety of biological activities. In this study, we report on the synthesis of disaccharide nucleosides utilizing the temporary protection of the 2′,3′-cis-diol of ribonucleosides, such as adenosine, guanosine, uridine, 5-metyluridine, 5-fluorouridine and cytidine, by a boronic ester. The temporary protection of the above ribonucleosides permits the regioselective O-glycosylation of the 5’-hydroxyl group with thioglycosides using a p-toluenesulfenyl chloride (p-TolSCl)/silver triflate (AgOTf) promoter system to afford the corresponding disaccharide nucleosides in fairly good chemical yields. The formation of a boronic ester prepared from uridine and 4-(trifluoromethyl)phenylboronic acid was examined by ¹H, ¹¹B and ¹⁹F NMR spectroscopy. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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1664 KiB

Open AccessArticle

Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position

by Wei Liu, Dongwon Shin, Martin Ng, Karissa Y. Sanbonmatsu, Yitzhak Tor and Barry S. Cooperman

Molecules 2017, 22(9), 1427; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22091427 - 29 Aug 2017

Cited by 3 | Viewed by 3893

Abstract

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base [...] Read more.

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5′- and 3′-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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993 KiB

Open AccessArticle

Identification of Optically Active Pyrimidine Derivatives as Selective 5-HT_2C Modulators

by Juhyeon Kim, Hanbyeol Jo, Hyunseung Lee, Hyunah Choo, Hak Joong Kim, Ae Nim Pae, Yong Seo Cho and Sun-Joon Min

Molecules 2017, 22(9), 1416; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22091416 - 26 Aug 2017

Cited by 2 | Viewed by 4893

Abstract

A series of pyrimidine derivatives 4a–i were synthesized and evaluated for their binding affinities towards 5-HT_2C receptors. With regard to designed molecules 4a–i, the influence of the size of alkyl ether and the absolute configuration of a [...] Read more.

A series of pyrimidine derivatives 4a–i were synthesized and evaluated for their binding affinities towards 5-HT_2C receptors. With regard to designed molecules 4a–i, the influence of the size of alkyl ether and the absolute configuration of a stereogenic center on the 5-HT_2C binding affinity and selectivity was studied. The most promising diasteromeric mixtures 4d and 4e were selected in the initial radioligand binding assay and they were further synthesized as optically active forms starting from optically active alcohols 5d and 5e, prepared by an enzymatic kinetic resolution. Pyrimidine analogue (R,R)-4e displayed an excellent 5-HT_2C binding affinity with good selectivity values against a broad range of other 5-HT receptor subtypes. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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779 KiB

Open AccessArticle

Synthesis of Compounds of the Pyrimidine Series Based on the Reactions of 3-Arylmethylidenefuran-2(3H)-ones with N,N-Binucleophilic Reagents

by Tatyana Aniskova, Vyacheslav Grinev and Alevtina Yegorova

Molecules 2017, 22(8), 1251; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22081251 - 28 Jul 2017

Cited by 3 | Viewed by 5335

Abstract

The arylmethylidene derivatives of furan-2(3H)-ones are important building blocks for the synthesis of various heterocyclic compounds containing pyrimidine and pyridazine structural fragments, analogues of nitrogen-containing bases of pyrimidine series. In order to continue the development of constructing of molecules containing pyridine [...] Read more.

The arylmethylidene derivatives of furan-2(3H)-ones are important building blocks for the synthesis of various heterocyclic compounds containing pyrimidine and pyridazine structural fragments, analogues of nitrogen-containing bases of pyrimidine series. In order to continue the development of constructing of molecules containing pyridine and pyridazine fragments, this article is devoted to the synthesis of new biologically active compounds with these moieties. The introduction of a heterocyclic chromenone fragment changes the previously observed 5-R-3-arylmethylidenefuran-2(3H)-ones route of reaction with guanidine carbonate and leads to 3-[(2-amino-4-(2-hydroxyphenyl)pyrimidin-5-yl)methylene]-5-phenylfuran-2(3H)-ones (2a–d). The structure of the reaction products depends on the nature of the aromatic substituent at the C-3 position of the furanone ring. The interaction of 5-aryl-3-arylmethylidenefuran-2(3H)-ones (1e–h) with thiourea in the basic medium leads to the isolation of 5-(2-oxo-2-phenylethyl)-6-aryl-2-thioxotetrahydropyrimidine-4(1H)-ones (3a–d), demonstrating pronounced plant-growth regulatory activity. Optimal conditions for all discussed processes were developed. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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2852 KiB

Open AccessArticle

Fluorination of Naturally Occurring N⁶-Benzyladenosine Remarkably Increased Its Antiviral Activity and Selectivity

by Vladimir E. Oslovsky, Mikhail S. Drenichev, Liang Sun, Nikolay N. Kurochkin, Vladislav E. Kunetsky, Carmen Mirabelli, Johan Neyts, Pieter Leyssen and Sergey N. Mikhailov

Molecules 2017, 22(7), 1219; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22071219 - 20 Jul 2017

Cited by 17 | Viewed by 4793

Abstract

Recently, we demonstrated that the natural cytokinin nucleosides N⁶-isopentenyladenosine (iPR) and N⁶-benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile [...] Read more.

Recently, we demonstrated that the natural cytokinin nucleosides N⁶-isopentenyladenosine (iPR) and N⁶-benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile of this class of compounds, we generated a series of fluorinated derivatives of BAPR and evaluated their activity on the replication of human enterovirus 71 in a cytopathic effect (CPE) reduction assay. The monofluorination of the BAPR-phenyl group changed the selectivity index (SI) slightly because of the concomitant high cell toxicity. Interestingly, the incorporation of a second fluorine atom resulted in a dramatic improvement of selectivity. Moreover, N⁶-trifluoromethylbenzyladenosine derivatives (9–11) exhibited also a very interesting profile, with low cytotoxicity observed. In particular, the analogue N⁶-(3-trifluoromethylbenzyl)-adenosine (10) with a four-fold gain in potency as compared to BAPR and the best SI in the class represents a promising candidate for further development. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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1773 KiB

Open AccessCommunication

Modified Nucleotides as Substrates of Terminal Deoxynucleotidyl Transferase

by Daiva Tauraitė, Jevgenija Jakubovska, Julija Dabužinskaitė, Maksim Bratchikov and Rolandas Meškys

Molecules 2017, 22(4), 672; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22040672 - 22 Apr 2017

Cited by 17 | Viewed by 9252

Abstract

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing [...] Read more.

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N⁴-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3’-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3’-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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2311 KiB

Open AccessArticle

Exploring the Antitumor Mechanism of High-Dose Cytarabine through the Metabolic Perturbations of Ribonucleotide and Deoxyribonucleotide in Human Promyelocytic Leukemia HL-60 Cells

by Zheng Li, Jian-Ru Guo, Qian-Qian Chen, Cai-Yun Wang, Wei-Jia Zhang, Mei-Cun Yao and Wei Zhang

Molecules 2017, 22(3), 499; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22030499 - 21 Mar 2017

Cited by 28 | Viewed by 10719

Abstract

Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide [...] Read more.

Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia cells (HL-60) after treatment with Ara-C to reveal its antitumor mechanism. The metabolic results revealed that four nucleotides (ATP, ADP, CDP, and dCTP) could be used as potential biomarkers indicating the benefit of high-dose Ara-C over lower dose Ara-C treatment. Combining metabolic perturbation and cell cycle analysis, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio induced by high-dose Ara-C can trigger AMP-activated protein kinase (AMPK) and subsequently Forkhead Box, class O (FoxO), to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C might further accelerate the reduction of dCTP, which then aggravates DNA synthesis disturbance. As a result, all of these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of potential mechanisms in the clinical benefits of high-dose Ara-C in therapy for AML. Full article

(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)

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