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1.
Expert Opin Ther Targets ; 24(2): 155-162, 2020 02.
Article in English | MEDLINE | ID: mdl-32005098

ABSTRACT

Background: The purpose of the present study was to examine the chemopreventive effect of stampidine, an aryl phosphate derivative of stavudine, in side by side comparison with the standard anti-breast cancer drug paclitaxel in the well-established 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine breast cancer model.Methods: Groups of 20 female mice were challenged with the DMBA. DMBA-challenged mice were assigned to various chemoprevention treatments, including stampidine, paclitaxel, and stampidine plus paclitaxel according to the same treatment schedules for 25 weeks.Results: Stampidine resulted in substantially reduced numbers of tumors, tumor weight as well as tumor size in DMBA-treated mice. Stampidine was as effective as paclitaxel in the model and their combination exhibited greater chemopreventive activity, as measured by reduced tumor incidence and improved tumor-free survival as well as overall survival of DMBA-treated mice. The length of time for the initial tumor to appear in DMBA-challenged mice treated with stampidine was longer than that of mice treated DMBA-challenged control mice. Tumors from mice treated with stampidine or stampidine plus paclitaxel displayed unique changes of a signature protein cassette comprised BRCA1, p21, Bax, and Bcl-2.Conclusion: Stampidine has potent chemopreventive activity and is as effective as the standard chemotherapy drug paclitaxel in the chemical carcinogenesis.


Subject(s)
Anticarcinogenic Agents/pharmacology , Breast Neoplasms/prevention & control , Dideoxynucleotides/pharmacology , Stavudine/analogs & derivatives , Thymidine Monophosphate/analogs & derivatives , 9,10-Dimethyl-1,2-benzanthracene , Animals , Breast Neoplasms/pathology , Disease Models, Animal , Disease-Free Survival , Female , Mice , Mice, Inbred BALB C , Paclitaxel/pharmacology , Stavudine/pharmacology , Survival Rate , Thymidine Monophosphate/pharmacology , Time Factors
2.
J Antimicrob Chemother ; 72(2): 504-510, 2017 02.
Article in English | MEDLINE | ID: mdl-27789684

ABSTRACT

BACKGROUND: In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level. OBJECTIVES: To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology. METHODS: This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm. RESULTS: Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ±â€ŠSD age was 35 ±â€Š11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106-461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N. CONCLUSIONS: This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.


Subject(s)
Anti-HIV Agents/therapeutic use , Drug Resistance, Viral/genetics , HIV Infections/drug therapy , HIV-1/drug effects , Thymidine Monophosphate/analogs & derivatives , Adult , Argentina , CD4 Lymphocyte Count , Cross-Sectional Studies , Female , Humans , Male , Reverse Transcriptase Inhibitors/therapeutic use , Thymidine Monophosphate/therapeutic use
3.
Biochemistry ; 52(51): 9167-76, 2013 Dec 23.
Article in English | MEDLINE | ID: mdl-24261692

ABSTRACT

Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >106-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 104-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH3). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²âº bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²âº bimetallo core.


Subject(s)
Adenosine Monophosphate/metabolism , Bacterial Proteins/metabolism , Models, Molecular , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Adenosine Monophosphate/chemistry , Amino Acid Substitution , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Biocatalysis , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Methylation , Molecular Conformation , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Nitrophenols/chemistry , Nitrophenols/metabolism , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/metabolism , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Protein Interaction Maps , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/chemistry , Thymidine Monophosphate/metabolism , Xanthomonas axonopodis/enzymology , Zinc/chemistry , Zinc/metabolism
4.
J Org Chem ; 78(19): 9560-70, 2013 Oct 04.
Article in English | MEDLINE | ID: mdl-24032477

ABSTRACT

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with "+1 interstrand zippers" of 2'-N-(pyren-1-yl)methyl-2'-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2'-O-(pyren-1-yl)methyl-RNA or 2'-N-methyl-2'-N-(pyren-1-yl)methyl-2'-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics.


Subject(s)
DNA/chemistry , Pyrenes/chemistry , Thymidine Monophosphate/analogs & derivatives , Magnetic Resonance Spectroscopy , Models, Chemical , Nucleic Acid Conformation , Oligonucleotides , Spectrometry, Fluorescence , Thymidine Monophosphate/chemistry
5.
Article in English | MEDLINE | ID: mdl-23217320

ABSTRACT

A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length×75.5µm (id)), electrokinetic injection for 60s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80µA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K(m) values for NPP1 and NPP3, K(i) values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.


Subject(s)
Electrophoresis, Capillary/methods , Phosphoric Diester Hydrolases/analysis , Pyrophosphatases/analysis , Thymidine Monophosphate/analogs & derivatives , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Dinitrocresols/chemistry , Electrophoresis, Capillary/instrumentation , Enzyme Assays , Hexadimethrine Bromide/chemistry , Humans , Hydrolysis , Limit of Detection , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Reproducibility of Results , Thymidine Monophosphate/analysis , Thymidine Monophosphate/chemistry , Thymidine Monophosphate/metabolism
6.
J Org Chem ; 77(23): 10718-28, 2012 Dec 07.
Article in English | MEDLINE | ID: mdl-23145501

ABSTRACT

Thymine intermediate 17 has been synthesized on a multigram scale (50 g, 70 mmol) from starting sugar 1 in 15 steps in an overall yield of 73%, with only 5 purification steps. The key thymine intermediate 18 was obtained from 17 in a single step in 96% yield, whereas the key 5-methylcytosine intermediate 20 was obtained from 17 in 2 steps in 58% yield. This highly efficient large scale route necessitates only 2 and 3 novel steps to obtain N2'-functionalized thymine and 5-methylcytosine amino-LNA phosphoramidites from these key intermediates, respectively.


Subject(s)
5-Methylcytosine/chemical synthesis , Nucleosides/chemical synthesis , Organophosphorus Compounds/chemistry , Thymidine Monophosphate/analogs & derivatives , Thymine/chemical synthesis , 5-Methylcytosine/chemistry , Molecular Structure , Nucleosides/chemistry , Thymidine Monophosphate/chemical synthesis , Thymidine Monophosphate/chemistry , Thymine/chemistry
7.
Article in English | MEDLINE | ID: mdl-22995377

ABSTRACT

Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis whose activity in serum is indicative of tumor proliferation and the severity of blood malignancies. 3'-deoxy-3'-fluorothymidine (FLT), a specific exogenous substrate for TK1, is phosphorylated by TK1 in the presence of a phosphorylating buffer, therefore the conversion of FLT to 3'-deoxy-3'-fluorothymidine monophosphate (FLT-MP) can be measured to assess serum TK1 activity. Here we describe a liquid chromatography-MS/MS (LC-MS/MS) method for quantification of FLT and FLT-MP from serum using protein precipitation and column switching followed by detection on an Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. The method was linear over the range of 0.5-500 ng/mL for FLT and 2.5-2000 ng/mL for FLT-MP with a mean correlation coefficient of 0.9964 and 0.9935 for FLT and FLT-MP, respectively. The lower limit of quantification was 0.5 ng/mL for FLT and 2.5 ng/mL for FLT-MP. Intra-assay accuracy and inter-assay accuracy was within ±12% for both FLT and FLT-MP. Intra-assay precision was 2.8% to 7.7% for FLT and 3.3% to 5.8% for FLT-MP. Inter-assay precision was 4.6% to 14.9% for FLT and 4.9% to 14.6% for FLT-MP. Serum TK1 activity was measured in serum from hepatocellular carcinoma patients and age-matched controls under standardized conditions. Elevated TK1 activity was detected in 26.3% of hepatocellular carcinoma samples compared to controls. This method provides a robust alternative to radiometric and immunochemical assays of serum TK1 activity.


Subject(s)
Chromatography, High Pressure Liquid/methods , Dideoxynucleosides/blood , Tandem Mass Spectrometry/methods , Thymidine Kinase/blood , Thymidine Monophosphate/blood , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/enzymology , Case-Control Studies , Dideoxynucleosides/metabolism , Drug Stability , Female , Humans , Linear Models , Liver Neoplasms/blood , Liver Neoplasms/enzymology , Male , Middle Aged , Phosphorylation , Reproducibility of Results , Sensitivity and Specificity , Thymidine Kinase/metabolism , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/metabolism
8.
Expert Opin Investig Drugs ; 21(4): 489-500, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22360744

ABSTRACT

INTRODUCTION: Pre-exposure prophylaxis (PrEP) is an evolving new approach to prevention of sexually transmitted HIV-1 that employs antiretroviral (ARV) agents prior to potential HIV-1 exposure in an attempt to reduce the likelihood of HIV-1 infection postexposure. The identification of new ARV agents with potent activity against multidrug-resistant HIV remains an unmet and urgent challenge in the field of PrEP. AREAS COVERED: This article reviews the preclinical and early clinical activity and safety profile of stampidine, a novel antiretroviral (ARV) drug candidate that exhibits remarkable subnanomolar to low nanomolar in vitro antiretroviral potency against genotypically and phenotypically nucleoside reverse transcriptase inhibitor (NRTI)-resistant primary clinical HIV isolates, non-nucleoside RT-resistant HIV-1 isolates. Stampidine has a favorable pharmacokinetic profile in mice, rats, dogs and cats with 25 or 50 mg/kg tolerable dose levels yielding micromolar plasma concentrations that are 1000-fold higher than its in vitro IC(50) value against HIV. Stampidine has a favorable, safety profile in mice, rats, dogs and cats and it showed significant in vivo ARV activity in HIV-infected Hu-PBL-SCID mice as well as FIV-infected domestic cats. Furthermore, it did not cause any maternal toxicity, developmental toxicity or teratogenicity in rabbits treated at 10 - 40 mg/kg/day dose levels. In a recently completed first-in-human Phase I clinical trial, stampidine did not cause dose-limiting toxicity at single dose levels ranging from 5 to 25 mg/kg. EXPERT OPINION: The favorable safety and activity profile of stampidine warrants its further development as a promising next-generation PrEP candidate to prevent the sexual transmission of HIV-1. The discovery of stampidine as a potent antiretroviral agent represents a significant step forward in the development of effective therapeutic as well as preventive strategies against HIV/AIDS.


Subject(s)
Anti-Retroviral Agents/pharmacology , Anti-Retroviral Agents/therapeutic use , Dideoxynucleotides/pharmacology , Dideoxynucleotides/therapeutic use , HIV Infections/prevention & control , HIV-1/drug effects , Stavudine/analogs & derivatives , Thymidine Monophosphate/analogs & derivatives , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase III as Topic , Drug Evaluation, Preclinical , Humans , Randomized Controlled Trials as Topic , Stavudine/pharmacology , Stavudine/therapeutic use , Thymidine Monophosphate/pharmacology , Thymidine Monophosphate/therapeutic use
9.
J Med Chem ; 55(6): 2649-71, 2012 Mar 22.
Article in English | MEDLINE | ID: mdl-22339166

ABSTRACT

Targeted molecular radiotherapy opens unprecedented opportunities to eradicate cancer cells with minimal irradiation of normal tissues. Described in this study are radioactive cyclosaligenyl monophosphates designed to deliver lethal doses of radiation to cancer cells. These compounds can be radiolabeled with SPECT- and PET-compatible radionuclides as well as radionuclides suitable for Auger electron therapies. This characteristic provides an avenue for the personalized and comprehensive treatment strategy that comprises diagnostic imaging to identify sites of disease, followed by the targeted molecular radiotherapy based on the imaging results. The developed radiosynthetic methods produce no-carrier-added products with high radiochemical yield and purity. The interaction of these compounds with their target, butyrylcholinesterase, depends on the stereochemistry around the P atom. IC(50) values are in the nanomolar range. In vitro studies indicate that radiation doses delivered to the cell nucleus are sufficient to kill cells of several difficult to treat malignancies including glioblastoma and ovarian and colorectal cancers.


Subject(s)
Neoplasms/radiotherapy , Radiopharmaceuticals/chemical synthesis , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/chemical synthesis , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Butyrylcholinesterase/metabolism , Cell Line, Tumor , Cell Survival , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/pharmacology , Colorectal Neoplasms , Drug Screening Assays, Antitumor , Female , Glioblastoma , Humans , Hydrolysis , Iodine Radioisotopes , Molecular Targeted Therapy , Neoplasms/diagnostic imaging , Neoplasms/enzymology , Ovarian Neoplasms , Radionuclide Imaging , Radiopharmaceuticals/pharmacology , Stereoisomerism , Structure-Activity Relationship , Thymidine Monophosphate/pharmacology , Uridine Monophosphate/pharmacology
10.
Anal Biochem ; 416(1): 112-6, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21620793

ABSTRACT

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5'-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3'- and 5'-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3'- and 5'-phosphotyrosyl linkage at the 3' and 5' ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5'-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5' activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC(50)=40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.


Subject(s)
Colorimetry/methods , Nuclear Proteins/metabolism , Transcription Factors/metabolism , DNA-Binding Proteins , Humans , Nuclear Proteins/antagonists & inhibitors , Phosphoric Diester Hydrolases , Substrate Specificity , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/chemistry , Thymidine Monophosphate/metabolism , Transcription Factors/antagonists & inhibitors , Vanadates/pharmacology
11.
Gen Physiol Biophys ; 30(1): 106-9, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21460419

ABSTRACT

Sporulation of the yeast Saccharomyces cerevisiae is negatively regulated by cyclic AMP (cAMP). This microbial cell differentiation process was applied for the screening of a substance that can elevate the intracellular cAMP level. Among nucleoside 5'-alkylphosphates, uridine 5'-eicosylphosphate (UMPC20) selectively and predominantly inhibited ascospore formation of the yeast cells. We suppose the inhibitory effect of UMPC20 could indeed reflect the elevation of the cellular cAMP level.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Cell Division/drug effects , Cyclic AMP/metabolism , Saccharomyces cerevisiae/drug effects , Thymidine Monophosphate/analogs & derivatives , Uridine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Saccharomyces cerevisiae/metabolism , Uridine Monophosphate/pharmacology
13.
J Med Chem ; 53(24): 8485-97, 2010 Dec 23.
Article in English | MEDLINE | ID: mdl-21090681

ABSTRACT

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinucleotides and thus control purinergic signaling. Enhanced NPP activity is implicated in health disorders such as osteoarthritis and cancer. We designed novel diadenosine polyphosphonate derivatives as potential NPP inhibitors. Analogues 1-4 bear a phosphonate and/or boranophosphate group and/or a 2'-H atom instead of a 2'-OH group. In comparison to ATP, analogues 1-4 were barely hydrolyzed by human NTPDase1, -2, -3, and -8 (<5% hydrolysis) and NPP1 and -3 (≤ 13%) and were not hydrolyzed by ecto-5'-nucleotidase, unlike AMP. These derivatives did not affect NTPDase activity, and analogues 1 and 2 did not inhibit ecto-5'-nucleotidase. All analogues blocked ∼80% of the NPP2-dependent hydrolysis of pnp-TMP, a specific NPP substrate, and inhibited the catabolism of pnp-TMP (K(i) and IC50 both found to be between 10 and 60 µM), Ap5A, and ATP by NPP1. The activity of NPP3 was inhibited to a lesser extent by the new analogues, with compounds 1 and 4 being the most effective in that respect. The analogues dramatically reduced the level of hydrolysis of pnp-TMP at the cell surface of both osteocarcinoma and colon cancer cells. Importantly, analogues 1-4 exhibited significantly reduced agonistic activity toward human P2Y1,11) receptors (except for analogue 1) and no activity with human P2Y2 receptor. Our data provide strong evidence that analogue 2 is the first specific NPP inhibitor to be described.


Subject(s)
Adenine Nucleotides/chemical synthesis , Boranes/chemical synthesis , Dinucleoside Phosphates/chemical synthesis , Pyrophosphatases/antagonists & inhibitors , Adenine Nucleotides/chemistry , Adenine Nucleotides/pharmacology , Adenosine Triphosphate/metabolism , Boranes/chemistry , Boranes/pharmacology , Calcium/metabolism , Cell Line , Dinucleoside Phosphates/chemistry , Dinucleoside Phosphates/metabolism , Dinucleoside Phosphates/pharmacology , Drug Screening Assays, Antitumor , Humans , Hydrolysis , Nucleotidases/metabolism , Phosphoric Diester Hydrolases , Purinergic P2Y Receptor Agonists/chemical synthesis , Purinergic P2Y Receptor Agonists/chemistry , Purinergic P2Y Receptor Agonists/pharmacology , Receptors, Purinergic P2Y/physiology , Structure-Activity Relationship , Substrate Specificity , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/metabolism
14.
Article in English | MEDLINE | ID: mdl-20706607

ABSTRACT

Nu-3 [butyl-phosphate-5'-thymidine-3'-phosphate-butyl] is a modified nucleotide that has been shown to have antimicrobial activity against a range of bacteria including Pseudomonas aeruginosa. However, data on the toxicological profile of Nu-3 are still lacking. In the present study, the toxicity of Nu-3 was evaluated by the following studies: acute oral toxicity, dermal and mucous membrane irritation, multiple-dose toxicity and genotoxicity in vivo and vitro. The acute oral toxicity test in mice showed that Nu-3 had an LD(50) of 2001 mg/kg body weight. The irritation tests on rats revealed that Nu-3 was not irritant, with an irritation scoring of 0. The multiple-dose toxicity study in rats showed that Nu-3 did not cause significant changes in histology, selected serum chemistry, and hematological parameters compared to the controls. Rats administrated with multiple-doses of Nu-3 showed no visible toxic symptoms. Both in vitro and in vivo, Nu-3 exhibited no notable genetic toxicity. Overall, the data suggest that Nu-3 is hypotoxic or nontoxic antimicrobial compound that warrants being further developed for treating Pseudomonas aeruginosa infection.


Subject(s)
Anti-Infective Agents/toxicity , Thymine Nucleotides/toxicity , Toxicity Tests, Acute/methods , Analysis of Variance , Animals , Anti-Infective Agents/administration & dosage , CHO Cells , Cell Proliferation/drug effects , Cricetinae , Cricetulus , Eye/drug effects , Female , Histocytochemistry , Kidney/drug effects , Kidney/pathology , Lethal Dose 50 , Liver/drug effects , Liver/pathology , Male , Mice , Mice, Inbred BALB C , Micronucleus Tests , Mucous Membrane/drug effects , Mutagenicity Tests/methods , Rats , Rats, Sprague-Dawley , Skin/drug effects , Skin Irritancy Tests/methods , Spleen/drug effects , Spleen/pathology , T-Lymphocytes/drug effects , Thymidine Monophosphate/administration & dosage , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/toxicity , Thymine Nucleotides/administration & dosage
15.
Org Lett ; 12(5): 916-9, 2010 Mar 05.
Article in English | MEDLINE | ID: mdl-20112966

ABSTRACT

A new and efficient route to the recently reported 3-nitro-2-ethyldibenzofuran caging group was developed. Furthermore, its installation on a thymidine phosphoramidite is described. This caging group is efficiently removed through light-irradiation at 365 nm.


Subject(s)
Benzofurans/chemistry , Benzofurans/chemical synthesis , Organophosphorus Compounds/chemistry , Photons , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/chemistry , DNA/chemical synthesis , DNA/chemistry
16.
Bioconjug Chem ; 21(3): 513-20, 2010 Mar 17.
Article in English | MEDLINE | ID: mdl-20099893

ABSTRACT

Incorporation of 2'-N-(coronen-1-yl)methyl-2'-amino-LNA monomer X or 2'-N-4-(coronen-1-yl)-4-oxobutanoyl-2'-amino-LNA monomer Y into short DNA strands induces high binding affinity toward DNA or RNA and a marked red-shift in steady-state fluorescence emission upon hybridization to cDNA or RNA.


Subject(s)
DNA/chemistry , Fluorescence , Polycyclic Compounds/chemistry , RNA/chemistry , Thymidine Monophosphate/analogs & derivatives , Models, Molecular , Molecular Structure , Nucleic Acid Denaturation , Spectrometry, Fluorescence , Temperature , Thymidine Monophosphate/chemical synthesis , Thymidine Monophosphate/chemistry
17.
Nat Prod Res ; 23(17): 1572-8, 2009.
Article in English | MEDLINE | ID: mdl-19662567

ABSTRACT

Nucleoside analogs have been evaluated as useful tools for the investigation of the mechanism of cell differentiation. We thus examined the effects of nucleoside 5'-alkylphosphates (1-10) on the morphogenetic development of starfish embryos. These nucleotide derivatives were all permissive for their development up to the blastula stage, but the derivatives with lauryl side chain selectively inhibited one of the following stages into bipinnaria larvae. Among them, uridine 5'-laurylphosphate (2) inhibited gastrulation of the blastula, as is the case with the antibiotic tunicamycin, suggesting its inhibitory activity on sulfated and non-sulfated glycoprotein syntheses. Unexpectedly, adenosine 5'-laurylphosphate (8) was evaluated as a novel class of inhibitor that can arrest the embryos exactly at the late gastrula stage, absolutely inhibiting cell differentiation involved in the development of gastrointestinal tract. This is the first report on the appearance of biological activity due to the structural modification of a naturally-occurring molecule, which is critical to the morphogenetic development of multicellular organisms.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Starfish , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/pharmacology , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/pharmacology , Adenosine Monophosphate/chemistry , Animals , Blastula/drug effects , Blastula/embryology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/embryology , Embryonic Development/drug effects , Gastrula/drug effects , Gastrula/embryology , Molecular Structure , Starfish/drug effects , Starfish/embryology , Thymidine Monophosphate/chemistry , Uridine Monophosphate/chemistry
18.
Arch Oral Biol ; 54(8): 730-6, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19473651

ABSTRACT

Xerostomia is commonly caused by antidepressant drugs and ATP can influence the saliva production. Adenosine is the product of extracellular hydrolysis of adenine nucleotides in submandibular gland cells, which occurs by the action of ectonucleotidases. In this study, we have evaluated the effect of three different antidepressants in ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP1-3) activities in cultured cells from salivary glands. Rats received imipramine (10mg/ml), fluoxetine (20mg/ml) or moclobemide (30mg/ml) by oral gavage. The drugs were administered once a day for 14 days. Our results have shown that the hydrolysis of p-nitrophenyl-5'-thymidine monophosphate increased in all treatments. These effects were not consequence of transcriptional control of E-NPP1-3 genes. The results reported here can highlight the importance of ectonucleotidases in the most common side effect caused by antidepressant therapy.


Subject(s)
Antidepressive Agents/pharmacology , Phosphoric Diester Hydrolases/drug effects , Pyrophosphatases/drug effects , Submandibular Gland/enzymology , Animals , Antidepressive Agents, Second-Generation/pharmacology , Antidepressive Agents, Tricyclic/pharmacology , Cells, Cultured , Fluoxetine/pharmacology , Hydrolysis , Imipramine/pharmacology , Male , Moclobemide/pharmacology , Phosphoric Diester Hydrolases/analysis , Phosphorylation , Pyrophosphatases/analysis , Rats , Rats, Wistar , Salivary Proteins and Peptides/analysis , Salivary Proteins and Peptides/drug effects , Submandibular Gland/cytology , Submandibular Gland/drug effects , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/analysis , Time Factors
19.
Epilepsy Res ; 84(1): 15-20, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19157784

ABSTRACT

Adenosine, a potent anticonvulsant, can be produced in the body by the hydrolysis of adenine nucleotides through the action of ecto- or soluble nucleotidases. Changes in nucleotide hydrolysis occur after pentylenetetrazol-induced epileptic events. We evaluated serum ATP, ADP and AMP hydrolysis rates and soluble nucleotide phosphodiesterase (PDEase) activity at 5, 10, 15, 30 and 60 min, and 12h following an epileptic event. Fifteen patients (seven female, eight male; mean age 15.5 years) were included in the study. The type of seizure was generalized in four patients and was localization related in the remaining 11. There were no differences in adenine nucleotide hydrolysis rates between patients and healthy subjects in the interictal stage. In comparison with controls, ATP, ADP and AMP hydrolysis rates were significantly increased at 5 min (53+/-1.4%, 79.2+/-2.8% and 37.0+/-2.6%, respectively) and up to 30 min following the epileptic event. In contrast to ADP and AMP, ATP hydrolysis remained significantly increased at 60 min (71.4+/-1.6%), returning to the basal level after 12h. Serum PDEase activity was also significantly higher in the patients than in healthy subjects, peaking at 15 min (61+/-2.9%) and remaining significantly increased up to 60 min (4.6+/-1.2%) following the epileptic episode. Globally, the variations in the postictal serum ADP hydrolysis rate almost overlapped those of AMP hydrolysis, whereas changes in the ATP hydrolysis rate overlapped those of PDEase activity. The clinical significance of this elevation in postictal soluble serum nucleotidase activity remains to be clarified. However, it is possible to hypothesize that the higher nucleotidase activity might play a role in the modulation of epileptic events.


Subject(s)
Epilepsy/blood , Nucleotidases/blood , Adenosine Diphosphate/blood , Adenosine Monophosphate/blood , Adenosine Triphosphate/blood , Adolescent , Adult , Child , Electroencephalography/methods , Female , Humans , L-Lactate Dehydrogenase/blood , Male , Statistics, Nonparametric , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/blood , Time Factors , Young Adult
20.
J Org Chem ; 74(3): 1070-81, 2009 Feb 06.
Article in English | MEDLINE | ID: mdl-19108636

ABSTRACT

Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting.


Subject(s)
DNA/chemistry , Intercalating Agents/chemistry , Thymidine Monophosphate/analogs & derivatives , Drug Delivery Systems , Intercalating Agents/chemical synthesis , Models, Molecular , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/chemistry , Pyrenes/chemistry , Thymidine Monophosphate/chemical synthesis , Thymidine Monophosphate/chemistry
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