The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Karen S. Anderson - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship towards design of cryptosporidium specific Thymidylate Synthase inhibitors
European Journal of Medicinal Chemistry, 2019Co-Authors: Daniel J Czyzyk, William L. Jorgensen, M Valhondo, Luca Deiana, Julian Tiradorives, Karen S. AndersonAbstract:Abstract Cryptosporidiosis is a human gastrointestinal disease caused by protozoans of the genus Cryptosporidium, which can be fatal in immunocompromised individuals. The essential enzyme, Thymidylate Synthase (TS), is responsible for de novo synthesis of deoxythymidine monophosphate. The TS active site is relatively conserved between Cryptosporidium and human enzymes. In previous work, we identified compound 1, (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl- l -glutamic acid), as a promising selective Cryptosporidium hominis TS (ChTS) inhibitor. In the present study, we explore the structure-activity relationship around 1 glutamate moiety by synthesizing and biochemically evaluating the inhibitory activity of analogues against ChTS and human TS (hTS). X-Ray crystal structures were obtained for compounds bound to both ChTS and hTS. We establish the importance of the 2-phenylacetic acid moiety methylene linker in optimally positioning compounds 23, 24, and 25 within the active site. Moreover, through the comparison of structural data for 5, 14, 15, and 23 bound in both ChTS and hTS identified that active site rigidity is a driving force in determining inhibitor selectivity.
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understanding the structural basis of species selective stereospecific inhibition for cryptosporidium and human Thymidylate Synthase
FEBS Letters, 2019Co-Authors: Daniel J Czyzyk, William L. Jorgensen, M Valhondo, Karen S. AndersonAbstract:Thymidylate Synthase (TS), found in all organisms, is an essential enzyme responsible for the de novo synthesis of deoxythymidine monophosphate. The TS active sites of the protozoal parasite Cryptosporidium hominis and human are relatively conserved. Evaluation of antifolate compound 1 and its R-enantiomer 2 against both enzymes reveals divergent inhibitor selectivity and enzyme stereospecificity. To establish how C. hominis and human TS (ChTS and hTS) selectively discriminate 1 and 2, respectively, we determined crystal structures of ChTS complexed with 2 and hTS complexed with 1 or 2. Coupled with the previously determined structure of ChTS complexed with 1, we discuss a possible mechanism for enzyme stereospecificity and inhibitor selectivity.
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Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis Thymidylate Synthase-Dihydrofolate Reductase
Bioorganic & medicinal chemistry letters, 2014Co-Authors: Vidya Prasanna Kumar, Kathleen M. Frey, Yiqiang Wang, Aleem Gangjee, José A. Cisneros, Alejandro Castellanos-gonzalez, A. Clinton White, William L. Jorgensen, Karen S. AndersonAbstract:Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate Synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional Thymidylate Synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis Thymidylate Synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.
Howard L Mcleod - One of the best experts on this subject based on the ideXlab platform.
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novel Thymidylate Synthase enhancer region alleles in african populations
Human Mutation, 2000Co-Authors: Sharon Marsh, Howard L Mcleod, Margaret M Ameyaw, Jessie Githanga, A A Indalo, David OforiadjeiAbstract:Thymidylate Synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A polymorphic tandem repeat sequence in the enhancer region of the TS promoter was previously described, where the triple repeat gives higher in vitro gene expression than a double repeat. We recently identified ethnic differences in allele frequencies between Caucasian and Asian populations. We now describe assessment of genotype and allele frequencies of the TS polymorphism in 640 African (African American, Ghanaian and Kenyan) and Caucasian (UK, USA) subjects. The double and triple repeat were the predominant alleles in all populations studied. The frequency of the triple repeat allele was similar between Kenyan (49%), Ghanaian (56%), African American (52%), American Caucasian (54%) and British Caucasian (54%) subjects. However, two novel alleles contained 4 and 9 copies of the tandem repeat. These novel alleles were found at a higher allele frequency in African populations (Kenyan 7%, Ghanaian 3%, African American 2%) than Caucasians (UK 1%, USA 0%). The novel alleles identified in this study decrease in frequency with Western migration, while the common alleles are relatively stable. This is a unique example suggesting the influence of multiple selection pressures within individual populations. Hum Mutat 16:528, 2000.
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ethnic variation in the Thymidylate Synthase enhancer region polymorphism among caucasian and asian populations
Genomics, 1999Co-Authors: Sharon Marsh, Elaina Susan Renata Collieduguid, Xiehe Liu, Howard L McleodAbstract:Thymidylate Synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.
Sharon Marsh - One of the best experts on this subject based on the ideXlab platform.
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Thymidylate Synthase pharmacogenetics
Investigational New Drugs, 2005Co-Authors: Sharon MarshAbstract:Thymidylate Synthase (TYMS) is an important target for chemotherapy drugs, such as 5-fluorouracil (5FU) and methotrexate. Over-expression of TYMS is linked to resistance to TYMS-targeted chemotherapy drugs. Currently there is no protocol for selecting cancer patients at risk for drug resistance prior to chemotherapy treatment. Three polymorphisms in the 5' and 3' untranslated regions (5'UTR and 3'UTR) of the Thymidylate Synthase gene have been shown to influence TYMS expression. Preliminary data has suggested a poorer response rate to 5FU or methotrexate is seen in patients with 3 copies of a 28 bp tandem repeat in the 5'UTR enhancer region (TSER polymorphism) and this relationship may be further clarified by the presence of a single nucleotide polymorphism (SNP) with the second repeat of the 3 repeat (TSER(*)3) allele. A 6 bp deletion in the 3'UTR of the TYMS gene has also been shown to affect TYMS RNA expression and has a significant association with poor outcome in 5FU treated patients. Evidence linking all 3 TYMS polymorphisms with TYMS expression and patient response to TYMS-targeted chemotherapy treatment will be highlighted.
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novel Thymidylate Synthase enhancer region alleles in african populations
Human Mutation, 2000Co-Authors: Sharon Marsh, Howard L Mcleod, Margaret M Ameyaw, Jessie Githanga, A A Indalo, David OforiadjeiAbstract:Thymidylate Synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A polymorphic tandem repeat sequence in the enhancer region of the TS promoter was previously described, where the triple repeat gives higher in vitro gene expression than a double repeat. We recently identified ethnic differences in allele frequencies between Caucasian and Asian populations. We now describe assessment of genotype and allele frequencies of the TS polymorphism in 640 African (African American, Ghanaian and Kenyan) and Caucasian (UK, USA) subjects. The double and triple repeat were the predominant alleles in all populations studied. The frequency of the triple repeat allele was similar between Kenyan (49%), Ghanaian (56%), African American (52%), American Caucasian (54%) and British Caucasian (54%) subjects. However, two novel alleles contained 4 and 9 copies of the tandem repeat. These novel alleles were found at a higher allele frequency in African populations (Kenyan 7%, Ghanaian 3%, African American 2%) than Caucasians (UK 1%, USA 0%). The novel alleles identified in this study decrease in frequency with Western migration, while the common alleles are relatively stable. This is a unique example suggesting the influence of multiple selection pressures within individual populations. Hum Mutat 16:528, 2000.
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ethnic variation in the Thymidylate Synthase enhancer region polymorphism among caucasian and asian populations
Genomics, 1999Co-Authors: Sharon Marsh, Elaina Susan Renata Collieduguid, Xiehe Liu, Howard L McleodAbstract:Thymidylate Synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.
F T Boyle - One of the best experts on this subject based on the ideXlab platform.
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design and synthesis of potent non polyglutamatable quinazoline antifolate Thymidylate Synthase inhibitors
Journal of Medicinal Chemistry, 1999Co-Authors: P R Marsham, F T Boyle, J M Wardleworth, Laurent Francois Andre Hennequin, R Kimbell, M Brown, Ann L JackmanAbstract:The synthesis is described of a series of analogues of the potent Thymidylate Synthase (TS) inhibitor, N-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzoyl]-L-glutamic acid (4, ZM214888), in which the glutamic acid moiety is replaced by homologous amino acids and alpha-amino acids where the omega-carboxylate is replaced by acylsulfonamides and acidic heterocycles. In general these modifications when compared to 4 gave compounds with increased potency as inhibitors of isolated TS and as cytotoxic agents against murine tumor cell lines. The new compounds require transport by the reduced folate carrier for entry into cells but are not converted intracellularly into polyglutamated species. Agents with this profile are expected to show activity against tumors that are resistant to classical antifolates due to low expression of folylpolyglutamate synthetase. The analogue (S)-2-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzamido]-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid (35, ZD9331) has been selected as a clinical development candidate and is currently undergoing phase I studies.
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the renal effects of the water soluble non folylpolyglutamate synthetase dependent Thymidylate Synthase inhibitor zd9331 in mice
British Journal of Cancer, 1998Co-Authors: Michael I Walton, F T Boyle, F Mitchell, G W Aherne, C J Medlow, A L JackmanAbstract:The renal effects of the water-soluble, non-folylpolyglutamate synthetase-dependent Thymidylate Synthase inhibitor ZD9331 in mice
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use of murine l5178y lymphoma thymidine kinase mutants for in vitro and in vivo antitumour efficacy evaluation of novel Thymidylate Synthase inhibitors
Advances in Experimental Medicine and Biology, 1993Co-Authors: T C Stephens, M N Smith, S E Waterman, M L Mccloskey, A L Jackman, F T BoyleAbstract:Antitumour efficacy studies with Thymidylate Synthase (TS) inhibitors are complicated in mice by a high level of thymidine in plasma (~lμM) which may be salvaged by tumour cells and prevent cytotoxicity when the de novo pathway of nucleotide synthesis is blocked1. Thymidine salvage involves the enzyme thymidine kinase (TK) which phosphorylates the nucleoside to produce thymidine monophosphate (dTMP) which is then further phosphorylated to the triphosphate (dTTP) for incorporation into DNA.
Amnon Kohen - One of the best experts on this subject based on the ideXlab platform.
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caught in action x ray structure of Thymidylate Synthase with noncovalent intermediate analog
Biochemistry, 2021Co-Authors: Svetlana A Kholodar, Janet Finermoore, Robert M. Stroud, Katarzyna świderek, Kemel Arafet, Vicent Moliner, Amnon KohenAbstract:Methylation of 2-deoxyuridine-5′-monophosphate (dUMP) at the C5 position by the obligate dimeric Thymidylate Synthase (TSase) in the sole de novo biosynthetic pathway to thymidine 5′-monophosphate ...
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parallel reaction pathways and noncovalent intermediates in Thymidylate Synthase revealed by experimental and computational tools
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Svetlana A Kholodar, Katarzyna świderek, Vicent Moliner, Ananda K Ghosh, Amnon KohenAbstract:Thymidylate Synthase was one of the most studied enzymes due to its critical role in molecular pathogenesis of cancer. Nevertheless, many atomistic details of its chemical mechanism remain unknown or debated, thereby imposing limits on design of novel mechanism-based anticancer therapeutics. Here, we report unprecedented isolation and characterization of a previously proposed intact noncovalent bisubstrate intermediate formed in the reaction catalyzed by Thymidylate Synthase. Free-energy surfaces of the bisubstrate intermediates interconversions computed with quantum mechanics/molecular mechanics (QM/MM) methods and experimental assessment of the corresponding kinetics indicate that the species is the most abundant productive intermediate along the reaction coordinate, whereas accumulation of the covalent bisubstrate species largely occurs in a parallel nonproductive pathway. Our findings not only substantiate relevance of the previously proposed noncovalent intermediate but also support potential implications of the overstabilized covalent intermediate in drug design targeting DNA biosynthesis.
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folate binding site of flavin dependent Thymidylate Synthase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Eric M Koehn, Laura L Perissinotti, Salah Moghram, Arjun Prabhakar, Scott A Lesley, Irimpan I Mathews, Amnon KohenAbstract:The DNA nucleotide Thymidylate is synthesized by the enzyme Thymidylate Synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH2H4folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic Thymidylate Synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent Thymidylate Synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs’ mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.
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trapping of an intermediate in the reaction catalyzed by flavin dependent Thymidylate Synthase
Journal of the American Chemical Society, 2012Co-Authors: Tatiana V Mishanina, Eric M Koehn, Scott A Lesley, Bruce A. Palfey, John A Conrad, Amnon KohenAbstract:Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme Thymidylate Synthase (TSase). Several human pathogens rely on an alternative flavin-dependent Thymidylate Synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an importan...
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trapping of an intermediate in the reaction catalyzed by flavin dependent Thymidylate Synthase
Journal of the American Chemical Society, 2012Co-Authors: Tatiana V Mishanina, Eric M Koehn, Scott A Lesley, Bruce A. Palfey, John A Conrad, Amnon KohenAbstract:Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme Thymidylate Synthase (TSase). Several human pathogens rely on an alternative flavin-dependent Thymidylate Synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics.
