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Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.

ABSTRACT: The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.

SUBMITTER: Llona-Minguez S

PROVIDER: S-EPMC4753678 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.

Llona-Minguez Sabin S Höglund Andreas A Jacques Sylvain A SA Johansson Lars L Calderón-Montaño José Manuel JM Claesson Magnus M Loseva Olga O Valerie Nicholas C K NCK Lundbäck Thomas T Piedrafita Javier J Maga Giovanni G Crespan Emmanuele E Meijer Laurent L Morón Estefanía Burgos EB Baranczewski Pawel P Hagbjörk Ann-Louise AL Svensson Richard R Wiita Elisee E Almlöf Ingrid I Visnes Torkild T Jeppsson Fredrik F Sigmundsson Kristmundur K Jensen Annika Jenmalm AJ Artursson Per P Jemth Ann-Sofie AS Stenmark Pål P Berglund Ulrika Warpman UW Scobie Martin M Helleday Thomas T

Journal of medicinal chemistry 20160127 3

The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, includi ...[more]

PMID: 26771665

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Project description:Factor XIIIa (FXIIIa) is a cysteine transglutaminase that catalyzes the last step in the coagulation process. An anion-binding site inhibition of FXIIIa is a paradigm-shifting strategy that may offer key advantages of controlled inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We previously reported a flavonoid trimer-based allosteric inhibitor of FXIIIa with moderate potency and selectivity. To further advance this approach, we evaluated a series of 27 variably sulfonated heparin mimetics against human FXIIIa. Only 13 molecules exhibited inhibitory activity at the highest concentration tested with IC50 values of 2-286 μM. Specifically, inhibitor 16 demonstrated an IC50 value of 2.4 ± 0.5 μM in a bisubstrate, fluorescence-based trans-glutamination assay. It also demonstrated a significant selectivity over other clotting factors including thrombin, factor Xa, and factor XIa as well as other cysteine enzymes including papain and tissue transglutaminase 2. Inhibitor 16 did not affect the viability of three human cell lines at a concentration that is 5-fold its FXIIIa-IC50. The molecule had a very weak effect on the activated partial thromboplastin time of human plasma at a concentration of >700 μM, further supporting its functional selectivity. Importantly, molecule 16 inhibited FXIIIa-mediated polymerization of fibrin(ogen) in a concentration-dependent manner as shown by the gel electrophoresis experiment. Michaelis-Menten kinetics revealed that the molecule competes with the Gln-donor protein substrate, i.e., dimethylcasein, but not with the Lys-donor small substrate, i.e., dansylcadaverine. Molecular modeling studies revealed that this type of molecule likely binds to an anion-binding site comprising the basic amino acids of Lys54, Lys61, Lys73, Lys156, and Arg244 among others. Overall, our work puts forward a new anion-binding site, selective, nontoxic, sulfonated heparin mimetic FXIIIa inhibitor 16 for further development as an effective and safer anticoagulant.

Project description:Human DCTPP1 (dCTP pyrophosphatase 1), also known as XTP3-transactivated protein A, belongs to MazG-like nucleoside triphosphate pyrophosphatase (NTP-PPase) superfamily. Being a newly identified pyrophosphatase, its relevance to tumorigenesis and the mechanisms are not well investigated. In the present study, we have confirmed our previous study that DCTPP1 was significantly hyperexpressed in breast cancer and further demonstrated its strong association with tumor progression and poor prognosis in breast cancer. Knockdown of DCTPP1 in breast cancer cell line MCF-7 cells remarkably retarded proliferation and colony formation in vitro. The capacity of mammosphere formation of MCF-7 was suppressed with the silence of DCTPP1, which was consistent with the enhanced mammosphere-forming ability in DCTPP1-overexpressed MDA-MB-231 cells. To further dissect the mechanisms of DCTPP1 in promoting tumor cell growth and stemness maintenance, its biochemical properties and biological functions were investigated. DCTPP1 displayed bioactive form with tetrameric structure similar to other MazG domain-containing pyrophosphatases based on structure simulation. A substrate preference for dCTP and its methylated or halogen-modified derivatives over the other canonical (deoxy-) NTPs was demonstrated from enzymatic assay. This substrate preference was also proved in breast cancer cells that the intracellular 5-methyl-dCTP level increased in DCTPP1-deficient MCF-7 cells but decreased in DCTPP1-overexpressed MDA-MB-231 cells. Moreover, global methylation level was elevated in DCTPP1-knockdown MCF-7 cells or mammosphere-forming MCF-7 cells but decreased significantly in DCTPP1-overexpressed MDA-MB-231 cells and its mammospheres. Our results thus indicated that human DCTPP1 was capable of modulating the concentration of intracellular 5-methyl-dCTP. This in turn affected global methylation, contributing to a known phenomenon of hypomethylation related to the cancer cell growth and stemness maintenance. Our current investigations point to the pathological functions of DCTPP1 overexpression in breast cancer cells with aberrant dCTP metabolism and epigenetic modification.

Dataset Information

Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.

Publications

Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.

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