dUTPase is the unique enzyme that catalyses the pyrophosphorolysis of dUTP, thus regulating the extent of uracil incorporation into DNA. Massive uracil incorporation may lead to cell death. dUTPase has therefore been recognized as a high-potential drug target in cancer, viral and bacterial disease control. The present proposal focuses on the Mycobacterium tuberculosis (MTB) dUTPase that plays a central role in the mycobacterial dTTP biosynthesis and thus it is likely to be essential for the viability of MTB. MTB is the pathogen that causes tuberculosis, which imposes an increasing global threat with the high-rate emergence of novel multidrug (MDR) and extensively drug-resistant (XDR) strains. Several research agendas (including one at NIH) articulated new measures needed for successful tuberculosis management, which involves intensive research on new drug targets and the development of novel drugs. The present proposal has three Specific Aims all directed towards the evaluation of MTB dUTPase as a valid tool in fighting tuberculosis: 1. Study of the effect of dUTPase functional ablation on the viability of Mycobacterium, 2. Elucidation of the enzymatic mechanism of MTB dUTPase with regards to the mechanistic differences between the human (host) and MTB (pathogen) dUTPases, 3) Determination of the catalytic role of the two structural elements that may be species-specifically targeted in dUTPase. To address the above issues, several transient kinetic and equilibrium enzymological as well as spectroscopical methods will be employed using wild- type and mutant MTB dUTPase enzymes. The physiological effect of functional ablation of dUTPase in Mycobycterium will be investigated in a non-pathogenic Mycobacterium model subjected to dUTPase gene replacement. The expected results of the proposed project will be highly useful in effective species-selective inhibitor design for MTB dUTPase and in the prediction of the in vivo mechanism of such inhibitors. The combination of approaches of this project (cf.
Aims 1 -3) may serve as a useful concept for the investigation of further potential dUTPase targets such as dUTPases from Plasmodium falciparum;Trypanosoma;vaccinia, herpes and Epstein-Barr viruses.
The present proposal focuses on an important DNA repair enzyme called dUTPase of the bacterium causing tuberculosis. Tuberculosis imposes an increasing threat on global health. Therefore several research agendas (including a robust NIH program) articulated new measures needed for successful tuberculosis management, which involves the intensive research of physiological targets for new drugs.
The aims of this proposal are directed towards the evaluation of dUTPase as a drug target in tuberculosis control.
|Lopata, Anna; Leveles, Ibolya; Bendes, Ãbris ÃdÃ¡m et al. (2016) A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes. J Biol Chem 291:26320-26331|
|Pecsi, Ildiko; Hirmondo, Rita; Brown, Amanda C et al. (2012) The dUTPase enzyme is essential in Mycobacterium smegmatis. PLoS One 7:e37461|
|Erdelyi, Peter; Borsos, Eva; Takacs-Vellai, Krisztina et al. (2011) Shared developmental roles and transcriptional control of autophagy and apoptosis in Caenorhabditis elegans. J Cell Sci 124:1510-8|
|Meszaros, Balint; Toth, Judit; Vertessy, Beata G et al. (2011) Proteins with complex architecture as potential targets for drug design: a case study of Mycobacterium tuberculosis. PLoS Comput Biol 7:e1002118|
|Pecsi, Ildiko; Szabo, Judit E; Adams, Scott D et al. (2011) Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination. Proc Natl Acad Sci U S A 108:14437-42|
|Merenyi, Gabor; Kovari, Julia; Toth, Judit et al. (2011) Cellular response to efficient dUTPase RNAi silencing in stable HeLa cell lines perturbs expression levels of genes involved in thymidylate metabolism. Nucleosides Nucleotides Nucleic Acids 30:369-90|
|Pecsi, Ildiko; Leveles, Ibolya; Harmat, Veronika et al. (2010) Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase. Nucleic Acids Res 38:7179-86|
|Kovacs, Erika; Toth, Judit; Vertessy, Beata G et al. (2010) Dissociation of calmodulin-target peptide complexes by the lipid mediator sphingosylphosphorylcholine: implications in calcium signaling. J Biol Chem 285:1799-808|
|Kovacs, Erika; Harmat, Veronika; Toth, Judit et al. (2010) Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. FASEB J 24:3829-39|
|Takacs, Eniko; Nagy, Gergely; Leveles, Ibolya et al. (2010) Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases. FEBS Lett 584:3047-54|
Showing the most recent 10 out of 11 publications