Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants’ improved kinetics with the HIV prodrug metabolite AZTMP 1 1Edited by J. Karn

Ostermann, Nils; Lavie, A.; Padiyar, S.; Brundiers, R.; Veit, T.; Reinstein, Jochen; Goody, Roger S.; Konrad, Manfred; Schlichting, Ilme

doi:10.1006/jmbi.2000.4175

Cited by 44 publications

(31 citation statements)

References 16 publications

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“…The threedimensional structure of human TK-2 has not been solved experimentally but a reliable molecular model has been built using a profile-based -fold recognition method (Hernandez et al, 2006). A Mg 2ϩ -bound ATP molecule was added to this TK-2 structure, and the complex was further refined by remodeling the loop that closes over the reaction substrates using as a template the X-ray crystal structure of human thymidylate kinase cocrystallized with the inhibitor P 1 -(5Ј-adenosyl)P 5 -(5Ј-(3Ј-azido-3Ј-deoxythymidyl))pentaphosphate (PDB code 1E9A) (Ostermann et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

Human Mitochondrial Thymidine Kinase Is Selectively Inhibited by 3′-Thiourea Derivatives of β-Thymidine: Identification of Residues Crucial for Both Inhibition and Catalytic Activity

Balzarini

Daele²,

Negri³

et al. 2009

Mol Pharmacol

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Substituted 3Ј-thiourea derivatives of ␤-thymidine (dThd) and 5Ј-thiourea derivatives of ␣-dThd have been evaluated for their inhibitory activity against recombinant human cytosolic dThd kinase-1 (TK-1), human mitochondrial TK-2, herpes simplex virus type 1 (HSV-1) TK, and varicella-zoster virus TK. Several substituted 3Ј-thiourea derivatives of ␤-dThd proved highly inhibitory to and selective for TK-2 (IC 50 value, 0.15-3.1 M). The 3Ј-C-branched p-methylphenyl (compound 1) and 3-CF 3 -4-Cl-phenyl (compound 7) thiourea derivatives of ␤-dThd showed competitive inhibition of TK-2 when dThd was used as the variable substrate (K i values, 0.40 and 0.05 M, respectively), but uncompetitive inhibition in the presence of variable concentrations of ATP (K i values, 15 and 2.0 M, respectively). These kinetic properties of compounds 1 and 7 against TK-2 could be accounted for by molecular modeling showing that two hydrogen bonds can be formed between the thiourea nitrogens of compound 7 and the oxygens of the ␥-phosphate of ATP. The importance of several active-site residues was assessed by site-directed mutagenesis experiments on TK-2 and the related HSV-1 TK. The low K i /K m ratios for compounds 1 and 7 (0.38 and 0.039 against dThd, and 0.75 and 0.12 against ATP, respectively) indicate that these compounds are among the most potent inhibitors of TK-2 described so far. In addition, a striking close correlation was found between the inhibitory activities of the test compounds against TK-2 and Mycobacterium tuberculosis thymidylate kinase that is strongly indicative of close structural and/or functional similarities between both enzymes in relation to their mode of interaction with these nucleoside analog inhibitors.

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Section: Methodsmentioning

confidence: 99%

Human Mitochondrial Thymidine Kinase Is Selectively Inhibited by 3′-Thiourea Derivatives of β-Thymidine: Identification of Residues Crucial for Both Inhibition and Catalytic Activity

Balzarini

Daele²,

Negri³

et al. 2009

Mol Pharmacol

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“…Indeed, the use of bifunctional fusion enzymes, such as HSVTK/MGMK and CD/UPRT, overlaid with previously described enzyme variants has recently been validated as a viable way to increase prodrug mediated cytotoxicity at low prodrug doses [26; 35]. Furthermore, prior mutagenesis studies on various nucleoside monophosphate kinase family members have created variants with altered substrate specificity [36] or a significantly enhanced ability to activate clinically relevant prodrugs [37; 38]. The use of UCMK variants in cooperation with the dCK activity of our UCMK/dCK fusion may likewise allow for more efficient dFdC conversion at less toxic treatment levels.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

Johnson

Brown

Black

2011

Biochemical and Biophysical Research Communications

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While gemcitabine (2′-2′-difluoro-2′-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.

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“…These drugs alone or in combination presently being used to control the filarial infection, are microfilaricidal in nature showing little effect on the adult parasites (Critchley et al, 2005;Ramzy (dTDP) in the presence of Mg 2+ with ATP as the phosphate donor (Li de la Sierra et al, 2001). TMK is found in all organisms for de novo pyrimidine synthesis and crystal structures of the TMK from a number of species have been determined (Li de la Sierra et al, 2001;Lavie et al, 1997Lavie et al, , 1998aOstermann et al, 2000a;Kotaka et al, 2006). The protein is a homodimer containing five-stranded parallel ␤-sheets surrounded by 7-11 ␣-helices in each subunit.…”

Section: Introductionmentioning

confidence: 99%