Due to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, and we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.

Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding / Cardinale, Daniela; O. M. H., Salo Ahen; Guaitoli, Giambattista; Ferrari, Stefania; Venturelli, Alberto; Franchini, Silvia; Battini, Renata; Ponterini, Glauco; R. C., Wade; Costi, Maria Paola. - In: PROTEIN ENGINEERING, DESIGN & SELECTION. - ISSN 1741-0126. - STAMPA. - 23:2(2010), pp. 81-89. [10.1093/protein/gzp075]

Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding.

CARDINALE, Daniela;GUAITOLI, GIAMBATTISTA;FERRARI, Stefania;VENTURELLI, Alberto;FRANCHINI, Silvia;BATTINI, Renata;PONTERINI, Glauco;COSTI, Maria Paola
2010

Abstract

Due to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, and we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.
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Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding / Cardinale, Daniela; O. M. H., Salo Ahen; Guaitoli, Giambattista; Ferrari, Stefania; Venturelli, Alberto; Franchini, Silvia; Battini, Renata; Ponterini, Glauco; R. C., Wade; Costi, Maria Paola. - In: PROTEIN ENGINEERING, DESIGN & SELECTION. - ISSN 1741-0126. - STAMPA. - 23:2(2010), pp. 81-89. [10.1093/protein/gzp075]
Cardinale, Daniela; O. M. H., Salo Ahen; Guaitoli, Giambattista; Ferrari, Stefania; Venturelli, Alberto; Franchini, Silvia; Battini, Renata; Ponterini, Glauco; R. C., Wade; Costi, Maria Paola
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/623036
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