Abstract
Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Binding Sites
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Cell Line, Tumor
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Cell Proliferation / drug effects
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Cisplatin / pharmacology
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Cisplatin / therapeutic use
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Crystallography, X-Ray
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Drug Design
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / metabolism
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Enzyme Inhibitors / pharmacology*
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Enzyme Inhibitors / therapeutic use
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Female
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Fluorouracil / pharmacology
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Fluorouracil / therapeutic use
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Humans
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Kinetics
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Models, Molecular
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Molecular Sequence Data
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Molecular Targeted Therapy*
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Ovarian Neoplasms / drug therapy
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Ovarian Neoplasms / enzymology*
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Ovarian Neoplasms / pathology
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Peptides / chemistry
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Peptides / metabolism*
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Peptides / pharmacology*
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Peptides / therapeutic use
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Protein Binding / drug effects
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Protein Conformation
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Protein Multimerization / drug effects
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Thermodynamics
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Thymidylate Synthase / antagonists & inhibitors*
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Thymidylate Synthase / chemistry
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Thymidylate Synthase / metabolism
Substances
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Enzyme Inhibitors
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Peptides
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Thymidylate Synthase
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Cisplatin
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Fluorouracil