Structural Bases for the Synergistic Inhibition of Human Thymidylate Synthase and Ovarian Cancer Cell Growth by Drug Combinations

SIMPLE SUMMARY: Drug combinations may help overcome drug resistance, a relevant cause of failure of ovarian cancer therapy. However, designing successful combinations requires a lengthy preclinical validation process. We have analyzed combinations of 5-fluorouracil and raltitrexed, two anticancer drugs that target thymidylate synthase, a key enzyme for the nucleotide synthesis. We have observed administration sequence specific and synergistic combined effects of the two drugs against cisplatin sensitive and resistant ovarian cancer cells. However, the focus of this work was to show that a high stability of the complex of the enzyme with the two drugs, as highlighted by X-ray crystallography, and synergistic inhibition of the enzyme represent indicators, if not prerequisites, for this drug combination to be synergistically active against sensitive and resistant ovarian cancer cells. We thus propose that structural and mechanistic information acquired during the preclinical research can help predict a successful therapeutic application of a drug combination. ABSTRACT: Combining drugs represent an approach to efficiently prevent and overcome drug resistance and to reduce toxicity; yet it is a highly challenging task, particularly if combinations of inhibitors of the same enzyme target are considered. To show that crystallographic and inhibition kinetic information can provide indicators of cancer cell growth inhibition by combinations of two anti-human thymidylate synthase (hTS) drugs, we obtained the X-ray crystal structure of the hTS:raltitrexed:5-fluorodeoxyuridine monophosphate (FdUMP) complex. Its analysis showed a ternary complex with both molecules strongly bound inside the enzyme catalytic cavity. The synergistic inhibition of hTS and its mechanistic rationale were consistent with the structural analysis. When administered in combination to A2780 and A2780/CP ovarian cancer cells, the two drugs inhibited ovarian cancer cell growth additively/synergistically. Together, these results support the idea that X-ray crystallography can provide structural indicators for designing combinations of hTS (or any other target)-directed drugs to accelerate preclinical research for therapeutic application.