TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy.

5-Fluorouracil (5-FU) is an antimetabolite and exerts antitumor activity via intracellularly and physiologically complicated metabolic pathways. In this study, we designed a novel small molecule inhibitor, TAS-114, which targets the intercellular metabolism of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addition, TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addition of TAS-114 was retained in the presence of a potent DPD inhibitor containing oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment. Mol Cancer Ther; 17(8); 1683-93. ©2018 AACR.

Dynamic profile analysis to characterize dynamics-driven allosteric sites in enzymes.

We examine the dynamic features of non-trivial allosteric binding sites to elucidate potential drug binding sites. These allosteric sites were previously found to be allosteric after determination of the protein-drug co-crystal structure. After comprehensive search in the Protein Data Bank, we identify 10 complex structures with allosteric ligands whose structures are very similar to their functional forms. Then, possible pockets on the protein surface are searched as potential ligand binding sites. To mimic ligand binding to the pocket, complex models are generated to fill out each pocket with pseudo ligand blocks consisting of spheres. Normal mode analysis of the elastic network model is performed for the complex models and unbound structures to assess the change of protein dynamics induced by ligand binding. We examine nine profiles to describe the dynamic and positional characteristics of the pockets, and identify the change of fluctuation around the ligand, ΔMSFbs , as the best profile for distinguishing the allosteric sites from the other sites in 8 structures. These cases should be considered as examples of dynamics-driven allostery, which accompanies significant changes in protein dynamics. ΔMSFbs is suggested to be used for the search of potential dynamics-driven allosteric sites in proteins for drug discovery.

1,2,3-Triazole-containing uracil derivatives with excellent pharmacokinetics as a novel class of potent human deoxyuridine triphosphatase inhibitors.

Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as a 5-fluorouracil-based combination chemotherapy. We describe the design and synthesis of a novel class of human dUTPase inhibitors, 1,2,3-triazole-containing uracil derivatives. Compound 45a, which possesses 1,5-disubstituted 1,2,3-triazole moiety that mimics the amide bond of tert-amide-containing inhibitor 6b locked in a cis conformation showed potent inhibitory activity, and its structure-activity relationship studies led us to the discovery of highly potent inhibitors 48c and 50c (IC(50) = ~0.029 µM). These derivatives dramatically enhanced the growth inhibition activity of 5-fluoro-2'-deoxyuridine against HeLa S3 cells in vitro (EC(50) = ~0.05 µM). In addition, compound 50c exhibited a markedly improved pharmacokinetic profile as a result of the introduction of a benzylic hydroxy group and significantly enhanced the antitumor activity of 5-fluorouracil against human breast cancer MX-1 xenograft model in mice. These data indicate that 50c is a promising candidate for combination cancer chemotherapies with TS inhibitors.

Discovery of highly potent human deoxyuridine triphosphatase inhibitors based on the conformation restriction strategy.

Human deoxyuridine triphosphatase (dUTPase) inhibition is a promising approach to enhance the efficacy of thymidylate synthase (TS) inhibitor based chemotherapy. In this study, we describe the discovery of a novel class of human dUTPase inhibitors based on the conformation restriction strategy. On the basis of the X-ray cocrystal structure of dUTPase and its inhibitor compound 7, we designed and synthesized two conformation restricted analogues, i.e., compounds 8 and 9. These compounds exhibited increased in vitro potency compared with the parent compound 7. Further structure-activity relationship (SAR) studies identified a compound 43 with the highest in vitro potency (IC(50) = 39 nM, EC(50) = 66 nM). Furthermore, compound 43 had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1 breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage.

Synthesis and discovery of N-carbonylpyrrolidine- or N-sulfonylpyrrolidine-containing uracil derivatives as potent human deoxyuridine triphosphatase inhibitors.

Recently, deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as part of a new strategy of 5-fluorouracil-based combination chemotherapy. We have initiated a program to develop potent drug-like dUTPase inhibitors based on structure-activity relationship (SAR) studies of uracil derivatives. N-Carbonylpyrrolidine- and N-sulfonylpyrrolidine-containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (12k) having excellent potencies (IC(50) = 0.15 µM). The X-ray structure of a complex of 16a and human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a hydrophobic region, respectively, and are stacked on each other. Compounds 12a and 16a markedly enhanced the growth inhibition activity of 5-fluoro-2'-deoxyuridine against HeLa S3 cells in vitro (EC(50) = 0.27-0.30 µM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemotherapeutic strategies when used in combination with TS inhibitors.

Discovery of a novel class of potent human deoxyuridine triphosphatase inhibitors remarkably enhancing the antitumor activity of thymidylate synthase inhibitors.

Inhibition of human deoxyuridine triphosphatase (dUTPase) has been identified as a promising approach to enhance the efficacy of 5-fluorouracil (5-FU)-based chemotherapy. This study describes the development of a novel class of dUTPase inhibitors based on the structure-activity relationship (SAR) studies of uracil derivatives. Starting from the weak inhibitor 7 (IC(50) = 100 µM), we developed compound 26, which is the most potent human dUTPase inhibitor (IC(50) = 0.021 µM) reported to date. Not only does compound 26 significantly enhance the growth inhibition activity of 5-fluoro-2'-deoxyuridine (FdUrd) against HeLa S3 cells in vitro (EC(50) = 0.075 µM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound 26 is a promising candidate for clinical development.