TAS1553, a small molecule subunit interaction inhibitor of ribonucleotide reductase, exhibits antitumor activity by causing DNA replication stress.

Ribonucleotide reductase (RNR) is composed of two non-identical subunits, R1 and R2, and plays a crucial role in balancing the cellular dNTP pool, establishing it as an attractive cancer target. Herein, we report the discovery of a highly potent and selective small-molecule inhibitor, TAS1553, targeting protein-protein interaction between R1 and R2. TAS1553 is also expected to demonstrate superior selectivity because it does not directly target free radical or a substrate binding site. TAS1553 has shown antiproliferative activity in human cancer cell lines, dramatically reducing the intracellular dATP pool and causing DNA replication stress. Furthermore, we identified SLFN11 as a biomarker that predicts the cytotoxic effect of TAS1553. Oral administration of TAS1553 demonstrated robust antitumor efficacy against both hematological and solid cancer xenograft tumors and also provided a significant survival benefit in an acute myelogenous leukemia model. Our findings strongly support the evaluation of TAS1553 in clinical trials.

Discovery of 3-Ethyl-4-(3-isopropyl-4-(4-(1-methyl-1 H-pyrazol-4-yl)-1 H-imidazol-1-yl)-1 H-pyrazolo[3,4- b]pyridin-1-yl)benzamide (TAS-116) as a Potent, Selective, and Orally Available HSP90 Inhibitor.

The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth, and survival. A novel series of HSP90 inhibitors were discovered by structure-activity relationship (SAR)-based optimization of an initial hit compound 11a having a 4-(4-(quinolin-3-yl)-1 H-indol-1-yl)benzamide structure. The pyrazolo[3,4- b]pyridine derivative, 16e (TAS-116), is a selective inhibitor of HSP90α and HSP90ß among the HSP90 family proteins and exhibits oral availability in mice. The X-ray cocrystal structure of the 16e analogue 16d demonstrated a unique binding mode at the N-terminal ATP binding site. Oral administration of 16e demonstrated potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body weight loss.

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.

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.

The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses.

Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).

Expression and molecular characterization of spherical particles derived from the genome of the hyperthermophilic euryarchaeote Pyrococcus furiosus.

Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.

The crystal structures of semi-synthetic aequorins.

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.

Novel mechanisms of pH sensitivity in tuna hemoglobin: a structural explanation of the root effect.

The crystal structure of hemoglobin has been known for several decades, yet various features of the molecule remain unexplained or controversial. Several animal hemoglobins have properties that cannot be readily explained in terms of their amino acid sequence and known atomic models of hemoglobin. Among these, fish hemoglobins are well known for their widely varying interactions with heterotropic effector molecules and pH sensitivity. Some fish hemoglobins are almost completely insensitive to pH (within physiological limits), whereas others show extremely low oxygen affinity under acid conditions, a phenomenon called the Root effect. X-ray crystal structures of Root effect hemoglobins have not, to date, provided convincing explanations of this effect. Sequence alignments have signally failed to pinpoint the residues involved, and site-directed mutagenesis has not yielded a human hemoglobin variant with this property. We have solved the crystal structure of tuna hemoglobin in the deoxy form at low and moderate pH and in the presence of carbon monoxide at high pH. A comparison of these models shows clear evidence for novel mechanisms of pH-dependent control of ligand affinity.

The structure of importin-beta bound to SREBP-2: nuclear import of a transcription factor.

The sterol regulatory element-binding protein 2 (SREBP-2), a nuclear transcription factor that is essential for cholesterol metabolism, enters the nucleus through a direct interaction of its helix-loop-helix leucine zipper domain with importin-beta. We show the crystal structure of importin-beta complexed with the active form of SREBP-2. Importin-beta uses characteristic long helices like a pair of chopsticks to interact with an SREBP-2 dimer. Importin-beta changes its conformation to reveal a pseudo-twofold symmetry on its surface structure so that it can accommodate a symmetric dimer molecule. Importin-beta may use a similar strategy to recognize other dimeric cargoes.

Structure of the carboxyl-terminal Src kinase, Csk.

The carboxyl-terminal Src kinase (Csk) is an indispensable negative regulator for the Src family tyrosine kinases (SFKs) that play pivotal roles in various cell signalings. To understand the molecular basis of the Csk-mediated regulation of SFKs, we elucidated the crystal structure of full-length Csk. The Csk crystal consists of six molecules classified as active or inactive states according to the coordinations of catalytic residues. Csk assembles the SH2 and SH3 domains differently from inactive SFKs, and their binding pockets are oriented outward enabling the intermolecular interaction. In active molecules, the SH2-kinase and SH2-SH3 linkers are tightly stuck to the N-lobe of the kinase domain to stabilize the active conformation, and there is a direct linkage between the SH2 and the kinase domains. In inactive molecules, the SH2 domains are rotated destroying the linkage to the kinase domain. Cross-correlation matrices for the active molecules reveal that the SH2 domain and the N-lobe of the kinase domain move as a unit. These observations suggest that Csk can be regulated through coupling of the SH2 and kinase domains and that Csk provides a novel built-in activation mechanism for cytoplasmic tyrosine kinases.

Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri.

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.