Our findings suggest that, as a result of synergy, the doses of these agents needed to achieve an antitumor effect may be reduced by twofold to eightfold when these agents are given in combination. The present quantitative data analyses for synergism or antagonism provide a basis for a rational design of clinical protocols for combination chemotherapy in patients with advanced germ cell tumors.
Edited by M. J. van Raaij, Centro Nacional de Biotecnología -CSIC, Spain Keywords: indoleamine 2,3-dioxygenase 1; cancer immunotherapy; heme proteins; tryptophan metabolism; crystal engineering.PDB references: indoleamine 2,3-dioxygenase 1, complex with epacadostat, 6e40; complex with sulfur analog of epacadostat, 6e41; complex with 4-chlorophenyl imidazole, 6e42; complex with anolog of BMS-978587, 6e43; free enzyme (ferric), 6e44; free enzyme (ferrous), 6e45; complex with tryptophan (ferrous), 6e46Supporting information: this article has supporting information at journals.iucr.org/f High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form
Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-a), resulting in the inhibition of estradiol (E2)-stimulated ER-a transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1-302) and the ligand-binding domain/ activation function-2 (LBD/AF-2) region (within aa 282-420) of ER-a. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-a interaction. We found that the N-terminal RING domain (aa 20-64) is not required for the BRCA1 : ER-a interaction. We identified two separate contact points for ER-a, one within aa 1-100 and the other within aa 100-200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 Nterminus, we found that aa 67-100 and 101-133 are required for the interaction with ER-a, but that aa 1-67 and 134-302 are dispensible. Previously, we showed that BRCA1 aa 1-302 does not inhibit E2-stimulated ER-a transcriptional activity but does bind to ER-a and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1-100 and BRCA1 aa 101-200 (but not aa 201-300) each inhibited ER-a activity, although not as efficiently as fulllength BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86-95) impaired the ability of both truncated (aa 1-100) and full-length (aa 1-1863) BRCA1 proteins to interact with and/or repress ER-a activity. Based on these findings, a partial BRCA1 : ER-a threedimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-a interaction are discussed.
Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex®, BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drugresistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC 50 values 40-and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26-and 97-fold in IC 50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg 50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.Thymidylate synthase (TS, 1 EC 2.1.1.45) catalyzes the de novo biosynthesis of thymidylate, which is necessary for DNA synthesis and repair (1). The mechanism of TS activity involves the reductive methylation of the substrate, 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) by transfer of a methylene group from the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate), to generate 2Ј-deoxythymidine 5Ј-monophosphate (dTMP) and 7,8-dihydrofolate. Human TS has been sequenced (2), purified (3, 4), and crystallized (5). As an attractive target for anti-cancer drug design, since the 1950s, many TS analogues of both the substrate, dUMP, and the cofactor, CH 2 H 4 folate, have been synthesized and tested as potential anti-cancer therapeutics. Until recently, 5-fluorouracil and fluorodeoxyuridine (FdUrd) were the sole TS-targeted drugs approved for clinical application. In vivo, 5-fluorouracil and FdUrd are metabolized to 5-fluoro-2-deoxyuridylate (FdUMP), a compound that subsequently occupies the pyrimidine binding site forming a ternary complex with TS and the folate cofactor, resulting in inhibition of enzyme function. The recent determination of the three-dimensional structure of human TS has allowed the design of highly specific inhibitors, leading to the emergence of novel folate analogues, such as Tomudex (ZD1694), BW1843U89, and Thymitaq (AG337) (Fig. 1) (6). These promising compounds have entered clinical trials in recent years (7).Previous studies h...
Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site-directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC 50 values 54-and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC 50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC 50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs.Thymidylate synthase (TS), 1 which catalyzes the conversion of dUMP to dTMP, is an attractive target for drug design (1-5). TS inhibitors, which occupy either the substrate or cofactorbinding site, have been designed based on the structure and properties of the enzyme. Fluoropyrimidines, such as 5-fluorouracil (5-FU) and fluorodeoxyuridine (FdUrd), are metabolized to 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and compete subsequently with the substrate, dUMP, for its binding site and have been used in the clinic for over 40 years to treat breast and gastrointestinal cancers. However, fluoropyrimidines, due to their incorporation into DNA and RNA, are not pure TS inhibitors. Also, they are susceptible to metabolic degradation in vivo. In contrast, the cofactor CH 2 H 4 folate is a relatively large molecule and has a variety of binding sites that may be altered in drug design. In recent years folate analogues have been designed as highly specific and stable TS inhibitors (6). The inhibitor CB3717 was the first folate analogue inhibitor of TS tested in the clinic and although anti-tumor activity was demonstrated, its further development was abandoned due to renal and hepatic toxicity (7,8). The information provided by the crystal structures of TS from bacterial and mammalian sources (9 -18) has led to the design and synthesis of novel analogues of CH 2 H 4 folate, e.g. raltitrexed (Tomudex ® , ZD1694), BW1843U89, Thymitaq (AG337), and AG331 (19 -27). These new and promising agents have entered clinical trials...
Elevated AR expression or mutations in tumor cells, causing resistance to first generation AR antagonist agents, are considered the main driver of castration resistant prostate cancer (CRPC). MDV3100, a more potent AR antagonist ( 5x vs bicalutamide) without residual agonism, has demonstrated excellent efficacy in CRPC patients and has been approved by FDA for treating CRPC in clinic. More recently, AR antagonists demonstrated anti-tumor activities in preclinical AR+ breast cancer models and are being tested in clinical trials. Although MDV3100 is generally well-tolerated, there is a need to eliminate the clinically observed CNS AE's ( 1% incidences of seizure) and revserse the resistance to MDV3100 in CRPC patients. Suzhou Kintor previously reported a preclinical candidate compound Proxalutamide (GT0918), a potent androgen receptor (AR) antagonist with ability to down regulate AR protein level in prostate cancer cells. In biochemical assay, GT0918 more potently inhibits androgen binding with AR's ligand binding domain than Bicalutamide (11.4x) and MDV3100(3.5x). In both hormone-sensitive (LNCaP) and CRPC (C4-2) cancer cells, GT0918 demonstrated stronger potency to block AR function of gene transcription than Bicalutamide (∼5-10) and MDV3100(2-5x) while maintaining full antagonism in CRPC cells. GT0918 impairs androgen stimulated AR translocation to cell nuclei hence blocks its binding DNA and shuts down the downstream oncogenic signaling. Moreover, GT0918 induced AR down regulation in prostate cancer cells. As the AR up-regulation remains to be the main driver of CRPC resistance, its feature of AR down regulation may enable GT0918 to retain efficacy against CRPC that have developed resistance to current AR-blockage treatments. In vitro, GT0918 not only inhibits proliferation of hormone-sensitive CaP cells, but also more potently inhibits proliferation of CRPC cells than MDV3100 (6.5x). In addition, GT0918 inhibits the growth of AR positive breast cancer cells. In contrast, GT0918 has minimum effects on the growth of AR-negative CaP cells (PC-3 and DU145), indicating it is a selective AR pathway inhibitor. In vivo, GT0918 demonstrated good anti-tumor efficacy among hormone sensitive, CRPC and AR+ breast cancer animal models with efficacious drug exposure lower than MDV3100. ADME profile was optimized for once a day dosing schedule in human to reduce drug accumulation as seen in MDV3100. IND-enabling GLP toxicology studies have been completed for GT0918, which was well-tolerated with minimum safety findings except for the effects expected from anti-androgens. Toxicokinetic studies confirmed the drug exposures in toxicology studies were significantly higher (rat: 11; dog: 25x) than the exposure required for maximum efficacy. No seizure was observed across all the animal safety studies. An IND application has been filed for starting Phase I clinical trial to test the selective AR pathway blocker GT0918 in patients. Citation Format: Youzhi Tong, Chunyun Chen, Juan Wu, Jiangtao Yang, Huihui Zhang, Xiaojun Wu, Yanmei Duan, Wei Gao, Weidong Qian, Xiaoxia Niu, Lili Mi, Chuangxing Guo. Proxalutamide (GT0918), a potent androgen receptor pathway inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 614. doi:10.1158/1538-7445.AM2014-614
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