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...