The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of >400 iAM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo.Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regresions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.We have previously reported our efforts to develop a therapeutic approach using gene transfer technology for the treatment of primary and metastatic tumors (1-4). This approach, virus-directed enzyme/prodrug therapy, exploits transcriptional differences between normal and neoplastic cells to selectively kill cancer cells. An artificial chimeric gene is created that is composed of tissue-specific transcriptional regulatory sequences linked to the coding domain of a nonmammalian enzyme. The nonmammalian enzyme can metabolically activate a nontoxic prodrug to a cytotoxic anabolite. Ifthe tissue-specific regulatory sequences are from a tumor-associated marker gene such as the carcinoembryonic antigen gene (5, 6), the prostate-specific antigen gene (7,8), or the a-fetoprotein gene (9, 10), then the artificial chimeric gene will result in tumor-specific expression of the nonmammalian enzyme and, consequently, in tumor-specific production of the cytotoxic metabolite.A critical issue influencing the therapeutic benefit of this approach will be the efficiency of gene transfer into a solid tumor. However, the efficacy of this gene therapy approach is dependent not only on the efficiency of gene transfer but also on the enzyme/prodrug system. Various enzyme/prodrug systems have been reported (1)(2)(3)(11)(12)(13)(14). A prodrug that is metabolically converted into a toxic anabolite that is not readily diffusible from one tumor cell into another will require very high gene transfer efficiency. However, if the toxic anabolite is readily diffusible, then the efficiency of gene transfer into the tumor mass may be quite low but still be able to achieve a significant therapeutic effect. Cells were lysed by freeze-thawing and centrifuged for 10 min at 8000 x g. The conditioned medium and the cell extract supernatant were evaporated to dryness, dissolved in deionized water (1 ml for medium; 100 !A for extract). Aliquots (20 1.l) of each sample were analyzed by HPLC on a Partisphere C18 column (4.6 mm x...

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G 2 /M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G 2 /M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.