INTRODUCTION:A large number of drugs from a variety of pharmacological classes have been demonstrated to cause adverse effects on cardiac rhythm, including the life-threatening arrhythmia Torsades de Pointes. These side effects are often associated with prolongation of the QT interval and are mediated via blockade of the human ether-a-go-go related gene (hERG) encoded potassium channel. In order to manage this risk in the pharmaceutical industry it is desirable to evaluate QT prolongation as early as possible in the drug discovery process.METHODS:Here we describe the development of a 384-well fluorescence polarization (FP) binding assay compatible with high-throughput assessment of compound blockade of the hERG channel during the lead optimisation process. To characterise the fluorescent ligand that was developed, competition binding studies, kinetic studies and electrophysiology studies were performed. Furthermore, to validate the assay as a key screening method a series of competition binding studies were performed and correlated with functional data obtained via patch-clamp.RESULTS:Evaluation of the assay indicates that high quality data is obtained (Z'>0.6), that the K(i) values determined are equivalent to more traditional radiometric methods and that it is predictive for functional hERG blockade as assessed by patch clamp.DISCUSSION:Whilst FP assays, utilizing a variety of fluors, have become well established for the evaluation of G-protein-coupled receptor (GPCRs) and kinase ligand interactions, this technique has not been applied widely to the study of ion channels. Therefore, this represents a novel assay format that is amenable to the evaluation of thousands of compounds per day. Whilst other assay formats have proven predictive or high throughput, this assay represents one of few that combines both attributes, moreover it represents the most cost effective assay, making it truly amenable to early assessment of hERG blockade.

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide. When bound to cell membranes expressing the hERG channel, 10e shows a robust increase in fluorescence polarization (FP) signal. In a FP binding assay using 10e as tracer ligand, Ki values for several known hERG channel blockers were measured and excellent agreement with the literature Ki values was observed over an affinity range of 2 nM to 3 muM. 10e blocks hERG channel current in electrophysiological patch clamp experiments, and computational docking experiments predict that the dofetilide core of 10e binds hERG channel in a conformation similar to that previously predicted for 1. These analogues enable high-throughput hERG channel binding assays that are rapid, economical, and predictive of test compounds' potential for prolonged QT liabilities.

3801 Urokinase plasminogen activator (uPA) and its receptor (uPAR) are overexpressed in many cancer types compared to normal tissue. The presence of both uPA and uPAR in cancer tissue may potentially be exploited in cancer therapy for specific and targeted activation of prodrugs. We have constructed a modified form of anthrax protective antigen, PrAg-U2, in which a furin activation site is shifted with an uPA cleavage site. PrAg-U2 administered together with the fusion protein FP59, consisting of lethal factor residues 1–254 and the ADP ribosylation domain of Pseudomonas exotoxin A, constitutes a potent and uPA-activity dependent cytotoxic drug (OTP-001). In vitro binding of pro-uPA to uPAR is necessary for the cytotoxicity of OTP-001 (Liu et al., J. Biol. Chem, 276:17976–84, 2001) and local administration of OTP-001 in mice with transplanted tumors had a potent antitumor effect (Liu et al., PNAS, 100:657–62, 2003). The aim of the present studies was i) To examine the antitumor effect of OTP-001 after systemic administration, ii) To find the optimal ratio between the two components PrAg-U2 and FP59, iii) To define the “therapeutic window” of OTP-001. Five separate in vivo experiments were performed with three different transplanted tumors, Lewis lung carcinoma, T241 fibrosarcoma and B16 melanoma. In all studies, C57BI/6J mice where transplanted with 10 6 tumor cells. When the tumors had reached a volume of approximately 50 mm 3 , a dose range of OTP-001 was administered systemically to the mice at day 0, 3, and 6. Different ratios of PrAg-U2 and FP59 were tested. Tumor sizes were obtained by daily recording of two orthogonal diameters during the growth phase. All experiments unequivocally demonstrated that OTP-001 has significant antitumor effect after systemic administration in mice with transplanted B16, T241 or Lewis lung tumors. These studies showed that OTP-001 displays a clear dose-response relationship with regard to anti-tumor effect and systemic toxicity. The dose-limiting toxicity appears to be due to mucosal reactions in the gut. The anti-tumor effect was most pronounced with a ratio of the two components PrAg-U2 and FP59 of 25:1. Intraperitoneal administration of 30 μg PrAg-U2 and 1.2 μg FP59 day 0, day 3 and day 6 was found to have minimal toxic effects and still had a significant antitumor effect.

We have constructed a capillary electrophoresis (CE) system with UV detection and have successfully interfaced it to an electrospray ionization mass spectrometry (ES-MS) system. A synthesized fragment of heregulin-beta (212-226) was thought to be a single component by re-injection into an HPLC system, but results from CE-UV-ES-MS indicated that a dehydration product was present in the desired peptide sample. A synthetic heregulin-alpha (177-241) was isolated by preparative HPLC, but re-injection on an analytical system indicated a tailing peak. CE-UV-ES-MS indicated a mixture whose two major components were of the same nominal molecular mass (within experimental error), suggesting the presence of an isomer or a deamidation product. The results show that CE-UV-ES-MS can be used as an orthogonal analytical technique to solve practical problems encountered in peptide synthesis laboratories.