Rita Schmitz

Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue.

Background Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. Results Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1) inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. Conclusion The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

Cloning and pharmacological characterization of CCR7, CCL21 and CCL19 from Macaca fascicularis

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play an important role in lymphocyte homing and have also been associated with inflammatory, allergic and lung disorders. Cloning of the cynomolgus monkey genes encoding CCR7, CCL19 and CCL21 revealed 93-97% sequence identity of the deduced proteins with their respective human homologs. In chemotaxis assays, B300-19 cells transfected with the cynomolgus (c) CCR7 receptor migrated in response to cCCL19 and cCCL21 in a dose-dependent manner with EC(50) values of 324+/-188nM and 247+/-29nM, respectively. cCCL19 and cCCL21 also elicited calcium responses in stable cell CHO-K1 lines expressing the cCCR7 receptor with EC(50) values of 227+/-4nM and 484+/-163nM, respectively. Although both human (h) CCL19 and hCCL21 elicited increases in intracellular calcium at the cCCR7 receptor, hCCL19 almost completely inhibited subsequent stimulation by hCCL21 whilst hCCL21 failed to inhibit subsequent stimulation by hCCL19. These results identify novel cynomolgus monkey genes and provide a model system for pre-clinical studies of potential drug candidates.

Automation for higher throughput in protein expression: visions, facts and fictions

Down-scaling, parallelization and automation are newtrends in the field of recombinant protein expression inthe post genomic era [1-3]. During the past years manycompanies and academic institutions have heavilyinvested in process and automation technologies. Doesthis trend keep its promise? Can post genomic proteinproduction issues be overcome with few automated proc-esses?This abstract wants to highlight two years of experience inrunning a Protein Production Center in an industrial envi-ronment applying the expression systems BEVS,