Xiao-Yong Li

Cell Lines for Drug Discovery: Elevating Target-Protein Levels Using Engineered Transcription Factors:

Drug discovery requires high-quality, high-throughput bioassays for lead identification and optimization. These assays are usually based on immortalized cell lines, which express the selected drug target either naturally or as a consequence of transfection with the cDNA encoding the target. Natural untransfected cell lines often fail to achieve the levels of expression required to provide assays of sufficient quality with a high enough signal-to-noise ratio. Unfortunately, the use of cDNA is increasingly restricted, as the sequences for more and more genes become subject to patent restrictions. To overcome these limitations, the authors demonstrate that engineered transcription factors with Cys2-His2 zinc finger DNA-binding domains can be used to effectively activate an endogenous gene of interest without the use of isolated cDNA of the target gene. Using this approach, the authors have generated a cell line that provides a high-quality and pharmacologically validated G-protein-coupled receptor bioassay. In principle, this technology is applicable to any gene of pharmaceutical importance in any cell type. (Journal of Biomolecular Screening 2004:44-51)

Pharmacological analysis of CCK2 receptors up-regulated using engineered transcription factors.

Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.