High-throughput siRNA-based functional target validation.
The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.
Xin
Hong
H
Johnson & Johnson Pharmaceutical Research & Development LLC, Welsh and McKean Roads, Spring House, PA 19477-0776, USA.
Bernal
Alejandro
A
Amato
Frank A
FA
Pinhasov
Albert
A
Kauffman
Jack
J
Brenneman
Douglas E
DE
Derian
Claudia K
CK
Andrade-Gordon
Patricia
P
Plata-Salamán
Carlos R
CR
Ilyin
Sergey E
SE
D001483:Base Sequence; D002460:Cell Line; D019295:Computational Biology; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D023281:Genomics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D034741:RNA, Small Interfering / Q000235:genetics*; D012371:Robotics; D014162:Transfection
D034741:RNA, Small Interfering