Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as
cancer. We report a new cell-based high-throughput technology for screening
chemical libraries against several potential
cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in
cancer cells was used to screen 9000 chemicals and known drugs in the human
prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind
DNA,
cell surface receptors, and components of intracellular signaling pathways. A class of
steroids related to the
cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-
ATPase resulting in the inhibition of four of the prostate target genes including
transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis,
survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA
methyltransferase, and
cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific
indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid
drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.