Understanding Drug Response
“Genomics and bioinformatics have always focused on target discovery,“ said Ian Humphrey-Smith, CEO of the Biosystems Informatics Institute (BII, www.biiuk.com). “Until we are able to reduce adverse effects and enhance target selectivity, we are not adding value.“
BII takes a three-pronged approach to systems biology that incorporates bioinformatics screening, near-target kinetic modeling, and molecular modeling of protein-protein interactions. Also critical, says Humphrey-Smith, are the interactions among alternate pathways and positive/negative feedback loops. “Unless you understand these accurately, you are not understanding the drug response.“
At a recent conference at the New York Academy of Sciences (www.nyas.org),four speakers presented their versions of the pathway approach. Yan Feng, Ph.D., a scientist at the Novartis Institute for Biomedical Research (www.nibr.novartis.com), presented data on cell cycle inhibitors, generated through a pathway approach.
Dr. Feng first identified genes that control various stages of the cell cycle through a genome-wide siRNA knockdown. Most knockdowns affect one major slice of the cell cycle and one or two others to a lesser extent. Some crossover also exists among the siRNAs, as some work on more than one gene. Cells were sorted and identified using an imaging cytometer, which classified cells according to cell cycle status through such parameters as DNA content, nuclear size, or levels of PH3 (a market for mitosis).
In much the same way, Dr. Feng was able to identify compounds from a small library that activate or knock down genes associated with individual cell cycle stages.
Also, two speakers offered results from experiments to design a drug screen, based on patterns of activated or inactivated genes or proteins.
Paul Young, Ph.D., vp of research at Avalon Pharmaceuticals (www.avalonrx.com), demonstrated that disrupting specific targets or cellular pathways with siRNAs causes reproducible changes in the gene expression profile of treated cells. Events downstream of knocked-down genes, as well as the gene itself, are affected.
Dr. Young selected 5-20 genes whose changes were most stable and from them constructed a panel or barcode that serves as a molecular signature specific to the disrupted pathway or target. Panels appear like zig-zag graphs with some genes showing lower activity and others higher activity.
Compounds tested against this panel of genes in a cell-based assay fall into two basic categories: those that more or less match the gene activity pattern, or barcode, of the siRNA knockdown and those that do not. The former constitute the hits for that screening experiment. What is exciting about this approach is it assumes nothing about the specific target or targets on which the compound acts, which is almost certainly a protein or receptor. Such barcodes can uncover new drugs as new disease-related pathways and targets and even pick up off-target activity.
Ellen Berg, Ph.D., CSO at BioSeek (www.bioseekinc.com), offered an approach similar to Dr. Youngs, but with proteins as the readout instead of genes.
BioSeeks BioMAP discovery system, of which the protein signature panel is a part, employs cell-based assays in which cells are stimulated by multiple inputs and measured for multiple readouts. The signatures consist of measuring levels of clinically relevant biomarkers, such as receptors, cytokines, chemokines, enzymes, and lipid mediators, whose concentrations change when a drug is administered. Subsequent experiments utilize those same signature panels to screen new molecules. Those that produce the same signature panel are considered hits.
Dr. Berg described an experiment in which eight signature proteins were measured in the presence and absence of antitumor necrosis factor-alpha, or anti-TNF-alpha, a drug used to treat rheumatoid arthritis. Drugs that act through the same mechanism as anti-TNF-alpha must produce the same protein signature as the drug and are likely to be good starting points for a new drug.
According to Dr. Berg, Bioseek scientists can detect and distinguish hundreds of distinct mechanisms of action for every important therapeutic category using similar protein expression profiles. In addition, the profiles can help uncover activity against specific subtypes of, say, inflammatory disease. Again, all this is possible without knowledge of a specific chemical mechanism.
Peter Krutzik, a graduate student working in the lab of www.stanford.edu) discussed a cytometry technique, phospho-specific flow cytometry, or phospho flow, which was pioneered by the Nolan group. As its name implies, phospho flow is a flow method that primarily targets phosphorylated proteins. The group says the technique is rapid, scalable, and highly multiplexing, taking on up to 10 different cellular characteristics from a heterogeneous cell population, for example from peripheral blood.
Krutzik presented his work on the Jak-Stat signaling cascade, defects in which are implicated in systemic lupus erythematosus. Using phospho flow, Krutzik identified compounds active against the Jak-Stat pathway activity from a natural product library and demonstrated a dose-response effect among the hits.