Table 1.

Local versus Global Methods for Mechanism of Action Studies

Class	Technique	Description	Advantages	Disadvantages	Ref
Proteomic	Affinity chromatography		Target proteins maintains the 3D strucutre necessary for protein binding	Requires high affinity small molecules and an abundance of target protein
	Yeast 3 hybrid system		Small molecule-protein target interactions occur within living cells rather than in vitro	False positives may be identified if the reporter gene is activated without the small molecule-protein interaction.
	Phage display	Bacteriophage expressing a library of proteins are exposed to immobilized small molecules. The solid support is washed extensively to remove nonspecific binders, bound proteins eluted, and the process repeated for further enrichment of binding proteins and isolation of high-affinity sequences.	Repeated cycles of selection allow the detection of low abundance proteins.	Library must be sufficiently large and diverse or it may not contain the protein target responsible for investigated drug activity.
	mRNA display	A protein is chemically attached to its own mRNA at the 3′ end through a puromycin linker. mRNA-protein fusions are subjected to repeated cycles of in vitro selection using an immobilized small molecule affinity column. Binding mRNA-protein fusions will be amplified to a cDNA library that codes for fewer proteins but with greater binding affinity for the small molecule.	Early rounds of selection can identify protein targets with low affinity binding	Target proteins may not be in their native form or possess post-translational modifications.
	Protein microarrays	Proteins immobolized to chips are incubated with a labeled small molecule and repeatedly wased. Labeled drug remains bound to target proteins.	Proteins expressed at low levels natively receive equal exposure to the small molecule on the microarray	If target proteins must complex with other proteins for drug interaction or require post-translational modification, the target may not be identified
Biological	NCI60 IVCLSP using COMPARE	The cytotoxic response of 60 cancer cell lines to an agent is used in a pattern recognition algorithm to relate small molecules and their mechanisms.		Chemosensitivity profiles do not always match existing small molecules with defined mechanisms of action.
Genomic	Yeast deletion library screening			Compounds active in mammalian organisms may not be active in yeast
	DNA microarray			Not all changes in gene expression associated with drug effect are under transcriptional control
	Murine haplotype based genetic mapping		Can directly identify numerous individual genes responsible for drug action
	Ex vivo famililial genetics		CEPH cell lines have been extensively genotyped and the data is publicly available allowing the identification of many loci influencing drug action	Tissue specific phenotypic effects such as hepatotoxicity can not be studied in these lymphoblastoid cell lines