Molecular Pharmacology

Cellular Mechanism of Action

Drug target identification and mode of action studies for molecules discovered through phenotypic screens usually represent a bottleneck in drug discovery. The limited abundance of compound available for characterization studies is an additional fundamental concern in natural products drug discovery, particularly if the structural complexity precludes chemical synthesis in a timely manner. Genomic and proteomic profiling techniques which require only minute amounts of natural products are being applied in our lab to gain insight into how the compounds act globally on the molecular and cellular level. We systematically transfer genome-wide studies used in yeast to infer targets of bioactive compounds into more relevant mammalian systems in order to interrogate more complex pathways.  Specifically, we use chemogenomic profiling in yeast as well as RNAi-based drug susceptibility screens in mammalian cells to identify pathways that are modulated by the compound.  We use classical biochemical approaches to study affected molecular pathways and to validate hypotheses derived from global strategies.

In vitro Binding and Efficacy

Identifying actions at a diversity of receptor targets is a critical step toward developing a compound as a therapeutic. CNPD3 capabilities include the ability to express human receptors individually in cell culture to establish the binding affinity of small molecules and peptides at those receptors. The ability of the test compound to displace a radiolabeled ligand with known affinity for the receptor of interest, and a comparison of these data across different receptor types, helps define the selectivity of the compound at receptor(s). Current infrastructure focuses on a subset of G protein-coupled receptors or ion channel receptors expressed in CHO or HEK cells and compounds are studied at a range of concentrations to get a precise estimate of binding affinity. Once binding is confirmed, the degree to which the novel compound stimulates signaling at the receptor(s) of interest is established to provide an estimate of efficacy. This is accomplished through measurement of G protein activation (GTPgammaS stimulation and cyclic AMP turnover), arrestin recruitment, ion flux, or other signaling mechanisms associated with the target. Evaluation of ligand biased activity and allosteric modulation as well as traditional measures of agonist activity are a focus of this core area.  For very early stage compounds lacking sufficient information to guide selection of putative targets, CNPD3 investigators partner with a commercial laboratory that has the ability to screen across hundreds of receptors, and these preliminary data guide follow up hits to be interrogated in CNPD3 laboratories.