Overview of MOA in Drug Discovery

From Assay Guidance Wiki

The purpose of a mode of action study is to characterize the interaction of a compound with its target to understand how the compound interacts with the target and how natural substrates at physiologic concentrations will modulate this activity. These compounds are often inhibitors of enzymes but only rarely become drugs due to the requirement of a drug to not only inhibit the target but to have acceptable solubility, permeability, protein binding, selectivity, metabolism and toxicity profiles. This potential of the compound to become a drug is slowly revealed through the analysis and tracking of these characteristics, as chemistry elaborates the structure activity relationship (SAR). As described in the body of this document, certain types of biochemical behavior are associated with good drug-like properties both in vitro and in vivo.

Most biochemical screens are designed to provide a chemical starting point based upon the most robust, simple and inexpensive modality for screening. This is due to the required reproducibility in the screening process and the potentially large number of molecules to be run through the screen. Most enzymatic screens are designed to identify inhibitors regardless of their mode of action. Thus, screens are usually run at or below the Km for the substrate(s). In the case of an enzyme with two substrates, the screen is often designed to run under pseudo-first order kinetics by running the assay under conditions where one substrate is at saturation, well above its Km, and the second is at or below its Km for the enzyme. One can therefore identify inhibitors that have competitive, noncompetitive and uncompetitive behavior with regard to the substrate at or below Km and noncompetitive or uncompetitive behavior with regard to the other substrate at well above its Km for the enzyme.

In the drug discovery process, the screening phase casts a wide net and the ability to further analyze compounds in more detail is limited, therefore the number of actives isolated from a screen for follow-up are determined by the overall hit rate, the repeat rate upon retesting and determination of the IC50 in a concentration response curve (CRC) test. In general, activities range from mid-micromolar to sub-micromolar for enzyme inhibitors right out of the screen. It is this piece of information (the IC50), along with an analysis of the structural classes of active molecules by a medicinal chemist, which defines the initial SAR, if there is one in the data. It is after this initial analysis that MoA studies can prove valuable by further defining the nature of the inhibitor from a biochemical point of view. Mechanism of action studies at this point in the drug discovery process define the nature of the SAR by elucidating the type of inhibition by which the discovered molecules operate. Thus, one can define if the discovered inhibitor is competitive with substrate, for example, and as described below, potentially suffers from certain liabilities associated with this mechanism.

Cell based assays of biochemical actives are usually utilized to identify promising molecules in a second round of low to medium throughput screening. If a molecule shows significant activity in a cell based assay, then it continues through the flow scheme. The lack of cell based activity of biochemically potent actives is usually attributed to lack of cellular permeability, with a wave of the hand; however, an understanding the MoA of a compound at this stage can add depth to the interpretation of cellular activity or its absence. Knowing a compound is competitive with a substrate helps establish the binding pocket and in combination with structural and SAR information provide an immediate direction for further chemical synthesis. However, these competitive compounds with promising structure and potent biochemical activity might compete with a cellular substrate present at high intracellular concentration thus show no significant cell based activity. Alternatively, more potent cell based activity than is biochemically predicted from IC50 curves might correlate with unusual kinetic behaviors such as slow binding behavior and/or slow off rates (tight binding). As there is no single unique answer, biochemical MoA studies help in interpretation of cell based activities, and provide further support for molecules with desirable characteristics to move forward in the flow scheme. Traditionally, as MoA studies were slow, laborious efforts, only a few selected molecules could be readily analyzed. With the advent of laboratory automation and enhanced data processing, it is now possible to assess a larger number of compounds rapidly. Therefore, it is feasible (and desirable) to examine the results of a screening campaign, in addition to standard cell based assays in the second tier, by an analysis of MoA.