Application of Mechanistic Principles to Assay Design

In cell-based assays where the inhibitor is present for a long incubation time, such as with the BaF3 assay (discussed above), a slow allosteric inhibitor can be identified. However, with other faster readout cell-based assays, it may be necessary to include a long pre-incubation step to allow the compound to diffuse into the cell and bind to the target. 

When working with purified enzymes, it can be useful to perform a close examination of their phosphorylation states and molecular masses. Mass spectrometry is often useful for this purpose. Post-translational modifications or sequence truncations can potentially alter the compound binding sites available and can also change the structure of potential inhibitory sites. For example, with protein kinases, phosphorylations distal from the ATP binding site can inactivate the kinase whereas phosphorylations near the ATP binding site can activate the catalytic activity. Often, practice does not permit control of such situations because the purified systems are often mixtures and cannot be controlled in the commonly used recombinant expression technologies. 

To reduce the likelihood of competitive inhibitors, one should run the assays with high concentrations of substrates relative to Km levels. To favor competitive inhibitors, one should run the assay below the Km values for the substrates. Thus, for making suitable conclusions for assay design, knowledge of the kinetic and binding constants of receptors and enzymes, such as Kd, kCM, Km, Bmax, is useful. Stoichiometric information, such as the number of enzyme molecules per assay, may also be useful because it can serve as a guideline to ensure that the assays are maximally sensitive to the mechanism of action one wants to discover. Problems in assay development often occur when the conditions required for sensitivity to the desired mechanism of action do not yield the best conditions for statistical reproducibility therefore, compromises and balances of these two opposing factors must be often made. 

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