Drug Discovery & Development

Application Notes

Conference Review

Hit to Lead Selection

It is common for approximately 1% of the compounds from high throughput screening (HTS) to demonstrate some level of antagonistic or agonistic effects during the assay campaign, hence being categorized as hits. The next step is to validate these by repeating the primary screening assay.  The hits that repeat will go into a secondary screening hit selection process to help choose the best candidates to go into lead optimization.

Data courtesy of Stephanie Leavitt, Gilead Sciences

Secondary screening will yield more detailed dosed response information, allowing the comparison of the affinities of the compounds for the target. This is usually represented as IC50 data, a good hit having <10 µM potency. Among the challenges at this point, is to cull the false positives and assign a level of quantitative intelligence to help guide the decision making process.  This is accomplished through the use of counter screens and selectivity screens to help reduce the risk of off-target effects. Mechanism of action studies may also be required to ensure that an inhibitor is intervening with the correct pharmacology.

Isothermal Titration Calorimetry (ITC) offers unique benefits to help guide the process of lead selection.  ITC provides a direct readout of binding affinity, but it is also a technology that provides insights into the mechanism of binding. A single experiment can provide measurements of attractive forces such as hydrogen bonds, van der Waals interactions, and ionic bonds (∆H), hydrophobic interactions (∆S), and stoichiometry (n). This information can be invaluable for rank order determination and can eliminate false positives, thereby offering an excellent compliment to traditional counterscreen techniques.  

ITC has established itself as the gold standard for measuring binding affinities. Given its sensitivity and the ability to universally measure biological reactions, it has been widely utilized to perform mechanism of action (MOA) studies.  In addition, because an assay “readout” is not required, even endogenous reagents can be used. 

Differential Scanning Calorimetry (DSC), which measures the thermal transition midpoint (T_m) of the protein target in the presence or absence of ligand, can also be used to rank order the binding potency of hits.

Microcalorimetry is an excellent compliment to traditional counterscreen techniques. In a similar fashion, both ITC and DSC are being utilized in fragment-based screening.