Screening is not a one-and-done exercise. The high-throughput screen is just the beginning.

Screening usually involves many thousands to millions of tests performed in a relatively short amount of time. Because of this, each small molecule drug candidate is measured a single time to determine activity.

After screening, potential active compounds are cherry picked from the larger selection and retested to confirm activity. Once the validated compounds are determined, potency must be measured to rank the activity of active compounds. Potency is determined by retesting active compounds in dose response by diluting compounds and testing in multiple different concentrations to determine the EC50/IC50 for each compound.

Artifacts & False-Positives
Almost every assay used in high-throughput screening is susceptible to artifacts or false-positives due to drugs interacting with the assay components instead of the intended target.  To remove drugs with artifactual data, secondary or even tertiary screening is implemented to confirm drugs have the intended effects. The key here is to use what is called orthogonal assays; these are assays which use a different technology compared to the original screen.

Counter-Screening
Counter-screening is often necessary to rule out non-specific drugs or drugs which have undesirable effects. For target-based assays, with enzymes for example, a counter screen would use an unrelated enzyme to make sure drugs of interest don’t act through non-specific mechanisms. Alternatively, for reporter assays, a different target with the same reporter protein is often used to counter-screen for compounds that specifically alter the activity of the reporter used in the screen. 

Cytotoxicity
Cytotoxicity is a major concern in high-throughput screening. Many compounds from commercial drug libraries contain compounds that are highly toxic to many cell types. Cellular viability/toxicity must be measured in dose response for all compounds of interest. The goal is to find compounds that have a good separation between their primary activity and any potential cytotoxicity. To determine this, we compare the EC50/IC50 and what is called the tox50 and ideally, they should be separated by tenfold.

Assay Validation and Quality
High-throughput screening normally involves testing each compound a single time at a single concentration. Combine this with the fact that there are many plate-based artifacts which lead to incorrect data; it is of the utmost importance to have a robust and reproducible assay before beginning screening.

Additional Resource: A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

Compound Transfers

The ACDD uses a pintool to transfer very small amounts of compounds (nanoliters, nl) into assay plates for testing. The pintool contains either 96 or 384 tiny metal pins that are manufactured to a precise width which directly correlates to a range of volumes that can be transferred. The exact volume transferred is dependent on the compound source volume, the destination volume (how much buffer or media in the assay plate), how far the pin travels into the compound well, and the pin retraction speed out of the source compound well.

The ACDD currently has three pin sizes:

• FP1 ~ 15nl
• FP8 ~ 30-50nl
• FP200SH ~150-250nl

DMSO %
Our standard assay plate for screening is 384 well which uses between 10ul (biochemical) up to around 50ul (cellular) of buffer or media. Because the pintools allow us to transfer from 15-250nl per well, we are able to minimize the amount of DMSO resulting in the assay reaction. In general cellular assays should not have more than 0.5% DMSO, while biochemical reactions can tolerate 1-2% DMSO. In most of our cellular pinning experiments we are transferring 40nl into 50ul media volume resulting in a DMSO % of 0.08%.