Hi,

i have a rather general question: what exactly is meant by "active hit" for some indication? I read something like EC50<100m. Are there any other definitions or ist that a term that everybody uses differently?

Sorry in case this is not the correct forum for such a question, I didn't find a better place.

Thanks!

Hi,
Yes, The cut-off for Active hit varies case-by-case, I have seen EC50 values ranging from 1m-120m for various active compounds.

Here is an excerpt from a 2007 Nature Chemical Biology paper "Reporting data from high-throughput screening of small-molecule libraries:
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After HTS, the primary screening data are analyzed and follow-up assays are carried out to confirm the activity of compounds that score as 'positives' in the primary screen. Chemical structures of active compounds should be verified before being reported. Addressing the following questions should provide the audience with an understanding of how the investigator arrived at the compounds of interest: [I] how were compounds selected as active in the primary screen? [/I] How were the initial active compounds retested to confirm activity? How was compound chemical structure confirmed? Have active compounds been further purified or resynthesized? Two examples:

Data were normalized as percentage activity relative to positive control. Active compounds were defined as those in the 99.5th percentile. Individual samples of actives were rearrayed in 384-well plates from separately maintained master samples and resubjected to the original screening assay, at 10 M fixed concentration, with triplicate sampling for each active. Those giving a reproducible (2 of 3 or 3 of 3) activity were used to produce 10-point dose-response curves ranging from 30 M to 1 nM, with triplicate sampling. [I] Active compounds of interest for further study were defined as those with a reproducible EC50 of less than 1 M [/I]. These compounds were subjected to further secondary screening including the use of a second biochemical test of activity, a cellular measure of pathway activity, cytotoxicity measurements using four human cell lines, and solubility and permeability measurements.

All compounds of interest were subjected to LC/MS/ELSD analysis using a sample from the original screening stock and a sample from the medium remaining after completion of the dose-response study (at the highest concentration). Each compound used for subsequent studies was either repurchased from the original vendor and purified in-house, or synthesized and purified in-house.
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http://www.nature.com/nchembio/journal/v3/n8/full/nchembio0807-438.html

Thanks a lot!