Hit identification

“The single most important factor determining the likelihood of success of a project is the quality of the starting lead”, Anon

The Hit confirmation phase is follows:

• Exclusion of hits with potential reactivity, assay interference or aggregation
• Re-testing: compounds that were found active against the selected target are re-tested using the same assay conditions used during the HTS.
• Dose response curve generation: an IC50 or EC50 value is then generated
• Are related analogues available, check for genuine Structure-Activity Relationships
• Check for irreversible binding
• Orthogonal testing: Confirmed hits are assayed using a different assay which is usually closer to the target physiological condition or using a different technology.
• Secondary screening: Confirmed hits are tested in a functional assay (agonist/antagonist) or in a cellular environment.
• Assessment of drug-like properties using computational analysis and early physicochemical and ADME measurements
• Chemical tractability: Medicinal chemists will evaluate compounds according to their synthesis feasibility and flexibilty towards chemical diversification or library synthesis.
• Intellectual Property evaluation: Hit compound structures are quickly checked in specialized databases to define patentability and freedom to operate.
• Hit ranking and clustering, preliminary SAR.

Building up a sample collection for High-throughput screening is a major undertaking and for a small company or academic group submitting a proposal to the European Lead Factory might be an attractive alternative.

There is an editorial in ACS Central Science DOI that I would encourage everyone involved in hit identification to read.

A couple of quotes will give you an idea of the content

Alarmingly, up to 80–100% of initial hits from screening can be artefacts if appropriate control experiments are not employed.

it is important to realise that no PAINS-containing drug has ever been developed starting from a protein-reactive PAINS target-based screening hit

They also emphasise the critical need for experimental validation for any screening hit.

Such validation experiments include classic dose response curves, lack of incubation effects, imperviousness to mild reductants, and specificity versus counter-screening targets. If a molecule is flagged as a potential PAINS or aggregator using published patterns but is well-behaved by these criteria, it may be a true, well-behaved ligand. Ultimately, genuine SAR combined with careful mechanistic study provides the most convincing evidence for a specific interaction. Covalent and spectroscopic interference molecules act via specific physical mechanisms, for which controls are known. Colloidal aggregation, fortunately, is readily identified by rapid mechanistic tests and by counter-screening.

In addition you need to consider compound identify and purity, reproducing the activity with an authentic sample is essential.

Whilst time-consuming this validation work will save a fortune in the future.

Updated 8 March 2017