During high–throughput screening you have identified series of biologically active compounds against the desirable target. These compounds are called primary hits.
Step 1: Confirmatory assays
We can start from this point. In order to confirm that biological effect is real, that desired bio–target is affected, that there is no unexpected toxicity or other side–effects (like specific reaction on high–throughput screening components, not related to biotarget) we need to perform a number of confirmatory assays. Hit confirmation can also involve deconvolution of the biotarget, using iTRAQ.
Step 2: Hits prioritization, scaffold selection
After the hits are confirmed, they should be prioritized according to perspective of becoming a lead compound. Hits should be analyzed in terms of chemotype synthetic accessibility, potency, Lipinsky and drug–lead like parameters. For example compounds with smaller Mw, higher LogP, lower PSA and higher potency will be more desirable for the start in case of good synthetic accessibility. After hits are prioritized, the best chemotype and scaffold for further development is selected. It can correspond to several compounds. Then we use Computer–assisted drug design methods in order to find closest analogues with desired properties for each of the hits, spending most of the resources for top ranked hits. Then we prioritize initial hits list again and select from one to several chemotypes those, which analogues which will be studied further.
Step 3: Hit2lead development
After selection of the chemotypes, we start Structure–Activity relationships work. This involves the work of experienced medicinal chemists teamed with computer chemists (QSAR, Drug design), who take hit compounds from selected chemotypes and suggest their analogs. Ordinarily this starts from analogues from commercial chemical libraries and then goes into the synthesis rounds. The most potent compounds, having combination of the best potency, synthetic accessibility, pharmacokinetics & toxicity perspectives will be selected as leads for the further optimization.
Step 4: Lead optimization
Lead optimization may involve: