After hits are identified from a high throughput screen, the hits are confirmed and evaluated using the following methods:
* Confirmatory testing: compounds that were found active against the selected target are re-tested using the same assay conditions used during the HTS to make sure that the activity is reproducible.
* Dose response curve: the compound is tested over a range of concentrations to determine the concentration that results in half maximal binding or activity (IC or EC value respectively).
* 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 cellular assay to determine efficacy.
* Synthetic tractability: medicinal chemists evaluate compounds according to their synthesis feasibility and other parameters such as up-scaling or cost of goods.
* Biophysical testing: nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), surface plasmon resonance (SPR), dual polarisation interferometry (DPI), microscale thermophoresis (MST) are commonly used to assess whether the compound binds effectively to the target, the kinetics, thermodynamics, and stoichiometry of binding, any associated conformational change and to rule out promiscuous binding.
* Hit ranking and clustering: Confirmed hit compounds are then ranked according to the various hit confirmation experiments.
* Freedom to operate evaluation: hit structures are checked in specialized databases to determine if they are patentable.