Hit to lead

Hit to lead (H2L) also known as lead generation is a stage in early

• Target validation (TV) → Assay development → High-throughput screening (HTS) → Hit to lead (H2L) → Lead optimization (LO) → Preclinical development → Clinical development

The hit to lead stage starts with confirmation and evaluation of the initial screening hits and is followed by synthesis of

On average, only one in every 5,000 compounds that enters drug discovery to the stage of preclinical development becomes an approved drug.^[5]

Hit confirmation

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 (IC₅₀ or EC₅₀ 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:
  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.^[6]

• high
  affinity
  towards the target (less than 1 μM)
• selectivity versus other targets
• significant efficacy in a cellular assay
• druglikeness (moderate molecular weight and lipophilicity usually estimated as ClogP). Affinity, molecular weight and lipophilicity can be linked in single parameter such as ligand efficiency and lipophilic efficiency.
• low to moderate binding to human serum albumin
• low interference with
  P450 enzymes and P-glycoproteins
• low cytotoxicity
• metabolic stability
• high cell membrane permeability
• sufficient water solubility (above 10 μM)
• chemical stability
• synthetic tractability
• patentability

The project team will usually select between three and six compound series to be further explored. The next step will allow the testing of analogous compounds to determine a

The objective of this drug discovery phase is to synthesize lead compounds, new analogs with improved potency, reduced off-target activities, and physiochemical/metabolic properties suggestive of reasonable in vivo pharmacokinetics. This optimization is accomplished through chemical modification of the hit structure, with modifications chosen by employing knowledge of the structure–activity relationship (SAR) as well as structure-based design if structural information about the target is available.

Lead optimization is concerned with experimental testing and confirmation of the compound based on animal efficacy models and

Best Practices for Hit Finding

For educational purposes the European Federation for Medicinal Chemistry and Chemical Biology (EFMC) shared a series of webinars including 'Best Practices for Hit Finding' as well as 'Hit Generation Case Studies'.^[7]

See also

References