Hit and Lead Validation

Introduction of Hit and Lead Validation

As the first committed step for drug discovery, hit identification allows the acquisition of small molecules which can bind to the interested target and modify its function. Once the hit is identified as active, more deep characterization of this compound is necessary to further improve drug efficacy, reduce off-target activity, and optimize physical and chemical/metabolic properties of pharmacokinetics in vivo.

High-throughput screening has been ubiquitous for the discovery of small compounds and peptides for therapeutic purposes. A variety of methods have been used for hit and lead validation, such as nuclear magnetic resonance, surface plasmon resonance, isothermal titration calorimetry, counter-screens, chemical assessment, as well as various computational approaches.

Scheme to find new competitive cruzain inhibitors by a parallel strategy of VS and HTS. Fig 1. Scheme to find new competitive cruzain inhibitors by a parallel strategy of VS and HTS. (Hevener, 2018)

Structure-Based Strategies

For the target protein with a known structure, virtual screening (VS) would be the basic tool as a supplement to HTS. Based on the structure information, a ligand with a suitable shape for binding can be quickly found. By combining VS and HTS, the accuracy of the screening results can be improved. In general, there are three integration strategies including parallel, focused, and sequential screening.

The fragment molecular orbital (FMO) protocol provides a more complete assessment of the interaction formed between ligand and protein. FMO calculation involves fragmenting the system into manageable small pieces and then calculating the paired interaction energy to obtain the total energy of the system. FMO method has been successfully used as part of drug design based on rational structure.

The Molecular Mechanics Poisson-Boltzmann or Generalized Born Surface Area (MM-PBSA or MM-GBSA) methods are important approaches for postprocessing of molecular docking. They are mainly used to estimate the binding free energies of small molecules to their biological targets.

Schematic workflow of the DOTS strategy. Fig 2. Schematic workflow of the DOTS strategy. (Hoffer, 2018)

NMR Spectroscopy

NMR is a sensitive biophysical technique to validate the target-ligand complex in solution. NMR techniques for hit validation typically involve three methods, which include target-based studies, ligand-based studies, and chemical reaction analysis. In summary, NMR methodology is widely applied in hit and lead validation especially for the identification of weakly binding inhibitors and agonists that cannot be detected by plate-based spectrophotometric assays. In most cases, NMR-assisted screening and verification are accompanied by combined analysis or parallel techniques.

Approaches for Hit Identification

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  1. Hevener, K.; et al. Hit-to-Lead: Hit Validation and Assessment. Methods in enzymology. 2018, 610: 265-309.
  2. Hoffer, L.; et al. Chemistry-driven Hit-to-lead Optimization Guided by Structure-based Approaches. Molecular informatics. 2018, 37(9-10): 1800059.
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