Hit Identification / Target Validation

During the Hit Identification / Target Validation process, a target needs a cavity/region to bind to a small molecule (for small molecule discovery). To select targets most likely to be useful in the development of new treatments for disease, researchers analyze and compare each drug target to others based on their association with a specific disease and their ability to regulate biological and chemical compounds in the body. Tests are conducted to confirm that interactions with the drug target are associated with a desired change in the behavior of diseased cells. This involves intensive in vitro, as well as in vivo studies that provide information on the effects of the pharmacological intervention.  The result of these efforts is to establish sufficient knowledge so that physiologically relevant model systems can be developed into assays for downstream screening. Research scientists can then identify compounds that have an effect on the target selected.

Ways To Identify a Hit (Hit Identification)

  • Start with a natural substrate and make it drug-like
  • Start with someone else’s hit (“patent bust”) – Getting involved with a late staged compound knowing its liabilities , and improving upon it., resulting in a better molecule.
  • Design a denovo hit by Structure- based Design – Design a hit from scratch, using structure based design if there is already extensive knowledge about the binding site.
  • Screening (High Throughput Screening or Fragment methods) – One of the most widely used methods of screening to find molecules as tools to investigate their biology.

Researchers Test Thousands of Compounds To Find a Drug

To find the right drug-like molecules, researchers begin with libraries of drug-like molecules. By utilizing these libraries they hopefully are left with less then 500,000 compounds.

There are three common places which these libraries are assembled from:

  • Natural Products – Natural products such as compounds that come out of extracts, or partially purified compounds that come from fungi and other species.
  • Structure Design – if there is already specific knowledge of the molecular targets of interest, there are designed libraries available for testing. These libraries are based on paticular biological and structural classes.
  • Diversity Libraries – Diversity libraries are selections of molecules, selected to be as different from each other as possible, falling within the drug-like properties of Lipinski’s Rules or other drug-like parameters that you may consider.

After screening the chosen compounds, researchers should be left with a smaller number of molecules which bind to the target in the desired way.

Assay formats

An assay is a procedure in molecular biology for testing or measuring the death rate of a drug or biochemical in an organism or organic sample.  A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized biochemical assays. Other assays measure processes such as enzyme activity, antigen capture, stem cell activity, and competitive protein binding.

Biochemical Assay

If researchers have a purified protein, and they are capable of making an activity that can be visualized, they can perform a biochemical based assay.

There are several instances where a biochemical assay is an appropriate high throughput screening assay to perform, instances such as:

  • When researchers know what protein they want to inhibit – such as when inhibiting an enzyme is going to inhibit a paticular disease progression
  • You can express and purify the protein in large enough quantities to run experiments – the protein needs to be similar enough to what it is in the cell to recognize
  • Selectivity among similar proteins is important – there are hundreds of similar proteins that can be tested against protein of interest

After the biochemical assay is completed, it shoud then be known what protein the target is binding to, known as the “molecular target” or “target validation”. However, after the biochemical assay is completed, it is not known whether a compound is going to be permiable to enter the cell, bind to the target inside the cell, or whether it is will even survive in the cell.

Cell Based Assay

When researchers perform a cell based assay, they will learn when a compound is going to be permiable to enter the cell, bind to the target, and whether it will survive in the cell.

When screening in whole cells using cell based assays, it is best that researchers know the following criteria:

  • Molecular target known, but not isolable from cell
  • Goal is to alter a cellular phenotype
  • Pathway is known, best target is not known
  • Results read out by whole well fluorescence/luminescence, and cell-by-cell properties

Handling Compounds for Testing

When handling so many compounds, researchers prepare the compounds for testing through miniaturization, and automation:

  • Miniaturization – by using plates with different standard well formats:
    • 96 well format – 96 experiments on a single plate
    • 384 well format – 384 experiments on a single plate
    • 1,536 well format – 1,536 experiments on a single plate
  • Automation – allows for prosecution of many examples of plates, one after another, often by utilizing:
    • Parallel pipetting – allows reagents to be added with high reproducibility, to each of these experiments
    • Robotics – to handle plates and pipettes
    • Plate readers – able to read all of these experiments in a high throughput fashion

Next Steps

  • Retest Compounds – Researchers determine if the compounds recapitulate. They run dose responses of those compounds to see how potent they are.
  • Determine Mechanism of Inhibition – Researchers must determine the mechanism of inhibition because many compounds will inhibit a cellular function or protein for reasons that are not drug-like.
  • Optimize – Once reserchers are confident that the compounds are truly active and act in the desired way, lead compound optimization.

Continue to Lead Identification (Hit to Lead) >

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