5 Target Deconvolution Approaches in Drug Discovery

The retrospective identification of the drug targets that underlie an observed phenotypic response is termed target deconvolution. Target deconvolution can be achieved by numerous methods including; affinity chromatography, expression-cloning, protein microarray, ‘reverse transfected’ cell microarray, and biochemical suppression.

This list highlights five approaches that can be applied to target deconvolution.

1. Affinity chromatography

Target deconvolution by affinity chromatography is achieved by modifying the small-molecule (ligand), a ‘linker’ is introduced that enables it to be immobilized, and the immobilized ligand is then incubated with protein extracts. Unbound proteins are removed by washing, leaving only bound proteins – with a strong enough affinity to the ligand. The bound proteins are then eluted using buffer conditions that interfere with the target protein-ligand interaction. The target protein is then identified – usually using mass spectrometry or immunodetection methods.

2. Expression-cloning

Phage display, mRNA display and three-hybrid systems are all examples of expression cloning technologies.

Phage display

Phage display enables you to present potential target proteins on the surface of phage in the form of a phage library. The phage (which are displaying different proteins) are then exposed to the small molecule drug candidate (immobilized). Phage that have affinity to the small molecule are ‘captured’ and unbound phage are removed via washing. The bound phage which display the target protein, are eluted and amplified using bacterial host cells. DNA sequencing is then used to identify the protein target.

mRNA display

mRNA display is an in vitro approach whereby a cDNA library is amplified and transcribed, the resulting mRNAs are ligated to a DNA Linker and in vitro translation is performed to generate protein-mRNA fusion molecules. These are purified, and reverse transcribed to create a cDNA template (used for amplification later on). mRNA display molecule libraries are incubated with the immobilized drug candidate and unbound protein-nucleic acid complexes are removed by washing. The cDNA is then amplified (PCR) to produce a library enriched for proteins that bind the drug. After subsequent selection, DNA sequencing is used to identify the cDNA.

Three-hybrid systems

Three-hybrid systems function via the interaction of 3 parts. The first is comprised of a DNA-binding domain linked to a ligand-binding domain. The second is comprised of the ligand linked to a small molecule drug candidate. The third component is made up of a transcriptional activation domain fused to a protein (a potential drug target). If the small drug candidate associates with the protein the three sections connect to form a trimeric complex, resulting in the expression of a reporter gene – which is used to measure the interaction.

3. Protein microarray

Protein microarrays enable high-throughput analysis of the molecular interactions between the target and drug candidate. The protein targets to be analyzed are first purified and subsequently immobilized on to a glass slide. The resulting array features numerous potential targets (fixed at specific positions). It is incubated with a labelled version of the small molecule drug candidate. The array is then washed thoroughly to remove any unbound molecules. After washing, any remaining labelling signal ‘spots’ indicate successful drug-protein binding. The location of the labeled drug-protein complex can then be mapped to the specific protein target fixed at that position.

4. Reverse transfected cell microarray

‘Reverse transfected’ cell microarrays are sometimes referred to as ‘living’ microarrays, as live cells (that are transfected with cDNAs) rather than proteins are used. The transfected cells express specific cDNAs at different locations on the array, the array is consequently covered in cell clusters that overexpress specific proteins at specific positions. As with protein microarrays, the cell microarray is incubated with a labelled version of the small molecule drug candidate, which enables detection of the target protein.

5. Biochemical suppression

Biochemical suppression is an alternative strategy for identifying drug targets and signaling pathway components. Unlike many other target deconvolution strategies, this approach does not depend on a molecule’s affinity for its biological target. Biochemical suppression involves the addition of a small molecule to protein extracts, which inhibits an activity of interest – inhibition is measured using an activity assay. Firstly, a protein extract is mixed with a molecule that is known to inhibit the activity of interest. An uninhibited protein extract is then fractioned and introduced to the inhibited extract, it is then possible to determine if any of the fractions suppress inhibition. If a fraction is identified, that suppresses the small molecule’s inhibitory activity, further rounds of fractionation are performed to purify the suppressor activity.

References

Terstappen, G., Schlüpen, C., Raggiaschi, R., & Gaviraghi, G. (2007). Target deconvolution strategies in drug discovery. Nature Reviews Drug Discovery, 6(11), 891-903. doi: 10.1038/nrd2410
Wermuth, C., Aldous, D., Raboisson, P., & Rognan, D. (2015). The Practice of Medicinal Chemistry (4th ed., pp. 45-70).