Question 11 of 20 Intermediate Practical
How would you show that a compound engages its target inside cells?
Short answer
Target engagement is measured, not assumed from a cellular phenotype. The cellular thermal shift assay uses ligand-induced thermal stabilization to show binding in intact cells, and it is backed up by a proximal pathway marker, a resistant target mutant or a competition experiment with a labeled probe.
Why
A compound can be potent on purified protein and never reach the target in a cell, or produce a phenotype through something else entirely. Martinez Molina and colleagues built the cellular thermal shift assay on a simple principle: a protein bound to a ligand usually resists heat denaturation better than the free protein. Heat intact cells across a temperature range, remove aggregated protein, and measure how much target remains soluble.
A rightward shift in the melting curve with compound means binding happened inside the cell. The same study used it to follow drug transport, activation, off-target effects and resistance, and to look at tissue distribution. Several other approaches answer the same question.
A proximal biomarker, such as the phosphorylation of the target's direct substrate, shows pathway modulation at a given exposure. Competition with a labeled or clickable probe measures occupancy directly. A target mutant that no longer binds the compound, but keeps function, is the strongest genetic test, because the phenotype should disappear.
Engagement is a separate claim from efficacy, and the dose at which it occurs should be compared with the dose that produces the phenotype. A phenotype appearing far below the engagement concentration points elsewhere.
A strong answer covers
- Ligand binding usually stabilizes a protein against heat
- Thermal shift measured in intact cells
- Proximal pathway biomarker at matched exposure
- Probe competition measures occupancy
- Resistant target mutant as a genetic test
Common mistakes
- Treating a cellular phenotype as evidence that the intended target was engaged
- Comparing engagement in lysate with phenotype in cells without matching exposure
- Assuming every protein shows a measurable thermal shift on binding
The interviewer might ask next
What would you conclude if the phenotype appeared at a concentration ten times below measured engagement?