How would you design an orthogonal validation cascade for a proposed target-compound interaction?

Why

Orthogonality means the assays do not share an artifact. Two fluorescence assays agreeing proves little, because a fluorescent compound breaks both. A label-free binding method and a thermal shift in cells fail for different reasons.

The cascade also moves from simplest to most demanding, so weak candidates are dropped cheaply. Arrowsmith and colleagues make the same point for probes, recommending structurally distinct compounds and a combination of genetic, chemical proteomic and biophysical assays, and noting that engineering drug resistance is a powerful way to link target, compound and effect. The resistant mutant test is the most decisive because it changes the protein instead of the compound.

If a mutation that blocks compound binding, but preserves function, abolishes the phenotype, then the phenotype is attributable to that protein.

How to work it through

1. Confirm compound identity and purity before anything else, since a wrong or degraded sample invalidates the cascade.
2. Measure direct binding to purified protein by a label-free method such as surface plasmon resonance or isothermal titration calorimetry, with a dose response and a stoichiometry check.
3. Show engagement in intact cells by thermal shift or probe competition, and record the concentration at which it happens.
4. Measure a proximal pathway marker over a matched concentration range to link engagement with pathway modulation.
5. Test a structurally unrelated compound against the same target and an inactive close analog of the original.
6. Finish with a genetic test, either a resistant target mutant that abolishes the phenotype or target depletion that reproduces it.

Where the answer stops holding

Purified protein may lack partners, membranes or modifications that matter in cells, so biophysical affinity need not match cellular potency. Resistant mutants are not available for every target and may alter function themselves. The cascade confirms that the compound acts through the target, not that the target is the right one for the disease.

Agreement across methods still cannot exclude an additional off-target that contributes to the phenotype.