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• Some very strong points are:

  • The comparison between cell based and lysate based is positive, one cannot replace the other, and the lysate approach adds more validation to cell-based aspect. There is value in the pair to understand which are potential downstream biological effects. There is also value in isolation of the target without a cell environment.

  • The storytelling is very specific to certain targets and pathways from such a large starting dataset. This makes it easy to verify those specific points and the value in knowledge extraction.

  • They highlight specific stories that were expected but also highlighted some that were unexpected.

  • Coherent experimental design using cancer cell lines and then they used predetermined drugs that are known to act in those cells.

• The use of duplicate samples seems low in terms of replicates. This led to a requirement for a heuristic cutoff (Figure 2) due to lack of statistical power (lack of replicates), which is a limitation of the conclusions that can be drawn.

• Related to the point that many of the protein changes seem to be off target effects of the drugs

  • Were there any drugs that were confidently only affecting their known target?

    • they said each had at least one but unsure

  • How can one discriminate between off-target effects for a drug that may actually affect health outcomes?

  • if a key point in the paper is that we don't fully understand the mechanism of action of many common drugs, how should clinicians or regulatory agencies or drug manufacturers respond?

  • perhaps this is outside the scope of the paper

  • it would be useful to show whether these effects are context specific (in additional lines) but again this is outside the scope of one paper

• Is it possible that these drugs could also alter the total quantity of the protein and confound the interpretation? Should there be a control that assesses the protein quants without any heating? This would depend on the experimental design, and it appears the compounds were incubated for 30 minutes before lysis for the differential centrifugation. Its unclear if protein quantities may change in response to drugs on this timescale. Figure 5C does show that at least for CRKL the quant is not altered by any of the compounds of interest.

• It may be useful to add a discussion about the mechanism of aggregation and precipitation

• The authors state thermal stabilization is expected to occur more frequently than destabilization. Why? In terms of thermodynamics, why would destabilization ever be favorable? ostensibly, wouldn't a protein always favor the more thermally stable state? or are entropic/other structurally stabilizing effects at play that energetically allow destabilization?

• Figure 2F states that a range of 1-10 um palbociclib was sufficient to alter PLK1 activity but only 10 um showed a response.

• The structural clashes referenced in 2H seem subjective - can this statement be quantified?

• Point about widespread alterations from three drugs only in the lysate experiment

  • the authors provide a citation about artefactual effects but could perhaps provide or hypothesize a more specific example

  • a more specific example or hypothesis for line 27 pg 12 "complex biophysical rearrangement" would be helpful

• I like the analyses in figure 6 to understand the protein perspective of how they generally respond to drugs. It's hard to see the grey versus black in 6E.

• Figure 6 - comment on homology binding of drugs is interesting

Competing interests

The authors declare that they have no competing interests.