Ligand affinities for isolated proteins vs in cells and cell lysates

Earlier this year we highlighted a study that compared the affinities of kinase inhibitors for isolated proteins vs those same proteins in cells and found differences – sometimes dramatic. A new open-access paper in J. Am. Chem. Soc. by Blake Peterson and colleagues at Ohio State University addresses the same question, and explores insights from cell lysates as well.

 

To measure affinity in cells, the earlier paper used NanoBRET, which relies on a fusion protein consisting of the kinase and a luminescent protein. It is possible that this artificial protein may behave differently from the native protein, so the Peterson group developed the Fluorescent Probe Cellular Binding Assay (FPCBA). This technique makes use of a fluorophore conjugated to a ligand for the protein of interest. The protein of interest is overexpressed, the binding of fluorescent probe causes cells to become fluorescent, and the level of fluorescence can be quantified using flow cytometry. Adding a (non-fluorescent) ligand displaces the fluorescent probe, which is rapidly effluxed, and the loss of fluorescence can be used to calculate the affinity of the ligand.

 

For the sake of brevity I’m skipping lots of important details, such as detailed characterization of efflux and use of a second fluorescent protein to correlate expression of the target, but these are described extensively in the paper. The researchers focus on a fluorescent probe based on dasatinib, an approved drug that binds to dozens of kinases.

 

The researchers compared the affinity of dasatinib and imatinib, another approved drug, to the kinase ABL1 using both FPCBA and NanoBRET. The results were reassuringly similar, though dasatinib bound 3 or 4-fold more tightly in the PBCBA assay than the NanoBRET assay while imatinib bound up to 2-fold less tightly, which might be due to changes in the regulatory mechanism of the ABL1 fusion protein required for NanoBRET.

 

Whole cell assays tend to be lower throughput, so cell lysates are often used instead. For kinases, a common assay makes use of “kinobeads.” Native kinases are captured from cell lysates on beads and quantified using mass spectrometry. An inhibitor blocks binding of the kinase to the bead, and this reduction in binding can be used to calculate the affinity. The researchers measured the affinity of dasatinib and imatinib against a score of kinases using both FPCBA and kinobeads.

 

Of 21 kinases, 14 showed similar affinities (within four-fold) for dasatinib by both FPCBA and kinobeads. Six of the remaining seven showed up to 13-fold tighter binding in FPCBA than kinobeads, which the researchers attribute to the kinases being in a more active state in cells than in lysates. (A similar phenomenon has been seen for the helicase WRN.)  In contrast, the kinase SRC showed 11-fold weaker activity in cells than lysates, which the researchers attribute to autoinhibitory regulation.

 

The situation becomes even more discordant when comparing affinities in cells to the affinities of isolated recombinant kinase domains using the KINOMEscan assay. Since a picture is worth several hundred words (the length of this blog post), I’ve graphed the results. While possible to see a correlation, it is also possible to see dragons in the shapes of clouds.

 

The affinity of dasatinib was generally higher (often by more than 10-fold) in the KINOMEscan than the FPCBA assay, which is not surprising given the lack of ATP in the biochemical assay. But there were exceptions, most notably EPHA4, which binds slightly more tightly to dasatinib in cells than in the KINOMEscan assay. Interestingly, the affinity in the cell lysate assay was lower; the researchers suggest that in cells the kinase is in a more active form than it is in lysates, perhaps due to membrane localization.

 

In the end, this paper is another useful reminder that assays with isolated proteins do not necessarily translate to cells. And of course, activity in cells does not necessarily translate to activity in vivo. Understanding why is part of what makes drug discovery both frustrating and exciting.