One of the less commonly used
fragment-finding methods is microscale thermophoresis (MST). This measures the
movement of proteins in a temperature gradient; ligand binding changes the movement.
When we first described MST in 2012, we noted that the technique seemed
relatively low throughput. In a paper recently published in J. Biomol. Screen., Alexey Rak and
colleagues at Sanofi teamed up with Dennis Breitsprecher and researchers
at NanoTemper (which makes MST instruments) to try to increase this.
The researchers chose the kinase
MEK1 and carefully developed assay conditions; their detailed description is a
useful resource for those who decide to give MST a try. Adding nonionic
detergent to the assay proved to be essential for reproducibility and to
prevent the protein from sticking to the capillary or aggregating. Also, rather
than relying on the weak chromophores (such as tryptophan) in native proteins,
MEK1 was labeled with a fluorescent dye. The substrate ATP was used as a
positive control, and the measured affinity was in good agreement with previous
results.
The screen itself was performed
on a set of 193 fragments that had been computationally preselected as
potential ligands for the kinase MEK1 (work we blogged about here). These were
serially diluted using automated liquid handling and tested in 12-point
dose-response curves to try to determine dissociation constants (Kd values) for
each fragment. All together this run of more than 2000 capillary tubes required
only 90 micrograms of protein and took less than 7 hours. Retrospective
analysis suggested that a single-point screen at 150 µM of each fragment would
have caught most of the best hits and cut analysis time to 70 minutes, so it looks
like MST is becoming competitive with other biophysical screening methods in
terms of time and reagent consumption.
What about results? The overall
hit rate was nearly 38%, which is high, though not outrageously so given that
the fragments were computationally pre-selected. Of these, the best 25
fragments showed well-defined dose-response curves with
Kd < 200 µM and competition with ATP. One nice feature of the method is that pathological behavior such as aggregation or denaturation could be observed directly in the form of irregular or bumpy MST traces, thus allowing false positives to be rapidly weeded out. Similarly, a loss in fluorescence signal was interpreted as the protein unfolding and sticking to the wells or pipette tips.
Kd < 200 µM and competition with ATP. One nice feature of the method is that pathological behavior such as aggregation or denaturation could be observed directly in the form of irregular or bumpy MST traces, thus allowing false positives to be rapidly weeded out. Similarly, a loss in fluorescence signal was interpreted as the protein unfolding and sticking to the wells or pipette tips.
It is always useful to
cross-check hits in orthogonal assays. As we noted previously, these fragments
had previously been screened against MEK1 using surface plasmon resonance (SPR)
and differential scanning fluorimetery (DSF). Most of the best hits from DSF
were rediscovered by MST, though MST found many hits DSF had missed. In
contrast, most of the SPR hits did not confirm in MST. The rank order of hits
was also similar for MST and DSF but not for MST and SPR.
A picture is worth a thousand
words, and some of the best hits were subjected to crystallography. In fact, 7
of the top 15 MST hits had previously been characterized by crystallography,
and 7 new crystal structures could be determined out of 11 additional MST hits
for which crystallography was attempted.
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5 comments:
One Should point out that the SPR ccreening conditions are choosen rather oddly. The stated immobilization level of 1200 RUs (suppl. data) is too low to give any reliable screening results. This is also reflected in the comparison table were 1 RU is considered a hit (figure 2). Even with the most sensitive SPR devices this signal is hard to distinguish from noise. At best this comparison to SPR is flawed and leads to a wrong conclusion and at worst its intentionally deceptive.
From what I understood after a talk about this study at a conference, is that MEK1 generally behaves poorly in SPR, since it is very difficult to immobilize in a functional way. MEK1 - and many other kinases - are very sensitive towards the low pH values required for covalent attachement to the chips, so it seems the SPR assay that was performed could be as good as it gets. I think there is no published work (besides the present study) where MEK1 was successfully used for SPR binding studies with compounds. Besides, some hits were found by SPR, so SPR should be generally applicable for this kind of screening, even if the conditions are not optimal. Either way, the MST data (also the single point screening data in the supplement) are quite impressive!
I would not count this study as a successful screen for MEK1 with SPR, for the exact reasons stated in the first comment. What is written in the second comment only emphasizes the point. Since the assay window is non existant you get a random hit population, which explains the bad overlap of SPR with MST and DSF. It really upsets me that this wasnt noticed by the experimentators nor the reviewers.
Beyond the legitimate critizism concerning the SPR results, one should appreciate that the MST data look really, really good. The one major benefit of MST in FBLD is that the size of fragments does not determine the quality of the binding signal. Even double digit Da fragments can give huge binding signals.
The data for MST are good, and in line with their (Nanotempers) application note on MEK. But we cannot use 1 case study (1 target) to say anything general about MST vs SPR. Also, the authors have a clear interest in making a good case for MST. What would be needed is an independent open-minded evaluation of SPR and MST against a series of targets, where each assay is individually optimized. Any volunteers?
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