Last year we highlighted a fragment-finding technique called
weak affinity chromatography, or WAC. Those of you who were at FBLD 2012 saw
some nice updates on the approach, but if you missed the meeting you can read
two new papers. In the first of these (in Anal.
Bioanal. Chem.), Elinor Meiby and Sten Ohlson at Linnaeus
University, in collaboration with
researchers at Oxford
University, describe the
use of WAC against a kinase.
Recall that WAC works by immobilizing a protein onto an HPLC
column and then flowing ligands through the column; ligands that bind to the
protein will have longer retention times compared to their retention times in a
column without bound protein. Small amounts of fragments can be injected (2
picomoles in this case), and the fragments are at low concentration, minimizing
potential artifacts.
Here the researchers were interested in cyclin G-associated
kinase (GAK), a potential target for Parkinson’s disease. They used less than 1
mg of protein to prepare their column and used adenosine as a positive control
to show that at least 22% of the protein was still active. They then performed
a virtual screen of 3200 fragments (from TimTec), looking for molecules that
would bind to the ATP site of five different proteins; the 170 highest-scoring
fragments were then tested by WAC in mixtures of 13 compounds. Despite the use
of mixtures, run times were long (140 minutes per injection), so this
was not a high-throughput approach, although more compounds could potentially
be injected simultaneously (see below).
Mass-spectrometry was used as a detection method to identify
fragments. The change in retention time between the protein column and
reference column is related to the dissociation constant, and the researchers
found that 78 of the fragments had an estimated Kd of better than 0.2 mM, a
fairly high hit rate. (30 of the 170 molecules could not be detected on both
the GAK and reference column, so over half of the assessed molecules tested
positive.) However, it is possible that some of the fragments bind to but don’t
inhibit the protein. In fact, 23 fragments eluted more slowly from the
reference column than from the protein-containing column, illustrating that
non-specific effects are certainly possible (see also below). That said, many of
the best fragments are structurally similar to known kinase inhibitors.
One of the coolest features of WAC is that a couple of the
fragments were racemic, and these gave double peaks on the GAK column but only
single peaks on the reference column, suggesting that one of the two
enantiomers binds more tightly than the other.
As noted earlier, this campaign was not high-throughput, and
in the second paper (in J. Biomol. Screen)
Minh-Dao Duong-Thi, Sten Ohlson, and collaborators at Linnaeus and AstraZeneca sought
to speed things up. They took 590 fragments from their larger collection and
pooled them into 11 groups of 35-65 members in DMSO, with final concentrations
of each fragment as low as 0.023 mM. Fragments likely to be positively charged
were pooled together, and negatively charged fragments were pooled separately
to facilitate mass spectrometry analysis. Also, fragments in each pool were
chosen to have unique molecular weights to facilitate unambiguous
identification.
These researchers looked for hits against thrombin, a
protein they had previously used in developing WAC. The mixtures were screened
in 20-minute runs, so all 590 fragments were screened in under 4 hours. 60
fragments were not observed in the mixtures, but 36 of these could be detected
when injected singly under more optimized conditions.
The 30 best hits were then confirmed by injecting them
individually. To assess whether they bind to the active site of thrombin, the
enzyme was inactivated with an irreversible inhibitor, and the fragments
retested. Remarkably, only a single fragment showed a significant reduction in
binding to the inactivated protein, suggesting that the other fragments were
either promiscuous or bound to regions outside the active site. The selective
fragment contains an amidine moiety, a known thrombin-binding motif.
This paper demonstrates that WAC can be done in a fairly
high-throughput manner. Although the number of non-selective binders does raise
concerns, WAC could still be a valuable primary screening method, particularly
given that it can be done using standard laboratory equipment.
4 comments:
Hello Dan,
Thank you very much for reviewing our articles. I’m just adding some more to the discussion
The difficult part of the screening was DMSO in the sample. Actually the thrombin project was carried out first and we had recognized the detection problem with DMSO 100% before we did the screening on GAK. If the original library were at higher concentration, we could have diluted samples more to facilitate MS detection without compromise the throughput. The original TimTec concetration was 1.5 mM in 100 % DMSO. It was a little bit low, wasn't it? Do we have other better alternative than DMSO to store the library?
About non-selective binders, I think they are not trivial at least in my experience with thrombin. We could detect them by blocked target in WAC. If there is no covalent inhibitor available for a target, the experiment can be more complicated but still manageable, such as adding competitor into mobile phase, or using recombinant protein with modified active site, etc. I’m wondering how people deal with off-active site bindings in other screening techniques? Are there anything easier than those in WAC?
Thanks Minh Dao for your comments. I definitely agree that 1.5 mM is a pretty low concentration for fragments; I'd say most people have their stocks at 50 or even 100 mM. As far as solvents besides DMSO, at least one company uses methanol. The disadvantage is that one has to be more careful about evaporation changing the concentration, but I think methanol is far less problematic in doing MS.
I also agree that non-selective binders are an issue - one that all techniques need to deal with. Your suggestions are good, and some of the strategies used for SPR, BLI, CEfrag, and TINS might also be useful. Looking forward to seeing how WAC progresses!
I'm just thinking why water have not been tried for library storing. What can be the problem?
Water would be ideal, but the problem is solubility: it's sometimes difficult to find fragments that are soluble at 1 mM in water, let alone the 50-100 mM people typically use for stock solutions.
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