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.