Following up on our last post on the use of the NMR technique TINS applied to a membrane protein, the same research group has now compared TINS with other techniques on a more conventional target. In addition to TINS, they conducted fragment screens using another commonly used NMR technique as well as surface plasmon resonance (SPR) and high-concentration screening; the results appear in the Journal of Biomolecular Screening.
TINS involves immobilizing a target protein onto a resin, then flowing fragments across the resin and determining whether they bind to the target as assessed by a reduction in their NMR amplitudes. A reference protein is evaluated at the same time; only fragments that bind to the target protein and not the reference are considered hits. In this case, the researchers chose the viral protein RNA-dependent RNA Polymerase (RdRP) as the target and the PH domain from the human protein Akt1 as the reference. Using a total of 4 mg of RdRP, they screened a library of 1270 commercially available fragments in pools of 3 to 5 compounds each, resulting in 74 hits.
One of the most commonly used NMR techniques for fragment screening is saturation transfer difference (STD), in which the magnetization of the protein target is saturated, and so magnetization transfers to any ligands bound to the protein. The researchers tested 133 fragments (both hits and non-hits from the TINS experiments) and found a total of 49 hits, of which 40 had also been found by TINS.
The 83 fragment hits from both TINS and STD were tested for their ability to inhibit polymerase activity at concentrations up to 2 millimolar; 70 of them showed some activity, and a few of these seemed to actually activate the enzyme.
Finally, a selected set of 62 fragments (all of which were hits in at least one of the three assays) were tested in an SPR assay at concentrations up to 0.2 millimolar. Of these, around half showed binding, and these tended to be the fragments that showed the greatest activity in the enzymatic assay.
The authors conclude that TINS picks up more hits than the other assays, though high-concentration screening comes close. This may be true, but it would have been nice if they had run the entire set of 1270 fragments through each of the different methods; it is possible that there were false negatives in the TINS experiments that could have been picked up by the other techniques. Moreover, some of the TINS hits that didn’t confirm in other assays may well have been false positives. Still, there are lots of useful data in this paper, and it demonstrates yet again the importance of using multiple, orthogonal techniques to discover and properly validate fragments.