Guest Blogger: Brian Stockman

[DrZ: Most of you probably know Brian and his excellent work in NMR and drug discovery, especially fragments. I have asked Brian to summarize his most recent paper for us. Below is his contribution. The Editors would welcome others to do the same if they are so inclined.]


A recent paper from Pfizer [Chemical Biology & Drug Design 73, 179-188 (2009)] described the concerted use of NMR screening, competition binding, TROSY-based binding site mapping, and NMR-based activity assays to identify allosteric fragment activators of 3-phosphoinositide-dependent kinase-1 (PDK1). This protein kinase presented an interesting challenge since, in addition to the ATP site typically targeted by structure-based drug design efforts, it was known to have an allosteric site that could activate (or potentially inhibit) activity.

An STD-based NMR screen resulted in 372 fragment hits out of 10,237 fragments screened. Testing the compounds in an activity assay would normally eliminate the many false-positive artifacts of the STD assay. A first pass of the hits through a Kinase-Glo assay revealed that many were in fact inhibitors. Fragments without activity in this assay, however, could not be discarded since this assay was not capable of monitoring events at the allosteric site and could not distinguish ‘non-inhibitors’ from activators. Thus fragments that did not inhibit in the Kinase-Glo assay were also run in a Caliper assay. This assay uses a shorter peptide substrate and is capable of detecting inhibition and activation. Ultimately, a subset of the original fragment hits that were either inhibitors with high ligand efficiencies, activators, and/or had very novel chemical structures were chosen for further studies.

STD competition binding experiments using the known ATP-site binder staurosporine or a short peptide known to bind in the allosteric pocket were very useful to distinguish these two binding sites. TROSY-based binding site mapping, using 15N-labeled PDK1 expressed in baculovirus, was used to confirm the binding site for several key compounds. Finally, the biochemical assay data was complemented with 19F NMR-based activity assays. These assays used the 2-fluoro-ATP method described in a previous paper from Pfizer [Journal of the American Chemical Society 130, 5870-5871 (2008)].

NMR-based activity assays proved very valuable since they could easily handle high fragment concentrations, and, since they directly monitor conversion of substrate to product, were capable of detecting both inhibition and activation. NMR-based activity assays are single-enzyme assays. As such, they are quite useful as both primary fragment screening assays and as orthogonal HTS-triage assays. NMR-based activity assays have been characterized as the ‘uncola’ of biochemical assays because, as opposed to many HTS and bench top assays, they do not rely on any coupling enzymes for their detection. NMR-based activity assays should prove very valuable for accurately evaluating compounds in the 10 uM to 1 mM dynamic range of activity typical of fragments.