Two separate research groups were both interested in the core domain of HIV-1 integrase (IN). They both purchased 500-compound fragment libraries from Maybridge, though since they were purchased about six months apart they contained only 455 compounds in common. One group screened pools of 10 fragments by STD-NMR to identify 84 hits, of which 62 confirmed as single compounds both by STD-NMR and 15N-HSQC NMR. All of these were soaked into crystals of IN, resulting in 15 co-complexes.
The second group used SPR to screen each compound individually; compounds that showed a significantly stronger signal binding to IN than to a reference protein were confirmed by doing full dose-response curves. 16 hits were taken into crystallography, resulting in 6 co-structures, and another 3 gave ambiguous electron density.
The problem, as shown in the figure, is that there was no overlap between the confirmed NMR hits and the SPR hits, or between the crystallographically confirmed fragments!
Five of the crystallographically confirmed SPR hits were retested by STD-NMR, though at a lower concentration (0.3 mM) than the original screen (1 mM) due to solubility issues. Four of these gave good signals, while the fifth produced a weaker signal that could only be detected at the pH of the original SPR screen. The reason the others may not have been detected initially could be because of competition in the original pooled NMR screen: with a 17% hit-rate, many pools probably contained multiple binders.
The title of the paper is “Parallel screening of low molecular weight fragment libraries: Do differences in methodology affect hit identification?” Clearly the answer is yes. Nonetheless, it is important to note that, at the end of the day, this may not matter so much. As the researchers observe:
We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites… that provided a basis for fragment-based lead discovery and further lead development.
In other words, no matter what technique you use, as long as you have a tractable target and you’re careful (and a little bit lucky) you’ll be able to find useful fragments.