Among the many ways to find fragments, one of the relatively inexpensive newcomers is weak affinity chromatography, or WAC (see also here). The technique works by immobilizing a target protein onto a column and flowing fragments over it; molecules that bind to the target will elute more slowly than those that don’t. WAC has a number of potential benefits, but as with any technique the question is how well it really works. In a paper published a few months ago in Analytical Chemistry, Sten Ohlson at Linnaeus University and collaborators at Vernalis compared WAC with more established methods.
The protein they chose, HSP90, is sort of the fruitfly of FBLD: just about every technique has been tested on it. It’s also an oncology target with which Vernalis has many years of experience. The researchers chose 111 fragments from the Vernalis library and screened these using WAC. They also screened most of the fragments using surface plasmon resonance (SPR), fluorescence polarization (FP), thermal shift, and NMR (using three techniques: STD, waterLOGSY, and relaxation filtered spectra; only fragments that confirmed in all three NMR assays were considered hits).
The top 27 hits from WAC were also investigated with isothermal titration calorimetry (ITC), and 32 hits were soaked into crystals for X-ray crystallography.
The results were quite encouraging, with good agreement between the different methods:
NMR performed the best, though this could be due in part to the fact that three separate NMR techniques were used. Thermal shift performed the worst, with both false positives as well as false negatives, but even here the agreement was always greater than 50%. It is also important to note that assay conditions varied from technique to technique (for example, the pH ranged from 6.5 to 7.5), which could account for many of the discrepancies.
These results are in sharp contrast to some other comparisons of fragment finding methods (such as here and here), which showed little or no correlation between hits. Why the difference? One possibility is that the folks at Vernalis have worked out all the kinks in their assays and are very adept at separating the true hits from the chaff. Of course, it probably doesn’t hurt that they were working with a well-behaved and extensively characterized target.
The main focus of the paper is WAC, which performed admirably. Compounds could be screened in pools of up to 16 fragments when mass-spectrometry was used as a detection method, and less than 2 milligrams of HSP90 was used to prepare all three of the WAC columns made. One worry with immobilizing your protein is long term stability, but the columns seemed to be stable for at least 6 months through multiple runs.
Of course, no technique is perfect, and one area where WAC gets whacked is in determining dissociation constants. The correlation between KD values measured by SPR and ITC was excellent (R2 = 0.91) but much worse for WAC versus ITC (R2 = 0.38) and nonexistent for WAC versus SPR (R2 = 0.016), though some of this could possibly be explained by differences in buffer conditions.
Overall it looks like WAC is a great way to find fragments, though you may want to use other methods to actually quantify binding. This paper provides a detailed guide for using WAC, as well as good descriptions of other fragment-finding methods.