18 March 2011

Weak affinity chromatography (WAC)

There are many ways to find fragments: NMR and X-ray crystallography are old favorites, but SPR is quickly catching on. There are also more specialized approaches, such as ITC, MS, TINS, and biochemical screening. Now another 3-letter abbreviation has joined the list: a paper published online by Sten Ohlson and colleagues at Linnaeus University in Sweden in Analytical Biochemistry describes weak affinity chromatography, or WAC.

The principle is remarkably simple. First, a protein of interest is covalently immobilized onto a chromatography column packed with modified silica gel. This can be done on a standard high-performance liquid chromatograph (HPLC). Then each fragment to be tested is injected in buffer; those that have affinity for the immobilized protein will stay on the column longer than they would if they lacked affinity. The fragments can be detected with either UV spectrometry or mass spectrometry.

To demonstrate the technique, the researchers used two model enzymes, thrombin and trypsin, and a couple dozen fragments ranging in mass from 93 to 307 Da. Most of these fragments contained an amidine, a moiety known to bind to both proteins. Columns without any immobilized protein served as controls. Of course, fragments may associate non-specifically with proteins, so the researchers also treated protein-containing columns with irreversible or potent reversible inhibitors; a fragment that comes out later from a column containing an active protein than from a column containing an inactive protein is presumably binding specifically to the active site.

Remarkably, the technique appears to work: most of the amidine-containing fragments were retarded in columns containing active protein compared to columns containing inhibited protein. Moreover, the relative affinity ranking correlated with the inhibitory activity of fragments in enzymatic assays. Some of the fragments were quite weak, with calculated dissociation constants around 1 mM.

The researchers also demonstrated that they could screen a mixture of 11 fragments, using mass-spectrometry to follow each fragment, and that the change in retention time was comparable to that observed when running each fragment individually. In this case it was important to use low fragment concentrations so as to avoid saturating the protein active sites.

As with any technique, there are bound to be limitations. The immobilized protein needs to be stable for an extended time; in the current case there was some degradation in performance, albeit over the course of months and more than 200 injections. A more serious constraint is the need for a proper reference. Inactivating an enzyme provides an ideal solution, but one that won’t be so easily generalized to all targets.

In some ways WAC could be seen as a low-price cousin of TINS: both methods rely on an immobilized protein, but while TINS uses a custom modified NMR spectrometer, WAC can get by with a much less pricey HPLC (though a mass-spectrometer seems nearly indispensible). It will be fun to see how WAC develops, and in particular whether it can be used to discover novel fragments against more challenging targets.

1 comment:

Salam Al-Karadaghi said...

The method is actually used now (or finally!) at SARomics Biostructures in Lund, Sweden, in fragment screening services, and has demonstrated good success on a number of targets.