30 October 2014

That's Just WAC

We are all about new methods here.  One method I have never been a big fan of is Weak Affinity Chromatography, which has been reviewed here, here, and here.  In this method, a target is immobilized on a column and compounds are flowed through and retarded if they interact. Affinity is determined by retention time, which we all know is never influenced by experimental conditions.  In this paper, the KDs determined by WAC are compared to those by SPR, as well as efficiency and consumption of materials.

As a model system they used alpha-thrombin, either in the active form or the covalently inactivated form.  Figure 1 shows the results for one compound. 
Figure 1.  A. WAC: tspec is difference in retention time for active (solid) and inactive (dashed) protein.  B. Triplicate response generated by subtracting in situ inhibited protein from active protein.  C. Isotherm from SPR. 
SPR required 120 minutes for triplicate data at 10 concentrations; WAC required 175 minutes.  WAC however can require up to 525 minutes for a single compound.  The authors note that Mass Spec detection can speed this up greatly by allowing multiplexing of ligands.  SPR requires more compound, but far less protein (25ug vs. 0.5mg).  As a counterpoint, they claim their thrombin column has a far longer lifetime than the thrombin chip.  In terms of robustness, WAC does not suffer from DMSO interference, while SPR can be mucked with by SPR, resulting in poor isotherm fits.  They noted this in 13 of 27 compounds they studied.  All 27 compounds were able to have isotherms generated by WAC.
 There is a high degree of correlation between the KDs determined by SPR and WAC.  The authors note one major factor is that similar conditions were used for both studies, while in the past this was not true.  In summary, SPR is faster, consumes less protein, and has higher throughput.  WAC uses less sample.  WAC, for future development, can use Mass Spec can give a advantage in terms of being able to multiplex compounds.  I am still not a fan of the method, but I want to give kudos to the authors who continue to develop the method. 


Prof.Dr. Sten Ohlson said...

assThanks for the “kudos” but I would clearly state that it is not just WAC but it is a promising future for fragment screening. I think Dr. Zartler has misunderstood the purpose of the article that was published recently in Analytical Biochemistry (Duong-Thi et al., Anal. Biochem., 461, 57-59, 2014) and I would like to make a few clarifications. This work was purely a validation study to show the correlation between WAC and SPR and it was not intended to demonstrate throughput, consumption of targets and fragments etc. In fact it was performed in such a way to enable an accurate comparison of the techniques disregarding any other critical features such as throughput. It does seem that Dr. Zartler, based on his summary, has not followed the literature on WAC for fragment screening in any detail which if he did would possibly have changed his opinion about this new emerging technology. We, as the researchers on the WAC technology, has in a number of studies demonstrated some interesting characteristics which may lead to an establishment of WAC as primary fragment screening tool. I just want to mention three but important features which include:
- WAC has high throughput where KD’s (not ranking) of 3000-5000 fragments can be achieved per day. The reason is that multiplexing of fragments is done by running mixtures and detected by MS and only one low (10-50 µM) concentration of fragment is needed to establish a Kd measurement (no isotherm) even for very weak binders (mM)
- The consumption of target is now minimized in the high µg range by doing WAC in capillary columns. As the columns have in many case high stability, they can be reused for many fragment campaigns.
- As mixtures can be screened, WAC has a possible rather unique position. For example, it should be possible to screen directly natural product mixtures, stereoisomer libraries and evolved fragment synthetic mixtures without much sample preparation which possibly can lead to major savings.
I recommend the readers of this blog to read and try out the WAC technology to see for themselves how it is working and how it fits into the toolbox for fragment screening. WAC is easy to run if you have access to a standard LC/MS platform. These four papers could be a good introduction how WAC works for fragment screening:
[1] M.D. Duong-Thi, M. Bergström, T. Fex, R. Isakson, S. Ohlson, High-throughput fragment screening by affinity LC-MS, J Biomolecular Screening, 18 (2013) 160-171.
[2] E. Meiby, H. Simmonite, L. le Strat, B. Davis, N. Matassova, J.D. Moore, M. Mrosek, J. Murray, R.E. Hubbard, S. Ohlson, Fragment screening by weak affinity chromatography: comparison with established techniques for screening against HSP90, Anal Chem, 85 (2013) 6756-6766.
[3] E. Meiby, S. Knapp, J. Elkins, S. Ohlson, Fragment Screening of Cyclin G-associated Kinase by Weak Affinity Chromatography, Anal. Bioanal. Chem., 404 (2012) 2417-2425.
[4] M.D. Duong-Thi, E. Meiby, M. Bergstrom, T. Fex, R. Isaksson, S. Ohlson, Weak affinity chromatography as a new approach for fragment screening in drug discovery, Analytical Biochemistry, 414 (2011) 138-146.
Cheers /Sten Ohlson

Dave Stepp said...

Conceptually I like WAC, but agree that there aren’t enough benefits to preferentially use it over other, well-established methods, especially for screening. I also applaud the direct comparison of the two technologies under similar conditions. However, I want to clear up some misconceptions in the article, and in your summary.
1) You and the authors correctly state that SPR uses far less protein, but “several-fold” more sample compared to WAC. On a technical level, injection time can be shortened to somewhat reduce sample consumption. But more importantly, for small molecule work, I submit that it is MUCH easier to make/buy more compound than it is to make mg quantities of pure, active protein. So in my mind SPR has the advantage here.
2) The stability of the enzyme on the WAC resin was shown to be longer than the enzyme captured onto the SPR chip. This is certainly an advantage, but not an insurmountable one (and one that likely is target dependent). With optimization there may be ways to increase the stability of the active chip surface. Simple things like buffer optimization and even lowering temperature can provide dramatic increases in stability. Additionally, with optimized method parameters, 384 injections can be performed in less than 24 hrs on an instrument not designed for high-throughput (i.e. Biacore T200). Higher throughput instruments (i.e. Biacore 4000) can provide dramatically higher throughput. Even with a new surface being generated daily, protein consumption for SPR does not approach that required for WAC.
3) SPR does not “suffer from DMSO interference”. With proper experimental design & buffer preparation, and proper solvent correction during data analysis, DMSO can be used without any “interference” at all. If an enzyme is sensitive to DMSO concentration, that will be reflected in most any assay system, and is typically assessed during assay development.
I will state that as a practitioner of small molecule SPR, I admit an inherent bias. But I’d like to think I’m open minded enough to know the technology’s limitations, and embrace any method that has advantages over SPR.

Anders said...

@Dave Stepp and Ohlson
This is an interesting discussion. Guess we want as many tools available as possible. But the identification of pros and cons of different methods is difficult, as its subjective. In experienced hands, pros/cons are so and so, but in less experienced hands, the situation can be completely different. And instrumental availability is an issue too. Personally, I am (still) looking for the best/feasible screening strategies. So far I am focused on native MS and STD-NMR. I would like to do SPR, but machines are expensive (we are academics). Therefore, WAC seem like a good alternative (apparently standard LCMS is enough; which we have). To me, that´s a big advantage.

Stefan Geschwindner said...

Having worked myself with the most prominent fragment screening technologies, just wanted to add my 50 cents worth. I have to admit, that my experience with WAC is probably not matching up with other technologies, but as limited as it is, it gave me at least a possibility to compare it with SPR and NMR.

The inability to set up a suitable SPR assay to screen an attractive but challenging drug target resulted in an evaluation of WAC as an alternative strategy for the fragment screening of a large fragment library (5K) . A smaller, non-overlapping library was screened in parallel by NMR. Different but validated fragment series could be obtained with both methods thus providing the project with more choices. This tells me at least two things:

(1) WAC can be a valid alternative to SPR in cases where the SPR assay is not suitable for fragment screening of larger libraries

(2) The ability to screen effectively a larger fragment library by WAC can eventually result in more fragment series to start with, which is a great benefit.