06 January 2014

That's One Way How to Do It

There is no right way to do science, that's what makes science awesome.  However, when you are entering a new field, or trying something new the first thing you do is find a current review.  I remember in grad school whenever it was my turn to present literature at group meeting, I would search the topic in TIBS (Trends In Biochemical Sciences).  That was always the best starting point.  However, when you want to really do something, as in practical applications you looked for a Methods in Enzymology paper or Current Protocols.  This always give you a way to do something, with in depth technical hints and tricks of the trade.  On this blog, we discuss a lot of different techniques and oftentimes it is out of the users area of expertise.  We try to make it understandable and I think we are largely successful.  

In this paper, yours truly, Darren Begley, and colleagues from Emerald put forth one way to run and analyze Saturation Transfer Difference NMR for fragment campaigns.  STD is the subject of MANY posts here.  One of the things I want to point out is that opinions are like belly buttons, everyone has one.  So, in this Protocol we put forth a way to perform STD it is not the only way to do, but I think it is a rather robust method.  Not everything will translate to every company.  For example, most companies don't have extremely small, highly soluble fragments like the Fragments of Life and thus 500mM stocks will not be achievable.  I believe 100 mM is a better generic concentration.  However, I would love to hear in the comments what other people think.  Additionally, there are computational approaches that make the manual creation of pools unnecessary.  In terms of analysis, there are a million different approaches.  Most companies that make NMR software have some sort of automation.  I really like the implementation from Mnova.  However, it is important to keep in mind that your results are really only as good as your understanding of the experiment. 

What this all really comes down to is that NMR, and STD in particular, is not a black box.  You still need an expert user running the experiments and analyzing the data.  My goal with this paper though is to enable better understanding of the STD experiment for the lay user.  Hopefully, this leads to greater use of the experiment and concomitant increased success in screening.  I would really like to hear comments about what people do differently and why. 


Dan Erlanson said...

This is a really nice resource for folks getting into STD NMR, but I did have one question. It seems that the technique is particularly susceptible to aggregating compounds, so what do folks think of routinely running STD NMR in the presence of detergent?

Dr. Teddy Z said...

I would say it is very important, for any assay format.

Anonymous said...

I think detergent should only be left out of any assay in exceptional cases.

Dan Erlanson said...

That's my feeling too. Since the Current Protocols paper does not mention detergent, which one(s) work best for NMR?

Dr. Teddy Z said...

One interesting example is the rise in Mass-Spec methods. The cannot tolerate detergents at all. So, should we be more worried if they are used?

Darren B. said...

I think everyone agrees that aggregation is a critical factor in fragment screening, and can be more prevalent in the absence of detergent. But there is nothing about STD-NMR more inherently sensitive to aggregating compounds than other methods testing hydrophobic compounds at high concentrations in aqueous buffers. Moreover, there are drawbacks to using protonated detergents for STD-NMR, which Teddy, my team & I discussed while writing this protocol:

1) Detergents will show up in the aliphatic spectral region, potentially masking the only proton signals for certain fragments, such as those 3D fragments of the future;
2) Depending on their chemical structure, detergents may experience direct irradiation by the saturation pulse, creating potential for positive % STD due to binding events with the detergent and not the protein...detergents which bind the protein may have STD enhancement as well, and in turn indirectly generate false positives;
3) In certain cases, detergents can compete for fragment binding sites, preventing fragments from binding in important spots;
4) At sufficiently high concentrations, detergents lead to reduced NMR signal strength and lower quality data;
5) Some protein targets are incompatible with anything remotely denaturing.

With such complicating factors (and limited space for text), we felt it was not suitable to present in our method for introductory screening. Instead, we chose another solution: test your library. Screen the compounds under the same/very similar conditions by STD-NMR minus the protein, and eliminate false positives that way. Saves you much time and frustration, rather than following up on the false positives post-screen, as was presented in Vom's 2013 AJC paper.

Note that, in the same study (Table 3), detergent only eliminated 1 bad actor at high concentration of fragment (1.0 mM ± Tween), while simply reducing the fragment concentration (1.0 mM -> 300 µM) eliminated 6 false positives. We have found reduced concentration to be similarly superior to detergents for removing bad actors, and presented those instructions in the method.

I'm not saying detergents should be left out of the realm of buffer agents one tries when screening. But when it comes to minimizing downstream problems, at least for the type of work that I do, I tend to put vetting the library first, and working up the best conditions on a per-target basis, detergent or no detergent.

- db

Anonymous said...

Teddy Z

As far as I'm aware, mass spec can tolerate detergents, you just need to use synthetic ones that have a single peak rather than giving you a smeary mess (octylglucoside being the one we mostly use for screening).

Martin S said...

I'm not sure that there is a simple answer to this. The fragments that we reported in the Vom paper were from our first library. Many of those compounds wouldn't make it into cocktails now, and weeding out the badly behaved compounds using no-protein controls is clearly a good way to get rid of some of the false positives as Darren said.

However, we still see cases where fragments that have good experimentally measured aqueous solubility and do not give signals in STD spectra of the mixtures without protein behave as bad actors. We don't yet have enough data to indicate whether detergent makes a systematic difference to these.

I'm not sure that it would be wise to treat a detergent as a benign additive that simply prevents aggregation, so whether to add detergent and which detergent to add is probably dictated by the target.