09 August 2010

Fragment specificity

A frequent topic in fragment roundtable discussions concerns specificity: do fragments hit lots of targets, or just a few? Isabelle Krimm and colleagues at the Université de Lyon in France studied this question experimentally and report their results in a recent issue of J. Med. Chem. The paper provides data for the ongoing debate of whether and how much specificity a fragment should exhibit before being pursued for further lead development.

The researchers assembled a diverse set of 150 fragments and used NMR techniques to determine whether they bind to five different proteins. Three of the proteins, Bcl-xL, Bcl-w, and Mcl-1 are related members of the Bcl-2 family of antiapoptotic proteins, and at least the first of these has been successfully targeted using fragment-based methods. The fourth protein, PRDX5, has proven to be much less yielding to inhibitor discovery, while the fifth, human serum albumin (HSA), binds a wide variety of small molecules.

After applying 1D-NMR techniques (WaterLOGSY and STD) to all of their fragments against each of the five proteins, the researchers used more rigorous but less sensitive 2D-NMR (HSQC) to determine the binding sites of the hits. (This later study revealed, in agreement with previous results from the same lab, that the fragments all bind in the “hot spots” or active sites of the proteins.)

More than two-thirds of the fragments bound to at least one protein, a rather high hit rate. However, the hit rates for each protein varied considerably, with only 7 hits for PRDX5 and 72 for HSA (with a close second of 71 for Bcl-xL). Within the Bcl-2 family there was little specificity observed: Mcl-1, with 29 hits, shared all but one hit with either Bcl-xL or Bcl-2 or both; such non-specificity among related proteins has been discussed previously. In the case of HSA and Bcl-xL, although both proteins had similar numbers of hits, just over half of these were in common, demonstrating that fragment specificity is not difficult even with small-molecule sponges such as HSA. That said, many fragments were remarkably nonspecific, with 22 hitting four of the 5 proteins. Amazingly, all 7 of the hits against PRDX5 also hit all four other proteins.

The physicochemical properties of the fragments that hit one or more proteins were compared with those of the library as a whole, and although most of the parameters were similar, the ClogP values (a measure of hydrophobicity) were considerably higher for hits, and highest of all for the non-specific hits.

These findings are more evidence that, as predicted almost a decade ago, fragments can bind to more proteins than can larger, more complex molecules. The follow-up question, how much does this matter, is still up for debate. There are plenty of examples of developing specific inhibitors from non-specific starting points during the course of fragment optimization. But how non-specific is too non-specific? Would you feel comfortable pursuing any of the fragments that hit all of the proteins?


Donnie Berkholz said...

I'm curious whether many of the nonspecific hits might go away if assayed in the presence of detergent.

Morten G said...

I should probably do this myself and probably also will at some point but... those anyone know if the hot spots they find are the same ones found by http://ftmap.bu.edu/ < presumably a hot spot finding web service?

Dan Erlanson said...

Good question. Unfortunately FT-Map is only available to academics, but please post if you find the answer.

Morten Grøftehauge said...

Okay, ran 2YXJ (Bcl-xL with the ABT compound), 1R2D, and 1MAZ (two apo structures) in FTMap.

Now there's a considerable conformational change on binding of ABT in the residues from 100 to 129. But the results were still pretty good.

Now in 2YXJ the top ranked hot spot is where the benzylthioether is and it extends to the nitro group. In both 1R2D and 1MAZ this spot is blocked by residues Arg100 and Tyr195 but in 1MAZ there is still a cluster composed of just acetone, formamide, and formamine (ranked 7th).
Still in 2YXJ the 2nd, 3rd, and 5th cluster covers the chlorine and the two rings attached to it. In 1R2D and 1MAZ Phe105 covers the chlorine and the first ring but in 2YXJ Phe105 switches into an area ranked 5th (and partially 1st) in 1MAZ and 4th in 1R2D.
Moving down to the 6th ranked site, it is placed in the same spot as the two 6-membered rings of which one has two nitrogens. This is the top ranked site in 1R2D and 2nd in 1MAZ.
Bizarrely the 3rd ranked site in 1MAZ/1R2D is not detected in 2XYJ at all. The only difference is that His177 and Asn128 move slightly away and Tyr173 moves slightly closer in the inhibitor bound conformation.

So basically the inhibitor covers ~3 hot spots by binding to them and 2 allosterically. And it remodels all but one of the hot spots it binds to.

Now unfortunately I'm between jobs so I don't access to the article and can't tell if it's the same spot =(

I can send you the files if you want to have a look.

Morten G

Sarah said...
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