17 July 2017

Native mass spectrometry revisited

Native electrospray ionization mass spectrometry (ESI-MS) is one of the less-commonly used fragment finding methods. The technique relies on gently ionizing a protein-fragment complex without causing denaturation; bound fragments reveal themselves as shifts in mass. The technique is truly label-free, and can use very small amounts of protein and fragments. In practice the technique can work really well, reasonably well, or quite poorly. Two new papers shed light on factors that influence success.

The first paper, by Kevin Pagel (Freie Universität Berlin), Benno Kuropka (Bayer), and collaborators, examines four different cancer-related proteins. Let me say up-front that that the paper is remiss in not disclosing the chemical structures of any of the fragments, so in a very real sense this work is not reproducible. It is a shame the editors of ChemMedChem were not more demanding. That said, there is some useful information here.

Most of the focus is on the protein MTH1, screened at 10 µM concentration with 100 µM of each fragment. This was not a naïve screen; the fragments were previously identified from a thermal shift assay (TSA): 24 stabilized the protein, 4 destabilized it, and 5 had no effect. Remarkably, all of the fragments showed complexes in ESI-MS ranging between 6 – 66%, even those that had no effect in the TSA! Choosing an (admittedly arbitrary) 20% cutoff weeded out most of the false positives: 16 of the 24 stabilizers passed, while none of the destabilizers or neutral molecules did.

The best hit by ESI-MS also gave the strongest thermal shift, and a titration curve revealed an impressive dissociation constant of 1.7 µM. However, even at high concentrations of fragment the amount of bound complex did not exceed 70%, meaning that interpretation of single-dose experiments (for example, from a primary screen) could be problematic.

The results were similar for the protein KDM5B: 8 of 9 stabilizing fragments were hits by ESI-MS, as were two of 7 destabilizing fragments. Note that fragments that destabilize proteins can still be tight binders, as illustrated here.

For two additional proteins, however, ESI-MS was disappointing. For BRPF1, ESI-MS didn’t find any of the 11 hits from TSA, while for UHRF1 it found only a single hit – though this hit was not one of the 10 stabilizers identified by TSA. One could argue that the TSA hits were false positives were it not for the fact that, in the case of BRPF1, 6 of them were confirmed by crystallography.

The second paper, in Angew. Chem., comes from Chris Abell and coworkers at the University of Cambridge, and focuses on the protein EthR, a potential target for tuberculosis that we’ve previously discussed.

EthR binds to DNA, so rather than look for direct binding of fragments to EthR the researchers instead looked for fragments that could disrupt the EthR-DNA complex. A small library of 73 fragments was tested (at 0.5 mM each, in 2% DMSO), yielding 8 hits. The same library was screened under the same conditions using differential scanning fluorimetry (DSF), yielding 7 hits, 4 of which had also been identified using ESI-MS. All 11 of these molecules were then tested under the same conditions in an SPR assay to see if they could disrupt the interaction between EthR and chip-bound DNA. The 7 best SPR hits were all fragments that had been identified by ESI-MS. Moreover, two fragments – one identified solely by ESI-MS and one identified by both ESI-MS and DSF – were characterized bound to EthR crystallographically, and these represent new chemotypes for this target.

So what are we to make of all this? In common with other techniques, ESI-MS works well for some targets and less well for others. The problem is that it is not clear what distinguishes the two classes of targets. If you have access to the equipment and expertise you might consider adding ESI-MS to your screening cascade. But if you can only afford to buy one instrument for fragment screening, you’d probably be better off investing in NMR or SPR.

10 July 2017

Reagents as covalent fragments

Covalent drugs are a thing these days: as long as you can get selectivity, there’s nothing like a covalent bond to juice up affinity for a target. Screening for covalent fragments is thus a reasonable approach, and multiple researchers have assembled libraries of fragments containing either irreversible or reversible covalent “warheads”. The latest example, by Marion Lanier, Mark Hixon, and collaborators at Takeda, appears in J. Med. Chem.

Boronic acids can form reversible covalent bonds with the side chains of serines or threonines in proteins, with a predilection for the highly reactive active-site residues found in hydrolytic enzymes. Indeed, three approved drugs – bortezomib, tavaborole, and ixazomib – contain boronic acids.

When medicinal chemists think of boronic acids, they probably think of them as reagents for the Suzuki coupling, a useful method for forming carbon-carbon bonds. Because the reaction is so popular, some 6000 boronic acids are commercially available, many of them fragment-sized. Thus, the researchers assembled a set of 650 into a boronic acid library (BAL).

To determine whether this BAL would be useful, the researchers screened it against autotaxin, a phospholipase with anti-cancer and anti-inflammatory potential. Fragments were tested at 100 µM in a functional assay, with hits retested in 11-point dose-response curves. This yielded a whopping 51 molecules with IC50 values better than 10 µM, some as good as 200 nM.

The researchers also screened autotaxin against a set of 1750 non-boronic acid containing fragments, this time at 500 µM. Not surprisingly, hits tended to be significantly weaker despite the similar sizes of the fragments. The BAL fragments had average ligand efficiencies of 0.61 kcal mol-1 per heavy atom, while the conventional fragments averaged a lower but still respectable 0.41. Some of the BAL fragments were also crystallized bound to autotaxin, revealing that they do in fact form bonds with the catalytic threonine. 

This is a nice paper, though I do wish that the researchers had tried to calculate the inherent reactivities of the boronic acids to determine how these differences affected their affinities for the protein, as has been done for other warheads such as aryl acrylamides. Also, it would be interesting to see how a docking program such as DOCKovalent performs against this target with the same set of fragments. Hopefully we’ll see these questions addressed in the future. In the meantime, expect to see commercial vendors start offering libraries of boronic acid fragments.

03 July 2017

Fragment events in 2017 and 2018

The year is halfway behind us, but there are still a couple upcoming fragment-based events, and next year is already taking shape.


July 23-28: Australia is coming into its own as a destination for fragment experts, many of whom will be participating in the Royal Australian Chemical Institute's Centenary Congress in Melbourne. The entire event should be huge - think ACS with wombats - so if you've been looking for yet another reason to travel Down Under, this is it.

September 25-29: CHI's Discovery on Target in Boston includes an Inaugural Lead Generation Strategies track (September 26-27), and it looks like fragments will play a major role - as well they should!


January 28 - February 1: The First Alpine Winter Conference on Medicinal and Synthetic Chemistry will take place in St. Anton am Alberg, Austria. This looks like a fun event and includes a section on FBDD.

April 2-6: CHI’s Thirteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of this year's meeting here, last year's meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

June 6-9: Although not exclusively fragment-focused, the fifth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and will be held for the first time in Boston. You can read my impressions of the recent Strasbourg event here and Teddy's impressions of the 2013 event herehere, and here.

October 7-10: Finally, FBLD 2018 returns to San Diego, where it was born back in 2008. This will mark the seventh in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2016, FBLD 2014,  FBLD 2012FBLD 2010, and FBLD 2009.

Know of anything else? Add it to the comments or let us know!