31 July 2017

Fragments in the clinic: PF-06650833

Of the more than 30 fragment-derived drugs that have entered clinical development, more than a third target kinases. While most of these are being developed against various types of cancer, a new paper in J. Med. Chem. by Katherine Lee, Stephen Wright, and their Pfizer colleagues describes the discovery of a compound that inhibits interleukin-1 receptor associated kinase 4 (IRAK4), a target for chronic autoimmune diseases. (Katherine also spoke about this project at FBLD 2016.) This details the earliest screens through development of the active clinical candidate.

The researchers started by screening their 2592-member Global Fragment Initiative library at 236 µM using STD NMR, resulting in 169 hits. A biochemical screen of the same library at 909 µM produced 160 hits, with 95 in common. Further triage using another assay along with modeling prioritized 15 fragments, of which 10 produced structures in co-crystallization trials. Fragment 51 was particularly interesting due to its impressive ligand efficiency and unusual binding mode to the hinge region of the kinase.

The crystal structure suggested that fragment growing could be productive, and indeed simply expanding the phenyl ring to a naphthyl improved the affinity to low micromolar for compound 10. Adding a nitrogen into the ring to lower lipophilicity while also adding a substituent to pick up additional interactions improved the affinity another order of magnitude (compound 14).

Guided by a co-crystal structure of compound 14 bound to IRAK4, the researchers used parallel chemistry to further improve the molecule, resulting in compound 20, which crystallography confirmed makes multiple interactions with the protein. Compound 20 also had promising selectivity and pharmacokinetic properties, but despite low nanomolar activity in a biochemical assay it had only high nanomolar potency in human peripheral blood mononuclear cells (PBMC).

At this point the medicinal chemistry began in earnest, again guided by structure and with a keen eye on maintaining good physicochemical properties. To a non-chemist the changes between compound 20 and PF-06650833 may appear subtle, but chemists will appreciate that you don’t introduce two new stereocenters without darn good reasons, which are discussed in depth in the paper. The results paid off, with the final molecule showing low nanomolar potency in the PBMC assay, excellent selectivity against a broad panel of kinases and other targets, and attractive ADME properties. It was also orally active in an acute rat inflammation model.

Sometimes publications only appear after a compound has dropped out of development, but that is not the case here. Indeed, after completing four phase 1 studies, PF-06650833 is currently being tested in a phase 2 trial for rheumatoid arthritis. Watch this space!

24 July 2017

Fragments vs Trypanosoma parasites

Last month we highlighted how fragments could be used to discover inhibitors of protein-protein interactions (PPIs). Today we continue the theme of fragments vs PPIs, in this case the interaction between PEX14 and PEX5, proteins which are important for glucose metabolism in disease-causing protists such as Trypanosoma.

The research, published recently in Science, was done by a large multinational team led by Grzegorz Popowicz, Michael Sattler (both at Helmholtz Zentrum München), and Ralf Erdmann (Ruhr University Bochum). They started by solving the NMR structure of the N-terminal domain of PEX14 from T. brucei, the organism that causes sleeping sickness. Previous work had shown that PEX5 binds to this domain, with two aromatic side chains of PEX5 binding in adjacent hydrophobic pockets. With this information in hand, the team performed a virtual screen of several million (non-fragment-sized) molecules. Eight of the best-scoring hits were tested, and four showed binding in an NMR assay, with compound 1 having the highest affinity.

Next, the researchers screened a library of 1500 fragments (each at 1 mM in pools of 5) using 1H, 15N HMQC NMR. This led to 12 hits with affinities better than 2 mM. Strikingly, all of these fragments contained fused bicylic aromatic ring systems, three of which were substituted naphthyls. Appending these onto compound 1 led to compound 4, with low micromolar affinity. Introducing an amine to interact with a glutamic acid residue in PEX14 led to compound 5, with high nanomolar affinity. This compound also showed activity against several species of pathogenic Trypanosoma. Further tweaking led to a molecule with activity in a mouse model of infection.

This example of fragment-assisted drug discovery (FADD) is reminiscent of other cases (described here, here, and here) in which fragments were used to replace elements of a previously identified molecule. While it is possible that traditional medicinal chemistry could have achieved the same result, fragments probably helped winnow down the number of molecules to be synthesized. It is also nice to see this technology applied to understudied diseases. 

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 13-15: 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!