15 January 2018

Fragments vs USP7, two ways, both allosteric

Proteins in cells are constantly synthesized and degraded in a complex, highly regulated manner managed in part by the ubiquitin proteasome system. Simplistically, a ubiquitous small protein called ubiquitin is conjugated to other proteins, targeting them for destruction, and some of the proteins thus targeted control the stability of still other proteins. But ubiquitination is not destiny: ubiquitin can be removed by more than 100 deubiquitinating enzymes, or DUBs.

As I said, this is complex. But complexity has never stopped folks from pursuing drug targets, and multiple groups are interested in a particular DUB called USP7, which is implicated in cancer and other indications. USP7 is one of more than 50 members of a subfamily of DUBs that use cysteine as a catalytic residue. Selectivity is an obvious challenge, and since cysteine is chemically reactive, any screening result carries a high risk of being an artifact. Two recent papers describe how fragment-based approaches led to potent, selective inhibitors.

The first, published in J. Med. Chem. by Paola Di Lello, Vicki Tsui, and coworkers at Genentech, started with an NMR fragment screen. This identified molecules such as compound 1, which NMR data suggested bound near the active-site cysteine. This and other fragments were used to conduct virtual screens of the much larger Genentech library, and 21 of these were then tested experimentally. Most of these either didn’t bind, bound to multiple sites, or caused protein aggregation, but four of them, including compound 2, showed clear binding to a specific site on USP7 and also inhibited the enzyme in a biochemical assay.

Surprisingly, protein-detected NMR suggested that these four molecules did not bind in the active site as expected but rather in an adjacent “palm site”, a hypothesis that was confirmed by a crystal structure of compound 2 bound to USP7. This led the researchers to reexamine other hits from the original NMR screen, where they identified several aminopyridinephenols, such as compound 13.

Meanwhile, a biochemical HTS against USP7 had identified 76 hits, but most of these turned out to be artifacts, and none of them yielded co-crystal structures with the enzyme. The fragment findings led the researchers to revisit some of the weaker hits that had been overlooked, such as compound 15. This led to a crystal structure showing binding in the palm site, and further medicinal chemistry ultimately led to molecules such as compound 28 (GNE-6640), with nanomolar activity in both biochemical and cell-based assays. A separate paper in Nature characterizes the biology in more detail, revealing that molecules in this series interfere with ubiquitin binding and are highly selective for USP7.

Another fragment effort on this target was reported by Timothy Harrison and collaborators at Almac and Queen’s University, Belfast in Nat. Chem. Biol. An SPR screen of 1946 fragments against the catalytic domain of USP7 led to compounds such as fragment B. This was combined with molecules from other groups that had been reported in the literature, leading to compound 1. Subsequent medicinal chemistry, informed by crystallography, led to compound 4, with low nanomolar biochemical and cell-based activity and excellent selectivity. The enantiomer is much less active, and compound 4 should be a useful chemical probe to further understand the biology of USP7.

Remarkably, not only do the two series of molecules bind some distance away from the active site cysteine (yellow, upper right), they bind in completely different, non-overlapping sites!

These papers illustrate the importance of allosteric sites for tackling specific members of large protein families. They are also both cases of “fragment-assisted drug discovery.” Unlike many success stories we’ve highlighted, it is difficult or impossible to find the initial fragment in the final molecules. Heck, Genentech’s best molecules bind in a completely different site from where the first fragment hits bound. Being open to such possibilities, and using all available data from every possible source, are keys to success.

08 January 2018

Fragments vs Lp-PLA2 – third time’s the charm?

The enzyme lipoprotein-associated phospholipase A2 (Lp-PLA2) cleaves phospholipids into inflammatory molecules. As such, it has been pursued as a target for several indications, from atherosclerosis to Alzheimer’s disease. In 2016 we highlighted two fragment success stories against this target (here and here). A recent paper in J. Med. Chem. provides a third, this one by Jianhua Shen, Yechun Xu, and colleagues at the Shanghai Institute of Materia Medica, ShanghaiTech University, and the University of Chinese Academy of Sciences. The fact that all the scientists are from China illustrates the growth of FBLD in that country, as we reported last November.

The researchers started by screening a 500 fragment library in an enzymatic assay. Compound 10 was a weak hit but had good ligand efficiency and was unlike known Lp-PLA2 binders. Moreover, crystallography revealed multiple interactions between the sulfonamide and the protein. This information was used to perform a similarity search followed by docking of 200,000 compounds. The top 500 were manually inspected and 100 were purchased and tested, with compound 11 showing low micromolar activity.

A crystal structure of compound 11 bound to the protein revealed a similar binding mode as the initial fragment, and also suggested further improvements, such as adding substituents to fill a small pocket (as in compound 14a). Further optimization for both affinity and stability ultimately led to compound 37, which inhibited Lp-PLA2 in human and rat plasma. It also exhibited good oral bioavailablilty in rats and promising pharmacokinetics. The researchers state that further optimization is ongoing.

How far will this go? The most advanced Lp-PLA2 inhibitor to make it to the clinic, darapladib, failed two phase 3 clinical trials (with nearly 30,000 patients!) for coronary diseases, casting a pall over the target. Darapladib, which was not fragment derived, can fairly be described as molecularly obese. Molecules such as compound 37 and the other fragment-derived series we previously mentioned do appear more attractive, but whether anyone will invest the massive resources needed to move them forward remains the billion yuan question.

03 January 2018

Fragment events in 2018

The new year has finally arrived, and brings quite a few interesting events.


January 24: There will be a one-day FBLD workshop in Barcelona. This is part of FRAGNET, a European Commission training program for the next generation of fragment scientists. Registration is free but you need to email fragnetworkshop@gmail.com. The subject should be Surname, Name – Institution (e.g. Potter, Harry – Hogwarts) and the body of the email should contain the word "register."

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 last year's meeting here, the 2016 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. Mary Harner and I will be presenting a FBDD short course on the afternoon of April 2.

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 last year's Strasbourg event here and Teddy's impressions of the 2013 event herehere, and here.

August 19-23: The 256th National Meeting of the American Chemical Society, which will also be in Boston, includes a session on "Best practices in fragment-based drug design", currently scheduled for August 20.

October 7-10: Finally, FBLD 2018 returns to San Diego, where it was born way 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 2016FBLD 2014,  FBLD 2012FBLD 2010, and FBLD 2009.

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