FragNet: The next generation

The first fragment event of 2018 was held in Barcelona last week. This was part of FragNet, established “to train a new generation of researchers in all aspects of FBLD.” Fifteen graduate students from 13 European countries are participating over the course of three years. This meeting marked the midway point for them. I was privileged to serve as a scientific advisor, and was impressed at how much they’ve been able to accomplish in just 16 months. They’ll be on the market next year, so you’ll definitely want to prioritize them if they apply to your institution.

One interesting feature of the program is that, in addition to their primary research, each student completes two “secondments” in other labs – one in academia and one in industry. This is unusual (in the US), and gives them a much broader range of experiences than is typical in graduate school.

The projects themselves are diverse, ranging from synthetic chemistry through computational approaches and biophysics. Fragment library design is a major theme: David Hamilton is building substituted cyclobutanes, Hanna Klein is focusing on pyrrolidines and piperidines, and Aaron Keely is exploring covalent fragments. Darius Vagrys, Sebastien Keiffer, Edward FitzGerald, Pierre Boronat, Lorena Zara, Eleni Makraki, Bas Lamoree, and Lena Muenzker are applying multiple (mostly) biophysical techniques against a variety of different targets. Andrea Scarpino, Moira Rachman, and Maciej Majewski are focusing on computational approaches. Finally, Angelo Romasanta is exploring the diffusion of FBLD techniques through industry. Often multiple students work on one problem from different angles: for example, Andrea is using modeling to explain some of the experimental results produced by Aaron. Plenty of interesting data are being generated in the projects, and I look forward to seeing the eventual publications.

In addition to the student presentations, there was a one day workshop open to the public, with a strong focus on computational approaches. Chris Murray (Astex) discussed how these play a role in all aspects of FBLD, from library design to finding related compounds using the Fragment Network (discussed here). Having a good set of validated experimental data is essential for benchmarking computational methods, and Astex has contributed one of these. But not every computational approach is applicable to every problem. Free energy perturbation (FEP), a rigorous method for predicting SAR, worked well retrospectively for the target XIAP but was not useful prospectively for a target in which the researchers were trying to find a less lipophilic replacement for a phenyl ring. Chris also pointed out that computational methods have a high hurdle – not just to make predictions but to do so better than experienced scientists.

Jenny Sandmark (AstraZeneca) discussed structure-guided design, with a heavy focus on crystallography. She emphasized the importance of quality control: resolution better than ~2.4 Å, with good electron density and low B factors. (Computation can help: Maciej gave an example where dynamic undocking was able to clarify an ambiguous crystal structure.) Jenny also highlighted a set of 52 crystal structures of fragments bound to the capacious binding site of soluble epoxide hydrolase that has been made publicly available for the benefit of modelers.

Chun-wa Chung (GlaxoSmithKline) discussed the importance of understanding your screening technologies and all their limitations. How to establish a cascade assay depends on the needs: if crystallography is challenging, you may want to limit the hits to those that confirm in multiple methods, as these are more likely to confirm crystallographically. If, on the other hand, you have the capacity to do lots of structures you should examine hits from all screens, as those that don’t repeat may be false negatives. Chun-wa also discussed the importance of biophysics for HTS (though this may require different protein constructs for different methods). An HTS screen of 1.7 million molecules against ATAD2 produced a 1% hit rate, of which 444 were studied using a variety of methods including fluorescence polarization, SPR, and NMR. Ultimately only 16 compounds turned out to be useful – all in a single series. (See here for their fragment efforts.)

John Overington (Medicines Discovery Catapult) gave an overview of the open-access database ChEMBL, which holds data from publications and patents on more than 11,000 targets and 14.5 million molecules, including 13,000 clinical candidates and 1500 drugs. Of course, the entries are only as good as the underlying publications: biochemical assays can vary by about 10-fold, cell-based assays can differ by about 100-fold, and in vivo results can vary by 1000-fold. Still, studying these data can produce interesting insights. For example, the observation that antibacterial compounds tend to be larger and more polar appears to be due to the fact that many antibiotics bind to bacterial RNA – those that just bind to bacterial proteins have more standard properties.

Finally, Anthony Bradley described the computational resources at XChem. We’ve recently discussed some of these, including their open-access version of Fragment Network for analog searching. XChem uses extremely high concentrations of fragments for soaking – DMSO stocks are 500 mM and are soaked at 30-50%, so the final concentration can be as high as 250 mM! This often results in multiple fragments binding to a crystal, many of which are of uncertain functional relevance; Anthony used the term “putosteric” for putative allosteric site. Achieving functional activity can be challenging, but it is encouraging that of 16 targets initiated in the past 12 months, 7 have produced compounds with IC₅₀ values better than 100 µM.

All in all a great start to the year – and lots of good events ahead – hope to see you at some!

Linking fragments on DNA

DNA-encoded chemical libraries are one of the sexier new approaches for lead discovery. Typically, small molecules are synthesized while covalently linked to DNA and then screened for binding to a target. The structure of the molecule is encoded in the sequence of the DNA, and since incredibly tiny amounts of DNA can be sequenced (wooly mammoth genome, anyone?) you can fit massive libraries into a single Eppendorf tube. Indeed, some companies boast 100-billion compound libraries, nearly three orders of magnitude more than the number of molecules indexed by Chemical Abstract Service.

One might think this has no relevance for fragments. Indeed, the only mention of DNA-encoded libraries I recall on Practical Fragments was a comment by Teddy back in 2012 that the approach is “as opposite from FBDD as you can go”. A recent paper by Dario Neri, Filippo Sladojevich, and their collaborators at the ETH Zürich and Philochem in ChemMedChem suggests otherwise.

The researchers have developed an approach called DNA-encoded self-assembling chemical (ESAC) libraries (see also their earlier paper in Nat. Chem.). Rather than synthesizing a single molecule on each strand of DNA, this approach involves assembling two separate sub-libraries of DNA-linked molecules, one attached to the 5’-end and the other attached to the 3’-end. These are then mixed together, allowed to hybridize in a combinatorial mixture, and screened against the target; if a specific combination of fragments is identified (through elegant PCR experiments), this indicates that the two fragments bind to the target in close proximity.

The researchers have focused on the protein alpha-1-acid glycoprotein (AGP), a prominent plasma protein whose function is poorly understood. In their Nat. Chem. paper, a library of 111,100 members (550 x 202 fragments) identified fragments A-117 and B-113. Neither of these fragments showed any binding themselves, but when linked together the resulting compound 1 bound with low micromolar affinity as assessed by isothermal titration calorimetry (ITC).

The linker connecting the two fragments is long, flexible, and not particularly drug-like; its improvement is the focus of the ChemMedChem paper. The researchers increased the size of their second fragment library from 202 to 428 elements, and an ESAC screen revealed that the pair of fragments A-117 and B-217 – both still attached to DNA – had a dissociation constant of 110 nM; B-217 itself (attached to DNA) was around 9900 nM.

To find out how these fragments could be productively linked, the researchers coupled them to 11 different scaffolds, each of which was attached to DNA. All of these bound to AGP, with dissociation constants ranging from 9.9 to 1300 nM. The moment of truth came when the researchers resynthesized some of the molecules no longer attached to DNA. Compound A₁₁₇-L1-B₂₁₇ bound with a K_d of 76 nM as assessed by SPR, while the weakest on-DNA binder (K_d = 1300 nM) showed no binding by itself. Although no explanation is provided for this discrepancy, it could be due to low solubility.

This is an interesting approach, though the molecules reported do tend towards molecular obesity (A₁₁₇-L1-B₂₁₇ weighs 765 Da and has a ClogP approaching 8). Indeed, this may be an inherent liability – the minimum allowable distance between two fragments that are each attached to DNA may be larger than desirable for most targets. Still, it will be fun to watch this develop.

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.

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.

Fragment events in 2018

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

2018

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 here, here, 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 here, here, 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 2016, FBLD 2014,  FBLD 2012, FBLD 2010, and FBLD 2009.

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