Fragments in China

The 2017 International Symposium on Fragment Based Lead Discovery (pdf here) was held in Shanghai, China last week. I was fortunate to be able to attend what I believe was the first significant FBLD meeting in Asia. Antimicrobials were a major theme, particularly against drug-resistant pathogens. The two days were filled with nearly 20 talks, so I’ll just try to capture a few impressions.

Ian Gilbert discussed the fragment-based efforts underway at the University of Dundee, focusing especially on library design. Among initially purchased commercial compounds, only 56% passed quality control, with 26% insufficiently soluble (at least 2 mM in water) and most of the rest either unstable or impure, similar to what has been seen by others. Ian has also enlisted undergraduate students to make “capped” fragments ready for optimization, as well as novel heterocycles.

Biophysics was a major theme of the conference, and Ian made a strong case for biolayer interferometry (BLI), one of the lesser-used fragment finding techniques. A screen can be completed in just a few days with less than a milligram of protein. In particular, BLI may be useful for assessing ligandability: Ian tested 31 targets, 13 known to be ligandable and 5 known to be not ligandable, and found good agreement with previous research. Ligandable targets generally gave primary hit rates >4.5%.

Ismail Moarefi (Crelux, now part of WuXi AppTec) highlighted microscale thermophoresis (MST) and differential scanning fluorimetry (DSF). NMR had identified ten hits against Pim1, but only six had yielded crystal structures, despite considerable effort. Of the four that didn’t, three had no activity by MST, while the fourth was very weak. Ismail also discussed the Prometheus nanoDSF instrument, which is sufficiently sensitive that it can resolve two-stage melting curves for a two-domain protein.

Another lesser used fragment-finding technique, affinity mass spectrometry, was described by Wenqing Shui (ShanghaiTech University). This uses ultrafiltration to separate protein-bound ligands from unbound molecules and mass spectrometry to identify hits; up to 1000 molecules can be screened in a single assay! Wenqing provided several success stories, including fragment hits with very weak (millimolar) affinity. She also demonstrated that the technique works against a membrane preparation of a GPCR.

Among more common biophysical methods, NMR was represented by Ke Ruan (University of Science and Technology of China). The challenge was characterizing a low-solubility ligand which caused extensive line-broadening of the protein due to intermediate exchange rates. This was solved by examining the distance between a fluorinated ligand and a paramagnetic label on the protein and using this to model the binding mode.

But by far the star of the show was crystallography. We’ve previously mentioned the high-throughput capabilities developed at the Diamond Light Source, and part of the impetus for this conference was to bring these technologies to China. Frank von Delft (Diamond and University of Oxford) noted that since the XChem platform launched in late 2015 more than 50,000 crystals have been screened against more than 40 targets, resulting in more than 1000 fragment structures. The group is committed to removing barriers and bottlenecks and today can process 1000 crystals per week through compound soaking, harvesting, data collection, and processing (using specially developed programs such as PanDDA). More than 30 external groups have used the facility, and every target has yielded at least one hit.

Of course, to collect data on 1000 crystals requires you to reproducibly grow lots of well-diffracting crystals that can handle the rigors of soaking, and Diamond has released a handy list of tips and tricks. Getting the right crystals was also the theme of two talks, one by Sheng Ye (Chinese Academy of Sciences) and the other by Carien Dekker (Novartis). Sheng emphasized the importance of optimizing the protein construct, which could include trimming flexible termini or disordered loops, mutating flexible surface residues, or considering different species. He also noted that adding heavy metal ions can actually improve the quality of the crystals as well as making the structures easier to solve. Carien also emphasized the importance of getting the construct right and discussed how seeding (crushing a hard-won crystal and using this to seed new drops) can be very useful. As we’ve noted, screening fragments at extremely high concentrations seems to be the current state of the art, with Novartis moving to 50 mM in the final soak and Diamond going beyond 200 mM! (In contrast to other types of screens at high concentrations, crystallography should not yield false positives, though hits might bind so weakly as to be undetectable by any other method.)

Such a wealth of structures can be daunting, and Anthony Bradley (Diamond) described the construction and use of a “poised library” for follow-up studies. The 768 fragments are (mostly) soluble to 500 mM in DMSO and are designed such that simple chemistry could generate 1.4 million analogs based on reagents currently in stock at Enamine. Potential analogs can be searched using the Fragment Network approach described here, and I was happy to see that Diamond has released their own open-source version (updated link as of 3 Jan 2018).

Jianhua He (Chinese Academy of Sciences) described the facilities at the Shanghai Synchrotron Radiation Facility (SSRF). This is the first third-generation synchrotron in China and has hosted more than 200 research groups since it opened in 2009. Feng Ye, who works at SSRF, gave a talk (in Mandarin) about screening a bacterial protein at XChem; the movies showing liquid handling and robotics would be impressive in any language. Renjie Zhang (Diamond), who also spoke in Mandarin, gave a talk describing (I’m told) not just XChem but how outside users can apply for access. Although there is currently a long waiting list, this should be addressed within the next year or so when SSRF gains Diamond status.

At the 2015 Pacifichem meeting there were only a few speakers from China. Given the level of interest and expertise I saw last week, I predict that the 2020 meeting will see many more.

Essays in Biochemistry special issue: Structure-based drug design

Structure-based drug design is often an integral part of fragment-based drug discovery. Indeed, a majority of respondents in a recent poll would not work on a fragment without experimental structural information. Given the close relationship between SBDD and FBDD, I was pleased to learn that a recent issue of Essays in Biochemistry is completely devoted to SBDD.

The collection begins with an editorial by issue editors Rob van Montfort and Paul Workman, both at the Institute of Cancer Research. It briefly introduces SBDD and FBDD and provides an overview of the rest of the issue. It also contains a laudable call for rigor, awareness of artifacts, and making data publicly available.

The first full review, by Martin Noble and collaborators at Newcastle University, discusses the role of SBDD in discovering inhibitors of cyclin-dependent protein kinases (CDKs), with a particular focus on selectivity. Several small molecules are discussed, though I do wish the paper included the fragment-derived compound AT7519, which made it to phase 2 clinical trials.

The following paper, by Bas Lamoree and Rod Hubbard (University of York), is completely devoted to FBLD. This is a concise and self-contained review of the field, and is also sufficiently up to date that it provides a good primer on the state of the art.

Chris Abell and collaborators at the University of Cambridge discuss mass spectrometry for fragment screening in the next paper, including ultrafiltration, WAC, HDX-MS, and native mass spectrometry (though not Tethering). The review also includes a handy table summarizing the advantages and limitations of commonly used fragment-finding methods.

Next up is another review devoted to FBDD, this one from Benjamin Cons and his Astex colleagues. The focus is on challenging drug targets such as BCL-family proteins and KEAP1 where SBDD was pivotal, and the researchers particularly emphasize the utility of X-ray crystallography.

NMR was the first experimental technique used for FBDD, and this is the topic of a paper by Gregg Siegal and colleagues at ZoBio. The review includes examples where NMR revealed that crystallographically-determined binding sites were not biologically relevant. Newer techniques, such as NMR², are also discussed.

Frank von Delft and collaborators describe the fourth funding phase of the Structural Genomics Consortium (SGC), which includes generating a couple dozen “target enabling packages” around new genetic targets. The ten year goals are certainly ambitious: “no crystal structure is complete without a careful analysis of the target’s disease linkage, a fully analysed fragment screen, and a series of follow-up compounds with demonstrated potency and rationalized SAR.” Given the tools and partnerships they have already established, I wouldn’t bet against them.

Hitting a single protein target can be difficult enough, but Scott Hughes and Alessio Ciulli (University of Dundee) focus on ternary interactions, in which a small molecule acts as a “molecular glue” to bring proteins together. PROTACS, molecules designed to target proteins for degradation, comprise one class that has garnered significant attention recently, and as we’ve noted previously FBDD could play a role in discovering and optimizing them. Targeted protein degradation is also the subject of the next paper, by Honorine Lebraud and Tom Heightman (Astex). In particular, the researchers focus on the use of click chemistry to rapidly build chemical probes that degrade specific target proteins.

Crystallographers have steadily been shrinking how big a crystal must be for analysis, in part due to brighter X-ray beams. Michael Hennig and collaborators at leadXpro discuss X-ray free electron lasers, which were experimentally realized less than a decade ago. The energy of these photons is more than a billion times higher than in the newest synchrotrons – so powerful that they destroy the crystals almost instantaneously, but not before producing a diffraction pattern. This means that tens of thousands of individual crystals need to be studied in order to obtain a full dataset. Needless to say the technical and computational demands are intense and still being optimized. The rewards include being able to use weakly-diffracting microcrystals, such as those of membrane proteins, and the ability to collect data at physiological temperatures, as opposed to the cryogenic temperatures typically used.

The last paper, by David Barford and collaborators at the MRC Laboratory, discusses the use of cryo-electron microscopy – which was recognized by a Nobel Prize this year. Single particle cryo-EM does not require a crystal at all, and recent advances have made near-atomic resolution possible. The idea is to image thousands of individual proteins and then computationally reconstruct them. The review discusses multiple protein-ligand complexes, and although none of these are from fragment programs, some of the ligands are approaching the size of fragments.

This collection of papers nicely captures where SBDD currently stands and illuminates the path ahead. For at least a while all the articles are free to download – so check them out now!

Quantitative native MS identifies a new zinc binder

Last week we highlighted a case where undetected zinc contamination turned out to be completely responsible for the observed activity of a fragment hit. But zinc plays many essential roles in biology, and several groups have sought fragments that target metals; drugs such as vorinostat derive most of their affinity from such interactions. In a recent paper in J. Med. Chem., Thomas Peat, Sally-Ann Poulsen, and collaborators at Griffith University and CSIRO have identified a new zinc-binding fragment. 

The researchers previously screened human carbonic anhydrase II (hCA II) against a library of 720 fragments, which yielded seven hits that bind to the catalytic zinc, as described here. Most of these fragments were either known zinc binders or had modest (high micromolar) affinities. In the new paper, the researchers reveal an eighth fragment that is both novel and potent.

Surface plasmon resonance (SPR) and native electrospray ionization mass spectrometry (ESI-MS) identified compound 10, which has an affinity and ligand efficiency approaching that of sulfonamides such as compound 3, a well-known class of zinc binder.

The researchers determined the crystal structure of compound 10 bound to hCA II, which revealed an interaction between the catalytic zinc and the deprotonated nitrogen, whose pKa is ~5.5. The oxazolidinedione core of the fragment has previously been used as a carboxylic acid bioisostere, but a search of the protein data bank (pdb) revealed no precedents as a zinc binder. In addition to the primary interaction with the metal, the fragment also formed a couple hydrogen bonds with the protein, helping to explain the high affinity.

Next the researchers made or purchased a series of 18 analogs to assess the SAR using both SPR and MS. Native ESI-MS results are usually assessed qualitatively, but the researchers were able to get quantitative data by holding protein concentration constant (at 14.5 µM) and varying the fragment concentration from 0.5 to 120 µM. Plotting the percentage of protein bound and curve-fitting revealed dissociation constants remarkably similar to those determined using SPR.

Nine of the new fragments showed at least some activity, though none were significantly more potent than compound 10. Crystal soaking experiments led to seven new structures, with all the fragments binding in a similar manner as compound 10. (There was one surprise: a bit of extra electron density in one structure led the authors to reexamine the fragment by high-resolution MS, revealing that about 5% had oxidized, and that this was in fact the bound species.)

Combing through the PDB revealed that some of the SAR compounds had not previously been reported as zinc binders. Interestingly, the key pharmacophore in one of the inactive molecules – hydantoin 15 – has been reported to be a zinc binder. The fact that it was inactive against hCA II augers well for achieving selectivity with metal-binding moieties. It will be fun to watch this story develop.

Heavy metals suck!

Much of the early work of fragment screening involves avoiding artifacts. For high-concentration assays, compound purity is absolutely essential. However, this is not always easily assessed, as demonstrated in a recent paper in J. Med. Chem. by Alessio Ciulli, Helen Walden, and co-workers at the University of Dundee (see here for Derek Lowe’s discussion).

The researchers conducted a screen against Ube2T, a ubiquitin-conjugating enzyme involved in DNA repair and thus of interest as an anti-cancer target. About 1200 fragments were screened using both differential scanning fluorimetry (DSF) and biolayer interferometry (BLI). Most of the hits were quite weak (millimolar), but one showed low micromolar activity. Although this fragment was a destabilizer in the DSF assay, other destabilizers have turned out to be useful starting points.

Two-dimensional (HSQC) protein-detected NMR experiments suggested that the fragment binds near the catalytic cysteine residue, possibly with some protein rearrangement. The binding was reversible, as expected by the chemical structure of the fragment. The fragment was also active in a functional assay. Finally, isothermal titration calorimetry (ITC) revealed an impressively tight dissociation constant of 17.7 µM for the 16-atom fragment. All of these orthogonal assays suggested the researchers had a winning fragment on their hands, so they started acquiring and making analogs to further optimize the affinity. Then things went awry.

Of 14 molecules tested, some quite similar to the initial fragment, only two showed any activity, and these were way down. Concerned, the researchers examined the fragment itself by ¹H and ¹³C NMR as well as high-resolution mass spectrometry, all of which revealed that the compound had the desired structure and appeared to be quite pure (not necessarily a given!) So what the heck was going on?

The mystery was finally resolved, after considerable effort, by a co-crystal structure of the fragment with the protein. Unlike previous structures of Ube2T, this one revealed an unusual domain-swapped architecture, in which a domain of one Ube2T protein interacts with a different molecule of Ube2T rather than with the rest of its own protein. More alarmingly, there was no electron density for the expected fragment, but there was a small, strong area of density connected to the catalytic cysteine residue. The researchers speculated that this could be a zinc ion, and sure enough, zinc chloride itself turned out to have essentially the same affinity for the protein as judged by ITC. Adding the zinc chelator EDTA to the fragment abolished activity, and a colorimetric probe revealed the presence of zinc in the original fragment as well as – to a lesser extent – the two active analogs.

Metal contamination is actually not uncommon – we mentioned a case where residual silver accounted for the apparent activity of many HTS hits. Enzymes with an active-site cysteine are particularly susceptible.

This type of artifact is particularly insidious because it is so difficult to discover. In this case, it was uninterpretable SAR that made the researchers suspicious, and crystallography that revealed the culprit. But SAR can be wonky, and crystallography often fails. What else can be done? Elemental analysis could have helped, but people usually only turn to this if they’re already suspicious.

Of the various fragment-finding methods, I think the only two besides crystallography that could have given warning are native mass spectrometry (MS) and ligand-detected NMR. The former is relatively specialized and doesn’t work for all targets, but it would be interesting to know whether standard NMR techniques such as STD, WaterLOGSY, or CPMG would have revealed that the initial fragment was not binding. Of course, there can be all sorts of reasons for negative results. Publications like this one are useful reminders that simply ignoring such data is unwise.