17 February 2020

Fragments vs MNK1 and MNK2: take three

Mitogen-activating protein kinase-interacting kinases 1 and 2, or MNK1 and MNK2, are implicated in several cancers while seeming to be dispensable for normal cells, making them attractive oncology targets. Indeed, we’ve previously written about two FBDD-derived clinical compounds against these kinases, eFT508 and ETC-206. In a J. Med. Chem. paper published last month, Alvin Hung and colleagues at A*STAR describe a third series.

The researchers started by screening 1700 fragments in a biochemical assay, resulting in 11 molecules that inhibited both MNK1 and MNK2 with decent ligand efficiency. Four of these had a meta-substituted pyridine, as in compound 6. Making a few analogs led to compound 13, with low micromolar potency against both enzymes.


Crystallography proved unsuccessful, but making the reasonable assumption that the pyridyl nitrogen binds to the kinase hinge region led to two models, only one of which was consistent with the SAR. Decoration of the phenyl ring led to compound 21, with submicromolar activity. Although still early, the researchers collected both in vitro and in vivo ADME data on this molecule, which turned out to be quite promising.

Next the researchers turned to the pyridyl ring and found that appending small (5-membered) heterocycles could also boost potency, as in compound 36. At this point, after multiple attempts with previous compounds, crystallography finally yielded a structure that confirmed the proposed binding mode. Combining elements from compounds 21 and 36 directly led to only a slight boost in activity, but further tweaking ultimately led to compound 47, the most potent member of the series. Unfortunately this molecule was unstable in mouse liver microsomes, but a related compound showed good mouse pharmacokinetics as well as impressive selectivity in a panel of 104 kinases.

No pharmacodynamic studies are described, and perhaps this series was deprioritized to focus on ETC-206, which was also developed at A*STAR. Indeed, the later compounds in this paper reveal a frustrating struggle between potency and stability often seen in medicinal chemistry. This is captured in a nice timeline that shows a rapid improvement in potency over about 8 months, followed by a slight drop as the researchers tried to improve exposure. Although no Goldilocks molecule is reported, this paper is nonetheless a lovely example of fragment to lead optimization done for the most part without the aid of crystallography.

10 February 2020

Toward SAR by SFX

As our poll last year revealed, X-ray crystallography has inched out ligand-detected NMR to become the most popular fragment-finding method. One criticism often leveled at crystallography is biological relevance: very few proteins in nature are found in the crystalline state. Moreover, crystallography is a dish usually served cold, with crystals typically frozen in liquid nitrogen. The reason for this is that the powerful X-ray beams used to elucidate the structure of molecules also rip them apart, and freezing them slows the damage. But protein-ligand complexes at low temperature may not always reflect our more temperate world.

One approach to collecting crystallographic data at room temperature is to do so very quickly, before radiation damage can occur. This is done using serial femtosecond crystallography (SFX), in which many crystals are individually examined using brief, intense beams from X-ray free-electron lasers (XFELs). The X-ray pulses last less than 20 femtoseconds, a time so breathtakingly short that light only travels the width of a typical human cell. In a recent IUCrJ paper, Robin Owen, Michael Hough, and collaborators at the Diamond Light Source, the University of Essex, and elsewhere describe a high-throughput version.

The protein crystals themselves can be quite small, just 1-20 µm across, compared with the > 50 µm crystals typically used in crystallography. These microcrystals are mounted in silicon “chips” containing 25,600 little apertures; the X-ray beam can then be swept across each of the positions.

The researchers demonstrated that they could collect high-quality data for three proteins that are particularly sensitive to radiation damage: two heme peroxidases and a copper nitrite reductase. For all three proteins, they were able to determine high-quality structures of bound fragment-sized ligands. Indeed, some of the ligands were even smaller than all but the smallest fragments: imidazole (five non-hydrogen atoms) and nitrite (three non-hydrogen atoms). The latter case was particularly impressive given that the nitrite displaces a bound water molecule, so the difference between empty and liganded protein is even more subtle.

The first word of this blog is “Practical,” so how does this technique stack up? The researchers used 2-4 chips for each structure, and data collection took about 14 minutes per chip. Despite the miniaturization, sample consumption is not trivial: 1.4 to 6 mg of protein and 4-40 µmol of ligand for each data set. However, the researchers showed that could get by with less data – in some cases significantly so – and state that a 4-5-fold improvement in throughput would be straightforward. Using processing software such as PanDDa could further improve results. I suspect it is only a matter of time before we see the first FBLD by SFX screen. It will be fun to see how useful it turns out to be compared with established methods.

03 February 2020

Fragments vs RIP2: from flat fragment to shapely selectivity

Last week we highlighted the utility of shapely fragments. However, as the latest review of fragment-to-lead success stories again shows, starting with a “flat” fragment does not condemn a lead to flatland. This is illustrated in a recent J. Med. Chem. publication by Adam Charnley and colleagues at GlaxoSmithKline.

The researchers were interested in receptor interacting protein 2 kinase (RIP2), which is implicated in various inflammatory diseases. A fluorescence polarization screen of 1000 fragments at 400 µM yielded 49 hits with inhibition constants ranging from 5-500 µM. Thirty of these confirmed in a thermal shift assay, and 20 were characterized crystallographically bound to the enzyme. Hit-to-lead chemistry was pursued for five series; the most successful started with compound 1a.


The crystal structure revealed that the carboxamide of compound 1a makes interactions with the hinge region of the kinase, with the phenyl group in the back pocket. A search of related molecules available in-house led to compound 2a, with a satisfying boost in potency. Interestingly, the crystal structure of this molecule bound to RIP2 revealed that the binding mode of the pyrazole moiety had flipped to keep the phenyl ring in the back pocket (compound 1a in cyan, 2a in gray). Enlarging the phenyl group to better fill the pocket led to compound 2k.


This molecule had relatively poor selectivity against several other kinases, but introducing a ring as in compound 8 improved the situation. Crystallography suggested that installing a bridged ring would pick up further interactions with the protein, and although the resulting molecule did not have better affinity, selectivity improved. Finally, a hydroxyl group was introduced (compound 11) to try to pick up interactions with a non-conserved serine residue. This addition did not improve biochemical activity, and in fact a crystal structure revealed that the hydroxyl group was pointing towards solvent, but the activity in human whole blood improved. Importantly, compound 11 was remarkably selective for RIP2: just 1 of 366 other kinases tested at 1 µM showed >70% inhibition.

This is a lovely fragment-to-lead success story that reiterates several important lessons. First, a generic (in this case commercial) and nonselective fragment can lead to novel, selective series. Second, as has been seen multiple times, fragment binding modes can flip unexpectedly, especially during early optimization. Finally, despite the relative flatness of fragment 1a (Fsp3 = 0, though the two aromatic rings are slightly twisted), it could be optimized to a more shapely lead, and the increased complexity is likely responsible for the impressive selectivity. Left unreported is the stability and pharmacokinetics of compound 11: the hydroxyl and all those sp3-hybridized carbons are likely metabolic hotspots. As is so often the case in lead discovery, what solves one problem can too often create another.

27 January 2020

Three dimensional fragments revisited

A long-running debate in the fragment world centers on the utility of “three dimensional” fragments. Proponents argue that these (often aliphatic) fragments may be more novel, have better physicochemical properties, and have more vectors for elaboration than “flatter” (mostly aromatic) molecules. Skeptics retort that hit rates are likely to be lower for these more complex molecules, and good luck making analogs. Two papers published late last year add more data to the debate.

The first paper, published in J. Med. Chem. by William Pomerantz and collaborators at the University of Minnesota and Eli Lilly, describes the results of a fragment screen against the bromodomain BRD4(D1), a popular member of the BET family. The 467 fragment library was enriched for shapely fragments as assessed by plane of best fit (PBF), which is the “average distance of a non-hydrogen atom from a plane drawn through the compound such as to minimize the average.” For example, "flat" benzene has a PBF of 0 while the cofactor NADPH has a PBF of 1.53.

The library was screened using ligand-observed (CPMG) NMR, and 34 hits were confirmed using protein-observed fluorine (PrOF) NMR. All of these were competitive with the known ligand (+)-JQ1, consistent with binding at the acetylated lysine recognition site. The average PBF of the hits was 0.44, essentially the same as the library itself (0.46). This is higher than the average PBF (0.36) of all fragments crystallized with BRD4 in the protein data bank.

Structures of all the hits are provided, and some of them are indeed quite unusual. The researchers characterized a substituted thiazepane crystallographically and were able to optimize this to a 32 µM binder with good ligand efficiency. This fragment was also selective against a handful of other bromodomains.

The researchers had previously screened BRD4(D1) under identical conditions with a more traditional, “flatter” library with an average PBF of 0.26. Interestingly, in that case the hits were less shapely than the library as a whole, with an average PBF of 0.17. The confirmed hit rate was also higher: 20% vs 7%. That said, the fragments in the traditional library tended to be smaller (averaging 180 Da vs 241 Da), so the molecular complexity of this library was likely to be lower, which could account for the higher hit rate.

The second paper, published in Bioorg. Med. Chem. Lett. by Ulrich Grädler and collaborators at Merck KGaA, EMD Serono, Edelris, and Proteros, focuses on cyclophilin D (CypD), which has been implicated in cardiovascular disease and multiple sclerosis. Unlike BRD4, this is a tough target: an HTS screen of 650,000 compounds in a biochemical assay yielded just 178 hits, none of which confirmed. Undeterred, the researchers screened 2688 fragments by SPR at 2 mM, resulting in 58 confirmed hits, all quite weak (millimolar). Crystallography was attempted on most of them, yielding six structures, including such shapely specimens as compounds 3 and 7.


Compound 3 binds in the lipophilic S2 pocket of CypD, overlapping with the aniline moiety of previously reported compound 2. Fragment merging led to compound 14, with nearly 40-fold improved affinity over compound 2. A similar strategy merging compound 3 with fragment 8 led to low micromolar compound 27, two orders of magnitude more potent than the starting fragments. Perhaps most impressively, fragment linking compound 3 with compound 7, a shapely fragment which binds in the S1’ pocket, led to submicromolar compound 39, with affinity more than 10,000-fold higher than either fragment.

So in the end, fanciers of shapely fragments and detractors alike can feel vindicated by these papers. Hit rates might be lower for three dimensional fragments, but the resulting hits are likely to be less precedented. In the case of CypD, a shapely fragment led to three different series for a target that had resisted HTS. Of course, there is still some way to go: no cell, permeability, or stability data are provided for any of the molecules, and medicinal chemists may blanch at the seven stereocenters in compound 39. But these are interesting starting points, and it will be fun to see where they end up.

20 January 2020

Fragments in the clinic: AMG 510

Few cancer targets are as prominent as KRAS, a molecular switch that has been called “the beating heart of cancer.” Mutations that cause the switch to be stuck in the “on” state occur in roughly a quarter of human tumors. The protein’s role in driving cancer has been known for nearly forty years, but for most of that time it has been considered undruggable.

Until now.

When bound to GTP, KRAS is turned on, promoting cell proliferation until the GTP is hydrolyzed to GDP. Activating mutants impair this hydrolysis. An obvious approach to targeting KRAS would be to develop small molecules that bind in the nucleotide-binding pocket, as has been done successfully for kinases. Unfortunately, the extremely high affinity of KRAS for GTP, along with the high intracellular concentration of GTP, makes this impossible.

One activating mutant replaces a glycine with cysteine (G12C), providing a convenient handle for covalent inhibitors. In 2013, Kevan Shokat and colleagues at University of California San Francisco described in Nature how they used Tethering to identify fragments such as 6H05 that bound to this cysteine through a disulfide bond. Because disulfides are not stable in cells, they replaced this “warhead” with an acrylamide moiety and made a few other tweaks to arrive at compound 12. Extensive optimization described by Yi Liu and collaborators at Kura, Wellspring, and Janssen in a 2018 Cell paper led to molecules such as ARS-1620, which showed potent biochemical and cell-based activity and promising results in mouse xenograft models.


In 2014, Carmot Therapeutics began a collaboration with Amgen to discover covalent inhibitors of the G12C mutant of KRAS. Carmot’s technology, Chemotype Evolution, entails rapid synthesis and testing of large libraries around an existing molecule such as a fragment (see here for a nice animation). In this case, fragments chosen included simple acrylamides, the thought being that – unlike the disulfides in Tethering – these could be carried through into the final molecule.

Initial hit 2 was truncated and cyclized to compound 4, which had comparable activity but with fewer atoms and rotatable bonds. Additional iterations of Chemotype Evolution and medicinal chemistry ultimately led to compound 1, with nanomolar activity in cells containing KRASG12C. Crystallography revealed that the tetrahydroisoquinoline moiety bound in a previously cryptic pocket formed by movement of a histidine side chain. These interactions contributed to the high affinity of the molecule; more details are provided in a paper published last year in ACS Med. Chem. Lett. by Victor Cee, myself, and our collaborators at Amgen and Carmot.


Unfortunately, although compound 1 was more potent in cell assays than ARS-1620, it had low oral bioavailability and rapid clearance in mice and rats. However, as described in J. Med. Chem., Brian Lanman and colleagues at Amgen superimposed ARS-1620 with compound 1 and realized that it would be possible to access the cryptic pocket from the former compound. This strategy proved successful, ultimately leading to AMG510, which entered the clinic in August 2018. And although it is still early, a paper in Nature by Jude Canon, J. Russell Lipford, and collaborators at Amgen and elsewhere describes promising responses to the drug by a handful of patients with non-small-cell lung carcinoma.


Amgen is not the only company to have capitalized on the Tethering results: Mirati used the information to develop their clinical-phase MRTX849. And Janssen has also entered the clinic with JNJ-74699157.

There are multiple lessons here. First, as we’ve seen previously, a single fragment can lead to multiple clinical compounds. Second, progress often requires considerable changes to the initial fragment. This story is clearly a case of fragment-assisted drug discovery, and in the interest of space I’ve had to omit most of the lovely medicinal chemistry, not to mention biology and biophysics, detailed in these five papers.

Another lesson is that covalent fragments can enable lead discovery for targets not accessible through other means. But this enablement may require non-conventional molecules; the initial fragments violate the rule of three, and the disclosed clinical compounds have molecular weights in excess of 500 Da. Despite these challenges, remarkably rapid progress is possible: less than five years elapsed between the first Nature publication and the entry of AMG 510 into the clinic.

Most important, this research has led to possibly life-extending molecules – one of the responders described in the second Nature paper had been on drug for 42 weeks. Practical Fragments wishes her or him, and everyone else involved with the trials, the best of luck.

13 January 2020

FBLD by Cryo-EM

X-ray crystallography is the most popular fragment-finding method according to our latest poll. This is in part due to the previously unmatched level of detail a crystal structure can provide, and in part due to the increasing speed and automation for data collection and processing. But many proteins can never be coaxed into crystals, and while it is possible to advance fragments in the absence of structure, it is rarely easy.

In the past few years, cryogenic electron microscopy (cryo-EM) has come to rival crystallography in terms of resolution (see here for details). The first mention of the technique on Practical Fragments was in 2013, when Teddy wrote that he “could not figure out how you would use [it] in screening/FBHG. However, the point of emerging technology is to emerge…” And emerge it has. At FBLD 2018 researchers from Astex presented the first cryo-EM structure of a bound fragment, and in a new paper in Drug Disc. Today Harren Jhoti and collaborators at Astex and Isohelio provide details for two proteins.

The first target, β-galactosidase (Bgal), is a model protein that has previously been characterized by cryo-EM. The researchers solved the bound structures of three small molecules, two of them fragment-sized, to resolutions of 2.2-2.3 Å. The quality of the maps is such that they could easily be mistaken for crystallographic data: the density is clear and includes ordered water molecules. Induced conformational changes are evident, stereochemistry is unambiguous for all the ligands, and one piperidine ring even shows a hole in the middle. Even more impressive, the structures were solved using automated software.

But three structures do not a screen make. For this, the researchers chose the oncology target pyruvate kinase 2 (PKM2). They designed a small library of 68 highly soluble fragments and screened these at 5 mM. The structures of two complexes are shown, and while the resolution is lower than for Bgal, clear contacts with the protein are evident. Next, the researchers screened cocktails of four fragments, each at 25 mM. Fragments in mixtures were chosen to have diverse shapes, and two structures are shown demonstrating that the technique can distinguish the binders.

Throughput is still an issue: each round of data collection and processing – whether individual compound or cocktail – took about a day. However, the researchers expect that improvements in software and hardware should enable 400 fragments to be screened in less than a month. Indeed, given that much of the focus of cryo-EM has been on generating novel structures, one can imagine various shortcuts for screening, such as collecting smaller numbers of images to look for any evidence of bound ligands. I wonder too if something like PanDDA could be developed for cryo-EM.

Both proteins described have been previously characterized by crystallography, but these are still early days, so it is only a matter of time before we see fragment structures in proteins that haven’t been crystallized. That will open thousands of proteins heretofore inaccessible for structure-based design. The researchers conclude by predicting that “cryo-EM will have a transformative impact on the pharmaceutical industry in the near future.” I would not bet against this.

06 January 2020

Fragment events in 2020

Welcome to 2020! The calendar this year is not overly crowded, and each event should be excellent - hope to see you at one.

April 13-17CHI’s Fifteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego April 14-15. This is part of the larger Drug Discovery Chemistry meeting, and includes a short course on FBDD taught by Ben Davis and me on April 13. You can read impressions of the 2019 meeting here, the 2018 meeting here, the 2017 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.

June 2-4: CHI is also launching an inaugural Expanding Chemical Space program as part of their World Pharma Week in Boston, and it looks like there will be several talks on FBDD.

June 15-17:  Although not exclusively fragment-focused, the Eighth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and returns to Boston this year. You can read my impressions of the 2018 event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event herehere, and here.

September 20-23: FBLD 2020 will be held for the first time in the original Cambridge (UK). This will mark the eighth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2018FBLD 2016FBLD 2014,  FBLD 2012FBLD 2010, and FBLD 2009.

December 17-18: What better way to close the year than in Hawaii? The second Pacifichem Symposium devoted to fragments will be held in Honolulu. Pacifichem conferences are held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. Here are my impressions of the 2015 event. Abstract submission is now open.

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

31 December 2019

Review of 2019 reviews

The year ends, and with it the awkward teenage phase of the twenty-first century. As we have done since 2012, we're using this last post of the year to highlight conferences and reviews over the previous twelve months.

There were some good events, including CHI’s Fourteenth Annual Fragment-based Drug Discovery meeting in San Diego in April, their Discovery on Target meeting in Boston in September, and the third Fragment-based Drug Design Down Under 2019 in Melbourne in November, which also saw the launch of the Centre for Fragment-Based Design. Our updated schedule of 2020 events will publish next week.

Turning to FBLD reviews, Martin Empting (Helmholtz-Institute for Pharmaceutical Research Saarland) and collaborators published a general overview in Molecules. This is a nice up-to-date summary, covering library design, methods to find, confirm, and rank fragments, and optimization approaches. It’s also open access so you can read it anywhere.

Targets
Protein-protein interactions can be particularly challenging drug targets, and these are covered in a Eur. J. Med. Chem. review by Dimitrios Tzalis (Taros Chemicals), Christian Ottmann (Technische Universiteit Eindhoven) and colleagues. The focus is on clinical compounds, and several of these – including venetoclax, ASTX660, mivebresib, onalespib – are discussed in detail. The article is particularly useful in discussing late-stage optimization of pharmacokinetic and pharmacodynamic properties. It also provides a nice summary of physicochemical properties for fragment hits and derived candidates.

Target selectivity is always important, and this is the focus of a review in Exp. Opin. Drug Disc. by Rainer Riedl and collaborators at the Zurich University of Applied Sciences and the Università degli Studi dell’Insubria. Although the broader topic is de novo drug design, fragment-based methods are prominent, and include case studies we’ve discussed on nNOS, pantothenate synthetase, and MMP-13.

In terms of specific targets, Fubao Huang, Kai Wang, and Jianhua Shen at the Shanghai Institute of Materia Medica provide an extensive review of lipoprotein-associated phospholipase A2 (Lp-PLA2) in Med. Res. Rev. This serine hydrolase has been studied for four decades but – as the researchers note – “divergence seems to be ubiquitous among Lp-PLA2 studies.” At least this is not for lack of good chemical tools, fragment-derived (see here, here, and here) and otherwise.

Methods
Although NMR has fallen behind crystallography in our latest poll, that is certainly not reflected in terms of reviews. In particular, 19F NMR is covered in three papers. CongBao Kang (A*STAR) manages to pack a lot (including 261 references!) into a concise review in Curr. Med. Chem. Topics include protein-observed 19F NMR, in which one or more fluorine atoms are introduced into a protein genetically, enzymatically, or chemically, as well as ligand-observed methods, in which fluorine-containing small molecules are directly observed or used as probes that are displaced by non-fluorine-containing molecules.

Protein-observed 19F NMR (PrOF NMR) is covered in Acc. Chem. Res. by William Pomerantz and colleagues at the University of Minnesota. Although the first example was published 45 years ago, only in the past few years has the technique been used for studying protein-ligand interactions. The researchers note that introducing fluorines into aromatic residues is ideal because they are relatively rare, simplifying interpretation, and overrepresented at protein-protein interactions, maximizing utility. Several case studies are described, and even proteins as large as 180 kDa are amenable to the technique.

Ligand-based fluorine NMR screening is simpler and more common than techniques that focus on proteins, and this topic is thoroughly reviewed by Claudio Dalvit (Lavis) and Anna Vulpetti (Novartis) in J. Med. Chem. After a section on theory, the researchers discuss library design, including a long section on quality control (which involves assessing solubility, purity, and aggregation of the molecule in a SPAM filter). Direct and competition-based screening approaches are covered in detail; for the latter, a new method for determining binding constants is provided. The paper concludes with more than a dozen case studies. Clearly much has changed in the ten years since I wondered “why fluorine-labeled fragments are not used more widely.” This perspective is a definitive guide to the topic.

Moving to less common methods for characterizing fragments, György Ferenczy and György Keserű (Research Center for Natural Sciences, Budapest) cover thermodynamic profiling in Expert Opin. Drug Disc. After discussing several case studies, they conclude that “thermodynamic quantities are not suitable endpoints for medicinal chemistry optimizations” due to the complexity of contributing factors. This is consistent with another recent paper on the subject (see here), though the information provided is still interesting for understanding molecular interactions.

And although you might have thought the 2017 VAPID publication was the last word on the limitations of ligand efficiency (LE), Pete Kenny has published a splenetic jeremiad on the topic in J. Cheminform. (see also his blog post on the topic, which includes a sea serpent). This is largely a retread of a 2014 article on the same topic (reviewed by Teddy in his inimitable manner here). Pete also describes a more complicated alternative to LE involving residuals, though unfortunately he provides no evidence that it provides more useful information. Pete is of course correct to remind us that metrics have limitations, but assertions that LE “should not even be considered to be a metric” are overwrought.

Chemistry
Two articles discuss virtual chemical libraries. In J. Med. Chem., W. Patrick Walters (Relay Therapeutics) describes efforts to measure, enumerate, and explore chemical space. He notes that false positives could quickly overwhelm a virtual screen of a hundred million molecules, but as we saw earlier this year, progress is being made. Indeed, Torsten Hoffmann (Taros Chemicals) and Marcus Gastreich (BioSolveIT) focus on navigating the vastness of chemical space in Drug Disc. Today. They note that the Enamine REAL Space is up to 3.8 billion commercially accessible compounds, more than double the number of stars in the Milky Way. But this pales in comparison to the 1020 potential compounds in Merck’s MASSIV space. Just storing the chemical structures of these in compressed format would require 200,000 terabytes – and searching them exhaustively is beyond current technology.

Ratmir Derda and Simon Ng (University of Alberta) discuss “genetically encoded fragment-based discovery” in Curr. Opin. Chem. Biol. This involves starting with a known fragment that is then coupled to a library of peptides and screened to find tighter binders. The researchers provide a number of case studies, though adding even a small peptide to a fragment will generally have deleterious effects on ligand efficiency. And – Rybelsus not withstanding – oral delivery of peptides is challenging.

Finally, Vasanthanathan Poongavanam, Xinyong Liu, and Peng Zhang, and collaborators at Shandong University, University of Bonn, University of Southern Denmark, and K.U. Leuven review “recent strategic advances in medicinal chemistry” in J. Med. Chem. Among a wide range of topics from drug repurposing to antibody-recruiting molecules is a nice, up-to-date section on target-guided synthesis. As I opined a couple years ago, I still doubt whether this will ever be generally practical, but from an intellectual standpoint I’m happy to see work continue on the approach.

And with that, Practical Fragments says goodbye to the teens and wishes you all a happy new year. Thanks for reading and commenting. May 2020 bring wisdom, and progress.

16 December 2019

Fragments in the clinic: S64315 / MIK665

Earlier this year we highlighted the discovery of AZD5991, a phase 1 compound from AstraZeneca that inhibits the anti-apoptotic cancer target Mcl-1. Those efforts made use of a fragment previously published by a different research group. Mcl-1 has been a popular target for some time; the first mention on Practical Fragments dates to 2010. The story behind another investigational drug is described in a couple papers from earlier this year.

The first, in ACS Omega by Rod Hubbard and colleagues at Vernalis, University of York, and Servier, describes fragment screening efforts against both Bcl-2 and Mcl-1. The proteins are related both structurally and functionally, and Bcl-2 is the target of venetoclax – the second fragment-derived drug approved. Some of the early fragment hits bound to both proteins, but selective and potent inhibitors were ultimately developed. In the interest of space only those against Mcl-1 will be discussed here.

Both proteins required considerable protein engineering, which is described in detail. Ultimately one form of human Mcl-1 was used for crystallography, while mouse protein was used for NMR screening due to its better stability. A total of 1064 fragments were screened at 0.5 mM each (in pools of eight) using ligand-observed NMR; 39 confirmed using STD NMR, WaterLOGSY, and CPMG. Additionally, fluorescence polarization, 2-dimensional (HSQC) NMR, ITC, and SPR were used to validate hits. Crystallography proved challenging in the beginning but ultimately helped drive optimization of more potent molecules. The large number of different assays employed is consistent with our recent poll results.

Protein-observed NMR was particularly useful in providing information on the quality of both the ligand and protein (reminiscent of the “validation cross” discussed here). Before crystallography was able to play a meaningful role, “NMR-guided models,” combining partial protein assignments with flexible docking, were used to drive SAR.


While the first paper focuses on protein optimization and biophysics, the second (in J. Med. Chem.), by András Kotschy and collaborators, focuses on chemistry. Fragment 1a was one of several hits pursued, initially by looking for analogs, but most of these had comparable (weak) activity. In the absence of a crystal structure a systematic chemistry campaign was conducted, varying elements of the core and sidechains. Many of these molecules had comparable activity against both Mcl-1 and Bcl-2, but replacing the nitrogen linker with an oxygen led to selectivity against the former. The addition of hydrophobic substituents led to compound 10c, with submicromolar activity.

Anticipating poor cell permeability for a negatively charged, lipophilic molecule, the researchers introduced a positively charged methylpiperazine moiety at various positions around the molecule, ultimately leading to compound 18a. In addition to potent Mcl-1 binding, this molecule is active in cells and shows reasonable pharmacokinetic properties in mice. Further optimization to S64315 does not appear to have been published yet, though the structure was disclosed earlier this year, and the fragment origins remain clear.

Together these papers provide a thorough description of drugging a difficult target. They also provide insights into the investment required. The Mcl-1 project began around 2007, and it took a decade before S64315 entered the clinic. Enabling drug discovery against protein-protein interactions required multiple biophysical techniques in addition to all the standard components of pharmaceutical research. The researchers note that “establishing such a platform can take some time and resource – a tool compound is usually needed to validate the assays, but the assays are needed to identify the tool compound.” In the end they have succeeded, and Practical Fragments wishes them – and the patients being treated – the best of luck.

09 December 2019

A new library of fluorinated Fsp3-rich fragments

Among fragment-finding methods, ligand-based NMR ranks near the top in terms of popularity. Of its many variations, fluorine (19F) NMR appears to be gaining in popularity. Fluorine NMR has several advantages, including high sensitivity and the fact that many fragments can be screened simultaneously because of the wide chemical shift range for fluorine. Although more commercial fluorine-enriched libraries are available now than when we first wrote about the approach a decade ago, the diversity of these libraries is still somewhat limited. This problem has been tackled by Mads Clausen at the Technical University of Denmark and an international team of collaborators in a new Angew. Chem. Int. Ed. paper.

The researchers wanted to create a fluorinated fragment library that would be not just diverse but also contain a high fraction of sp3-hybridized carbons (high Fsp3). Some of the early claims around “three dimensional” fragments have been questioned, and there seems to be little if any correlation between the shapeliness of fragments and that of derived leads, but if you’re going to make new fragments in academia it makes sense to explore interesting molecular architectures.

Starting from just six simple building blocks, each containing a trifluoromethyl group, the researchers generated nine different cores which were further derivatized at multiple positions to yield 115 diverse fragments. Consistent with diversity-oriented synthesis, no more than five synthetic steps were used for any molecule. All molecules were made as racemates in order to further increase the diversity of the library.

The resulting “3F Library” is mostly rule-of-three compliant, though given that the trifluoromethyl moiety alone adds 69 Da the fragments do tend to be larger, with an average molecular weight of 284 Da. They are, however, less lipophilic than two commercial fluorinated fragment libraries. And with an average Fsp3 = 0.7 and 3.3 chiral centers they are also quite shapely as assessed by principal moment of inertia.

Building a library is nice, but will it provide hits? To find out, the researchers screened the 102 fragments that passed quality control against four targets. They used a transverse (T2) relaxation assay (specifically, CPMG) in which fragments bound to a protein tumble more slowly, causing a reduction in 19F signal intensity. Hit rates ranged from 3% to 11%, and about two thirds of these confirmed in STD or WaterLOGSY assays. As seen by the examples shown here, the fragments are quite diverse.

Whether these hits will lead to more potent molecules remains to be seen. Laudably the paper ends with the statement: “we hope that the 3F library will find use for other researchers and we encourage anyone interested in screening the fragments to contact us.” If you are looking for interesting new fragments that are tailored for follow-up chemistry, I encourage you to take the team up on their offer.

02 December 2019

Poll results: affiliation and fragment-finding methods in 2019

The fourth iteration of our fragment-finding methods poll has just closed. If you want to jump right to the results feel free to skip the next paragraph, which focuses on methods.

The poll was run using Crowdsignal, the successor to Polldaddy, and ran from 20 October through 30 November. This free polling software tabulates total number of votes for a question but not the number of individual respondents. To determine individual respondents, we included a question on “workplace and practice.” Of the 137 individual respondents to this question, 116 identified themselves as practicing FBLD, and we assumed they also answered the second question. The overall number of responses is slightly higher than in 2013 but a bit lower than in 2016.

Readership demographics have shifted from previous years, with about two thirds of respondents hailing from industry, up from just over half historically. The fraction of respondents who actively practice FBLD is also up modestly, to 85%.


But the question probably of most interest is on screening methods, summarized here.


As we also saw in 2013 and 2016, nearly all fragment-finding techniques are being used more, with the average respondent employing 6 methods today compared with 4.1 in 2016, 3.6 in 2013, and 2.4 in 2011.

X-ray crystallography has leapt to first place, likely driven in part by increasing speed and automation as well as by studies suggesting that crystallography can give impressively high hit rates.

As in 2016, ligand-detected NMR, SPR, and thermal shift assays are all very popular. Use of computational approaches has increased, though perhaps not as much as might be expected given recent advances. Functional screening is the only technique for which use has remained constant, or perhaps even declined very slightly from 2013.

For the first time we asked about use of literature to identify fragments, and nearly a third of respondents said they incorporate previously published fragments into their work. As the amount of publicly available information continues to increase it will be interesting to see whether this number grows.

More niche methods such as mass spectrometry, MST, affinity selection, and biolayer interferometry are gaining adherents; 30 respondents reported using mass spectrometry, for example. While fewer than 20% of respondents are using affinity chromatography (including WAC), CE, or ultrafiltration, that proportion has nearly quadrupled from our previous three polls, though we can’t say which of these related methods accounts for the increase.

Finally, only four respondents reported using “other” methods, such as SHG. Perhaps we’ll ask about this and other emerging methods explicitly next time.

Do the results surprise you, or are they consistent with what you are using at your organization?

25 November 2019

Reverse micelle encapsulation for measuring low affinities

NMR is among the more sensitive fragment-finding techniques: the starting point for clinical compound ASTX660 had low millimolar affinity at best. Now, three papers by A. Joshua Wand and colleagues at University of Pennsylvania have taken sensitivity to a new level, enabling the detection of fragments that bind hundreds of times less tightly. (Derek Lowe recently wrote about one of them, and I highlighted a talk last year.)

All three papers focus on a method called reverse micelle encapsulation, in which an aqueous solution of protein and ligand is encapsulated in nanoscale reverse micelles measuring less than 100 Å in diameter. At this size, each micelle will contain at most just a single protein and a few thousand water molecules. Because of the small volume, the protein concentration – and that of any fragments – will be extraordinarily high. The micelles have polar groups pointed inwards towards their watery interior, and their hydrophobic tails point out towards solvent, typically pentane. The overall water content of the sample is typically around 2%.

Various NMR techniques can be used to study the proteins. Although the reverse micelles are larger than the proteins themselves and thus would be expected to tumble more slowly, the low viscosity of the pentane solvent makes up for this, providing high-quality spectra.

The primary paper, in ACS Chem. Biol., focuses specifically on fragments. To establish that the technique can detect weak interactions, the researchers show that they can measure the 26 mM dissociation constant of adenosine monophosphate to the enzyme dihydrofolate reductase.

Next, they turned to the protein interleukin-1β (IL-1β), an inflammatory target with no reported small-molecule binders. One challenge of the method is that hydrophobic fragments could partition into the micelles or even diffuse into the pentane, thus reducing their concentration. To avoid this, the researchers assembled a library of 233 very polar, water-soluble fragments with cLogP values < 0.5. A 2-dimensional NMR screen (15N-TROSY) using standard conditions (100 µM protein and 800 µM fragment) yielded no hits.

In contrast, NMR screening using reverse micelles with the protein at an effective concentration of 5 mM and fragments at 40 mM yielded 31 hits. Chemical shift perturbations (CSPs) were used to determine where they were binding. Ten of the fragments didn’t show clear binding to specific sites on the protein, but the remaining 21 did, with all but one binding to multiple sites. Of these, 13 also showed non-specific interactions with other regions of the protein. Altogether, the fragment binding sites covered 67% of the protein surface, with the receptor-binding interface particularly well-represented.

Concentration-dependent CSPs were used to determine dissociation constants, which ranged from 50 mM to over 1 M. An SAR-by-catalog exercise was able to improve the affinity of one fragment from 200 mM to 50 mM at one site, though it also binds three other sites with slightly weaker affinity.

The second paper, also in ACS Chem. Biol., uses IL-1β but focuses on the interaction of even smaller molecules such as pyrimidine, methylammonium, acetonitrile, ethanol, N-methylacetamide, and imidazole. Not surprisingly, the dissociation constants are even weaker, averaging 1.5 – 2.5 M.

Finally, a Methods in Enzymology paper goes into depth on how to actually run the experiments, including details on choosing detergents and making the micelles. At high fragment concentrations, for example, pH needs to be carefully controlled.

Five years ago we asked “how weak is too weak” for a fragment. In terms of practicality, I’d say that these fragments qualify. Indeed, the ligand efficiency for the best fragment mentioned above is just 0.15 kcal mol-1 atom-1.

But the findings do raise the almost philosophical question of what exactly constitutes a small molecule binding site. Astex researchers reported several years ago that most proteins have more than one, and their more recent work with MiniFrags suggest on average 10 sites at high enough concentrations. Similar results were also reported earlier this month from Monash. Whether or not the fragments from such screens turn out to be immediately useful, they could certainly advance our understanding of molecular recognition.

18 November 2019

Fragment-based Drug Design Down Under 2019

The last major fragment meeting of 2019 took place at the Monash Institute of Pharmaceutical Sciences, Monash University, in marvelous Melbourne last week. This was the third Australian meeting devoted to fragments; you can read about the first, in 2012, here. With some 125 participants from four continents, two dozen talks, and nearly as many posters I’ll just try to capture major themes.

Biophysics played a starring role – if you haven’t already voted (right side of page) on which fragment-finding techniques you use please do so. Sarah Piper (Monash) discussed cryo-electron microscopy and showed some lovely high-resolution structures of proteins with bound ligands, though not yet with fragments. Sally-Ann Poulsen (Griffith University) described using native-state ESI mass spectrometry to discover new carbonic anhydrase binding fragments (see here). She uses a 96-well “nanoESI” chip to generate 5 µm droplets as opposed to the ~100 µm droplets typically fed into the instrument. Smaller droplets contain fewer molecules of salt and buffer, and thus generate cleaner spectra.

NMR screening is the go-to method for screening at Monash University, as highlighted by Martin Scanlon and multiple other speakers. Indeed, Monash has built their own version of Astex’s MiniFrag library – their MicroFrags include 92 compounds with 5-8 non-hydrogen atoms. Rebecca Whitehouse has screened these at 300 mM (yes, millimolar) by 15N-1H HSQC against the E. coli protein DsbA (EcDsbA) and found numerous hits, including at an internal cryptic site previously identified by Wesam Alwan (Monash). Encouragingly, the results were consistent with a crystallographic screen of the same library done at 1 M.

SPR was highlighted by Nilshad Salim (ForteBio) and in a separate Biacore user day, and is an essential tool for off-rate screening (ORS). ORS facilitates screening of crude, unpurified reaction mixtures, since the off-rate of a compound bound to a protein is not dependent on compound concentration (see here). Compound purification is a major time-sink, and avoiding it is a key component of REFiL, or Rapid Elaboration of Fragments into Leads.

As Bradley Doak (Monash) discussed, REFiL entails the parallel synthesis of compound libraries around a selected fragment in 96-well plates using diverse reagents and high-yielding chemistries such as amide bond formation, alkylation, and reductive amination. Reaction mixtures are evaporated, resuspended in DMSO, and screened using ORS; this has led to affinity improvements of ten-fold or better compared with the original fragment for four projects tested thus far.

Beatrice Chiew (Monash) presented a case study against the oncology target 53BP1. Screening 1198 fragments led, after catalog-mining and rescreening, to 25 hits, all quite weak. Applying REFiL improved affinities by up to 15-fold, with the best molecules around 10 µM. Beatrice noted that because SPR provides “on-chip purification,” active compounds could be identified even when the reaction yields were less than 10%. She did note that examining the raw data (sensorgrams in SPR-speak) is important to recognize and avoid false positives.

Similarly, Luke Adams (Monash) applied REFiL to the bromodomain BRD3-ET. After two cycles, he was able to improve a 230 µM fragment to a 1.5 µM binder. Importantly, the off-rates were similar for the purified molecules and the crude reaction mixtures.

And Mathew Bentley (Monash) is exploring the potential of REFiL using crystallography, or REFiLX. This led to a 60 µM binder against the notoriously difficult EcDsbA. That affinity is more impressive given that the previous structure-based design and synthesis of more than 100 compounds – aided by 25 crystal structures – had failed to break 250 µM.

Vernalis pioneered off-rate screening, and Alba Macias described the company’s latest developments in this area. In the case of tankyrase, a 700 µM fragment was used to generate 80 compounds, which took one chemist a couple days. This yielded a 350 nM binder, the structure of which bound to the enzyme was solved using the crude reaction mixture for soaking.

Following up on this success, Vernalis is exploring the limits of crude reaction mixtures for high-throughput crystallography. Although promising, Alba noted caveats for the two proteins tested. Unlike off-rates, crystallographic success is dependent on compound concentration, so low-yielding reactions can lead to false negatives. And as anyone who has spent time working with fragments can attest, a beautiful co-crystal structure is no guarantee of high affinity, so false positives (ie, no improvement in affinity over the starting fragment) can be a problem too.

Alba also gave a brief summary of the discovery of S64315/MIK665, a fragment-derived MCL-1 inhibitor discovered by Vernalis, Servier, and Novartis that is currently in phase 1.

MCL-1 is a member of the BCL-2 of family proteins, and BCL-2 itself is targeted by the second fragment-derived drug to be approved. Guillaume Lessene (Walter & Eliza Hall Institute) spoke about both of these proteins, as well as BCL-xL. Long-time readers may remember this selective BCL-xL inhibitor, discovered using second-site NMR screening. Blocking this protein leads to platelet cell death, but AbbVie researchers are ingeniously side-stepping this liability by conjugating a related small molecule to an antibody to reduce systemic exposure. The resulting ABV-155 may be the first antibody drug conjugate derived from fragments, and was said to be in phase 1.

There was quite a bit more, though in the interest of time (and readers’ patience!) I’ll stop here. But I must note before closing that this meeting launched the Australian Research Council-funded Centre for Fragment-Based Design. This is in some ways an Antipodean version of FragNet, though with a longer (five-year) funding period and the opportunity to include a few postdocs as well as graduate students. If you’re interested, please contact them.

10 November 2019

A new tool for detecting aggregation

Historically the most popular method for finding fragments has been ligand-detected NMR. Preliminary results of our current poll (to the right) suggest crystallography has pulled ahead. (Please do vote if you haven’t already done so.) However, NMR has many uses beyond finding fragments, as illustrated in a recent J. Med. Chem. paper by Sacha Larda, Steven LaPlante, and colleagues at INRS-Centre Armand-Frappier Santé Biotechnologie, NMX, and Harvard.

Among the many artifacts that can occur in screening for small molecules, one of the most insidious is aggregation. A distrubing number of small molecules form aggregates in water, and these aggregates give false positives in multiple assays. Unfortunately, determining whether aggregation is occurring is not always straightforward. The new paper provides a simple NMR-based tool to do just that.

All molecules tumble in solution, but small fragment- or drug-sized molecules tumble more rapidly than large molecules such as proteins. The “relaxation” of proton resonances is faster in slower tumbling molecules, and in the NMR experiment called spin-spin relaxation Carr-Purcell-Meiboom-Gill (T2-CPMG) various delays are introduced and slower tumbling molecules show loss of resonances. Indeed, this technique has frequently been used in fragment screening: if a fragment binds to a protein, it will tumble more slowly, resulting in loss of signal.

The researchers recognized that an aggregate could behave like a large molecule, and they confirmed this to be the case for known aggregators, while non-aggregators did not. The experiment is relatively rapid (~30 seconds), and has been used to profile a 5000-compound library to remove aggregators.

One of the frustrations of aggregators is that it is currently impossible to predict whether a molecule will aggregate, and indeed, the researchers show several examples of closely related compounds in which one is an aggregator while the other is not. Even worse, the phenomenon can be buffer-dependent: the researchers show a fragment that aggregates in one buffer but not in another, even under the same pH.

Many fragment screens are done with pools of compounds, and the researchers find that molecules can show a “bad apple effect”, whereby previously well-behaved molecules appear to be recruited to aggregates.

The limit of detection for T2-CPMG is said to be single-digit micromolar concentration of small molecule, though the researchers note that double- or triple-digit micromolar concentrations are more practical, which is more typical of fragment screens anyway. And some compounds may show rapid relaxation due to non-pathological mechanisms, such as tautomerization or various conformational changes.

Still, this approach seems like a powerful means to rapidly assess hits, and pre-screening a library makes sense. Another NMR technique using interligand nuclear Overhauser effect (ILOE) has also been used to test for aggregation, though not to my knowledge so systematically. For the NMR folks out there, which methods do you think are best to weed out aggregators?

04 November 2019

Second harmonic generation (SHG) vs KRAS

Practical Fragments is currently running a poll on fragment-finding methods used by readers – please vote on the right-hand side. One biophysical method that perhaps we should have included is second harmonic generation (SHG). A recent paper in Proc. Nat. Acad. Sci. USA by Josh Salafsky, Frank McCormick, and collaborators at Biodesy, University of California San Francisco, and elsewhere describes the technique and its application to find fragments that bind to the oncogenic protein KRAS.

In SHG, two photons of the same energy are absorbed by a material which then emits a single photon with twice the energy. In the commercial instrument developed by Biodesy, a powerful 800 nm laser irradiates a dye, and the 400 nm photon it emits is detected. The intensity of the signal is exquisitely sensitive to the precise orientation of the dye. If a protein is labeled with an SHG-active dye and then immobilized on a glass surface, even subtle changes in conformation will be detected.

The researchers chose the G12D mutant form of KRAS, which is one of the most common variants and is associated with particularly aggressive tumors. They labeled the protein with a lysine-reactive SHG dye under conditions in which each protein would, on average, have one covalently-bound dye molecule (though some would have none and others would have more than one). Proteolysis and mass-spectrometry analysis revealed that the dye molecule labeled three different lysine residues, which the researchers viewed as a feature since a ligand causing a conformational change to any of the lysine residues would generate a signal. The researchers also demonstrated that the dye modification did not interfere with the ability of KRAS to bind to the RAS-binding domain of RAF.

Labeled KRAS was then immobilized and tested against several proteins known to bind it, including antibodies and the nucleotide exchange factor SOS. These produced SHG signals, presumably by causing conformational changes to KRAS, while non-binders such as tubulin did not.

Having established that the assay could detect binders, the researchers screened 2710 fragments at 250 and 500 µM, and obtained a whopping 490 hits. These were then triaged by screening at lower concentrations and performing dose-titrations, and 60 were then characterized by SPR.

Fragment 18, 4-(cyclopent-2-en-1-yl)phenol, showed binding by both SHG and SPR, and was further studied by 2-dimensional NMR (1H-15N HSQC). This technique allowed measurement of the weak 3.3 mM dissociation constant. More importantly, it allowed the researchers to establish the binding location as being near the so-called “switch 2” region where SOS normally binds. This is the same region where a previous NMR screen had identified the slightly more potent fragment DCAI. The current paper confirmed that finding, though the researchers found evidence that DCAI may bind to other sites too. Docking studies using SILCS suggested that fragment 18 likely binds in a similar orientation as DCAI. Not surprising given the low affinity, the new fragment did not show functional activity in a biochemical screen.

SHG is an interesting approach, and the ability to rapidly assess protein conformational changes distinguishes it from other biophysical techniques. Site-specific labeling would produce more informative data on which regions of a protein move. However, I wonder if SHG is perhaps too sensitive, as evidenced by the large number of hits. Indeed, the researchers demonstrated that the promiscuous lipophilic amine mepazine also generated a strong SHG signal with KRAS. It would be interesting to do a head-to-head comparison with other similarly rapid techniques such as DSF or MST. Have you tried using SHG, and if so, how did it perform for you?

20 October 2019

New poll: affiliations and methods

It has been three years since we last asked about fragment-finding methods, and a lot can change in that time – just compare the world today to the world in 2016. Our new poll has two questions (right-hand side, under Links of Utility). Please answer the first, and answer the second if you practice FBLD.

The first question asks your affiliation and whether you actively practice FBLD or whether you are interested in the topic (though hopefully the latter also applies to the former!) We’ve simplified the question from prior years to include just four categories: For-profit practice, For-profit interest, Non-profit practice, and Non-profit interest. For-profit includes pharma and biotech as well as venture capital and consulting. Non-profit includes academia, government labs, disease foundations, and retirement.

Please answer this question as it is the only way we can count the number of respondents, which is essential for determining how many fragment-finding methods people are using on average.

If you do practice FBLD, the next question asks which method(s) you use to find and validate fragments. Please click every method you use, whether as a primary screening technique or for validation. You can read about these methods below, and if you select “other” please describe in the comments.


Please forward this so we can get as many responses as possible.

Let the voting begin!