Review of 2018 reviews

As 2018 recedes into history, we are using this last post of the year to do what we have done since 2012 – review notable events along with reviews we didn’t previously cover.

This was a busy year for meetings, starting in January with a FragNet event in Barcelona, then moving to San Diego in April for the annual CHI FBDD meeting. Boston saw an embarrassment of riches, from the first US-based NovAliX meeting, to a symposium on FBDD at the Fall ACS meeting, followed closely by a number of relevant talks at CHI’s Discovery on Target. Finally, the tenth anniversary of the renowned FBLD meeting returned to San Diego. Look for a schedule of 2019 events later this month.

If meetings were abundant, the same can be said for reviews.

Lead optimization

Writing in J. Med. Chem., Dean Brown and Jonas Boström (AstraZeneca) asked “where do recent small molecule clinical development candidates come from?” For three quarters of the 66 molecules published in J. Med. Chem. in 2016 and 2017 the answer is from known compounds or HTS, though fragments accounted for four examples. Although average molecular weight increased during lead optimization, lipophilicity did not, suggesting the importance of this parameter.

The importance of keeping lipophilicity in check is also emphasized by Robert Young (GlaxoSmithKline) and Paul Leeson (Paul Leeson Consulting) in a massive J. Med. Chem. treatise on lead optimization. Buttressed with dozens of examples, including several from FBLD, they show that the final molecule is usually among the most efficient (in terms of LE and LLE) in a given series, even when metrics were not explicitly used by the project team. Perhaps with pedants like Dr. Saysno in mind, they also emphasize the complexity of drug discovery, and note that “seeking optimum efficiencies and physicochemical properties are guiding principles and not rules.”

Lipophilic ligand efficiency (LLE) is also the focus of a paper in Bioorg. Med. Chem. by James Scott (AstraZeneca) and Michael Waring (Newcastle University). This is based largely on personal experiences and provides lots of helpful tips. Importantly, the researchers note that calculated lipophilicity values can differ dramatically from measured values, and go so far as to say that “this variation is sufficient to render LLEs derived from calculated values meaningless.”

Turning wholly to fragments, Chris Johnson and collaborators (including yours truly) from Astex, Carmot, Vrije Universiteit Amsterdam, and Novartis have published an analysis in J. Med. Chem. of fragment-to-lead success stories from last year. This review, the third in a series, also summarizes all 85 examples published between 2015 and 2017, confirming and expanding some of the trends we mentioned last year.

Targets

Two reviews focus on specific target classes. Bas Lamoree and Rod Hubbard (University of York) cover antibiotics in SLAS Discovery. After a nice, concise review of fragment-finding methods, the researchers discuss a number of case studies, many of which will be familiar to regular readers of this blog, including an early example of whole-cell screening.

David Bailey and collaborators from IOTA and University of Cambridge discuss cyclic nucleotide phosphodiesterases (PDEs) in J. Med. Chem. The researchers provide a good overview of the field, including mining the open database ChEMBL for fragment-sized inhibitors. As they point out, the first inhibitors discovered for these cell-signaling enzymes were fragment-sized, so it is no surprise that FBLD has been fruitful – see here for an example from earlier this year. Interestingly though, although at least six fragment-sized PDE inhibitor drugs have been approved, none of these were actually discovered using FBLD.

PDEs are an example of “ligandable” targets, for which small molecule modulators are readily discovered. In Drug Discovery Today, Sinisa Vukovic and David Huggins (University of Cambridge) discuss ligandability “in terms of the balance between effort and reward.” They use a published database of protein-ligand affinities to develop a metric, LIG_exp, for experimental ligandability, and also describe their computational metric, Solvaware, which is based on identifying clusters of water molecules binding weakly to a protein. Comparisons with experimental data and with other predictive metrics, such as FTMap, reveal that while the computational methods are useful, there is still room for improvement.

We have previously written about how target-guided synthesis methods such as dynamic combinatorial chemistry have – despite decades of research – yielded few truly novel, drug-like ligands. Is this because the targets chosen were simply not ligandable? In J. Med. Chem., Anna Hirsch and collaborators at the University of Groningen, the Helmholtz Institute for Pharmaceutical Research, and Saarland University review some (though by no means all) published examples and examine their computationally determined ligandability scores. There seems to be no difference between these targets and a set of traditional drug targets.

Finding fragments

Crystallography continues to be a key tool for FBLD: as we noted in the review of the 2017 literature, 21 of the 30 examples made use of a crystal structure of either the starting fragment or an analog, and only 3 projects didn’t use crystallography at all. That said, FBLD is possible without crystallography, as illustrated through multiple examples in a Cell Chem. Biol. review by Wolfgang Jahnke (Novartis), Ben Davis (Vernalis), and me (Carmot).

In the absence of a crystal structure, NMR is best suited for providing structural information, and this is the subject of a review in Molecules by Barak Akabayov and colleagues at Ben-Gurion University of the Negev. The researchers provide a nice summary of NMR screening methods and success stories within a broader history of FBLD. They also include an extensive list of fragment library providers as well as a discussion of virtual screening.

Speaking of virtual screening, three reviews cover this topic. In Methods Mol. Biol., Durai Sundar and colleagues at Indian Institute of Technology Delhi touch on a number of computational approaches for de novo ligand design, though the lack of structures sometimes makes it challenging to read. A broader, more visually appealing review is published in AAPS Journal by Yuemin Bian and Xiang-Qun Xie at University of Pittsburgh. In addition to an overview and case studies, the researchers also provide a nice table summarizing 15 different computational programs. One of these, SEED, is a main focus of a review in Eur. J. Med. Chem. by Jean-Rémy Marchand and Amedeo Caflisch (University of Zürich). The researchers describe how this docking program can be combined with X-ray crystallography (SEED2XR) to rapidly identify fragments; we highlighted an example with a bromodomain. Their ALTA protocol uses SEED to generate larger, more potent molecules, as we described for the kinase EphB4. The researchers note that together these protocols have led to about 200 protein-ligand crystal structures deposited in the PDB over the past five years.

Rounding out methods, Sten Ohlson and Minh-Dao Duong-Thi (Nanyang Technological University) provide a detailed how-to guide in Methods for performing weak affinity chromatography, and how this can be combined with mass spectrometry (WAC-MS), as we noted last year.

Chemistry

One drawback of some computational approaches for fragment optimization is that they do not consider synthetic accessibility. In Mol. Inform., Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University and Institut Paoli-Calmettes focus on hit to lead approaches that do, and provide a handy table summarizing nearly a dozen computational methods. We highlighted one from the authors, DOTS, earlier this year.

DOTS is an example of using DOS, or diversity-oriented synthesis. In Front. Chem., David Spring and colleagues at University of Cambridge review recent applications of DOS for generating new fragments, some of which we recently highlighted. Only a couple examples of successfully screening these new fragments are described, but the authors note that this is likely to increase as virtual library screening continues to advance.

Perhaps the most productive fragment of all time is 7-azaindole, the origin of three fragment-derived clinical compounds. (The moiety appears in both approved FBLD-derived drugs, vemurafenib and venetoclax.) Takayuki Irie and Masaaki Sawa of Carna Biosciences devote their attention to this little bicycle in Chem. Pharm. Bull. The researchers count six clinical kinase inhibitors that contain 7-azaindole (not all from FBLD) as well as more than 100,000 disclosed compounds containing the fragment. More than 90 kinases have been targeted by molecules containing 7-azaindole, and the paper provides a list of 70 PDB structures of 37 different kinases bound to molecules containing the moiety.

Finally, in J. Med. Chem., Brian Raymer and Samit Bhattacharya (Pfizer) survey the universe of “lead-like” drugs. Among the most highly prescribed small molecule drugs, 36% have molecular weights below 300 Da. Only 28 of 174 drugs approved between 2011 and 2017 fall into this category, consistent with the increasing size of newer drugs. The researchers discuss 16 recently approved drugs, and find that 13 have very high ligand efficiencies (at least 0.4 kcal mol^-1 per heavy atom). As noted above, optimization often entails adding molecular weight by growing or linking, and the researchers suggest that alternative strategies such as conformational restriction and truncation also be investigated.

And with that, Practical Fragments wishes you a happy new year. Thanks for reading some of our 686 posts over the past decade plus, and please keep the comments coming!

New types of covalent fragments

As covalent drugs become more accepted, covalent fragment libraries are becoming more popular: we’ve previously written about both reversible and irreversible fragments. One potential limitation is the number of different types of covalent modifiers, or warheads. The program DOCKovalent, for example, only considers ten classes. György Keserű and collaborators at the Hungarian Academy of Sciences and the University of Ljubljana go some way towards expanding this list in a recent paper in Arch Pharm Chem Life Sci.

The researchers were interested in heterocyclic electrophiles. For heterocycles, they considered pyridines, pyrimidines, pyrazines, imidazoles, pyrazoles, oxazoles, isoxazoles, and thiazoles. For electrophiles, they considered chloride, bromide, iodide, nitrile, vinyl, and ethynyl groups, often at different positions around a given heterocycle. So for example, they chose 2, 3, and 4-chloropyridine. Not every electrophile was available or easily synthesized for every heterocycle, so in total they assembled a library of 84 different fragments.

The library was tested for aqueous stability, and all but six fragments had half-lives of at least 24 hours at pH 7.4. Next, the researchers examined the intrinsic reactivities of their molecules by reacting them with glutathione, a physiologically relevant thiol. As might be expected, the different molecules showed a wide range of different reactivities, all of which are reported in the paper. This is a useful list: ultimately one wants a warhead with low or modest reactivity for better selectivity.

Next, the researchers tested their fragments against MurA from Staphylococcus aureus and Escherichia coli; this enzyme is important for bacterial cell wall biosynthesis, and contains an active-site cysteine that has previously been shown to be sensitive to electrophiles. The reactivity patterns were similar between the two enzymes, but they did differ somewhat from glutathione reactivity, suggesting the possibility of molecular recognition. Dose response assays were performed on the most potent molecules, most of which had IC₅₀ values in the mid to high micromolar range. These results expand on research we highlighted six years ago showing that halopyridines could covalently modify a cysteine-dependent enzyme.

The researchers also examined the mechanism of their fragments by doing time-dependence and dilution experiments. Some of the results are quite unexpected, suggesting that, for example, 4-iodopyridine is a reversible modifier, which is hard to understand mechanistically. Perhaps, like the “universal fragment” 4-bromopyrazole, the molecule does not act covalently, though the time dependence observed suggests otherwise.

This is a nice example of how to create and assess a fragment library with a particular mechanism in mind, reminiscent of the metal-chelating fragments described by Seth Cohen and colleagues. Finally, I’d like to note that the first author, Aaron Keeley, is part of the FragNet training program. He and his fellow trainees will soon be looking for the next phase in their careers, so if you’re hiring keep them in mind!

Poll results: library vendors

Our latest poll has just closed, and the results are quite interesting. We asked three questions:

1) Have you bought fragments in the past few years?

2) Which of the following vendors would you RECOMMEND?

3) Which of the following vendors would you AVOID?

First, a paragraph on methodology. The poll ran from November 3 through December 7. Due to the limitations of the free version of Crowdsignal (formerly Polldaddy), I have no way of knowing how many individuals responded to questions 2 or 3 (respondents could choose multiple answers). This was the purpose of question 1; 37 people answered yes, and 12 people answered no. Assuming that only people who answered yes answered questions 2 and 3, I divided the responses to questions 2 and 3 by 37 to give percentages. So for example, 33 people would recommend Enamine, which is 89%. If some people who answered no to question 1 answered 2 and/or 3, or answered questions 2 and/or 3 but not 1, the percentages may be overestimates. This seems possible, as the total number of people who would recommend or avoid Enamine adds up to 36. So either nearly everyone who said they bought fragments did so from Enamine, or more people responded than were accounted for by answering “yes” to whether they purchased fragments.

The results are shown here.

The first thing that jumps out is the popularity of Enamine – which is recommended by nearly 90% of respondents. Life Chemicals, Maybridge, ChemBridge, and Key Organics are each recommended by more than 30%, while Vitas-M, ChemDiv, and Asinex are each recommended by 16-24% of respondents. Seven other vendors were recommended by 2 or fewer respondents.

The second observation is that, for the most part, people seem fairly happy with their vendors: each named vendor would be avoided by fewer than 10% of respondents. That said, the relative numbers vary considerably: only one respondent would avoid Life Chemicals, while 18 would recommend them. In contrast, for some of the less popular suppliers, the number of people who would avoid them was comparable to the number who would recommend them.

Finally, I was pleased to see that although a few respondents selected “other” for vendors they would recommend, these were outnumbered by the number selecting “others” they would avoid. That suggests the list provided in the poll captured most trusted vendors. That said, there is no way of knowing whether, for example, the 7 respondents who chose to recommend “other” vendors all had the same vendor in mind, or up to 7 different ones.

Of course there are caveats (and more in the methodology section above). First, the response rate is lower than most of our other polls, reflecting the fact that library generation is not something done lightly or frequently. Second, the first question was deliberately vague; people may have different definitions of “past few years,” and some vendors may have improved or deteriorated. Third, we have no way of knowing how many organizations are represented; if many people responded from a single company this could bias the results. Fourth, we are dependent on the honesty of respondents – we don’t know whether vendors recommended themselves.

Finally, please leave comments, positive or negative, especially if you would recommend vendors not included in the poll. Remember, you can comment anonymously.

Fragments vs lectins - allosterically

Carbohydrates are ubiquitous in nature but largely ignored in drug discovery. This is because interactions between carbohydrates and proteins, while important, tend to be quite weak; sugar binding sites in proteins rarely have deep, ligandable binding pockets. The few case studies we’ve highlighted (here, here, and here) have resulted in weak and/or large ligands.

However, you don’t need to target the active site to inhibit a protein: one of the most advanced fragment-derived drugs in the clinic is an allosteric inhibitor. Recognizing that many proteins contain secondary (and potentially allosteric) binding sites, Marc Nazaré (Leibniz Forschungsinstitut für Molekulare Pharmakologie), Christoph Rademacher (Max Planck Institute) and collaborators at Freie Universität Berlin and Berlin Institute of Health set out to find some, as they report in a recent paper in J. Am. Chem. Soc.

The researchers were interested in the protein langerin, a C-type lectin receptor involved in pathogen recognition. They screened the extracellular domain against a total of 871 fragments using a combination of NMR methods: STD, T₂-filtered, and ¹⁹F NMR. A total of 78 fragments confirmed in at least two of these assays, of which 53 also confirmed by SPR. Three of these fragments inhibited the binding interaction between langerin and the polysaccharide mannan.

Next, the researchers acquired or synthesized more than a hundred derivatives of the active fragments and tested them in their battery of assays. Throughout the process they were careful to look for and exclude compounds that showed bad behavior such as aggregation or instability.

Ultimately, the best compounds showed triple-digit micromolar affinity by SPR and double-digit micromolar inhibition in the mannan-binding assay. Interestingly, these compounds do appear to be allosteric: they reduce the affinity of langerin towards mannan but don’t appear to directly block binding. Moreover, two-dimensional (HSQC) NMR studies suggest that the compounds bind to a different binding site on the protein than the carbohydrate does.

Of course there is still a long way to go: the compounds are far too weak to be useful chemical probes at this point. Still, this is a nice tour-de-force of biophysics. And perhaps – as we’ve seen before – someone else will be able to improve the potency of these molecules.

From noncovalent to covalent fragment for BTK

The approved anti-cancer drug ibrutinib is a poster child for covalent modifiers, with projected 2018 sales of more than $1.2 billion. The molecule reacts with a cysteine residue in the kinase BTK as well as several other kinases, forming an irreversible bond. However, it is also a potent noncovalent inhibitor of multiple kinases, leading to various side effects. This is because the so-called “hinge-binding” moiety is quite promiscuous. To find a more selective and potentially safer molecule, researchers at EMD Serono, Constellation Pharmaceuticals, and Hoffmann-La Roche turned to fragments, and describe their results in two recent Bioorg. Med. Chem. Lett. papers.

In the first, Richard Caldwell and collaborators disclose Fragment A. Although no details are provided as to how this was discovered, the researchers were able to determine a crystal structure of the fragment bound to BTK, revealing that the carboxamide forms interactions with the hinge region. The binding mode also suggested how an acrylamide warhead could be positioned to react with the nearby cysteine residue, and indeed compound A7 turned out to be a nanomolar inhibitor. Further fragment growing ultimately led to compound A17, with subnanomolar biochemical activity and nanomolar cell activity. Unfortunately, this compound had poor oral bioavailability in mice.

The second paper, by Hui Qiu and collaborators, picks up the story. Hypothesizing that the number of nitrogen atoms in compound A17 could be deleterious, the researchers swapped the added portion of the molecule with the phenoxyphenyl moiety present in ibrutinib. Compound B7 did indeed show good permeability, albeit at a cost in potency. However, a crystal structure of this molecule bound to BTK revealed the potential for improving hydrophobic interactions.

The best molecule reported, B16, has picomolar activity in a biochemical assay and nanomolar activity in cells. Moreover, it is orally bioavailable in rats. While ibrutinib inhibits 35/270 kinases at 1 µM, compound B16 only inhibits 4. However, the compound does inhibit hERG, which can cause cardiac complications, so more work needs to be done. A crystal structure reveals that B16 (gray) binds in a similar manner to the initial Fragment A (cyan).

We have previously described examples of covalent fragments being used to target kinases, but these new papers are a useful reminder that it is also possible to start with ordinary non-covalent fragments and introduce a warhead later. Or not – as we highlighted in 2015 regarding a noncovalent BTK inhibitor from Takeda. The possibilities are limited only by your creativity.

Fragment chemistry roundup

Our current poll (please vote on the right-hand side of the page!) asks about commercial fragment libraries. However, there is much to be said about making your own fragments: you have total control over the quality, and it is easier to get into novel chemical space. We highlighted one example of custom fragments in 2016; here are a few more. Please feel free to share others in the comments.

James Bull and collaborators at Imperial College London and Eli Lilly describe a nice divergent approach to cyclopropane-containing compounds in a Eur. J. Org. Chem. paper published last year. As they note, cyclopropane is the tenth most common ring found in small-molecule drugs: common enough to be validated, but sufficiently rare as to quickly yield novel compounds. They start with an efficient cobalt-catalyzed cyclopropanation that can be conducted on gram-scale to produce scaffolds A1 and A2. The sulfur atoms can be oxidized to sulfoxides or sulfones, and the esters can be converted to amides. Perhaps more interestingly, the sulfoxides can be converted to Grignard reagents that can be reacted with electrophiles or used in cross coupling reactions, ultimately generating diverse molecules such as A24 and A25.

The researchers also calculated various parameters of the 56 molecules they synthesized, and found that many of them were quite “shapely” as assessed by calculating principal moments of inertia.

A more recent paper, by Adam Nelson and colleagues at the University of Leeds and published in Bioorg. Med. Chem., also looked at more “three-dimensional” fragments based on bridged bicyclic lactams found in certain alkaloids. Intermediates such as compound B11 could be rapidly assembled and diversified at multiple points to generate very different molecules such as B17b and B19. All in all, 22 fragments with < 17 non-hydrogen atoms and clogP < 2.5 were generated.

Finally, Nicola Luise and Paul Wyatt at the University of Dundee describe the synthesis of semi-saturated bicyclic pyrazoles in Chem. Eur. J. As the researchers point out, at least five fragment-derived drugs that have gone into the clinic contain pyrazole moieties, and 4-bromopyrazole seems to be a universal fragment. Although many pyrazoles are commercially available, the number drops considerably with partially aliphatic bicycles, and these may also have improved physicochemical properties.

As we noted in 2016, Paul Wyatt is having students build libraries of novel fragments. Given the range of chemistries explored in this paper, this seems like good training. Starting from 3-bromopyrazole, the researchers generated 25 different molecules, all conforming roughly to the rule of three, and also characterized whether they met criteria for purity, stability, and solubility at 2 mM in phosphate buffer. Only a single molecule dropped out, supporting the design criteria, and the molecules have been added to the Dundee fragment library.

Papers like these do not get as much attention as they deserve, in part because the biological properties of new molecules are by definition unknown. Still, it is refreshing to see chemists coming up with creative new classes of fragments. Hopefully we will revisit some of them as hits in future posts!

Library vendor poll!

A good fragment library is essential for generating good fragment hits. Earlier this year we asked about fragment size and library size, and summarized the results here.

Some folks make their own fragments, but this is expensive, and it’s probably fair to say that most fragment libraries contain a large fraction of commercially available compounds. Unfortunately, what you buy is not always what you get: sometimes the wrong compound is sent, or the compound is not as pure as advertised. As this post makes clear, different vendors have different track records.

Two years ago we highlighted a number of fragment library vendors. The current poll first asks whether you’ve bought fragments in the past several years. (Please answer this question as otherwise I have no idea how many people actually vote.)

If the answer to the first question is yes, the second question asks which vendors you would recommend buying from – presumably because you’ve had good experiences with them in the past few years. You can vote for as many as you’d like.

Finally, the third question asks which vendors you would avoid.

Please vote on the right-hand side of the page, and feel free to leave comments below, particularly if you've used vendors not on the list.

And on the subject of voting, if you are eligible to vote in the United States, please make sure to do so before the polls close on November 6.

Democracy atrophies when citizens don’t exercise their rights.

Capillary electrophoresis revisited

Among the various fragment-finding methods, capillary electrophoresis (CE) seems to be among the least-used, at least according to our polls. Indeed, we last wrote about CE in 2012, and since then the company that was popularizing the technique seems to have quietly dropped it from its website. A new paper by Marianne Fillet and collaborators at the University of Liege and the University of Namur in Analytica Chimica Acta presents a how-to guide for CE.

As we previously discussed, the most general CE assay involves filling a capillary with a protein solution as well as a “probe ligand” with affinity for the target protein. Interactions between the probe ligand and the protein will increase the migration time of the probe ligand compared to its progress through a capillary without protein (i.e., the probe ligand will move more slowly through the capillary in the presence of protein).

If a “test ligand” is introduced into the capillary and displaces the probe ligand, the migration time of the probe ligand will again decrease. By changing the concentration of test ligand and measuring the shift in migration time of the probe ligand, the affinity of the test ligand can be determined.

The researchers applied CE to thrombin, a drug target that is often used for validating fragment-finding methods. The low nanomolar inhibitor NAPAP was chosen as the probe ligand due to its strong chromophore (simplifying detection) and positive charge (allowing it to move in the electric field of the capillary). They tested three literature compounds with inhibition constants ranging from high nanomolar to high micromolar and found good agreement with published results.

Next, the researchers applied CE to a small library of fragments, generating several hits. They also describe a method for detecting irreversible binding: this involves screening the protein with an even higher concentration of probe ligand to see whether the test ligand itself can be displaced.

This is a nice study, but it perhaps also illustrates why the technique hasn’t caught on. First and most importantly is throughput; the runs shown are on the order of 14 minutes. Second, it does require a probe ligand. (Screening test ligands directly only works if they are positively charged.) On the other hand, CE can work with native protein, unlike immobilization-based techniques such as SPR and WAC.

Have you tried CE yourself – and if so how did it perform?

Fragments vs Ras – part 3

Six years ago we highlighted papers from Genentech and Steve Fesik’s group reporting fragments that bind to Ras-family proteins, which are among the best validated but most difficult anti-cancer targets. The fragments bind some distance from the GTP-binding site, but can block Ras signaling by interfering with important protein-protein interactions. However, the most potent molecules reported bound at this site with just ~200 µM affinity, and we concluded by musing that “it still remains to be determined whether this is a ligandable site on the protein.” As reported recently in Nature Communications by Terence Rabbitts and collaborators at the University of Oxford, St. James University, Domainex, and the University of Aberystwyth, the answer appears to be yes.

The researchers screened HRas against 656 fragments, each at 200 µM, using SPR, resulting in 26 initial hits. These were tested again by SPR against active-form protein (bound to the GTP mimetic GTPγS) or inactive protein (bound to GDP). A single compound, Abd-1, was selective for the activated form of the protein, and did not bind when the protein was complexed to an antibody the researchers had previously generated that binds at the same PPI site.

Abd-1 had low affinity and was not particularly soluble, so the researchers looked for analogs with better properties, resulting in Abd-2, which binds to both HRas and KRas. Further growing in the direction taken by the Fesik group did not lead to significant improvements, but a breakthrough occurred when the researchers grew off a different region of the fragment, towards what looked to be the wall of the small pocket. As Trevor Perrior mentioned at the DOT meeting last month, this led to the opening up of a new channel and a substantial boost in affinity for Abd-5. Further growing allowed the researchers to trim off the right-hand portion entirely, leading to Abd-7, with mid-nanomolar activity and good ligand efficiency. Crystallography revealed that, despite the conformational changes, the core of Abd-7 still binds in the same location as Abd-2.

Not only did Abd-7 bind tightly to KRas, it also inhibited the pathway in cell-based assays (albeit at 100-fold higher concentrations), presumably by blocking interactions with Ras-effector proteins. The compound also showed low micromolar activity against cancer lines with different Ras mutations in cell viability assays. The researchers note that “the observed discrepancy between affinity (in vitro Kd) and efficacy (IC₅₀ in cells) is a known challenge that can be addressed through chemistry.” Other possible challenges include metabolic stability and oral bioavailability, neither of which is discussed. Nonetheless, the paper reveals that this site in Ras family proteins is ligandable. It is also a useful reminder that proteins can be remarkably plastic, and sometimes the best route forward really is by slamming into what appears to be a solid wall.

FBLD 2018

Ten years ago, Vicki Nienaber (Zenobia) enlisted a small group of fellow enthusiasts to help her organize an independent fragment-based lead discovery conference in San Diego. That event was so successful that it was repeated in York in 2009, Philadelphia in 2010, San Francisco in 2012, Basel in 2014, and Cambridge (USA) in 2016. Last week, to celebrate its first decade, Derek Cole (Takeda), Rod Hubbard (University of York) and Chris Smith (COI) brought FBLD 2018 back to San Diego, along with some 200 fragment fans. With around 30 talks, more than 40 posters, and nearly 20 exhibitors, I won’t attempt to present a comprehensive overview, but just focus on broad themes.

Success Stories

I estimate that, in 2008, 14 fragment-based programs had entered the clinic, none of which had advanced beyond phase 2. That list has now grown to more than 40, so naturally success stories were a focus.

Andy Bell (Exscientia) discussed NMT inhibitors for malaria and the common cold (see here); the AI-driven approach took < 500 molecules to get to molecules with animal efficacy. Steve Woodhead (Takeda) revealed potent inhibitors of TBK1, a kinase involved in the innate immune response. It took just three months to go from a fragment hit to an animal-active lead, though unfortunately that molecule also showed apparent on-target toxicity. And Rosa María Rodríguez Sarmiento (Roche) described the discovery of COMT inhibitors (see here).

Mary Harner (BMS) described the discovery of sub-micromolar KAT II inhibitors in just a few months, enabled by parallel chemistry and the synthesis of 833 compounds. Several series turned out to be aggregators, and BMS has instituted a routine β-lactamase screen (an enzyme particularly sensitive to aggregators) to catch these early.

Keith McDaniel (AbbVie) described the discovery of the BET-family bromodomain inhibitor ABBV-075. This program also made rapid progress: just six months from the initial fragment hit, although the team did spend another year trying to find better molecules. This effort eventually paid off, as the same fragment has now led to a BD2-selective molecule, ABBV-744, that has recently entered the clinic.

And Paul Sprengeler (eFFECTOR) described the discovery of eFT508. This too was a rapid success: just 1 year and 170 compounds, enabled by 30 co-crystal structures, and in the end a dozen molecules competing for candidacy.

Notice that many of these projects moved quickly. Feel free to send this summary to anyone who worries that fragment programs move too slowly to be practical.

Technologies

Technologies have always had a starring role in FBLD conferences, and this one was no exception. Ben Cravatt (Scripps) discussed his fragment-based target discovery methods (see here and here). As I speculated recently, he is now using these approaches to discover new protein degraders. And his "fully functionalized fragments" are being adopted by others, as described in a poster by Emma Grant and collaborators at GlaxoSmithKline and University of Strathclyde.

Surface plasmon resonance (SPR) was used routinely by many of the speakers, but there is plenty of room for innovation. John Quinn (Genentech) described how to extend kinetic measurements to the very fast and the very slow. John also noted that gathering kinetic data earlier to deprioritize series with slow on-rates may be wise. And for those who wonder about the limits of detection for SPR, John measured the affinity of imidazole for NTA: just 13.6 mM!

Miles Congreve (Sosei Heptares) described multiple methods applied to GPCR targets along with a number of success stories. He also noted that, in the PAR2 program we mentioned recently, fragments were able to identify a buried pocket that could not be found using DNA-encoded libraries of several billion members, presumably because the pocket would not be accessible to a DNA-bound ligand. Interestingly, this pocket could be detected computationally using FTMap, as shown in a poster presented by Amanda Wakefield (Boston University).

Pedro Serrano (Takeda) described a variety of biophysical methods applied to GPCRs, the most stunning of which is an SPR microscope capable of performing kinetic binding assays on whole cells. He has tested this Biosensing Instrument on four different GPCRs, and although there are technical challenges, the data seem usable.

But the light shone most brightly on crystallography, illuminated by Stephen Burley (Protein Data Bank) among others. In order to justify continued public funding and free access (yes, there were suggestions to put the PDB behind a paywall), the PDB was asked to demonstrate its usefulness to society. Their analysis found that of the 210 new molecular entities (NMEs) approved by the FDA from 2010 through 2016, 184 had PDB entries for the target and/or the NME – for a total of 5914 structures, 95% of which were crystallographic. Most of these structures had been deposited at least 10 years before the drug was approved, so in many cases they probably played an important role.

John Barker described how Evotec has jumped into high-throughput screening by crystallography in a collaboration with the Diamond Light Source, which is now capable of doing 700 soaks per day. They have run 10 screens over the past 18 months with a small library of 320 fragments, with hit rates typically around 8%.

We have written about how high concentrations can improve success in crystal soaking experiments, and both Chris Murray and Dominic Tisi of Astex described how they’ve taken this to an extreme: 1 M soaks, with the fragment dissolved directly in the soaking solutions. Obviously this requires highly soluble fragments, so they’ve built a library of 81 “MiniFrags” having on average just 6.4 non-hydrogen atoms. They have tested these against five targets that diffract to high resolution and have found impressively high hit rates of 20-60%, compared to the 2-20% in the original 100 mM soaks for the same targets. Some of the sites are exploited by previously reported inhibitors or substrates, while others are new. And while the “universal fragment” 4-bromopyrazole did well, 1,2,3-triazole did even better – binding to all five targets in a total of 22 sites.

Crystallographers should not become complacent. Gabe Lander (Scripps) gave an update on cryo-EM, which we’ve written about here. The number of cryo-EM structures deposited in the PDB eclipsed those from NMR in 2016, and resolution continues to improve, with the current (as of late September) record at 1.56 Å. Still, the technique is not nearly as fast as crystallography: best case is 8 hours from data collection to refinement, although Gabe did think that 10 structures per day would be possible within the next few years. And Chris Murray noted that, if present trends continue, “we’ll all be doing cryo-EM in five years’ time.” Backing this up, he showed what I suspect may be the first clear density map of a fragment bound to a test protein.

This was the last major fragment event of the year, but next year’s calendar is already shaping up nicely. And mark your calendar for September 2020, when FBLD 2020 will move to the original Cambridge (UK).

Fragments in the clinic: 2018 edition

To celebrate FBLD 2018, we're updating the list of FBLD-derived drugs. The current list contains 40 molecules - 25% more than the last compilation two years ago. As always, this table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and those that have been discussed on Practical Fragments are hyperlinked to the most relevant post.

Drug	Company	Target
Approved!
Vemurafenib	Plexxikon	B-Raf(V600E)
Venetoclax	AbbVie/Genentech	Selective Bcl-2
Phase 3
Asciminib	Novartis	Bcr-Abl
Erdafitinib	J&J/Astex	FGFR1-4
Lanabecestat	AstraZeneca/Lilly/Astex	BACE1
PLX3397	Plexxikon	CSF1R, KIT
Verubecestat	Merck	BACE1
Phase 2
AT7519	Astex	CDK1,2,4,5,9
AT9283	Astex	Aurora, JAK2
AUY-922	Vernalis/Novartis	HSP90
AZD5363	AstraZeneca/Astex/CR-UK	AKT
CPI-0610	Constellation	BET
DG-051	deCODE	LTA4H
eFT508	eFFECTOR	MNK1/2
Indeglitazar	Plexxikon	pan-PPAR agonist
LY2886721	Lilly	BACE1
LY517717	Lilly/Protherics	FXa
Navitoclax (ABT-263)	Abbott	Bcl-2/Bcl-xL
Onalespib	Astex	HSP90
PF-06650833	Pfizer	IRAK4
PF-06835919	Pfizer	KHK
Phase 1
ABT-518	Abbott	MMP-2 & 9
ABT-737	Abbott	Bcl-2/Bcl-xL
ASTX029	Astex	ERK1,2
ASTX660	Astex	XIAP/cIAP1
AT13148	Astex	AKT, p70S6K, ROCK
AZD3839	AstraZeneca	BACE1
AZD5099	AstraZeneca	Bacterial topoisomerase II
BI 691751	Boehringer Ingelheim	LTA4H
ETC-206	D3	MNK1/2
GDC-0994	Genentech/Array	ERK2
IC-776	Lilly/ICOS	LFA-1
LP-261	Locus	Tubulin
LY2811376	Lilly	BACE1
MAK683	Novartis	PRC2 EED
Mivebresib	AbbVie	BRD2-4
PLX5568	Plexxikon	kinase
SGX-393	SGX	BCR-ABL
SGX-523	SGX	Met
SNS-314	Sunesis	Aurora

I have no doubt that this list is incomplete, particularly in Phase 1. If you know of any others (and can mention them) please leave a comment.

Sixteenth Annual Discovery on Target

CHI’s Discovery on Target took place in Boston last week. With >1300 attendees from over two dozen countries, this is the older, larger cousin of the San Diego DDC meeting; at some points ten tracks were running simultaneously. Although more heavily focused on biology, there were still plenty of talks of interest to fragment folks.

Michael Shultz (Novartis) provocatively asked “do we need to change the definition of drug-like properties?” Long-time readers will recall that his earlier papers on ligand efficiency led to considerable debate, which seems to have been settled to everyone’s satisfaction with the exception of Dr. Saysno.

His new study, which has just published in J. Med. Chem., analyzes the molecular properties of all 750 oral drugs approved in the US between 1900 and 2017. Contrary to what strict rule of five advocates might expect, the molecular weight has increased over the past couple decades, as has the number of hydrogen bond acceptors. In contrast, the number of hydrogen bond donors (#HBD) has remained constant, suggesting that this may be more important for oral bioavailability. (Indeed, #HBD is the only Lipinski rule not broken by venetoclax.) Although Shultz did not examine “three dimensionality,” he laudably includes all the raw data – including SMILES – in the supporting information. This will be a useful resource for data-driven debates.

Molecular properties are carefully considered by Ashley Adams, who discussed the four fragment libraries used at AbbVie. The first is a 4000-member “rule of three” compliant library. For tougher targets, a 9000-member Ro3.5 library is available, as is a specialized fluorine library for ¹⁹F NMR (2000 members) and a 1000-member “biophysics” library, in which all compounds are less than 200 Da. Fragment optimization is often challenging, and since the C-H bond is most common but perhaps least explored, the AbbVie database is annotated with references on C-H bond activation relevant to each fragment.

Anil Padyana spoke about the metabolic enzymes being targeted at Agios. As we mentioned recently, these are very difficult targets, so the researchers often use parallel (as opposed to nested) screening using different techniques to minimize false negatives. Anil also described an interesting SPR assay in which fragments were introduced to the protein after the addition of an activating substrate.

High-quality protein constructs are essential for any fragment screen, and Jan Schultz described ZoBio’s technology for generating these. The company’s “protein domain trapping” approach entails high-throughput generation and screening of tens or hundreds of thousands of truncations of a given protein and rapidly selecting stable, high-expressing, and active variants.

Trevor Perrior mentioned that Domainex has a similar technology, which has been able to produce soluble protein domains in 90% of its attempts. Trevor also described a separate project in which a 656-fragment compound library was screened using SPR against the enzyme RAS. They found fragments that bind in a previously discovered site but, unlike the earlier work, the Domainex researchers were able to optimize these to nanomolar inhibitors.

Another success story was presented by Dean Brown (AstraZeneca), who described a collaboration with Heptares to discover inhibitors of protease-activated receptor 2 (PAR2). As the name suggests, this GPCR is activated when a protease cleaves the N-terminus, allowing the remaining N-terminal residues to fold back and activate the GPCR. The researchers used a stabilized form of PAR2 in an SPR screen of 4000 fragments and obtained >100 binders in multiple series. This led to AZ8838, which blocks signaling by binding in an allosteric pocket. It also has a slow off-rate, which is often an attractive feature – particularly in the context of intramolecular activation.

A number of talks were focused on protein degraders such as PROTACs (PROteolysis-TArgeting Chimeras). These are generally two-part molecules connected by a linker: one part binds to a target of interest, while the other engages the cellular degradation machinery to destroy the target. As Shanique Alabi, a graduate student in Craig Crews's lab at Yale demonstrated, the molecules are catalytic – a single PROTAC molecule can cause the destruction of multiple copies of a target protein. This “event-driven” pharmacology is thus different from most historical drugs, which are “occupancy-driven.” Is there a role for fragments?

One of the strengths of FBLD is that if a ligandable site exists, it can be found. As Astex demonstrated, the majority of proteins seem to have secondary sites, away from the active site. Although some of these may be allosteric, others probably have no functional activity, particularly in the case of protein-protein interactions where secondary sites may be located some distance from the interface. The power of degraders is that non-functional sites can be made functional. The power of FBLD is that it can find small-molecule binding sites, which could then be used as anchoring sites for one side of a degrader. Watch this space!

Fragments in the clinic: Asciminib

Imatinib is the early poster child of personalized medicine. The drug famously works by binding to the mutant kinase BCR-ABL1, and its approval by the FDA for chronic myelogenous leukemia in 2001 arguably launched hundreds of programs targeting kinases. Although imatininb is remarkably effective, resistance sometimes develops, usually caused by mutations that lead to loss of affinity for the drug. Imatinib and other approved drugs that target BCR-ABL1 all bind in the active (ATP-binding) site of the kinase, and they all have various off-targets that can lead to toxicity. To sidestep these issues, researchers at Novartis have developed an allosteric inhibitor, as described by Andreas Marzinzik and colleagues in a new paper in J. Med. Chem.

The ABL1 kinase is naturally autoinhibited by the binding of a myristoyl group to an allosteric pocket. Although the pocket exists in BCR-ABL1, the site that is normally myristoylated is lost. The researchers wanted to create a molecule that would mimic the function of the myristoyl group and exert its inhibitory effect within the allosteric pocket.

An NMR-based screen of 500 fragments yielded 30 hits – perhaps not a surprisingly high hit rate given the lipophilicity of the pocket. Compound 2, with low micromolar affinity, had a high ligand efficiency. Unfortunately, it and similarly high-affinity fragments showed no cell-based activity. A crystal structure of compound 2 bound to the protein revealed that, although the fragment binds in the myristate pocket, its binding mode would actually prevent the conformational change necessary for allosteric inhibition. Tweaking and growing the fragment led to molecules such as compound 4, which were still inactive.

To determine why, the researchers developed a clever NMR assay based on a specific valine residue located in a disordered region of the protein that becomes helical in the allosterically inhibited state of the protein. This assay allowed them to distinguish which protein conformation molecules bound and revealed that, contrary to design, compound 4 did not in fact bind to the inhibited form of the protein. Other researchers had found a different series of molecules that also bind in the myristate pocket, and these all contained a trifluoromethoxy group. When this moiety was grafted onto compound 4, the resulting compound 5 showed cell-based activity.

Now the medicinal chemistry began in earnest. Crystallography revealed a lipophilic cleft in the allosterically inhibited form of the protein which could be filled with a pyrimidine, and the cationic solubilizing group in compound 5 was replaced by the neutral moiety in compound 7. This compound showed some hERG channel inhibition, which could be fixed by replacing the pyrimidine with a pyrazole. Also, crystallography revealed that there was a little extra space near one of the fluorine atoms, which could be replaced with a chlorine in the clinical compound asciminib (ABL001). A crystal structure of this molecule shows it binding to the inactive conformation of the protein (the helix that forms is in the upper right).

Asciminib effectively inhibits proliferation of cells containing either wild-type or T315I BCR-ABL1, the latter being one of the more pernicious resistance mutations. The compound is also highly selective against > 60 other kinases, and is only active against CML cell lines in a panel of 546 cancer cell lines, suggesting that it should be well tolerated. Mouse xenograft models were also impressive, and the compound is currently in a phase 3 clinical trial.

This is a thorough, clearly written account combining biophysics, modeling, chemistry, and biology to discover a first-in-class drug. It is also a useful reminder that binding alone may not be sufficient to cause desired effects. As with all the clinical-stage programs, Practical Fragments wishes everyone involved the best of luck!

Fragments in the clinic: ASTX660

Three years ago we highlighted a paper from Astex describing the discovery of an extraordinarily weak fragment and its advancement to a dual inhibitor of the anti-cancer targets cIAP1 and XIAP. We ended that post by writing, “whether or not this leads to a drug, it does look like another candidate for a useful chemical probe.” As three papers now make clear, the program has indeed led to an experimental drug.

The first paper, by Emiliano Tamanini and colleagues, was published in J. Med. Chem. last year and describes the optimization of Compound 21, one of the best compounds from the 2015 report. The researchers noticed that compounds in the series were chemically unstable: the amide bond was subject to hydrolysis. Fortunately this was readily fixed by repositioning the pyridyl nitrogen.

Optimization of the benzyl group was complicated by the fact that it binds in the P4 pocket, which differs between cIAP1 and XIAP. In the end, adding a fluorine gave a slight potency improvement against both proteins. The bulk of the work was focused on elaborating the methoxy group of compound 21. Detailed modeling experiments were used to choose moieties that would fold back on the core of the molecules in solution, thus pre-orienting them for binding as well as shielding a critical hydrogen bond. These efforts led to AT-IAP, with low nanomolar cell activity against both proteins as well as activity in mouse xenograft models.

Although AT-IAP is orally bioavailable in mice and rats, the bioavailability is much lower in monkeys, and it also inhibits the hERG channel, which can lead to cardiac toxicity. Fixing these problems is the focus of a paper published last month in J. Med. Chem. by Christopher Johnson and colleagues.

Metabolite identification studies revealed that the morpholine ring of AT-IAP is cleaved by CYP enzymes, so this was one area the researchers tried to modify. Although somewhat successful, hERG was still a problem, and this correlated with lipophilicity. Knowing how the molecules bound allowed the researchers to introduce small hydrophilic substituents without disrupting critical interactions, ultimately leading to ASTX660. Not only did the added hydroxymethyl group decrease hERG binding, it also improved bioavailability – a reminder that decreasing lipophilicity can have useful effects even on distant parts of the molecule.

More characterization of ASTX660 is provided in a paper by George Ward and colleagues in Mol. Canc. Ther. This reports the crystal structure of the molecule bound to XIAP. As Johnson et al. note, the polar interactions made by the molecule are conserved from the original fragment – the additional protein interactions that improve affinity by more than a million-fold are all hydrophobic.

Ward et al. also provide more detailed mechanistic cell biology, pharmacokinetics, and xenograft data. In particular, ASTX660 is a much more potent antagonist of XIAP activity in vivo than other clinical-stage compounds, which will hopefully translate to better efficacy. The compound is currently in a phase 1-2 study.

Collectively these papers provide a valuable lesson in structure- and property-based drug design and illustrate just how much effort can be required to go from fragment to clinical compound. I’ll end this post with an echo of the original: whether or not this leads to an approved drug, it is a lovely story of perseverance combined with creative chemistry and biology. Practical Fragments wishes everyone involved the best of luck.