27 June 2016

Fragments vs Lp-PLA2 – less greasily

Human lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme involved in lipid metabolism that is implicated in multiple diseases, from atherosclerosis to Alzheimer’s. Because the natural substrates are lipophilic phospholipids, it is no surprise that reported inhibitors are also large and hydrophobic. A case in point is darapladib: with a molecular weight of 667 Da and a clogP of 8.3 this is a poster child for molecular obesity – and it also failed in phase 3 clinical trials. A new paper in J. Med. Chem. by Alison Woolford (Astex), Joseph Pero (GlaxoSmithKline) and colleagues describes an effort to discover less lipophilic inhibitors.

The researchers performed a screen of 1360 fragments using thermal shift and ligand-detected NMR and a smaller screen of 150 fragments using crystallography. This yielded 34 fragments that were ultimately characterized crystallographically; screening commercial and in-house collections for related fragments yielded another 16. Interestingly, rather than clustering at a single hot spot, these fragments bound to different regions of the extended active site, with some – such as fragment 6 – a full 13 Å from the catalytic center. This is reminiscent of a fragment campaign against soluble epoxide hydrolase, another enzyme with a long, hydrophobic active site.

In addition to binding in an interesting site, fragment 6 also has good affinity and ligand efficiency. Moreover, its binding site overlaps partly with that of fragment 5. Thus, the researchers merged the two fragments together, resulting in compound 7, with submicromolar activity.

Further structure-guided optimization, which included growing into a polar region of the protein, ultimately led to compound 16, with low nanomolar potency.





Compound 16 has a molecular weight of 411 Da and a clogP of 3.4 and is correspondingly reasonably soluble (> 0.3 mM). Whereas darapladib showed a dramatic 700-fold potency decrease upon addition of human plasma – presumably due to nonspecific binding to other proteins – the decrease in potency for compound 16 is only 13-fold. Indeed, though darapladib is a picomolar binder, compound 16 is slightly more potent in plasma.

Unfortunately, compounds in this series turned out to have high clearance in rats, proving once again that lead optimization is often a frustrating game of whack a mole. Still, the fact that the researchers were able to develop smaller, more soluble inhibitors of an enzyme with such a lipophilic substrate gives hope that the game is perhaps winnable.

20 June 2016

19F-NMR-guided fragment linking on BACE1

Fragment growing has been the dominant strategy of most of the recent posts involving lead optimization, consistent with our poll results. However, fragment linking can be powerful too, as illustrated by the recent approval of venetoclax, which was derived from fragment linking. A recent paper in J. Med. Chem. by Brad Jordan and colleagues at Amgen provides another nice case study.

Amgen researchers had previously used fragment growing to discover inhibitors of BACE1, an Alzheimer’s target which has been heavily tackled by fragments. However, the most potent molecules in the series also inhibited the related aspartic protease cathepsin D (CatD), which could cause serious side effects. The researchers sought to gain selectivity by building inhibitors to occupy the so-called S3subpocket of BACE1. To do so, they used 19F-NMR to find fragments that would bind to BACE1 in the presence of a “blocking compound” that filled most of the active site but not the S3subpocket. This led to the discovery of seven fragments, the most potent being compound 3. Interestingly, this fragment only bound in the presence of the blocking compound as assessed both by NMR and SPR. Also, it could be competed by a compound that binds in the S3subocket.


Having thus identified a fragment that bound in the presence of one of their inhibitors, the researchers used interligand NOE (ILOE) to determine how the two compounds bind relative to one another. This supported the idea that compound 3 binds in the S3subpocket, and also suggested how the fragment could be linked onto the existing lead series, exemplified by compound 5. Just four compounds were designed and synthesized, and all of them were more potent than either of the starting points, with compound 9 being the best. More importantly, this compound also proved to be ~2000-fold selective for BACE1 over CatD in enzymatic and cell-based assays.

Despite the excellent (high picomolar) affinity of compound 9 for BACE1, this is actually about 25-fold worse than would be predicted by a simplistic additivity of binding energies – a not uncommon occurrence when linking molecules. Still, with its combined used of multiple NMR techniques and structure-based design to solve a specificity challenge, this paper is worth perusing.

15 June 2016

Covalent fragments writ large

We’ve written previously about irreversible covalent fragment-based lead discovery. The nice thing about irreversible inhibitors is that they have an infinite no off-rate: once they bind and react with a target, that protein is permanently out of action. A paper published today in Nature by Keriann Backus, Benjamin Cravatt, and colleagues at Scripps Research Institute takes this approach to a whole new level.

The researchers assembled a library of just over 50 fragments containing cysteine-reactive electrophiles, such as chloroacetamides and acrylamides; the average molecular weight was 284 Da. These were then screened against human cells or cell lysates using a proteomic approach called isotopic tandem orthogonal proteolysis-activity based protein profiling (isoTOP-ABPP). This technique, previously developed by the Cravatt laboratory, uses mass spectrometry to differentiate contents of treated and untreated cells and identify specific regions of proteins that are modified.

In all, 758 cysteine residues in 637 different proteins were found to be modified by at least one of the fragments. These included targets (such as BTK) with known covalent drugs as well as many proteins with no small molecule inhibitors. Even more exciting, this set included some particularly challenging classes of proteins, such as transcription factors and various adapter and scaffolding proteins. Most proteins only had a single modified cysteine, and these were not necessarily in the active site (see also here). Happily, computational docking did a good job of (retrospectively) predicting the modified cysteine residues.

The fragments themselves ranged significantly in how many cysteines they modified, from < 0.1% to > 15%, with a median of 3.8%. Interestingly, the correlation with intrinsic electrophilicity – as measured by reaction with the small molecule thiol glutathione – was fairly weak. This suggests that the fragments are modifying proteins based on other properties, such as specific interactions between fragment and protein.

The initial studies were done using cell lysates at high (500 µM) fragment concentrations. Follow-up studies in whole cells using 50-200 µM fragment gave similar results, with 64% of the cysteines from the lysate experiments reacting with the same fragments in cells, even at the lower concentrations. Interestingly though, four fragment-cysteine interactions were found only in cells and not in lysates.

One class of proteins you might expect reactive fragments to hit are cysteine proteases, such as the caspases, and indeed one chloroacetamide-containing fragment reacted with the active site cysteine of caspase-8 (CASP8). Surprisingly though, this fragment showed only marginal activity in an inhibition assay, and subsequent experiments revealed that it is selective for the inactive zymogen (or proenzyme) form of the protein, thereby preventing activation. This fragment does not react with the related caspases 2, 3, 6, or 9, though it does hit CASP10. Modest modifications led to a compound that was also selective for CASP8 over CASP10. These two molecules were used to show that both CASP8 and CASP10 appear to be essential for extrinsic apoptosis in primary human T cells, but not in the immortalized Jurkat T-cell line.

Of course, it will be essential to rigorously characterize any covalent molecules used as probes. Chloroacetamides are well-known electrophiles – so well known in fact that they are generally excluded from screening libraries, including those that helped define the original PAINS filters. A single digit percentage hit rate means that any given covalent fragment could easily hit hundreds of proteins. The researchers here do careful control experiments – such as using an inactive enantiomer and extensive proteomic analyses – but someone less careful could easily mislead themselves and others. Done rigorously, though, this is an exciting approach that may well increase the number of ligandable targets.

13 June 2016

Fragments vs MetAP2: reversible inhibitors

Methionine aminopeptidases, or MetAPs, cleave the N-terminal methionine residue from newly translated proteins. The human enzyme MetAP2 is a potential target for obesity, as demonstrated by the impressive clinical results of beloranib. But this drug hasn't been approved, and patients have died while taking it. Beloranib is an irreversible inhibitor that may also hit other targets, so researchers at Takeda California have been seeking non-covalent inhibitors. They report their results in two recent papers in Bioorg. Med. Chem. Lett.

In the first paper, Zacharia Cheruvallath and colleagues describe a biochemical fragment screen of ~5000 fragments (11-19 non-hydrogen atoms) conducted at 0.1 mM. This produced an impressive number of hits (110 compounds with > 20 % inhibition), which were triaged based on both ligand efficiency and LLE, ultimately yielding 16 interesting fragments. In particular, fragment 6 is remarkably potent.
Crystallography was not successful for any of the fragments. Undeterred, the researchers performed classic “SAR by catalog” (and corporate collection) to develop a binding model. This quickly revealed that the hydroxyl group was unnecessary. It also suggested that one of the indazole nitrogen atoms might be interacting with an active site metal ion, and that the bromine might be pointing towards a hydrophobic pocket where the side chain of the methionine substrate normally binds. Growing led to compound 16, and a closely related compound was characterized crystallographically bound to the protein, confirming the model. Further optimization led to compound 38, with low nanomolar potency in both biochemical and cell-based assays, excellent selectivity against a panel of >100 other targets, good oral bioavailability, and reasonable pharmacokinetics. This compound caused dose-dependent weight loss in a mouse model of obesity.

The second paper, by Christopher McBride and colleagues, involved more dramatic changes to the fragment. The indazole 4 is very potent, but indazoles are quite common in the literature, so the researchers sought to scaffold-hop to a novel core. This led them to design compound 6’, and using some of the SAR from the previous series ultimately led to compound 10. As with compound 38 above, this compound showed good cell-based activity, acceptable pharmacokinetics, oral bioavailability, and a clean profile against > 100 off-targets at 10 µM. It also showed measurable weight loss in a rodent model of obesity.

The question sometimes arises as to how many fragment hits are necessary for a program to move forward. These two papers show that a single fragment can be elaborated to two very different lead series with animal efficacy. In contrast to some of our recent posts, these efforts did not initially require crystallography. There are many ways to advance fragments, and no single technique is essential.

06 June 2016

Fragments vs Dengue virus polymerase

Dengue fever, evocatively called “breakbone fever” for the severe pain it can inflict, is caused by a mosquito-borne virus that infects hundreds of millions of people each year. There are no approved antiviral treatments. Two papers from researchers at the Novartis Institute for Tropical Diseases and the University of Texas Galveston provide some promising early leads.

The first, in J. Biol. Chem., by Christian Noble, Pei-Yong Shi, and collaborators, describes a crystallographic screen of 1408 fragments against Dengue virus RNA-dependent RNA polymerase (DENV RdRp), which is highly conserved among the four serotypes of Dengue virus. Crystals were soaked in pools of eight fragments, with each present at only 0.625 mM, ten to one hundred times lower than other recent crystallographic screens. Perhaps because of this low concentration, only a single hit was identified – compound JF-31-MG46. The crystal structure revealed that the molecule binds in the “palm subdomain” of the protein, which is analogous to a druggable site on the hepatitis C virus protein.

Surface plasmon resonance (SPR) showed that this fragment had a dissociation constant of 0.21 mM against RdRp from serotype 3 and 0.61 mM against RdRp from serotype 4, suggesting weak but real binding. Isothermal titration calorimetry (ITC) was not successful, perhaps because of compound solubility, but replacing the terminal phenyl group with a thiophene led to more potent compounds which could be characterized both by SPR and ITC. The compounds were also active in an enzymatic assay, with IC50 values comparable to their affinities.


The second paper, by Fumiaki Yokokawa and collaborators and published in J. Med. Chem., describes the optimization of these fragments. Fragment growing was performed to try to displace a bound water molecule, resulting in the low micromolar compound 17. Compounds that contain carboxylic acids often have low cell permeability, so several bioiosteres were tested to try to replace this moiety, and compound 23 showed increased affinity. However, this compound was still quite polar, showed poor permeability, and no cell activity. Adding a lipophilic substituent and decreasing the acidity led to compound 27, with nanomolar affinity and enzymatic inhibition of all four Dengue virus serotypes. Importantly, this compound also showed low micromolar activity against all four serotypes in cell assays.

The J. Med. Chem. paper notes that a high-throughput screen against RdRp had been plagued with false positives. One validated low micromolar hit was optimized to nanomolar potency, but this was very lipophilic and displayed no cell activity. It is interesting that the fragment-derived leads initially displayed no cell activity for the opposite reason: they were too polar. This is a useful reminder that physicochemical properties matter. The successful optimization of the fragment-derived series suggests that it can be easier to make leads more lipophilic than less.

01 June 2016

Fragment library vendors - 2016 version

It's been two years since we last updated our list of commercial fragment libraries, and there have been several changes. The prompt for updating the list is a new Perspective published in J. Med. Chem. by György M. Keserű & György G. Ferenczy (Hungarian Academy of Sciences), Mike Hann & Stephen Pickett (GlaxoSmithKline), Chris Murray (Astex), and me. This covers all aspects of fragment library design, so definitely check it out.

One table in the Perspective compares various libraries, both commercial and proprietary. One of the manuscript reviewers asked if we could evaluate the various vendors, particularly given some negative experiences with commercial compounds. Such direct criticism (and praise!) can be awkward in the peer-reviewed literature, but is more acceptable in an online forum - think of Yelp for library suppliers. Please comment (anonymously if desired) if you've had experiences, positive or negative, with these vendors, and please feel free to add any we omitted.

Note that this list only includes companies that sell their libraries (as opposed to just using them internally).

ACB Blocks: 1280 compounds, 19F NMR-oriented, RO3 compliant, predicted to be soluble, purity >96%

Analyticon: 213 compounds, fragments from nature, RO3 compliant, high solubility, purity >95%

Asinex: >22,000 compounds

ChemBridge: >7000 compounds, RO3 compliant with predicted solubility; minimum purity 90% by 1H NMR

ChemDiv: >4000 3D fragments

Enamine: Multiple subsets including >18,000 RO3 compliant, ~1800 "Golden", and >126,000 with < 20 heavy atoms. Also separate fluorinated, brominated, sp3-rich, and covalent subsets.

InFarmatik: 1700 member consolidated library with different subsets (3D, GPCR, kinase)

IOTA: 1500 diverse, mainly RO3 compliant fragments

Integrex: 1500 compounds with diversity in shape and chemical structure, RO3 allowing one violation

Key Organics: ~26,000 compounds total with multiple subsets including 1166 with assured solubility and RO3 compliant as well as brominated, fluorinated, and CNS-directed fragments

Life Chemicals: 31,000 fragments of which 14,000 are RO3 compliant; also fluorinated, brominated, covalent, Fsp3-enriched, and covalent subsets

Maybridge: >30,000 fragments in total. The 2500 Diversity collection is guranteed soluble at 200 mM in DMSO and 1 mM in PBS.  NMR spectra are available (in organic solvent). It is available in many formats, from powder to DMSO-d6 solution. A smaller 1000-fragment subset is also available.

Otava: >12,000 fragments with various subsets including fluorinated, brominated, and metal-chelating

Prestwick: 910 mainly derived from drugs, RO3 compliant

Timtec: 3200 compounds, structurally diverse with predicted high solubility

Vitas-M: ~19,000 fragments, RO3 compliant

Zenobia:  968 fragments from different design paradigms, cores from drugs, higher Fsp3, flexible cores

30 May 2016

Fragments in Texas

The meeting Development of Novel Therapies through Fragment Based Drug Discovery was held last week in Houston, Texas, organized by the Gulf Coast Consortium for Quantitative Biomedical Sciences. Although it was only a single day, it was packed, with thirteen speakers, a couple vendor lunch talks, and some two dozen posters. Below is just a flavor – please add your own impressions if you were there.

I kicked off the first session by giving an overview of FBDD, highlighting both pitfalls and successes. Beth Knapp-Reed (GlaxoSmithKline) then discussed efforts against LDHA, a target previously tackled successfully using fragment linking (see here and here). In this case, an HTS screen of 1.9 million compounds produced only a single hit that resulted in a crystal structure, while fragment screens yielded 16 structures at three binding sites. An NMR-based functional screen (using 13C-labeled substrate) was key to obtaining robust SAR, and using information from both the HTS hit and the fragments ultimately led to nanomolar inhibitors. Next, Tom Davies provided an overview of Astex’s discovery platform, focusing on a success with KEAP1. We recently highlighted research suggesting that crystallography should be used as a primary screen, which Astex does for some targets. Tom noted that doing so currently takes about a month, though only after spending somewhere between 3 and 12 months establishing a robust protein construct as well as crystallization and soaking conditions.

Jane Withka opened the next session by discussing the continuing evolution and use of the Pfizer fragment library. Out of 32 targets screened, only one produced no hits, and this was a particularly flexible protein. Interestingly, despite being relatively balanced among basic, acidic, and neutral members, hits were strong enriched in neutral compounds and strongly depleted in basic fragments. Neutral fragments were exactly what was sought by Daniel Cheney (Bristol-Meyers Squibb), who discussed successful efforts to replace a basic amidine moiety in Factor VIIa inhibitors. And Brad Jordan (Amgen) discussed the successful application of 19F NMR screening to find fragments that could be linked to previously discovered inhibitors to obtain selective picomolar inhibitors of BACE1.

Alex Waterson (Vanderbilt) started the first afternoon session by discussing how fragments had been successfully applied to RPA, RAS, and MCL-1. In the last case, the best compounds now have dissociation constants in the picomolar range, are active in cells, and show activity in xenograft models. IND-enabling studies are slated to begin as early as this year, with the hope of developing a cousin of venetoclax. Inna Krieger (Texas A&M) described how fragments could be used to understand the mechanism of M. tuberculosis malate synthase, while Dawn George (AbbVie) described selective (but inexplicably toxic) PKCθ inhibitors, which are now being made available to researchers to probe the biology. Finally, Damian Young (Baylor) gave an update on his sp3-carbon enriched fragments. Jane had mentioned that following up on hits with multiple stereocenters was not always easy, but Damian’s DOS approach efficiently and systematically yields each possibility. Whether these will meet the Safran-Zunft challenge remains to be seen.

The last session was focused on success stories. Michael Mesleh (Broad Institute) discussed Cubist’s bacterial DNA gyrase inhibitors. Marion Lanier (Takeda) described how fragment screening and careful medicinal chemistry led to a low nanomolar, selective inhibitor of BTK. With a molecular weight of just 318, the molecule is scarcely larger than a Texas fragment, and has good pharmacokinetics and activity in a rat arthritis model. And Yi Liu (Kura) discussed the optimization of covalent KRAS inhibitors originally discovered using Tethering.

This was my first visit to Houston, and I was struck by the number of researchers who had relocated from around the world, particularly from (previously) large(r) pharma companies. Whenever scientists meet the talk often turns to funding shortages, but not here: everyone seemed to have plenty of money and resources, and one of the organizers announced that he was trying to fill several positions. This was the first major fragment meeting in Texas but likely not the last – there is talk of turning it into a recurring event. And there are still several good upcoming events this year; early registration for FBLD 2016 closes in just a few weeks.

23 May 2016

Calculating hotspots in detail

In the eight years since Practical Fragments first started, Moore’s law has held strong and computational power has increased accordingly. Last year we described how tools such as FTMap can be used to identify hot spots – regions on proteins where fragments are most likely to bind. Although FTMap is quite successful at identifying these, it is less able to point to specific interactions (such as hydrogen bond donors or acceptors) that are likely to drive binding. In other words, computational chemists have become adept at identifying where fragments might bind but lag in predicting how. A new paper in J. Med. Chem. by Chris Radoux at the Cambridge Crystallographic Data Centre and collaborators at UCB and the University of Cambridge addresses this challenge.

The approach starts with a set of three simple molecular probes: toluene, to look for hydrophobic interactions; aniline, to look for hydrogen bond acceptors; and cyclohexa-2,5-dien-1-one, to look for hydrogen bond donors. These probes are larger than those (such as ethanol) used in many other programs, the idea being that too-small molecules might find hot spots so small as to be useless. Indeed, with 7 non-hydrogen atoms, these probes are near the low end of the consensus size for fragments.

Calculations are performed on protein structures – either with no ligand bound or with a bound ligand computationally removed – to determine whether each surface atom of the protein is a hydrogen bond donor, acceptor, or hydrophobic, as well as how exposed the particular atom is. The three probes are then mapped onto the proteins to look for favorable interactions. Regions where multiple probes can bind are scored higher, with hotspots defined as those regions of the protein having the highest scores. The type of probe with the highest score also describes what type of interactions are likely to be favorable at various regions within a given hot spot. Although the researchers note that multiple software packages could be used for these calculations, they used a program called SuperStar, and calculations took just a few minutes on an ordinary laptop.

To validate the approach, the researchers used a previously published data set (discussed here) of 21 fragment-to-lead pairs against a variety of proteins for which crystal structures and binding affinities were available. In general, the method was able to identify the fragment binding site quite effectively; the one outright failure was on the fragment with the lowest affinity, which also had poorly resolved electron density in the crystal structure. Importantly, the fragments tended to have the highest scores, with added portions of the leads scoring lower. This data set was used to calibrate the scoring system for identifying hot spots, as well as specific molecular interactions within each hot spot.

Having thus validated the approach, the researchers took a more detailed look at two published fragment-to-lead programs for protein kinase B and pantothenate synthetase. In both these cases, group efficiency analyses had previously been performed to establish which portions of the ligands contributed most significantly to binding. Gratifyingly, the computations correctly predicted these.

Overall this approach appears promising. At a minimum, it is another tool for assessing the ligandability of potential targets. More significantly, by highlighting the hottest bits of hot spots, it could be useful for medicinal chemists trying to optimize and grow fragments and leads. Unfortunately, as currently described, the process will require a skilled modeler. It would be nice if the authors built a simple web-based interface for people to upload pdb files for analysis, as is the case for FTMap. Also, all the data presented are retrospective – a prospective example would be the true test. Does anyone have experience to share?

16 May 2016

Fragments vs JAK – but phototoxicity

The four members of the JAK family of kinases have received plenty of attention due to their role in inflammation. Two drugs that inhibit these targets, tofacitinib and ruxolitinib, have been approved for rheumatoid arthritis and myelofibrosis, respectively. Psoriasis is another possible indication, but for this disease a topical drug might be useful, particularly one with low systemic bioavailability. The search for such molecules is the subject of a paper recently published in ACS Med. Chem. Lett. by Andrea Ritźen and colleagues at LEO Pharma.

The researchers screened 500 fragments at 100 µM each using surface plasmon resonance (SPR) against JAK2. Hits were then tested in a biochemical assay against JAK1; in general, there was good correlation, suggesting a (desirable) lack of selectivity between the two family members. One of the more attractive hits was compound 1, which was characterized crystallographically bound to JAK2.


Compound 1 is an indazole, which is often seen in kinase inhibitors binding to the so-called hinge binding site where the adenine of ATP binds. Other indazoles have previously been reported as JAK2 inhibitors. Nonetheless, the sulfonamide moiety of compound 1 provides an interesting new vector. Moreover, a search of published structures suggested that adding a phenol moiety could make additional contacts to the proteins. This led to the design and synthesis of compound 2, which showed a dramatic boost in potency as well as measurable cell activity. Further optimization led ultimately to compound 34, with low nanomolar biochemical activity and mid-nanomolar cell activity. Compound 34 was also reasonably selective in a panel of 20 kinases.

A topical drug needs to be stable in sunlight, but unfortunately compound 34 showed phototoxicity. This led to the testing of a few other compounds, revealing that even the initial fragment 1 is unstable over a period of a few hours in simulated outdoor light. Indazole itself seems reasonably stable, suggesting that perhaps adding different substituents could fix the problem.

This is a brief but satisfying example of using published information as well as medicinal chemistry to advance a fragment hit. Although the program does not appear to have led to a drug lead, it is laudable that the researchers describe the photoinstability of the indazoles. With these moieties appearing so frequently in campaigns against kinases, this could be a valuable cautionary tale to others pursuing similar scaffolds.

09 May 2016

Fragments vs KEAP1 – crystallographically

Last week we discussed how soaking crystals in high concentrations of fragments could identify useful molecules. Indeed, last month’s Drug Discovery Chemistry conference featured a talk by Tom Davies (Astex) illustrating the power of this approach. In a recent paper in J. Med. Chem., Tom, Jeffrey Kerns (GlaxoSmithKline), and their collaborators provide the full story.

The researchers were interested in the protein KEAP1, which binds to and blocks the activity of the transcription factor NRF2. Small electrophilic molecules covalently react with KEAP1, causing NRF2 to dissociate and upregulate various cytoprotective genes, which could be useful for a variety of diseases. Indeed, this is at least partly how the approved drug dimethyl fumarate seems to work. However, dimethyl fumarate is quite reactive; a more specific molecule could have a better therapeutic profile, and would certainly be useful for probing the complex biology. With this in mind, the researchers sought a non-covalent inhibitor.

NRF2 interacts with a bowl-shaped “Kelch domain” of KEAP1 largely through electrostatic interactions. Thus, not only is this a challenging protein-protein interaction, coming up with a cell-permeable molecule is all the more difficult. The researchers soaked crystals of the Kelch domain against 330 fragments overnight at concentrations of 5-50 mM. Fragments were observed binding in three adjacent hot-spots, and although no functional activity could be detected, the binding modes suggested a path forward.

Growing from fragment 1 toward a hot-spot occupied by an aromatic fragment (magenta below) led to compound 4, with detectable activity in a fluorescence-polarization assay as well as clear binding in an isothermal titration calorimetry (ITC) experiment. Growing compound 4 toward another hot-spot occupied by a sulfonamide-containing fragment (green below) led to the sub-micromolar compound 6, and further optimization resulted in the low nanomolar compound 7. As is often (but not always) the case, the fragment portion of the final molecule binds in virtually the same position as the initial fragment 1 (blue).


Comparison with the structure of the NRF2 peptide reveals that the carboxylic acid in compound 7 binds in a very similar fashion to a glutamate residue in the peptide, and some of the other peptide contacts are also mirrored, but with very different moieties. Importantly, compound 7 has only a single negative charge, balanced lipophilicity, and fills the binding pocket more effectively than the peptide. These properties translate to good biological activity in multiple different cell-based assays, where the compound causes NRF2 translocation to the nucleus, upregulates appropriate gene expression, and prevents glutathione depletion when cells are treated with an organic peroxide. Although the pharmacokinetics have yet to be optimized, it also shows encouraging activity in a rat model of ozone exposure. Finally, it is reasonably selective in a panel of 49 undesirable off-target proteins. All these properties make this at least an excellent chemical probe.

There are several important lessons in this paper. First, as we’ve noted previously, it is possible to replace a highly polar peptide with a much more drug-like molecule. Second, fragments don’t need measurable affinity to be useful starting points. Crystallography is ideally suited for finding such fragments, though Astex researchers reported a similar success story with NMR last year. Finally, progressing these fragments won’t necessarily be easy, as reflected in the 25 authors on the paper. That said, such intensive efforts can pay off, as illustrated last month by the approval of venetoclax against another difficult protein-protein interaction.

02 May 2016

A strong case for crystallography first

We noted last week that one theme of the recent CHI FBDD meeting was the increasing throughput of crystallography. Crystal structures can provide the clearest information on binding modes, and a key function of standard screening cascades is to whittle the number of fragments down to manageably small numbers for crystal soaking. Only a few groups have used crystallography as a primary screen. A team led by Gerhard Klebe at Philipps-Universität Marburg argues in ACS Chem. Biol. that crystallography should be brought to the forefront.

The researchers were interested in the model protein endothiapepsin. As discussed last year, they had previously screened this protein against a library of 361 compounds using six different methods, and the agreement among methods was – to put it charitably – poor. Nonetheless, many hits that did not confirm in orthogonal assays produced crystal structures when soaked into the protein. Thus emboldened, the researchers decided to soak all 361 fragments individually into crystals of endothiapepsin. This resulted in 71 structures, a hit rate of 20%, higher than any of the other methods (which ranged from 2-17%). Even more shocking, 31 of the fragments were not identified by any of the other methods, and another 21 were only identified by one other method. Thus, a cascade of any two assays would have found, at best, only a quarter of the crystallographically validated hits.

In agreement with other recent work, the fragments bound in multiple locations, including eight subsites within the binding cleft as well as three potentially allosteric sites. Not all of these sites were found using other methods.

But are these fragments so weak as to be uninteresting? To find out, the researchers performed isothermal titration calorimetry (ITC) to determine dissociation constants for 59 of the crystallographic hits. Three of the 21 most potent (submillimolar) binders were not detected by any of the other methods, and another seven were only found by one.

What factors led to this crystallographic bonanza? First, the researchers used the very high concentration of 90 mM for each fragment (in practice sometimes <90 mM because of precipitation). Not surprisingly, solubility was important: 97% of the hits had solubilities of at least 1 mM in aqueous buffer, and the soaking solution contained 10% DMSO as well as plenty of glycerol and PEG. Achieving such high concentrations is harder when multiple fragments are present, and the researchers argue from some of their historical data that the common use of cocktails lowers success rates.

How did different methods compare? Interestingly, functional assays such as high-concentration screening or reporter-displacement assays fared best, while electrospray ionization mass-spectrometry (ESI-MS) and microscale thermophoresis (MST) were close to random. This is in marked contrast to other reports for ESI-MS and MST, and the researchers are careful to note that “the choice and success of the individual biophysical screens likely depend on the target and expertise of the involved research groups.”

Primary crystallographic screening was an early strategy at Astex, and although this may not have been fully feasible 15 years ago, it seems they were on the right track. Of course, not all targets are amenable to crystallography, and not everyone has ready access to a synchrotron beam with lots of automation. But for those that are, it might be time to drop the pre-screens and step directly into the light.

25 April 2016

Eleventh Annual Fragment-based Drug Discovery Meeting

The first major fragment event of 2016, CHI’s Drug Discovery Chemistry, was held last week in San Diego. FBDD was the main focus of one track, and fragments played starring roles in several of the others as well, including inflammation, protein-protein interactions, and epigenetics. Also, for the first time this year the event included a one-day symposium on biophysical approaches, which also included plenty of fragments.

In agreement with our polls, surface plasmon resonance (SPR) received at least a mention in most of the talks. John Quinn (Genentech) gave an excellent overview of the technique, packed with lots of practical advice. At Genentech fragments are screened at 0.5 mM in 1% DMSO at 10°C using gradient injection, which permits calculation of affinities and ligand efficiencies directly from the primary screen. Confirmation of SPR hits in NMR is an impressive 80%. A key source of potential error in calculating affinities is rebinding, in which a fragment dissociates from one receptor and rebinds to another. That problem can be reduced by increasing the flow rate and minimizing the amount of protein immobilized to the surface. Doing so also lowers the signal and necessitates greater sensitivity, but happily the baseline noise has decreased by 10-fold in the past decade.

Some talks focused on using SPR for less conventional applications. Paul Belcher (GE) described using the Biacore S200 to measure fragments binding to wild-type GPCRs. In some cases this provided different hits than those detected against thermally stabilized GPCRs. And Phillip Schwartz (Takeda) described using SPR to characterize extremely potent covalent inhibitors for which standard enzymatic assays can produce misleading results. These screens require exotic conditions to regenerate the chip, so it helps that the SensiQ instrument has particularly durable plumbing.

In theory, SPR can be used to measure the thermodynamics of binding by running samples at different temperatures, but John Quinn pointed out that enthalpic interactions dominate for most fragments, so the extra effort may not be worthwhile. Several years ago many researchers felt that enthalpically driven binders might be more selective or generally superior. Today more people are realizing that thermodynamics is not quite so simple, and Ben Davis (Vernalis) may have put the nail in the coffin by showing that, for a set of 22 compounds, enthalpy and entropy of binding could vary wildly simply by changing the buffer from HEPES to PBS! (Free energy of binding remained the same with either buffer.)

Thermal shift assays (TSA or DSF) continued to be controversial, with Ben finding lack of agreement between the magnitude of the shift and affinity, though there was a correlation with success in crystal trials. In contrast, Mary Harner (BMS) reported good agreement between thermal shift and affinity. She also found that it seemed to work better when the fragments bound in deep pockets than when they bound closer to the surface. However, Rumin Zhang (Merck), who has tested more than 200 proteins using TSA, mentioned that some HCV protease inhibitors could be detected despite the shallow active site. Rumin also pointed out that a low response could indicate poor quality protein – if most of the protein is unfolded it might be fine for biochemical assays but not for TSA. Negative thermal shifts are common and, according to Rumin, sometimes lead to structures, though others found this to be the case less often.

What to do when assays don’t agree was the subject of lively discussion. Mary Harner noted that out of 19 targets screened in the past two years at BMS using NMR, SPR, and TSA, 45% of the BMS library hit in at least 1 assay. However, 68% of hits showed up in only a single assay. Retesting these did lead to more agreement, but even many of the hits that didn’t confirm in other assays ultimately led to leads. All techniques are subject to false negatives and false positives, so lack of agreement shouldn’t necessarily be cause for alarm. Indeed, Ben noted that multiple different soaking conditions often need to be attempted to obtain crystal structures of bound fragments.

Crystallography in general is benefiting from dramatic advances in automation. Jose Marquez described the fully automated system at the EMBL Grenoble Outstation, which is open to academic collaborators. And Radek Nowak (Structural Genomics Consortium, Oxford) discussed the automated crystal harvesting at the Diamond Light Source, which is capable of handling 200 crystals per hour. He also revealed a program called PANDAA (to be released soon) that speeds up the analysis of crystallographic data.

Crystallography was used as a primary screen against KEAP1, as discussed by Tom Davies (Astex). A subset of 330 of the most soluble fragments was tested in pools of four, which revealed several hot spots on the protein. Interestingly, an in-house computational screen had not identified all of these hot spots, though Adrian Whitty (Boston University) noted that they could be detected with FTMap. The fragments themselves bound exceptionally weakly, but intensive optimization led to a low nanomolar inhibitor.

Another case in which extremely weak fragments turned out to be useful was described by Matthias Frech (EMD Serono). A full HTS failed to find any confirmed hits against cyclophilin D, but screening by SPR produced 168 fragments, of which six were characterized crystallographically. Although these were all mM, with unimpressive ligand efficiencies, they could be linked or merged with known ligands to produce multiple leads – a process which took roughly one year from the beginning of the screen. Matthias noted that sometimes fragment efforts are started too late to make a difference, and that it is essential to not be dogmatic.

Huifen Chen discussed Genentech's MAP4K4 program. Of 2361 fragments screened by SPR, 225 had affinities better than 2 mM. Crystallography was tough, so docking was used instead, with 17 fragments pursued intensively for six months, ultimately leading to two lead series (see here and here), though one required bold changes to the core. This program is a nice reminder of why having multiple fragment hits can be useful, as the other 15 fragments didn’t pan out.

Finally, George Doherty (AbbVie) gave a good overview of the program behind recently approved venetoclax, which involved hundreds of scientists over two decades. He also described intensive medicinal chemistry which led to a second generation compound, ABT-731, with improved solubility and oral bioavailability.

We missed Teddy at this meeting, and there is plenty more to discuss, so please add your comments. If you did not attend, several excellent events are still coming up this year. And mark your calendar for 2017, when CHI returns to San Diego April 24-26.

18 April 2016

Native mass spectrometry vs SPR

Native state electrospray ionization mass spectrometry (ESI-MS) is, in theory, a fast and easy way to find fragments: just mix protein with fragment, shoot it on the MS, and look for complex. As a bonus, the exact mass tells you whether your fragment is what you think it is (or at least whether it has the right mass). However, published examples are relatively rare, and not always favorable. A new paper in J. Med. Chem. by Tom Peat, Sally-Ann Poulsen, and their colleagues at CSIRO and Griffith University seeks to change this.

The researchers chose the fragment-friendly model protein carbonic anhydrase II (CA II) as their target. They first screened a library of 720 fragments, each at 100 µM, using surface plasmon resonance (SPR). This yielded 7 hits, with affinities ranging from 1.35 to 1280 µM. These seven hits were then assessed by ESI-MS using equimolar concentrations of protein and fragment (10 or 25 µM each). Encouragingly, all seven hits confirmed. Soaking these fragments into crystals of CA II yielded structures for six of them.

This is nice, but of course the real question is how well ESI-MS works as a primary screen. To address this, the researchers chose 70 compounds structurally related to the 7 hits and independently tested these using both SPR and ESI-MS. This yielded 37 hits, of which 24 were detected both by SPR and ESI-MS. In fact, every SPR hit was confirmed by ESI-MS. Of 14 fragments subsequently soaked into crystals of CA II, 7 provided interpretable electron density.

This is impressive, and the researchers note that the level of agreement between SPR and ESI-MS might be better still, since some of the ESI-MS hits did give signals by SPR – they were just weaker than the chosen cutoff (KD ≤ 3 mM). Thus, in contrast to a paper discussed last year, ESI-MS does seem to be a sensitive detection method. In fact, given the low concentration of fragment needed, the researchers suggest that it could be useful for screening fragments with lower solubilities.

So what’s the secret to success? One difference from some previous reports is that the researchers used a 1:1 ratio of protein to fragment. Others have used excess fragment, which could lead to nonspecific binding and aggregate formation. And of course, CA II is a pretty forgiving model protein. I look forward to seeing ESI-MS used as a primary screen on more difficult targets.

12 April 2016

Second fragment-based drug approved

Yesterday the US FDA approved venetoclax (VenclextaTM) for certain patients with chronic lymphocytic leukemia (CLL). This drug, which readers may know more familiarly as ABT-199, was co-developed by AbbVie and Genentech. The drug binds to BCL-2 and blocks its interaction with other proteins.

The first fragment-derived drug approved, vemurafenib, illustrated how quickly FBDD could move: just six years from the start of the program to approval. In contrast, venetoclax is the culmination of a program that has been running for more than two decades; Steve Fesik and his colleagues at Abbott published the X-ray and NMR structure of the protein BCL-xL back in 1996! The original SAR by NMR work was done on this protein, leading to ABT-263, which hits both BCL-xL and BCL-2. Subsequent work revealed that a selective BCL-2 inhibitor might be preferable in some cases, and further medicinal chemistry led to venetoclax.
This drug illustrates the power of fragments to tackle a difficult target by accessing unusual chemical space. It also illustrates creative, fearless, data-driven medicinal chemistry: not only does venetoclax violate the Rule of five, it even contains a nitro group, a moiety red-flagged due to its potential for forming toxic metabolites. This is a useful reminder that in our business rules are more appropriately considered guidelines, to be discarded when necessary.

Clinical results were sufficiently impressive that the drug was given breakthrough status and granted priority review, accelerated approval, and orphan drug designation. The ultimate victory is for the thousands of patients with relapsed CLL who have the 17p deletion on chromosome 17. In the registration trial, 80% of patients showed a partial or complete remission. It is rare to create something that works this well. Congratulations to all who played a role.

11 April 2016

Fragments vs histone demethylases: docking and merging

Tweaking epigenetic machinery is increasingly popular as a therapeutic strategy. Epigenetics often involves modification to proteins – such as histones – that interact with DNA. One common type of modification is methylation of lysine or arginine residues. A couple months ago we highlighted how fragment-based approaches were used to discover inhibitors of a methyltransferase, one of many classes of protein-modifying enzymes that underlie epigenetics. Just as methyltransferases put methyl groups on, demethylases take methyl groups off. In a recent paper in J. Med. Chem., Udo Oppermann, Brian Shoichet, and Danica Fujimori and their collaborators at the University of Oxford and UCSF show that demethylases too can be successfully targeted with fragments. What’s more, the work exemplifies concrete contributions of computational approaches to both identify and advance fragments.

The demethylase KDM4C has been implicated in cancer. This enzyme uses iron, the cofactor α-ketoglutarate (α-KG), and oxygen as part of its mechanism. The researchers ran a computational screen (using DOCK 3.6) of more than 600,000 compounds in the ZINC fragment library. Top-scoring hits were triaged on the basis of novelty and good interactions with the iron atom, and 14 fragments were tested in a functional assay. Remarkably, all of them were active, with 7 showing IC50 values < 200 µM!

Several of the top hits were 5-aminosalicylates such as compound 4. Testing 80 commercial analogs led to low micromolar inhibitors, but these could not be further optimized. Moreover, despite the small size and polarity of these compounds, many of them showed signs of aggregation – a reminder that this type of artifact must always be considered.


Unfortunately, crystallography was also not successful for any of the fragments or analogs. But the researchers noticed that, according to the docking results, fragments such as compound 4 could assume two different binding modes: in one, the carboxylate and phenol interacted with the iron atom, while in the other the carboxylate interacted with lysine and tyrosine residues in the protein. This inspired several ideas for fragment merging, leading to molecules such as compound 45. Additional variations led to mid-nanomolar inhibitors such as compound 35.

As expected, these molecules are competitive with the α-KG cofactor (which normally binds to the iron atom) but not with the peptide substrate. Many also showed encouraging selectivity profiles against other demethylases, though no cell data are reported. Finally, crystallography mostly confirmed the predicted binding models for several of the merged compounds, including compound 35.

This is a lovely example of using computational approaches not just for fragment-finding, but for fragment merging as well. As the authors point out, this was done not to showcase computational methods but because crystallography didn’t initially work. Even in the short lifetime of Practical Fragments, in silico methods have made remarkable progress, and this is another milestone. It will be fun to see further optimization of these molecules.

06 April 2016

Biophysics: not just for fragments

Biophysics and fragment-based drug discovery go together like Nutella and strawberries. Indeed, SAR by NMR ushered in the dawn of fragment-based methods two decades ago, and most fragment-based programs today make use of NMR, SPR, and/or ITC – not to mention X-ray crystallography. Interestingly, the same is not necessarily true for high-throughput screening (HTS) programs. In a recent paper in Drug Discovery Today, Rutger Folmer makes a strong case for engaging biophysics early and often in HTS. He bolsters his argument with more than 20 examples from internal programs at AstraZeneca.

The first descriptions of using NMR to profile HTS hits were not published until several years after SAR by NMR, but they were rather shocking, with up to 98% of hits failing to confirm. Nor is this merely a historical problem, as discussed here. Aggregators, redox cyclers, generically reactive covalent modifiers – all of these are problems not just in fragment screening but in HTS as well. Sometimes the most potent hits are artifacts, particularly for more difficult targets. The key to triaging out pathological actors is to assess binding and not rely solely on inhibition.

That means bringing biophysics into hit profiling at the earliest stages, before trying to optimize fruitless hits. As Rutger points out, it is often difficult to rally colleagues to look at less active molecules after they have wasted months pursuing more potent dead ends.

And biophysics can make an impact even before running screens. Profiling published tool compounds or in-licensing opportunities with biophysical techniques can reveal unwelcome surprises. Testing the output of early HTS pre-screens (7000-10,000 compounds) before a full HTS (2 million compounds at AstraZeneca) can reveal whether an assay is particularly susceptible to false positives. In some cases this can result in reconfiguring the assay, for example by choosing a different detection technology or modifying the protein construct.

A key element to gaining such benefits is organizational commitment. At AstraZeneca, a biophysicist is assigned to a project team immediately after target selection – well before any screens are run. This seems like prudent practice. How many other organizations are doing this?

01 April 2016

An interview with Dr. Saysno

Readers of a certain age may fondly remember the interviews with Dr. Noitall that used to enliven the pages of Science. Sadly, he died a few years back. But his cousin, Dr. Saysno, is still very much alive. Practical Fragments caught up with him at a recent conference in Shutka.

Practical Fragments (PF): Dr. Saysno, you've stated that experts should never be trusted.

Dr. Saysno (DS): Niels Bohr defined an expert as a person who has made all the mistakes that can be made in a very narrow field. If someone has made every possible mistake, how could you possibly trust them?

PF: But don't you think they may have learned from their mistakes?

DS: Balderdash! Hegel was right: the only thing we learn from history is that we learn nothing from history.

PF: What's your opinion of ligand efficiency (LE)?

DS: Ligand efficiency is an abomination! It's mathematically invalid!

Worse, determining the free energy of binding from a dissociation constant is not even wrong: if you change your definition of standard state, you can make ΔG° any positive or negative number you want. Just how relevant do you think your definition of standard state is on the surface of Venus? Or Pluto?

For the same reason, pH is utterly meaningless. You really ought to throw out your pH paper, not to mention your pH meters, since they all assume an arbitrary reference state.

PF: But what about all the researchers who find pH and LE useful?

DS: Usefulness is the last refuge of the scoundrel!

Look, many of the best selling drugs on the market are antibodies, and when you calculate their ligand efficiencies, they are close to zero. How can you have a metric that doesn't work on some of the most important drugs out there?

I only believe in equations that are universal and apply in all situations, unsullied by the physical world. Anything that involves standard states is just mumbo-jumbo.

PF: What do you think of pan-assay interference compounds, or PAINS?

DS: Now that’s a topic that really gets my blood boiling! PAINS were defined on the basis of just six assays. Six assays I tell you!!! [DS vigorously pounds his shoe on the desk.] Just because something hits six assays – or six hundred for that matter – doesn’t mean it will hit the six hundred and first!

PF: But aren't there some chemical substructures that are so generically reactive they should never be used in probes?

DS: Nothing is universal! All molecules are unique, like little snowflakes. If a compound comes up as a hit in your assay, by all means publish it as a chemical probe in the best possible journal, and try to encourage suppliers to start selling it so other people can use and cite your brilliant discovery.

No one has a right to criticize your molecule unless they test it against every single protein in the human body and show that it hits all of them.

When the revolution comes, the imperialist PAINS stooges will be swept into the dustbin of history along with the lackeys of ligand efficiency!

PF: So if you don't trust experts, you don't like metrics, and you can't make generalizations, how can we move forward in science short of deriving every result ourselves from first principles?

DS: That's simple: just ask me!

21 March 2016

Fragments vs bacterial GyrB

Anti-bacterial targets are not common among fragment-based lead discovery efforts. We’ve written previously about AstraZeneca’s work on DNA gyrase, which led to a clinical candidate. In a recent paper in Bioorg. Med. Chem. Lett., Michael Mesleh and collaborators at Cubist and Evotec describe their efforts on this protein.

Bacterial DNA gyrase has two subunits, GyrA and GyrB, and is essential during DNA replication. It is also well-validated, being the target of decades-old antibiotics such as the fluoroquinolones. The researchers started by screening a library of 5643 fragments against Staphylococcus aureus GyrB using STD NMR, yielding 304 hits. These were winnowed down to 46 based on intensity of the STD signal, novelty, and ease of follow-up chemistry. These were then tested using chemical shift perturbations and crystallography. Although several crystal structures of fragments bound to GyrB were obtained, these did not suggest clear ways to advance the hits.

On the other hand, compound 5, which showed only weak binding by NMR and did not result in a crystal structure, was appealing because of its novelty and polarity. The researchers knew that most ligands that interact with GyrB make a pair of hydrogen-bond donor-acceptor interactions, and they used that knowledge to surmise a binding model. This suggested that growing the fragment towards a pair of arginine residues could improve affinity and led to the synthesis of compound 8, with low micromolar activity.


A crystal structure of a related molecule confirmed the model and also suggested that removing the methyl group would stabilize a more planar conformation better matched to the binding site. Doing so (compound 9) yielded a ten-fold boost in potency. (This methyl is also a nice example of Teddy’s “Sauron Atom”). In a separate paper published last year, the researchers further optimized this molecule to compound 2, with low nanomolar potency and activity in animal models.

Several things stand out about this paper. First, the researchers were willing to pursue a fragment with an affinity lower than other hits. Second, careful modeling and conformational analyses were critical in advancing the molecules. Finally, crystallography was not used in the initial fragment growing. Of course, it helped that the researchers were working with a well-characterized protein amenable to modeling. Still, it is nice to see another example of advancing fragments in the absence of experimentally-determined structures.

14 March 2016

EthR revisited again: fragment merging this time

Fragment linking, growing, and merging: these are the main methods for enhancing affinity. Two years ago we highlighted a fragment screening effort against the tuberculosis target EthR, which involved fragment linking. A few months later we discussed fragment growing against the same target by a different group. Now the first group, led by Chris Abell at the University of Cambridge, has published a new paper in Org. Biomol. Chem. describing fragment merging.

In the original paper, a thermal shift assay had led to the discovery of a few dozen fragments, several of which were characterized crystallographically bound to EthR. In some cases, two molecules of the same fragment could bind in the large lipophilic cavity of the protein and block binding to DNA, as assessed by SPR. Capitalizing on this, two copies of compound 1 were linked together to generate a micromolar binder.


In the new paper, the researchers tried merging compound 1 with another fragment, compound 2, which also binds at two positions within the protein. Several merging strategies were attempted, and although they all stabilized the protein against thermal denaturation and could be characterized crystallographically bound to the protein, most were no better at blocking DNA binding than the original fragments. Compound 5, however, did show enhanced activity, and was the subject of additional SAR. This led to compound 15, which showed low micromolar binding by isothermal titration calorimetry (ITC) and functional activity. (Oddly, compound 1 appeared to bind considerably more tightly by ITC than suggested by its functional activity, perhaps a result of having two binding sites.) The crystal structure of the optimized, merged compound bound to EthR revealed that compound 15 binds as expected (gray), overlaying with one copy each of compound 2 (magenta) and compound 1 (cyan). 

Unfortunately, aside from compound 1, none of the molecules showed activity in a cell-based assay. The researchers propose that this is due to poor permeability across the notoriously impenetrable envelope of the mycobacterial envelope. All in all this is a nice story, as well as a sobering reminder that while potency is important, it is just one of many properties that need to be optimized.

As to the question of whether one should apply growing. linking, or merging, a single case study does not really permit generalization. However, it is satisfying that all three techniques can lead to early leads.

07 March 2016

Fragments vs choline kinase alpha

Fragments and kinases have a long and successful association, as demonstrated by nearly half of FBLD-derived clinical candidates. Most of the attention has been on protein kinases, which transfer phosphate groups to other proteins. But there are more kinases out there, and in a recent paper in J. Med. Chem., Stephan Zech and colleagues at Ariad describe how they developed inhibitors against choline kinase α (ChoKα), a potential anticancer target involved in phospholipid synthesis.

A screen of known kinase inhibitors came up largely empty, so the researchers used STD NMR to screen a library of 1152 diverse, rule-of-three compliant fragments in pools of five, each at 3 mM. This yielded 55 hits, which were then tested in a fluorine-detected NMR (FAXS) assay to see whether they could displace molecules that bind in either the ATP or substrate binding sites. These experiments suggested that 13 fragments bind in the choline binding site while 21 bind in the ATP site; the remaining 21 either bind elsewhere or are artifacts.

Most of the fragments showed minimal activity in functional assays, but compound 11 was an exception. SPR confirmed binding, though it also seemed to bind to an unrelated protein and displayed super-stoichiometric behavior at high concentrations. Nonetheless, it could successfully be soaked into crystals of ChoKα, and the resulting structure revealed that it binds deep in the choline-binding pocket, with the terminal methyl group in a small pocket at the bottom. This is also consistent with STD epitope mapping of a related fragment, which showed that the azepane ring was closely associated with the protein.

An initial search for commercial analogs followed by several rounds of medicinal chemistry led to compound 43, with low micromolar potency, and several more rounds of optimization led to compound 65, with nanomolar binding (by SPR) and inhibition. A crystal structure of this molecule bound to the enzyme revealed that the fragment binding mode is roughly conserved, while the added basic moiety binds close to several acidic residues near the surface of the binding pocket. A very closely related molecule showed low micromolar activity in several cell-based assays (data for compound 65 is not reported). 
This is an interesting paper for several reasons. First, it reports the successful use of fragment screening against an unusual target. Second, although multiple fragments were found to bind in the ATP-binding site (a productive starting point for many conventional kinases), these fragments could not be optimized. On the other hand, a fragment that binds in the choline-binding site could rapidly be improved to nanomolar inhibitors. Third, although fragment 11 did show some red flags, it was ultimately optimizable – a reminder that some misbehavior in a fragment should not necessarily disqualify it. Finally, the iterative and structure-based nature of the medicinal chemistry – which is well beyond what this brief blog post can cover – makes a nice case study in fragment growing. Of course, the final molecule still has high hurdles to surmount, and it will be fun to see the story progress.