14 October 2019

Fragments vs Mycobacterium TrmD – and native ESI-MS revisited

The Mycobacterium genus of bacteria contains more than 190 species, including the pathogens that cause leprosy and tuberculosis. Mycobacterium abscessus (Mab) is not as famous, though it is a growing concern for people living with cystic fibrosis. The enzyme tRNA (m1G37) methyltransferase (TrmD) is essential for Mycobacterium growth. In a recent open-access paper in J. Med. Chem., Chris Abell, Tom Blundell, Anthony Coyne, and collaborators at University of Cambridge, Royal Papworth Hospital, and the US NIH describe potent inhibitors of this target.

The researchers screened 960 fragments using differential scanning fluorimetry (DSF). The 53 hits were soaked into crystals of TrmD, and 27 yielded electron density – all in the cofactor (S-adenosyl methionine, or SAM) binding site. One weak but particularly ligand-efficient fragment was advanced through fragment growing to low micromolar affinity, but further improvements proved challenging.

A powerful feature of FBLD is that screens often yield multiple starting points, and this proved useful here. Specifically, compound 16 bound in the pocket where the adenine of SAM normally sits, while compound 20 bound nearby in the ribose pocket. Merging these led to compound 23,  and a crystal structure suggested that the indole nitrogen would be a good vector for fragment growing, resulting in compound 24c. Addition of a positively charged moiety to engage a couple glutamic acid residues led to compound 29a, a mid-nanomolar compound as assessed by isothermal titration calorimetry (ITC).


Several of the molecules were characterized by native electrospray ionization mass spectrometry (ESI-MS) – an interesting but somewhat controversial technique in which protein-ligand complexes are gently ionized and detected in the gas phase. For large molecules such as proteins, even the gentlest ionization will generate highly charged species; in this case the dimeric TrmD protein had between 13 and 17 positive charges. Each of these charge states represents a different species, or perhaps several since the positive charges could be on different regions of the protein.

Compound 24c has modest affinity, but encouragingly, native ESI-MS at 100 µM ligand revealed two bound ligands, as expected for a protein dimer. However, for the much more potent compound 29a, native ESI-MS at 100 µM ligand revealed two bound ligands for a high charge state but largely unbound protein for a lower charge state. The researchers speculate this could be due to dissociation of the positively charged ligand in the gas phase.

Overall this paper is a nice exercise in fragment merging and growing, guided by crystallographic data. Some of the molecules elaborated from 24c (including 29a) inhibited growth of Mycobacteria species, though further improvements in affinity will be needed, and no DMPK properties are provided. Still, it's good to see solid work being done on new antibacterial agents.

The disconnect between observed affinity by native ESI-MS and that observed by ITC does make one cautious about the utility of the former technique. We’d welcome comments on your experiences with it.

07 October 2019

The story of erdafitinib – abridged

In April we celebrated the FDA approval of erdafitinib for certain types of urothelial carcinomas with genetic alterations in the FGFR2 or FGFR3 genes. This marked the third launch of a fragment-derived drug. However, although erdafitinib was mentioned in a 2016 review, the fragment origins have remained obscure. The full story has yet to be published, but Chris Murray (Astex), David Newell (Newcastle University) and Patrick Angibuad (Janssen) have recently published brief highlights in MedChemComm.

The story begins all the way back in 2006, with a collaboration between Astex and the Northern Institute of Cancer Research at Newcastle University. The four fibroblast growth factor receptors (FGFR1-4) are kinases whose aberrant activation was known to be associated with multiple cancers. In those days most FGFR inhibitors also inhibited VEGFR2, which was linked to unacceptable side effects. A fragment screen in 2008 led to a series of potent, selective molecules and enticed Janssen to join the collaboration. This particular lead series was ultimately discontinued, though, as discussed below, it turned out to be important.

Meanwhile, fragment 1 was identified, characterized crystallographically bound to FGFR1, and improved to low micromolar compound 2. Virtual screening and medicinal chemistry led to compound 4, with improved selectivity for FGFR3 over VEGFR2. Superimposing the crystal structure of this compound with the previous series suggested building off the aniline nitrogen to improve potency (and, one might assume, solubility). This ultimately led to erdafitinib.


The researchers highlight the cooperative nature of this project. “Each team brought their specific expertise and most importantly, wisely and collaboratively capitalized on each other’s strengths.”

The publication also illustrates that success can take time – thirteen years in this case. This is not unusual for drug discovery, and is in fact halfway between the remarkably fast six years for vemurafenib, the first fragment-based drug approved, and the two decades required for venetoclax, the second.

But in the end good things are worth waiting for, and the 15-20% of metastatic bladder cancer patients with an FGFR alteration now have a new treatment option. Erdafitinib is being tested in at least a dozen other clinical trials for various cancers. Practical Fragments wishes all the participants the best of luck.

30 September 2019

Combining fragments and HTS hits to target PHGDH

Boehringer Ingelheim has been on something of a tear reporting new chemical probes for difficult targets – see here for their NSD3 inhibitor and here for their RAS inhibitor. This is part of an ambitious effort to develop probes for the entire human proteome by 2035. In a new paper published in J. Med. Chem., Harald Weinstabl and collaborators at BI and Shanghai ChemPartner describe the discovery of BI-4924, a potent inhibitor of phosphoglycerate dehydrogenase (PHGDH).

The enzyme is the rate-limiting step in serine synthesis, and has been implicated in multiple types of cancers. However, metabolic enzymes such as PHDGH are particularly challenging drug targets for several reasons: cofactors such as NADH are present at high concentrations in cells, the substrate binding pocket is both shallow and polar, and one often needs near complete inhibition to see an effect. Thus, the researchers chose multiple approaches.

An STD NMR fragment screen was conducted against the apo form of the protein (250 µM fragment and 20 µM protein) to find compounds that would bind in the NAD+-binding site. Of 1860 fragments screened, 60 hits were identified, and 19 of these gave measurable dissociation constants in an SPR assay and were selective against two other proteins. Compound 9 was found crystallographically to bind in the adenine pocket of the NAD+-binding site. Fragment growing was challenging due to the “kinked shape” of this pocket: elaborated molecules tended to point out of the pocket into solvent. Careful design led to modest improvements in potency (compound 11), and adding a negatively charged moiety led to potent molecules such as compound 43. To avoid problems with permeability, the researchers tried various uncharged bioisosteres, but these were not tolerated. Interestingly, crystallography revealed that the carboxylic acid does not seem to make specific interactions with the protein; its necessity may be due to long-range electrostatic interactions with multiple nearby basic residues.


In parallel, a biochemical HTS screen of more than a million molecules yielded 27,000 hits, which were whittled down to 11,250 that confirmed and didn’t interfere with the assay. Removing PAINS and large, lipophilic molecules narrowed the set to 4750 compounds. Further rigorous assessment included biophysical methods, as recently recommended. Aware of the potential for metal contaminants to give false positives, the researchers examined select samples with inductively coupled plasma mass spectrometry and found that some contained mercury or copper, which inhibited the enzyme. Ultimately 77 hits were validated with dissociation constants better than 300 µM, including compound 8, which crystallography revealed binds in a similar manner to fragment 9.

Combining information from both campaigns and growing to engage an aspartic acid side chain ultimately led to BI-4924. This compound is soluble, stable, and selective against other dehydrogenase enzymes. Unfortunately, the carboxylic acid moiety does indeed impart low permeability, and perhaps because of this the molecule has only low micromolar activity in cells. However, the ethyl ester (BI-4916) transiently accumulates in cells and modulates serine levels.

Unfortunately, the researchers appear to have been scooped; as they politely note, “subsequently, these findings were independently confirmed….” As it stands BI-4916 is too unstable for use in vivo. Still, it could be useful for further unraveling the biology around serine biosynthesis and its role in cancer cells, and the paper itself stands as a nice example of structure-based lead design combining information from multiple sources.

23 September 2019

Seventeenth Annual Discovery on Target

Just as April brings CHI’s Drug Discovery Chemistry in San Diego, September brings CHI’s Discovery on Target in Boston, and last week saw more than 1100 attendees attend 14 tracks over three days, along with associated training seminars and short courses. Fragments made appearances throughout.

Perhaps the most notable new development was an entire session devoted to covalent fragments. We spent three blog posts in June covering five papers on this topic, so this was a timely addition.

Eranthie Weerapana (Boston College) described proteomics methods for analyzing cysteine modification in cells. (Some of this is similar to Ben Cravatt’s work, which we discussed here.) She noted that many mitochondrial proteins have low abundance and are thus hard to detect, but by isolating the mitochondria she has been able to observe about 1500 cysteines on 500 proteins. Selenocysteine-containing proteins are even less common and can thus be lost in the noise, but by lowering the pH these rare beasts can be labeled selectively.

Alexander Statsyuk (University of Houston) gave two wide-ranging talks, and he noted that (beyond acrylamides) there are about 50 warheads that have not been widely explored. We’ve previously covered his work with fragments containing the 4-aminobut-2-enoate methyl ester, and Katrin Rittinger (Francis Crick Institute) discussed her recent use of a small library of just 104 of these to find a covalent inhibitor of LUBAC. And on the subject of very recent papers, I presented work from Carmot and Amgen on the discovery of covalent KRASG12C inhibitors.

You know a field is becoming popular when suppliers start selling reagents, and this is certainly the case for covalent fragments: Enamine and Life Chemicals both sell covalent fragment libraries. If you’ve had experience with them or others, please leave comments!

Natalia Kozlyuk (Vanderbilt) gave a nice presentation that emphasized some of the challenges that can arise in FBLD. Screening 14,000 fragments against the protein RAGE by NMR resulted in a number of hits, and crystallography revealed that a couple of these bind just 4 Å apart. Linking these together with variable-length linkers led to one molecule that bound as expected, while in another one of the linked fragments bound in a third site. Unfortunately, even the best dimeric molecules are quite weak, and they also cause the protein to precipitate. This appears to be a different mechanism from “classic” small molecule aggregation, in which the small molecules first form aggregates that block protein activity, though it is not unprecedented.

In a similar vein, Stijn Gremmen and Jan Schultz (ZoBio) described work done with Gotham Therapeutics to discover inhibitors of the methyltransferase complex METTL3/METTL14. HTS-derived compounds caused aggregation of protein as assessed by size exclusion chromatography and multiangle light scattering, but a fragment screen followed by optimization led to potent, well-characterized molecules.

Continuing the theme, Beth Knapp-Reed (GSK) described an HTS assay against the anti-cancer target LDHA in which 1.9 million compounds yielded 560 hits, almost all of which turned out to be false positives due to oxalic acid contamination. Fragment screening was more productive, yielding 16 crystallographically validated hits, and the researchers were able to improve the affinity more than 10,000-fold. And Puja Pathuri discussed successful efforts at Astex to discover ERK1/2 inhibitors (see here for more details).

Finally, last year we noted the increasing number of talks on PROTACs and targeted protein degradation. This year for the first time the conference held a full day and a half long program on the topic. PROTACs typically consist of two-component molecules in which one piece binds to a target of interest and the other piece binds to a protein called an E3 ligase which ultimately causes proteolytic degradation of the target. For example, Michael Plewe described how he and his colleagues at Cullgen have modified the fragment-derived vemurafenib to target an oncolytic mutant form of BRAF.

One of the interesting features of targeted protein degradation is that a high affinity ligand is not always necessary: Craig Crews (Yale) described how an 11 µM p38α ligand was sufficient to degrade 99% of the protein in cells. And fragments can help not just with target proteins, but in identifying new E3 ligase ligands as well. Carles Galdeano (University of Barcelona) described how fragment screening has yielded a couple sub-micromolar affinity ligands of an E3 ligase called Fbw7.

In the interest of time I’ll stop here, but if you were particularly struck by anything please mention it in the comments. And if you missed the meeting, be sure to mark September 15-18 on your calendar for next year!

16 September 2019

Fragments find flexibility in fascin 1

Protein flexibility can be both an opportunity and a barrier – quite literally, when a solid wall of protein seems to block opportunities for fragment growing. But like secret doorways, protein domains can yawn open to expose tunnels and cavities. An example of this was published earlier this year in Bioorg. Med. Chem. Lett. by Stuart Francis and collaborators at the CRUK Beatson Institute.

The researchers were interested in fascin 1, which increases the invasiveness of multiple cancers by helping pack filamentous actin into bundles important for cell migration. The team began their search for an inhibitor by performing a surface plasmon resonance (SPR) screen of 1050 fragments, generating an impressive 53 hits. Although a number of these were reported to bind to multiple sites on the protein, only one is discussed.

Compound 1 binds between two domains of the protein in a pocket that does not exist in unbound fascin. However, the fact that the pocket completely envelopes the fragment “hampered attempts to develop the series.” Fortunately, the researchers were following the patent literature, and when they characterized compound 2 (not a fragment, and reported by a different group), they discovered that while it binds in the same pocket as compound 1, additional conformational changes occur to accommodate the larger molecule.


Next, the researchers looked for analogs of compound 2 and also performed a virtual screen against the enlarged pocket. Of 110 commercial compounds tested, three gave dissociation constants better than 100 µM, including compound 3. The researchers recognized that compound 3 lacks the halogens found on both the original fragment and compound 2, and by adding these they were able to improve the affinity more than ten-fold. Further optimization ultimately led to BDP-13176, with mid-nanomolar affinity by SPR and ITC as well as activity in a functional assay. Although the molecule has reasonable solubility and stability against liver microsomes, it has low permeability and high efflux.

This is a nice structure-based design story, and while the fragment did provide some information about the binding site, one could argue that the real breakthrough came with determining the binding mode of compound 2. Indeed, without this information, it would have been all too easy to assume that the pocket was not ligandable. This is an important reminder that crystal structures usually only reveal one form of a protein. The system is also a good test case for modelers who want to see how their algorithms perform against a dynamic protein. Breakthroughs are often unexpected, and it is always worth making a few compounds that don’t look like they’ll fit.

09 September 2019

Fragments vs sepiapterin reductase, via 19F NMR

It has been two years since we’ve had a post devoted to fluorine NMR. Though I don't share Teddy’s “fetish” for 19F-based screening, I do think the technique can be quite powerful, as demonstrated in a recent J. Med. Chem. paper by Jo Alen, Markus Schade, and their colleagues at Grünenthal GmbH.

The researchers were interested in sepiapterin reductase, which is abbreviated as SPR but which I’ll spell out to avoid confusion with surface plasmon resonance. This enzyme performs the last step in the production of tetrahydrobiopterin, an essential cofactor for multiple enzymes, including some that synthesize neurotransmitters and produce nitric oxide. Sepiapterin reductase has been proposed as a target for non-opioid-based pain medications.

The primary assay involved displacement of a fluorine-containing inhibitor that binds in the substrate site of the enzyme; thus, the researchers could use 19F NMR without requiring fluorinated fragments. A total of 4750 fragments were screened at 250 µM, initially in pools of 12. The 26 hits were then tested in an enzymatic assay, and 21 showed activity better than 75 µM. The best, compound 3, was sub-micromolar.

Crystal structures were obtained for six compounds, including compound 3, and all bound in the substrate pocket as predicted from the original displacement assay. The phenolate of compound 3 makes hydrogen bonds to two critical catalytic residues. Not surprisingly, capping this moiety with a methyl group led to an inactive compound. The researchers made dozens of variants, but aside from compound 26, most of these were disappointingly less active. Compound 26 does show good solubility and permeability, though no cell data are provided, and the phenol will likely be glucuronidated in vivo.

This is a nice story that illustrates a not-infrequent frustration: after identifying the initial nanomolar hit from a small library, the researchers likely thought improving potency still further would be easy. Instead, it took more than 60 analogs just to gain another order of magnitude. That said, 57 nM is nothing to sneeze at. And this situation is certainly preferable to the more common alternative of starting with a weak fragment that remains weak no matter what you do to it!

02 September 2019

Fragment vs hematopoietic prostaglandin D2 synthase: a chemical probe

Six years ago we highlighted work out of GlaxoSmithKline and Astex describing some of their efforts to find inhibitors of hematopoietic prostaglandin D2 synthase (H-PGDS), an enzyme implicated in asthma, lupus, and multiple other inflammatory diseases. A recent paper in Bioorg. Med. Chem. by David Deaton and collaborators describes another chemical series from that program.

As noted in the earlier publication, the researchers were graced with 76 crystallographic fragment hits, of which compound 1a was a weak but ligand-efficient member. Several other fragments that bound in the same region contained a methoxy group, and a quick survey of commercially available analogs led to compound 1b, with a nice bump in potency. Replacing the nitrile with an amide (compound 1d) improved activity further.


What do you do when you’ve got an amide? Make lots of them! This was effective, and the researchers show more than 60 analogs leading to low nanomolar inhibitors such as compound 1bg. One problem with amides is that they can be enzymatically cleaved in vivo, but this challenge was surmounted by tweaking the substituents.

The researchers also noted that the compounds are quite electron-rich, potentially leading to phototoxicity, and in fact some of the molecules degraded upon exposure to UV light. Also, methoxy substituents are prone to dealkylation in vivo. The researchers solved both problems by replacing the methyl with a difluoromethyl group, leading to GSK2894631A.

This molecule was put through a battery of tests and found to be orally bioavailable (at least in mice) with good pharmacokinetics. It is selective against related enzymes as well as a larger panel of receptors and transporters. Encouragingly, the compound showed potent activity in a mouse model of acute inflammation. In other words, this looks to be a useful chemical probe to explore the biology of prostaglandin signaling.

This is a nice story on several levels, and it also illustrates an important point that younger researchers and folks in academia sometimes overlook: it can take ages before work done in industry sees the light of day. Indeed, one of the authors on the paper left Astex more than five years ago, so the work described is likely several years older than that. Still, better late than never. Good science is always worth publishing, even if – like another paper we recently highlighted – it happened some time ago.

26 August 2019

Biophysics beyond fragments: a case study with ATAD2

Three years ago we highlighted a paper from AstraZeneca arguing for close cooperation of biophysics with high-throughput screening (HTS) to effectively find genuine hits. A lovely case study just published in J. Med. Chem. shows just how beneficial this can be.

Paul Bamborough, Chun-wa Chung, and colleagues at GlaxoSmithKline and Cellzome were interested in the bromodomain ATAD2, which is implicated in cancer. (Chun-wa presented some of this story at the FragNet meeting last year.) Among epigenetic readers, bromodomains are usually quite ligandable, but ATAD2 is an exception, and when this work began there were no known ligands.

Recognizing that they might face challenges, the researchers started by carefully optimizing their protein construct to be stable and robust to assay conditions. This included screening 1408 diverse compounds, none of which were expected to bind. Disturbingly, a TR-FRET screen at 10 µM yielded a 4.1% hit rate, suggesting many false positives. Indeed, when an apparently 30 nM hit from this screen was tested by two-dimensional 15N-1H HSQC NMR, it showed no binding. The researchers thus made further refinements to the protein construct to improve stability and reduce the hit rate against this “robustness set.”

This exercise illustrates an important point: make sure your protein is the highest quality possible!

Having done this, the researchers screened 1.7 million compounds and obtained a relatively modest 0.6% hit rate. Of these 9441 molecules, 428 showed dose response curves and were tested using SPR and HSQC NMR. In the case of SPR, the researchers also tested a mutant form of the enzyme that was not expected to bind to the acetyl-lysine mimics being sought. Most of the hits did not reproduce in either the SPR or the NMR assays, and at the end of the process just 16 closely related molecules confirmed – a true hit rate of just 0.001%!

Compound 23 is the most potent molecule disclosed, but the researchers mention a manuscript in preparation that describes further optimization. The compound shows promising selectivity against other bromodomains; it certainly doesn’t look like a classic bromodomain binder. X-ray crystallography revealed that the binding mode is in fact different from other bromodomain ligands. Trimming down compound 23 produced compound 35, which shows reasonable activity and ligand efficiency.

This paper nicely demonstrates the power of biophysics to discern a still small signal in a sea of noise. As the researchers note, PAINS filters and computational approaches would not have worked due to the sheer diversity of the misbehaving compounds. (That said, if the library had been infested with PAINS, the false positive rate would have been even higher!)

The paper is also a good argument for FBLD. Compound 35 is probably too large to really qualify as a fragment, but perhaps related molecules could have led to this series. And GSK also discovered a different series of potent ATAD2 inhibitors from fragments, which Teddy wrote about.

19 August 2019

Fragments in the clinic: Navoximod

A good fragment can be a useful starting point for creative scientists, no matter where it comes from. Indeed, we recently described how fragment-derived molecules discovered at one institution were used to discover a clinical compound at another. A similar story from Mario Mautino and collaborators at NewLink Genetics and Genentech has recently appeared in J. Med. Chem.

The researchers were interested in the protein indoleamine 2,3-dioxygenase 1 (IDO1), whose immunosuppressive properties may allow cancer cells to survive and proliferate. They took their starting point from a 2006 publication of a crystal structure of compound 1 bound to IDO1. Structure-based design led to potent molecules such as compound 11, but (not unexpectedly) these phenol-containing molecules tended to have high clearance.


Abandoning the phenols, the researchers instead began rigidifying the series, leading to modest improvements in potency as exemplified by compound 37. Modeling suggested that fragment growing could be productive, and this was confirmed by compound 46.

IDO1 is a heme-containing enzyme, and in fact the imidazole moiety of compound 1 interacts with the heme iron. Other human heme-dependent enzymes include the CYPs, and since these are often involved in metabolizing drugs, it is important to avoid inadvertently inhibiting them. The researchers spent considerable effort further optimizing their molecules for potency, selectivity against CYPs, and metabolic stability. This is described in extensive detail – it makes for an excellent case study in lead optimization. (The separation of stereoisomers and absolute assignment is an impressive piece of work.) Ultimately they arrived at navoximod (NLG-919 or GDC-0919), which showed activity in mouse xenograft models and suitable properties for oral dosing in humans, and entered the clinic in 2014.

Unfortunately, although several IDO1 inhibitors have entered clinical development, those that have made it to late stage trials have proven disappointingly ineffective. Whether some combination with other drugs or a new biomarker will reinvigorate interest in this target, or whether, like BACE1, a good idea meets an unforgiving reality, remains to be seen. There is still no shortcut to avoid the massively expensive experiment of putting a drug into the clinic to test a therapeutic hypothesis.

12 August 2019

Achieving maximum diversity with minimum size

One theoretical advantage of fragment-based drug discovery is the ability to efficiently explore chemical space: there are vastly fewer possible fragment-sized molecules than lead-sized molecules. That said, even fragment space is daunting; the number of possible molecules with up to 17 non-hydrogen atoms is about three orders of magnitude larger than the largest computational screen. Maximizing diversity is thus a key goal in designing fragment libraries, but how do you actually do this? A new open-access paper in Molecules by Yun Shi and Mark von Itzstein at Griffith University provides a practical new approach.

As the researchers point out, diversity itself can be a slippery concept. Functional diversity (ie, what targets are bound) is important but hard-won knowledge. Physicochemical diversity is by definition limited for fragments. That leaves structural diversity, as defined by “molecular fingerprints.” These can be as simple as the presence or absence of a fluorine atom, or can require complicated calculations involving, say, the distance between a hydrogen bond donor and acceptor in the lowest energy conformation of a molecule. In their paper the researchers focus on “extended-connectivity” fingerprints, which take into consideration the physical connectivity between different types of atoms.

But how can you actually quantify structural diversity? One possibility is by comparing molecules to see how different they are, as used for example in Tanimoto similarity assessments. Each additional molecule would be chosen to be least similar to those in a library. Alternatively, one could consider “richness,” how much of chemical space is covered, by calculating how many unique structural features (such as specific bond connectivities) are represented. Each additional molecule would be chosen to provide as many new molecular fingerprints as possible. Shi and von Itzstein propose a third approach, “true diversity,” that considers the number of unique features as well as their proportional abundances. In other words, a library with a higher true diversity would have a “more even distribution of proportional abundances.” The researchers note that this approach has been used in ecology for decades.

To see how their approach performs, the researchers started with a set of 227,787 commercially available fragments, all of which were roughly rule-of-3-compliant and scrubbed of undesirable functionalities. They also considered a subset of 47,708 fluorine-containing fragments. For both sets, they then assessed structural diversity as a function of increasing fragment library size using Tanimoto similarity, richness, and true diversity, as well as random sampling.

Naturally, as the size of a fragment library rose, the diversity increased. As expected, applying Tanimoto similarity or richness led to greater diversity at a smaller library size than did random sampling. This was even more true for true diversity. Interestingly, true diversity reached a maximum at 8.8% or 15.7% (for the full and fluorinated libraries) and then began to decline. This conceptually makes sense because commercial compounds themselves are unlikely to be truly diverse.

More importantly, just 1% or 2.5% of fragments were sufficient to achieve the same true diversity as the full sets. This corresponds to 2052 fragments for the complete commercial set, the structures of which are provided in the supplementary material. As the researchers note, this is comparable to the size of many commonly used fragment libraries.

The method is computationally inexpensive (it runs on a desktop), and should be a useful tool for both building and curating fragment libraries, real and virtual. Of course, diversity is not everything, and it probably makes sense to include privileged pharmacophores even at the cost of lower diversity. But as Lord Kelvin said, “when you can measure what you are speaking about, and express it in numbers, you know something about it.” This paper provides a quantitative approach for measuring diversity.

05 August 2019

Fragments vs RAS family proteins: A chemical probe

RAS family proteins are considered a holy grail of oncology research. Way back in 2012 we discussed a couple papers disclosing low affinity fragments that bind in a small, shallow, polar pocket found in KRAS, NRAS, and HRAS. At the time we wondered “whether this is a ligandable site on the protein.” Last year we highlighted a paper proving that the site is, in fact, ligandable, as exemplified by the mid-nanomolar molecule Abd-7. A paper just published in Proc. Nat. Acad. Sci. USA by Darryl McConnell and collaborators from Boehringer Ingelheim and Vanderbilt University (including Steve Fesik, who published one of the 2012 reports) describes successful development of another ligand. (See here for a fun animated description set to music.)

Consistent with the “undruggable” reputation of RAS family proteins, a high-throughput screen of 1.7 million compounds failed to find anything useful. In contrast, a library of just 1800 fragments screened using STD NMR and MST identified 16 fragments that bind to an oncogenic mutant form of KRAS, as confirmed by 2-dimensional (HSQC) NMR. A separate HSQC NMR screen of 13,800 fragments identified several dozen more, though all the fragments from both screens have dissociation constants weaker than 1 mM. SAR by catalog led to amine-substituted indoles such as compound 11, which modeling suggested could form a salt bridge to an aspartic acid side chain.


The pocket in which all of these molecules bind, between the so-called switch I and switch II regions of KRAS, is much smaller than typical drug-binding sites, but modeling suggested that fragment growing could pick up an additional hydrogen bond, leading to compound 15. Crystallography confirmed the predicted binding mode of this molecule, and informed additional structure-based design, leading first to compound 18 and ultimately to BI-2852, with low or sub-micromolar affinity for wild-type and mutant KRAS, NRAS, and HRAS as assessed by ITC. The researchers also confirmed that the enantiomer is about 10-fold less potent, thereby providing a control compound. Commendably, the researchers have made BI-2852 and the enantiomer available (for free!) to the research community as a chemical probe.

A crystal structure of KRASG12D bound to BI-2852 (cyan) compared with Abd-7 (magenta) reveals how shallow the pocket is; both molecules are largely surface-exposed. The conformational flexibility of the protein is also interesting: Abd-7 would not be accommodated by the protein conformation bound by BI-2852.

The biology is also quite interesting – and complicated. RAS family proteins behave as molecular switches, cycling between the “on” (GTP-bound) state and the “off” (GDP-bound) state, with these transitions assisted by other proteins. On-state RAS drives cell-proliferation and survival. Molecules that bind at the switch I/II pocket block the transition from off to on, but they also block the transition from on to off. Thus, cellular effects are modest. Moreover, BI-2852 hits all RAS isoforms, which could lead to unacceptable toxicity in animals.

This is a lovely paper, but I do quibble that the promise of the title – “drugging an undruggable pocket on KRAS” – remains to be demonstrated. First, both the biochemical and cell-based potency need to be further improved. As the molecule is already large, gaining this needed potency could come at the cost of physicochemical properties. Indeed, the researchers do not discuss the pharmacokinetics of BI-2852. And finally, as the authors themselves note, they will probably need to improve selectivity to spare one or more wild-type RAS isoforms.

What this work does establish indisputably is that the switch I/II pocket is ligandable, though not without effort, as indicated by the 42 authors. Whether or not the site is actually druggable may require another seven years to determine.

29 July 2019

SAR by WaterLOGSY?

Among ligand-based NMR methods, WaterLOGSY is nearly as popular as STD NMR. Normally the information obtained is limited: does a given small molecule bind to a protein or not? In a new paper in J. Enzyme Inhib. Med. Chem., Isabelle Krimm and collaborators at the Université de Lyon and University of York try to wring more data from this common experiment.

In WaterLOGSY, magnetization is transferred from water, to protein, and then to bound ligand. This can happen through multiple mechanisms, and even talented NMR spectroscopists have told me they have trouble understanding exactly what is going on. In short, the WaterLOGSY spectra of molecules bound to proteins show a change in sign compared to molecules that don’t bind. Examining ligands in the presence and absence of protein can thus provide evidence for whether a ligand binds.

The researchers go beyond this simple qualitative approach and look at changes in peaks corresponding to specific hydrogen atoms in each ligand. They define a “WLOGSY factor,” which shows an inverse correlation to solvent exposure. In other words, a smaller WLOGSY factor means that a given hydrogen atom in a ligand is more exposed to water, and thus less exposed to protein. If all the hydrogen atoms in a bound ligand have the same WLOGSY factor, this suggests either multiple binding modes, or that the ligand is completely enclosed by the protein. If, on the other hand, different hydrogen atoms in a bound ligand have different WLOGSY factors, this could provide information on the binding mode. This analysis is conceptually similar to the STD epitope mapping the Krimm lab described several years ago, and STD experiments were also run on the proteins here for comparison.

To validate the approach, the researchers tested six proteins (with molecular weights ranging from 22 to 180 kDa) for which fragment ligands had been previously identified with affinities from 50 µM to worse than 1 mM. Screens were done using 400 µM fragment and 5 to 20 µM protein. (NMR aficionados, please see the paper for details on the effects of mixing times and ligand exchangeable protons.)

The results look pretty impressive: for PRDX5, HSP90, Bcl-xL, Mcl-1, and glycogen phosphorylase, the ligand hydrogen atoms previously shown to be solvent exposed from crystallographic or two-dimensional NMR structures do in fact show reduced WLOGSY factors. In the case of human serum albumin, a ligand showed uniform WLOGSY factors, suggesting multiple binding modes, as expected given the multiple promiscuous binding sites on this protein.

To a non-NMR spectroscopist such as myself, this seems like a useful approach for obtaining binding information in the absence of crystallographic data. It also seems easier to run than the LOGSY titration we highlighted a couple years ago. But the first word of this blog is “Practical.” We recently discussed work demonstrating that STD NMR data is perhaps not as easily interpretable as many assume. Have you tried anything like this yourself, and if so how well does it actually work?

22 July 2019

Fragments vs the PWWP1 domain of NSD3: a chemical probe

Epigenetics is a topic we’ve covered frequently at Practical Fragments. Much attention has been focused on bromodomains, which recognize acetylated lysine residues. However, lysine side chains are also methylated to affect gene expression. The PWWP1 domain of the protein NSD3 (NSD3-PWWP1) recognizes these modified lysines. This protein is amplified in several tumour types, and so makes an intriguing cancer target. At the CHI DDC conference last year Jark Böttcher presented how Boehringer Ingelheim and a large multinational group of collaborators developed a chemical probe for NSD3. The story now appears in Nat. Chem. Biol. (and see here for a fun animated short set to music).

The researchers started by screening a library of 1899 fragments against NSD3-PWWP1. STD NMR (at 0.25 mM of each fragment, in pools of four) as well as differential scanning fluorimetry (at 0.5 mM of each fragment) resulted in 285 and 20 hits, respectively. Two-dimensional NMR was used to confirm hits. Interestingly, only three fragments were identified from both STD-NMR and DSF, and these did not confirm – a cautionary reminder that screening orthogonal methods is not necessarily the best path.

Fortunately, 15 fragments not only confirmed, but also caused the same changes to the 2D-NMR spectra as a histone-derived peptide containing a dimethyl-lysine residue, suggesting that the fragments bind at the recognition site for modified lysines. Those fragments with dissociation constants better than 2 mM were pursued crystallographically, and some of the successes included compound 4. This molecule was used in a virtual SAR-by-catalog screen of internal compounds. Of the 601 fragments experimentally tested, compound 8 was the most potent. Crystallography confirmed that the compound binds in the expected site, and further structure-based design ultimately led to BI-9321.


BI-9321 was put through a battery of tests. Affinity was confirmed in biochemical, SPR, and ITC assays, and crystallography revealed the binding mode to be similar to the initial fragment. BI-9321 was selective for NSD3-PWWP1 when tested against 14 other PWWP domains, and showed no activity against 35 protein methyltransferases, 31 kinases, and 48 bromodomains. Solubility, in vitro metabolic stability, permeability, and plasma protein binding all look good.

Multiple assays also demonstrated selective target engagement in cells at a concentration of around 1 µM. BI-9321 showed downregulation of MYC mRNA levels, though the effect was both modest and transient. Antiproliferative activity was also observed in cells, and the effects were synergistic with a bromodomain inhibitor. Moreover, these effects were only seen in NSD3-dependent cells, suggesting that the activity is on-target and that the compound is not generally cytotoxic.

All of this makes BI-9321 an attractive chemical probe, at least for cell-based assays. More work will need to be done to improve potency and further understand the biology. Laudably, to this end, the researchers have made the molecule publicly available.

15 July 2019

Fragments vs viral protein EBNA1

Epstein-Barr virus (EBV) infects more than 90% of adults. In most cases it remains latent, but even then it expresses genes that cause cellular proliferation, which can lead to cancer. In fact, up to 2% of human cancers are caused by the virus. In a recent paper in Sci. Transl. Med., Troy Messick, Paul Lieberman (both at the Wistar Institute) and a large group of collaborators (including Teddy Zartler) take aim at this pathogen.

The researchers focused on the viral protein EBNA1, a DNA-binding protein essential for viral replication as well as host cell survival. They started with a virtual screen of 1500 fragments from Maybridge, and then did fragment soaking of the top 100 hits. Happily, this resulted in structures of 20 fragments in four separate sites on the protein. Less happily for modelers, none of the fragments bound as predicted – more grist for the “crystallography first” argument.

A dozen fragments bound in a deep hydrophobic pocket, and most of them contained an acidic moiety that made hydrogen bond contacts to conserved threonine and asparagine residues that normally contact the DNA backbone. Merging two of these fragments led to VK-0497, which disrupts binding of EBNA1 to DNA at sub-micromolar concentrations. Crystallography confirmed that it bound as expected. The molecule contains a potentially unstable pyrrole, and replacing this with an indole and growing led to molecules such as VK-1248, with high nanomolar activity in the DNA-binding assay. Additional biophysical techniques including SPR, ITC, and 2-dimensional (HSQC) NMR confirmed binding for this and related compounds.


The carboxylic acid moiety likely reduces permeability across cell membranes, and indeed the compounds showed no activity in cells. However, methyl esters were found to be rapidly cleaved by intracellular esterases, and these prodrugs were tested in a variety of assays.

The prodrugs inhibited proliferation of EBV-positive human cells but had no effect on non-infected cells. The prodrugs also reduced expression of both viral and host proteins. More importantly, they inhibited tumor growth in xenograft models using four different cancer cell lines, two of which were patient-derived. Prolonged dosing over as long as eight weeks showed a sustained effect, which is reassuring in terms of drug resistance. Finally, the molecules could effectively be combined with existing drugs or radiation.

There is still much work to be done, not just in terms of potency but also further pharmacokinetics, pharmacology and toxicology. And as the researchers acknowledge, xenograft models are regrettably poor surrogates for humans. Still, this is an interesting approach, and hopefully further work will be done on this series, or at least the target.

08 July 2019

Stabilizing apolipoprotein E4 with fragments

Among fragment-derived drugs that have entered the clinic, BACE1 inhibitors are well-represented. Sadly, multiple drugs targeting this protein have failed to show efficacy against Alzheimer’s disease. That said, every drug that has been thrown at Alzheimer’s has failed to slow the disease, so perhaps we need to think more boldly. An example was published recently in J. Med. Chem. by Andrew Petros, Eric Mohler, and colleagues at AbbVie.

The researchers were interested in apolipoprotein E4 (apoE4), one of three isoforms found in humans. Folks who have two apoE4 alleles are at increased risk for Alzheimer’s, suggesting that the protein might make a good drug target. Unfortunately, although it is known to be a lipid carrier, its precise function is unclear. What is known is that apoE4 is less stable to thermal denaturation than apoE2 or apoE3, so the team set out to find molecules that would stabilize the protein. This being AbbVie, they used two-dimensional NMR to find fragments.

The methyl groups of all the isoleucine, leucine, methionine, and valine residues in apoE4 were 13C labeled, and the researchers looked for changes in the 13C-HSQC spectra upon addition of fragments; just over 4000 were screened in pools of 12, each at 1 mM. Of the dozen or so hits, compound 1 was among the best.


NMR titration studies revealed an affinity just under 1 mM, while SPR suggested slightly stronger binding. As hoped, compound 1 raised the melting temperature of apoE4. Adding the fragment also altered the kinetics of liposome breakdown, causing the protein to behave more like apoE2 and apoE3. Although this assay isn’t necessarily physiologically relevant, the reasoning is that causing apoE4 to behave more like the other isoforms may be useful.

A crystal structure of compound 1 bound to apoE4 revealed the fragment to be binding in a small pocket, and growing led to compound 2, with a slightly improved affinity. Introduction of polar substituents to interact with a nearby aspartic acid side chain led to compound 8, with low micromolar affinity (assessed by NMR). This molecule also stabilized apoE4 with respect to thermal denaturation.

As noted above, it is not entirely clear why apoE4 is associated with Alzheimer’s, but researchers had previously found that overexpression in a neuronal cell line caused release of the inflammatory cytokines IL-6 and IL-8. When human induced pluripotent stem cell (iPSC)-derived astrocytes carrying two copies of apoE4 were treated with compound 8, release of IL-6 and IL-8 cytokines was reduced to levels similar to those from iPSC-derived astrocytes carrying two copies of apoE3. The compound also showed no toxicity, even at relatively high concentrations (100 µM).

There is still a tremendous amount to do: affinity needs to be improved considerably, and permeability is also mentioned as an issue. Moreover, the highly polar nature of compound 8 will likely make transport across the blood-brain barrier challenging. Optimizing activity against a target whose function is poorly understood will present a host of problems. But if it were easy, Alzheimer’s disease would not be the scourge that it is. Practical Fragments salutes thinking outside the box, and wishes those involved the best of luck.

01 July 2019

Fragment events in 2019 and 2020

We're already halfway through 2019, but there are still some excellent upcoming events, and 2020 is taking shape, so start planning your calendar!

2019
September 1-4: BrazMedChem2019 will be held in the Brazilian holiday destination of Pirinopolis, and will include a section on FBLD.

September 17-19: CHI's Seventeenth Annual Discovery on Target takes place in Boston. Multiple biological targets are covered, and there are also more general talks on a variety of topics of interest to readers, including two sessions on fragments. You can read my impressions of last year's event here.

November 12-15: FBDD Down Under 2019 will take place in beautiful Melbourne. This is the third major FBDD event in Australia, and given the success of the first, I expect it to be excellent.

November 13-15: Although not exclusively fragment-focused, the Seventh NovAliX Conference on Biophysics in Drug Discovery will have several relevant talks, and will be held in the lovely city of Kyoto. You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event herehere, and here.

2020
April 13-17CHI’s Fifteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

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

December 15-20: The second Pacifichem Symposium devoted to fragments will be held in Honolulu, Hawaii. The Pacifichem conferences are held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. Here are my impressions of the 2015 event.

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

23 June 2019

New chemistries for covalent fragments

Good things come in threes, and since our last two posts have covered covalent fragments, we thought we’d continue the theme with two more papers on the topic.

The first, in MedChemComm by György Keserű and colleagues at the Hungarian Academy of Sciences, is actually a companion to a paper we covered at the end of last year. The researchers were interested in electrophilic heterocycles, and assembled a library of 84, of which 57 were commercial and the rest were synthesized. In their previous paper, the researchers focused on reactivity against proteins. This paper is focused more on aqueous stability and intrinsic reactivity with glutathione, a biologically important thiol. The paper includes handy figures and tables summarizing reactivity rate constants and half-lives (at pH 7.4). These should be useful for selecting warheads that are sufficiently reactive as to be able to label proteins, but not so reactive as to label many proteins nonselectively.

The researchers also did computational studies to try to understand different trends in reactivity. And if you’re interested in testing the compounds yourself, they note that the “library is available for screening against relevant targets upon request from the authors.”

The second paper, by Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University, was published in J. Chem. Inf. Model. Last year we highlighted their diversity-oriented target-focused synthesis (DOTS) approach, which combines virtual screening with automated synthesis to rapidly generate new compounds for testing. They have now expanded this approach (called CovaDOTS) to focus on covalent modifiers.

Conceptually, CovaDOTS is akin to fragment linking, in which one of the “fragments” is the nucleophilic residue in the target protein. The process starts with a known noncovalent ligand which is computationally grown by attaching it to commercially available building blocks that contain reactive warheads. These new molecules are then linked with the side chain of an amino acid (cysteine and serine in the paper), and the assemblies are docked against the protein to find molecules that fit well. Ultimately, the best would be resynthesized and tested.

The researchers applied CovaDOTS to three proteins for which covalent and non-covalent ligands had been previously characterized crystallographically. In the case of two kinases, EGFR and ERK2, the program performed well, with the “correct” (i.e., published) ligand observed in the top 14% and 4.4% of hits. For the serine peptidase PREP, the published ligand was the top hit of 303 molecules scored. In all three cases, the predicted binding mode also closely resembled the experimentally determined structure.

One limitation of CovaDOTS is that, as currently implemented, it considers only commercially available building blocks that also “possess both a warhead and an activated function compatible with the selected chemical reaction(s).” It would be interesting to combine the approach with the “make-on-demand” molecules we discussed a few months ago. And of course, it will be interesting to see real-world examples of how the program performs, in addition to these retrospective case studies.

This ends the June 2019 covalent fragment trilogy, but I think it’s fair to say that covalent fragments have a bright future. Look forward to many sequels!

17 June 2019

Screening irreversible covalent fragments, computationally and experimentally

Last week we discussed a paper that characterized commercially available irreversible fragments and screened them against ten proteins. The “warheads” used were either acrylamides or chloroacetamides. This week we’re continuing the theme of irreversible fragments with two papers, each using different warheads.

The first paper, published in Bioorg. Med. Chem. Lett. by Alexander Statsyuk and collaborators at University of Houston, Schrödinger, and Northwestern University, uses a computational approach.

Research we previously highlighted from the Statsyuk lab found that methyl vinylsulfones have a narrower range of reactivities than – for example – acrylamides. This property is important  to ensure that differences in binding to a target are caused by (specific) noncovalent interactions rather than mere differences in warhead reactivity. For the current campaign, the researchers constructed a virtual library in which 1648 commercially available carboxylic acids were coupled to H2N-CH2-CH=CH-SO2-CH3.

The researchers used the CovDock program from Schrödinger to dock the fragments. (Another computational tool for doing so is DOCKovalent, which we described back in 2014.) The specific target chosen was cathepsin L, a cysteine protease which has been implicated in a variety of diseases from cancer to osteoporosis. The virtual screen yielded 33 high-scoring compounds, five of which were synthesized based on price and diversity. Unfortunately, most of these had “solubility issues,” but compound 11 did show time-dependent inhibition of cathepsin L. The researchers also found that the racemic methyl substituent could be removed (compound 13), suggesting that compound growing might be productive. Compound 13 was also selective against three other cysteine proteases.

The second paper, by David House, Katrin Rittinger and collaborators at GlaxoSmithKline, the Francis Crick Institute, and Cellzome, was published in J. Am. Chem. Soc. The researchers were interested in protein ubiquitination, in which the small protein ubiquitin is conjugated to various other proteins to cause a variety of effects depending on the context. The biology is fiendishly complex, but the final step is done by an E3 ubiquitin ligase, of which there are more than 600 in human cells. Needless to say, selective chemical probes would be useful.

The researchers were specifically interested in LUBAC, an RBR E3 ubiquitin ligase which conjugates ubiquitin to proteins with an N-terminal methionine to modulate cellular pathways important in cancer and inflammation. The ligase itself actually consists of three protein subunits, with HOIP containing the catalytic cysteine residue. Although a couple inhibitors had been previously reported, the researchers found these to be nonspecific. Thus, they built and screened their own fragment library. For a warhead, they chose the 4-aminobut-2-enoate methyl ester, which Statsyuk had previously shown has a narrow range of reactivity and is about 10-fold less reactive than the vinylsulfones discussed above. The researchers constructed a small set of 104 fragments, grouped them into pools of 4 or 5, and screened these against 2 µM HOIP at 20 µM each using intact protein mass spectrometry. Compound 5 was one of the best hits.

Compound 5 functionally inhibited HOIP and was selective against about a dozen other cysteine-containing enzymes. The researchers obtained a crystal structure of the molecule bound to HOIP, which confirmed covalent binding to the active site cysteine. Limited SAR studies led to a slightly more potent analog (containing a six-membered ring instead of a five-membered ring), and this molecule showed pathway inhibition in a cell-based assay with EC50  = 37 µM. Activity-based profiling in two cell lines revealed only 8 or 11 proteins that were significantly modified by the compound in addition to HOIP.

The molecules in both of these papers still require considerable work to become chemical probes, let alone development candidates. Nonetheless, they are useful starting points, and together demonstrate the increasing interest and utility of irreversible covalent fragments.

10 June 2019

Characterizing and screening commercially available irreversible covalent fragments

A few years ago we highlighted the utility of irreversible fragments. Because these molecules form covalent bonds with their targets, they can be more effective than similarly sized noncovalent molecules at inhibiting proteins. However, compared with conventional fragments, the quality and quantity of commercial irreversible fragments is limited. This is changing, as described (open access!) by Nir London (Weizmann Institute of Science) and a large, multinational group of collaborators in J. Am. Chem. Soc.

The researchers assembled a collection of 993 fragments from Enamine, all of which contained a cysteine-reactive warhead, either a chloroacetamide (76%) or an acrylamide (24%). The molecules were largely rule of three compliant, even with the warhead included.

A major concern with screening irreversible fragments is that binding to the target protein can be dominated by the inherent reactivity of the warheads rather than non-covalent (and presumably target-specific) interactions from the fragment. Indeed, a previous study found that the reactivities of acrylamides ranged over more than three orders of magnitude. To assess fragments for this, the researchers developed a rapid, plate-based spectrophotometric assay based on labeling the reduced form of Ellman’s reagent. Not surprisingly, the chloroacetamides tended to be more reactive than the acrylamides, but overall the reactivity range across both classes was a relatively modest ~100-fold.

Next, the researchers screened their library against ten cysteine-containing proteins. Fragments were screened in pools of five (200 µM each) with 2 – 10 µM protein for 24 hours at 4 °C. As with Tethering, intact protein mass spectrometry was used to identify hits, which were found for seven of the ten proteins. Hit rates ranged from 0.2 to 4%.

Not surprisingly for fragments, some hits were promiscuous: they strongly labeled two or more proteins. However, these represented less than 3% of the library. Surprisingly, promiscuity did not correlate with reactivity, and in fact some of the most reactive fragments did not label any of the proteins. This suggests that non-covalent interactions are playing a role in promiscuity, and indeed many of the frequent hitters were aminothiazoles – which have previously been found to be promiscuous.

The researchers also screened their fragments (at 10 µM) against three cell lines, and here they did see a correlation with reactivity, with the most reactive fragments tending to be more toxic.

Next, the researchers began optimizing hits against two targets. The first, OTUB2, is a deubiquitinase (DUB) implicated in diverse diseases from amyotrophic lateral sclerosis to diabetes to cancer. The primary screen yielded 47 hits which labeled at least 50%, of which 37 were quite selective. Co-crystal structures were solved for 15 fragment-protein complexes, and two shared a hydrazide moiety (as in PCM-0102954) which made multiple hydrogen bonds with the protein. Two rounds of SAR-by-catalog eventually led to OTUB2-COV-1, which inhibited the enzyme with a respectable kcat/KI = 3.75 M-1 s-1. Despite containing a chloroacetamide, the molecule labeled just 26 of 2998 cysteines in proteins detected in a cell-based proteomic assay.

The researchers also found 36 fragment hits against NUDT7, a protein potentially associated with diabetes, and many of these stabilized the protein in a differential scanning fluorimetry (DSF) assay. Crystal structures were obtained for several, and compound PCM-0102716 showed an overlap with the non-covalent molecule NUDT7-REV-1 derived from a previous crystallographic fragment screen. When the researchers merged these, the resulting NUDT7-COV-1 showed low micromolar inhibition and rapid labeling (kcat/KI = 757 M-1 s-1). This is all the more impressive given that the original noncovalent hit showed no activity. NUTDT7-COV-1 also showed target engagement in a cell assay, and hit only 37 of 2025 detected cysteine residues in a proteomics screen.


This is a nice, thorough paper, though I suspect people in industry will be wary of the chloroacetamides that form the bulk of the library. Nonetheless, chemical structures and reactivity data for all the fragments are reported in the supporting information, making this a useful resource for anyone wishing to dip their toes into covalent fragment screening.