Fragments vs DHX8: towards a chemical probe

RNA helicases present an intriguing but challenging opportunity for drug discovery. Their importance for cell growth makes them attractive cancer targets. But to function, they must undergo considerable conformational changes, and this dynamism makes it difficult to find high-affinity inhibitors. In a new open-access J. Med. Chem. paper, Gurdip Bhalay, Rob van Montfort, and collaborators at The Institute of Cancer Research (ICR), Merck KGaA, and Proteros describe their efforts against DHX8.

 

DHX8 is one of 14 or so DEAH-box helicases and helps release mRNA from spliceosomes. Previous research had found it to be important for the cellular stress response as well as malignant cell transformation and progression, and siRNA-knockdown experiments showed that cancer cells were particularly dependent on DHX8 activity. Thus, the researchers set out to find a chemical probe.

 

The 2000-fragment ICR library was screened (at 300 µM) against the helicase domain of DHX8 using both surface plasmon resonance (SPR) and thermal shift assay (TSA). In addition, the full-length yeast homolog of DHX8, Prp22, was also screened by SPR to look for fragments that might bind outside the helicase domain. The TSA screen yielded 18 hits, while the SPR screen yielded 91, of which 63 also bound Prp22, while another 17 fragments bound Prp22 more strongly than the helicase domain of DHX8.

 

Eight-point dose response curves were collected for the hits, and 10/18 TSA hits gave a concentration-dependent response on both DHX8 and Prp22, along with 42/108 SPR hits, while another 5 SPR hits only bound to Prp22. Surprisingly, there were only 2 hits in common between the TSA and SPR hits; the researchers note the importance of using multiple orthogonal screens.

 

Next, the confirmed hits were tested in three ligand-observed NMR assays: CPMG, WaterLOGSY, and STD. Reassuringly, 39 were active in all three assays. Competition experiments suggested that the fragments did not bind in the ATP-binding site, but some of them were competitive with RNA.

 

Crystallography was attempted on several of the fragments, and compound 1 yielded a structure revealing that it did in fact bind in the “RNA-binding tunnel” of the helicase domain, with the carboxylic acid making contacts similar to a backbone phosphate. Although the dissociation constant (assessed by SPR) was > 1 mM, compound 1 was active in an ATP-hydrolysis assay and an RNA fluorescence anisotropy (FA) binding assay with triple-digit micromolar activity (values for the FA assays are shown here).

 

Much of the paper is devoted to the optimization of this fragment, which, true to the reputation of the helicase family, turned out to be difficult. Testing related compounds led to compound 6, with low micromolar activity, but despite extensive efforts it was impossible to improve the affinity much further. Thus, the researchers reexamined their larger compound collection for other similar compounds and identified compound 24. Although weaker than compound 6, a crystal structure revealed that the phenyl ring binds deeper into the RNA-binding tunnel, providing new opportunities for fragment growing. Structure-based design led to compound 34, with low micromolar activity, and further optimization ultimately led to compound 53, with submicromolar activity.

 

The mechanism of DHX8 requires the RNA-binding tunnel to open and close right where compound 53 sits, so its inhibitory activity can be attributed to blocking this motion. In addition to biochemical activity, the molecule also showed low micromolar activity in a cellular target engagement assay. However, no other cellular data are reported.

 

This is a textbook example of fragment finding and growing against a difficult target. The fact that the researchers returned to fragments after the initial series reached an affinity plateau is a useful reminder that it is all too easy to get stuck in a local minimum, and you may need to take a step back to make progress. Although compound 53 is just on the edge of what may be considered a chemical probe in terms of potency, it is the first reported nanomolar inhibitor of DHX8. The researchers also mention a second series derived from a different fragment, and it will be interesting to see if this yields even better molecules.

An unexpected lysine-reactive covalent fragment against a Pleckstrin Homology domain

Last week we highlighted a study in which an attempt to optimize a covalent fragment led to a non-covalent fragment that bound in a different location. A new open-access paper in J. Med. Chem. by David Spring, Marko Hyvönen and colleagues at University of Cambridge is almost the inverse – a surprisingly covalent binder from a conventional fragment screen.

 

The researchers were interested in Pleckstrin Homology (PH) domains. The 250 or so PH domains in humans bind to the intracellular side of the plasma membrane by interacting with phosphoinositides such as PIP3. Blocking membrane recruitment could be useful for multiple diseases, but the highly charged nature of the interaction (the “P3” in PIP3 means three phosphate groups) makes drugging PH domains difficult, a perfect challenge for fragments.

 

The study focuses on the PH domain from Bruton’s Tyrosine Kinase (BTK), an important oncology and immunology target with several approved drugs, all of which bind to BTK’s kinase domain. Its PH domain was screened against 720 fragments using differential scanning fluorimetry (DSF). Seven fragments stabilized the melting temperature by a whopping 5 degrees C or more. Crystallography was successful for compound 1, which surprisingly revealed that the ketone reacts with the terminal amine of lysine 12, which normally makes electrostatic interactions with two phosphates in phosphoinositides.

 

The crystal structure showed unoccupied space nearby; subsequent fragment growing and optimization led to compound 24, with low micromolar affinity as assessed by DSF. The researchers obtained some two dozen crystal structures, which they were able to correlate with structure-activity relationships (SAR). All of the molecules formed the imine with K12; reducing the ketone to an alcohol abolished stabilization in DSF.

 

Intact protein mass spectrometry was used for assessing SAR and confirming that the covalent bond formation is reversible: adding two fragments with different affinities led to the same distribution of products regardless of the order of addition of the fragments. Not surprisingly, the reaction was faster at higher pH, but nearly complete covalent modification still took place within an hour at pH 7.4.

 

Even at high compound to protein ratios the molecules largely gave single protein modification, and two separate computational studies of the pK_a values of the 15 lysine residues in the BTK PH domain revealed that K12 is an outlier, with a calculated pK_a of 7.1 or 8.8 compared to an average of 10.5 or 10.8 for the others. This means that K12 is largely unprotonated at physiological pH, increasing its reactivity. A similar analysis of four related PH domains suggested that the equivalent lysine residues also have anomalously low pK_a values.

 

The fact that K12 is conserved across related PH domains does raise the question as to whether selectivity will be possible with this type of ligand. Also, no in vitro ADME properties are provided, so it is not clear whether this warhead is advanceable. I wish the researchers had explored other heterocycle alternatives to the furan moiety to assess the tunability of the reactivity; perhaps those will come later. Overall though, this paper is a nice case study where following up on unexpected observations identified a new approach for covalently targeting lysine residues. As Isaac Asimov famously said, “The most exciting phrase to hear in science … is not ‘Eureka!’ but ‘That’s funny.’”

Noncovalent fragments vs WRN

Werner syndrome helicase, or WRN, is an interesting target both for its biological mechanism and its flexible structure. Two years ago we highlighted work out of Vividion describing the discovery of a clinical-stage covalent WRN inhibitor. In a Nat. Commun. paper published earlier this year, Sandra Gabelli, Daniel Wyss, and collaborators at Merck and Proteros describe their noncovalent efforts against this target.

 

Inhibiting WRN kills cancer cells that are already defective for certain DNA repair pathways. It is an example of a “synthetic lethal” approach to drug discovery that expands the number of cancer targets by focusing on oncogenic cells rendered vulnerable by pre-existing mutations. As an ATP-dependent helicase, WRN acts as a molecular machine to unwind DNA. This requires the multidomain protein to undergo dramatic conformational changes, which makes finding ligands challenging: how do you know which conformation(s) to target? Moreover, WRN enzymatic assays are particularly prone to false positives; a paper published in 2024 demonstrated that some previously disclosed inhibitors are at best nonspecific, and at worst downright artifacts. Thus, the researchers chose to use biophysics to identify fragments.

 

A library of 1020 fluorine-containing fragments was screened in pools of up to 21 compounds using ¹⁹F NMR T₂ CPMG experiments. The 31 primary hits were re-screened as pure compounds in this assay as well as three more ligand-detected NMR assays, leading to seven hits taken into crystallography, of which three yielded structures. A separate SPR screen of 500 non-fluorinated fragments followed by confirmation by NMR led to three additional fragments characterized crystallographically. None of the validated fragments from either screen showed functional activity in an enzymatic assay.

 

The fragments bound in three different sites on the protein, which itself underwent significant conformational changes to accommodate the fragments. Fragments 1 and 2 bound in the same site and could be partially superimposed on one another, and these were used to generate a virtual library, of which 17 compounds were made and tested. Compound 4 had the best affinity as assessed by SPR and was also active in a functional assay.

Crystal structures of some of the other compounds bound to WRN were also determined, and these showed significant protein domain rearrangements, even when the compounds themselves were structurally similar. The researchers include a nice movie (link to download here) and suggest that “these structures capture only a few states of WRN as it translocates along the DNA and conducts its helicase and exonuclease functions.”

 

This paper nicely illustrates the challenges of finding ligands, particularly noncovalent ones, against conformationally flexible proteins. We’ll revisit this topic next week.

Fragments vs DsbA: towards a chemical probe

Despite its ubiquitous use as a model organism, Escherichia coli causes nearly a million deaths each year worldwide. Antibiotics provoke rapid selection for resistance and are becoming ineffective. An interesting alternative is to inhibit virulence factors. Doing so won’t directly kill the bacteria but instead reduce its infectivity, a trait that might be subject to less evolutionary selection.

 

The oxidoreductase DsbA facilitates disulfide bond formation in other bacterial proteins and is a key regulator of resistance. Martin Scanlon’s lab at Monash University has been pursuing this enzyme for some two decades; we described some of their work in 2015. In two recent papers, he and his colleagues describe progress towards a chemical probe.

 

DsbA has more than 300 protein substrates that bind in a shallow, hydrophobic groove. The lack of deep pockets or specific recognition elements makes finding small molecule ligands particularly challenging. Three years ago we highlighted fragment screens that identified two dozen hits in this groove. Intriguingly, that screen also identified a couple fragments that bind in a cryptic pocket close to the groove. This pocket is the focus of a paper published in Angew. Chem. late last year by Martin and collaborators at Monash University, La Trobe University Bundoora, and Scripps.  

 

Crystallography revealed that compound 1 binds in a pocket that is completely enclosed by DsbA. Twenty commercial analogs were purchased and tested by protein-observed [¹⁵N,¹H]-HSQC NMR. Six bound to the protein, but NMR suggested all bound in the hydrophobic groove, not in the cryptic pocket. Undeterred, the researchers made and tested a few dozen analogs, some of which did indeed bind the cryptic pocket and also had slightly higher affinities as measured by NMR and SPR.

 

How do the fragments get inside a pocket with no apparent entrances? Computational, protein-observed NMR, SPR, and HDX experiments suggested that DsbA is dynamic and one region can open up to allow access of the fragments. Interestingly, the fragments bind preferentially to the oxidized (active) form of DsbA, a fact that makes sense given that this state is more dynamic, allowing readier access to the pocket.

 

Unfortunately, the affinity of the best fragments is only around 150 micromolar. The small size of the cryptic pocket makes further affinity improvements unlikely, so the researchers sought to break the bounds of this pocket to gain added affinity. This is the focus of a paper just published in J. Med. Chem. by Martin, Bradley Doak, and collaborators at Monash, Vernalis, University of Western Australia, and The University of Sydney.

 

The researchers first built a small set of compounds that would break out of the pocket. Compound 5 had slightly worse affinity, as measured by SPR, but crystallography confirmed that the alkyne does in fact protrude as designed. A small set of analogs led to compound 13, with mid micromolar affinity. This compound was nearly 30-fold more potent than its enantiomer, with the hydroxyl moiety displacing a conserved water to make hydrogen bond interactions with the protein.

 

To gain additional interactions in the hydrophobic groove, the researchers chose direct-to-biology, screening crude reaction mixtures without purification, an increasingly popular strategy as we noted last week. In this case the researchers used automated flow reactors, allowing air- and moisture-sensitive organometallic chemistry. A set of 92 compounds was made and tested by off-rate screening (ORS) SPR and affinity-selected mass spectrometry (ASMS). Four crude hits were remade, purified, and tested, and compound 17 came in as a low micromolar binder both by SPR and ITC. This molecule also inhibited the enzyme in a functional assay and even showed some activity in a bacterial swarming motility assay.

 

Further improvements in potency will be needed to obtain a chemical probe, let alone a drug, but these two papers describe meaningful progress. They also provide a useful reminder that proteins are far from static. Cryptic pockets are surprisingly common, and even if they are too small and enclosed to support high affinity binding, they can be used as footholds to build larger molecules.

Twenty-First Annual Fragment-Based Drug Discovery Meeting

Last week some 875 people attended the CHI Drug Discovery Chemistry (DDC) meeting in San Diego. I can’t do justice to the 40 or so presentations I attended over four days but can highlight some of the main themes.

 

Reversible fragments

Membrane targets such as G protein-coupled receptors (GPCRs) pose a challenge for biophysical methods, but three talks presented progress. Matthew Eddy (University of Florida Gainesville) described high-resolution magic angle spinning (HRMAS) NMR, which entails spinning isolated cellular membranes containing GPCRs at high speed (4 kHz!), which miraculously yields sharp NMR signals for bound ligands. Matthew demonstrated applications with the human adenosine A_2A receptor and weak (mM) ligands. He noted that the technique can work with native, poorly expressed proteins, though data collection times can be upwards of 30 minutes.

 

Kris Borzilleri described using ¹⁹F NMR to find ligands against an orphan GPCR at Pfizer. The 2287 fragments screened yielded 87 hits, of which 38 confirmed by SPR. SAR studies eventually yielded low micromolar ligands, but these were difficult to advance in the absence of structure (see here for a more successful example from Merck).

 

Vanessa Porkolab (Eurofins Cerep) described using the Nanotemper Spectral Shift technology to screen 826 fragments against the adenosine A_2A receptor at 300 µM, with a 9.2% hit rate. Many of these ligands stabilized the GPCR in a thermal shift assay and seven were even active (as antagonists) in a cellular assay.

 

Turning to soluble proteins, Paola Di Lello presented a case study from Genentech and Vernalis applying ligand-observed NMR to the protein phosphatase PTPN22. Subsequent protein-observed NMR revealed that most of the 16 validated hits bound to two pockets some distance from the active site. The fragments were optimized to mid-micromolar affinity but showed no functional activity.

 

And Charlotte Hodson presented the eIF4E story from Astex. As we discussed last year, this yielded a low nanomolar ligand that did not have the desired cellular effects. Charlotte noted that subsequent genetic experiments were consistent with the limited efficacy. Still, the target was sufficiently interesting that a chemical probe would have been pursued even knowing it would be high-risk.

 

Covalent ligands

Covalent approaches made appearances throughout the conference. Keriann Backus (UCLA) described chemoproteomic approaches to find cysteine-targeting ligands; she noted that gain of cysteine residues (such as G12C in KRAS) are the most common missense variants in cancer. Keriann also warned how covalent compounds can cause potentially misleading effects in cells, as she described in Nat. Chem. Biol. last year.

 

In 2021 we wrote about the SpotXplorer fragment library from György Keserű (Hungarian Research Centre for Natural Sciences). György has now prepared a PhotoXplorer library, which uses diazirine tags for photochemical screening, which we described here. The new library has produced high hit rates across a variety of targets. György also described a new sulfozone-based photoprobe that is easier to prepare than diazirines.

 

Kelly Craft recounted a DNA-encoded library (DEL) screen at AbbVie against the target BCL2A1, also known as BFL1. This produced an aldehyde-containing low micromolar binder that formed an imine with buried lysine 102. Uncomfortable progressing an aldehyde, the researchers sought to covalently engage cysteine 55, the same cysteine targeted by AstraZeneca, as we wrote about here. The progression included at least one dual-warhead molecule which was crystallographically confirmed to bind both the lysine and cysteine. The effort ultimately yielded cysteine-selective leads.

 

Earlier this year I described the dDRTC method we developed at Frontier Medicines for determining k_inact/K_I, and Svetlana Kholodar presented a nice overview of its scope and utility. My colleague Johannes Hermann spoke in more detail about our covalent technologies, particularly those using AI.

 

Chemical space and the exploration thereof

Brian Shoichet (UCSF) gave an entertaining and wide-ranging account of “directed and random walks in chemical space.” Brian has consistently been on the bleeding edge of high-throughput in silico screens, from 67,000 compounds in 2009 to 138 million molecules in 2019 to 4 billion molecules today. When docking artifacts are avoided (as we discussed here), bigger libraries consistently produce more potent hits for more targets – an observation strikingly consistent with Alex Shaginian’s in 2023 as HitGen expanded their DEL libraries from billions to more than a trillion molecules. Brian is developing methods to computationally screen the >4 trillion make-on-demand molecules now available from companies such as Enamine.

 

Direct-to-biology (DTB) approaches, which rely on microscale chemical reactions screened without purification, have become increasingly popular methods for exploring chemical space. Jack Sadowsky correctly stated that Carmot was the first company formed around this approach; we previously wrote about the role Chemotype Evolution played in the discovery of sotorasib. Jack described how Kimia, which spun out of Carmot, has continued to advance the technology, applying it to find inhibitors selective for single members of closely related kinase families.

 

Allan Jordan described how Sygnature Discovery is applying DTB in a variety of assays including microsome stability and crystallography. (We wrote about crude reaction screening by crystallography earlier this year.) Expanding beyond DTB, Allan called their platform direct-to-discovery, and discussed how it led to a preclinical candidate with STORM Therapeutics in just 18 months.

 

WuXi Apptec is also using DTB. Peichuan Zhang described starting with ligands derived from fragment and DEL screens against the E3 ligase GID4 to make PROTACs to degrade BRD4; DTB was used to explore a wide range of different linkers. And Daniel Blair (St. Jude) described using DTB and affinity selection-mass spectrometry (AS-MS) to find new molecular glues for the oncology target LCK.

 

Computers, DEL, and DTB are not the only way to explore chemical space. Last year we covered Tom Kodadek’s bead-based screening approach at University of Florida Scripps, and Tom presented two talks on the topic, one using macrocycles to find binders to difficult targets such as PTP1B and one using small molecules to find molecular glues.

 

Speaking of PROTACs and glues, plenary keynote speaker Alessio Ciulli (University of Dundee) discussed the “evolution and future of targeted protein degradation.” Alessio noted that there are >25 PROTAC degraders and >10 glues in the clinic, though these collectively target only a small number of E3 ligands, so there is plenty of opportunity for the area to expand.

 

For many of us in industry, drugs represent the most privileged points in chemical space, and these often look quite different than we assume, as Dean Brown (Jnana) noted in his recent analysis of 104 oral small molecule drugs approved by the FDA from 2020 to 2024 (which we mentioned here). Some drugs contain eye-raising moieties such as acetylenes, styrenes, N-O bonds, and nitro groups. Indeed, it is worth remembering that venetoclax, arguably the most successful fragment-derived drug, sports a nitro group.

 

But before getting too complacent, Jonathan Baell (Manas) warned about frequent hitters in libraries of FDA-approved drugs. He notes in Eur. J. Med. Chem. earlier this year that many commercial libraries are actually enriched for molecules that cause spurious biological activity. Jonathan calls on library vendors to remove particularly egregious compounds, though I’d settle for world peace.

 

I’ll close on that pleasant thought, but please feel free to comment. I hope to see you in San Diego next year April 19-22 for the twenty-second iteration of DDC.

Fragments vs the E3 ligase KLHL12

Last week we highlighted work out of Steve Fesik’s lab at Vanderbilt University about PLPro. This week we’ll highlight another paper just published (open access) in J. Med. Chem. on a different subject from Steve, Alex Waterson, and colleagues.

 

Targeted protein degradation has been receiving increasing attention. The most common approach uses bivalent molecules called PROTACs. Imagine a molecular barbell, where the weight plate on each end is a different ligand, one targeting an E3 ligase and the other targeting a protein to degrade. As he discussed at the DDC meeting in 2023, Steve has long been pursuing ligands against previously unexplored E3 ligases with desirable properties, such as tissue-specific expression. A PROTAC using an E3 found predominantly in cancer cells could degrade essential proteins while sparing proteins in normal cells, and so yield safer drugs. The E3 ligase Kelch-like protein 12 (KLHL12) is overexpressed in many cancers but not expressed in heart tissue. The new paper describes fragment screening and optimization of ligands for this ligase.

 

The researchers started with a protein-detected ¹H-¹⁵N correlation NMR screen of 13,824 fragments in pools of 12, each at 0.8 mM. This yielded just 35 hits, of which 15 showed similar chemical shifts to those caused by a peptide substrate, suggesting that they bind in the same site. Dose-response experiments were used to determine affinities, with compound 1 being the best.

A crystal structure of this molecule bound to KLHL12 confirmed that it binds in the substrate-binding cleft, but the resolution was insufficient to determine the precise orientation. Nonetheless, SAR around the aniline moiety led to more potent molecules such as compound 7c, and exploration around the benzimidazole led to compound 7k, with sub-micromolar affinity as assessed both by a fluorescence polarization anisotropy (FPA) assay as well as SPR.

 

A crystal structure of 7k bound to KLHL12 was solved at high resolution, explaining the SAR and also revealing tempting space for further growing. Disappointingly though, most of the dozens of analogs had at best low micromolar affinity, and the few that had comparable activity to compound 7k had significantly worse ligand efficiencies. KLHL12 is homologous to the protein KEAP1, which as we noted last year has also proven challenging for conventionally drug-like ligands.

 

In addition to KEAP1, KLHL12 has more than 35% sequence identity to nine other proteins, so selectivity is a potential concern. Unfortunately, these proteins proved difficult to express. However, one compound tested was selective for KLHL12 over KEAP1.

 

Finally, a small set of compounds was tested for in-cell KLHL12 engagement using a nanoBRET assay. Happily, compound 7k proved active at sub-micromolar concentrations, suggesting that cell permeability would not be an issue.

 

This is a nice fragment to lead story. The relatively flat SAR for many of the compounds, while undoubtedly frustrating, should be useful for further understanding molecular recognition. It is not clear whether compound 7k is sufficiently potent to be useful, but as the researchers conclude, the work “provides a promising foundation for the future design and synthesis of KLHL12-based PROTACs.”

From noncovalent fragment to (non)covalent leads against PLPro

The most successful drug against COVID-19, nirmatrelvir, targets the main protease of SARS-CoV-2. As we discussed just last year, this protein has received considerable attention. But the genome for SARS-CoV-2 also encodes a second cysteine protease, papain-like protease, or PLPro. Despite this enzyme being essential for viral replication, the only previously disclosed chemical series targeting it dates from 2008 efforts against the original SARS. Two new papers in J. Med. Chem. from Stephen Fesik and colleagues at Vanderbilt University introduce new molecules, covalent and non-covalent.

 

One reason progress has been slow against PLPro is that it is inherently challenging. It recognizes the sequence LXGG, where X = Arg, Lys, or Asn. The two glycine residues thread a narrow channel to access the catalytic cysteine, while leucine and the “X” residue bind in solvent-exposed subsites. In 2024 the Fesik group published an open-access paper in ACS Med. Chem. Lett. describing the results of a protein-observed NMR fragment screen. Out of 13,824 fragments screened in pools of 12 at 0.8 mM each, 77 confirmed when tested individually, and 22 had affinities better than 1 mM.

 

An attractive feature of protein-observed NMR is that it tells you where the fragments bind, and in this case there were two main binding sites. One set of fragments bound in the S3 and S4 pockets, where the leucine and lysine residues of the substrate would normally fit, while another group of fragments bound some distance from the active site. Representatives from both groups were tested for inhibition of enzymatic activity. Only those in the first group were active, so these were prioritized.

 

In the first open-access J. Med. Chem. paper this year, the researchers started with compound 11, which fulfills rule-of-three fragment criteria, binds in the S4 pocket, and inhibits the enzyme with high-micromolar activity. Adding a couple methyl groups (compound 15) improved the potency by roughly 10-fold, and building towards the S3 pocket yielded compound 37, with low micromolar activity. Addition of a basic nitrogen, as in compound 46, improved the potency to submicromolar activity, and crystallography revealed that the basic nitrogen interacts with a glutamic acid side chain. This molecule was active in a cellular assay at submicromolar concentration.

 

An increasingly popular strategy for addressing difficult targets is through covalent inhibitors, and this is the subject of the second open-access J. Med. Chem. paper this year. The researchers synthesized and tested 25 analogs based on molecules such as compound 37 in which various warheads were appended by flexible linkers. Although some of these were active at low micromolar concentrations, only a few showed time-dependent activity as would be expected for an irreversible covalent inhibitor.

 

These few were optimized with the aid of molecular dynamics and structure-based design to molecules such as compound 45. Interestingly, despite being nearly 10-fold more potent than the best non-covalent molecule, it was less active in cells; the researchers attribute lower-than-hoped activity to the two hydrogen-bond donors in the diacetylhydrzaine linker. Unfortunately, these turned out to be essential for covalent binding; crystallography revealed that the compound forms four hydrogen bonds in the glycine channel. Indeed, this particular linker-warhead combination had been previously reported, and the inability to improve on it emphasizes the restrictive requirements for this particular protein.

 

This is a nice series of papers that shows how a single fragment can lead to multiple leads. The last paper is also a useful reminder that adding a warhead to a high-affinity binder is not always easy, nor does it necessarily lead to superior molecules. Indeed, neither the covalent nor the noncovalent leads have any reported in vitro ADME or pharmacokinetic data. It would be fun to screen PLPro against a library of covalent fragments to look for even more starting points.

3D fragments vs the histamine H1 receptor

Last month we highlighted a paper from Iwan de Esch and colleagues at Vrije Universiteit Amsterdam about assessing molecular shapeliness. In a new open-access RSC Med. Chem. paper, de Esch and colleagues use a small, shapely library to find and develop potent antagonists of the histamine H₁ receptor (H₁R).

 

The G protein-coupled receptor H₁R is one of four histamine receptors and the target for dozens of antihistamines used for allergic and inflammatory conditions. Thus, it’s well understood and a nice model system.

 

In 2013 we discussed how the de Esch group screened a library of 1010 fragments against several targets, including H₁R. In the new paper, the researchers built a smaller library of just 80 compounds designed to be more shapely, as assessed by Fsp³, plane of best fit (PBF), and principal moment of inertia (PMI). A screen against H₁R using a radioligand displacement assay at 10 and 30 µM yielded a single hit, compound 1a.

 

Compound 1a deviates slightly from the rule of three due to some unusual elements: an azide as a synthetic handle and a butyl moiety to make the compound less explosive. Trimming these features led to compound 3a, a rule-of-three compliant fragment that – while having lower affinity – mostly maintained ligand efficiency and improved lipophilic ligand efficiency (LLE). Fragment growing led to compounds 13a and 13k, and merging with previously reported molecules led to compound VUF26691, with low nanomolar affinity and picomolar antagonistic activity in a cell assay. Throughout the process the cis isomers generally had equal or better potency than the trans isomers, an empirical observation difficult to explain by modeling.

 

This is a nice fragment-to-lead story, and the researchers note that fewer than 40 compounds were synthesized in the journey from compound 3a to VUF26691. Interestingly though, compound 1a was the only hit from the 80 compounds tested, while the hit rate from the larger, flatter library screened in 2013 was nearly three-fold higher, at 3.6%. Although it’s difficult to extrapolate from n=1, the results are consistent with our post last year that more shapely libraries will likely have lower hit rates.

 

Still, this shapely hit makes for a neat scaffold, and if nothing else perhaps it will be easier to establish intellectual property.

GAS41 revisited: a chemical probe

The YEATS domain of the protein GAS41 is an epigenetic reader that modulates gene expression by binding to acetylated lysine residues in chromatin. Multiple lines of evidence suggest it could be a useful target for various cancers, in particular non-small cell lung cancer (NSCLC). Four years ago Practical Fragments highlighted a paper from Jolanta Grembecka, Tomasz Cierpicki, and colleagues at the University of Michigan describing a fragment screen and subsequent optimization of a hit to molecules with some cellular activity. In a new J. Med. Chem. paper, the same team now describes molecules with better cell potency, as well as a negative control.

 

Compound 1, the initial fragment hit, had weak affinity for GAS41, but replacing the t-butyl group with a proline led to compound 7, with low micromolar activity in a fluorescence polarization assay. On the other side of the molecule, modification and growth of the amide moiety led to compound 16, also with low micromolar activity. In the 2021 paper, molecules related to compound 16 were dimerized to bind to two YEATS domains in close proximity in the GAS41 dimer. This yielded mid-nanomolar inhibitors, but the molecules were also large, with limited cell permeability. In the new paper, the researchers instead combined medicinal chemistry learnings and used structure-based design to generate monomeric molecules, culminating in DLG-41.

 

The affinity of DLG-41 for GAS41 was measured as 1 µM using isothermal titration calorimetry (ITC). In accordance with best practices for chemical probes, the researchers also developed a negative control by replacing the thiophene moiety with a thiazole; this compound, DLG-41nc, shows negligible activity in two different biochemical assays.

 

DLG-41 showed high nanomolar activity in a NanoBRET assay, demonstrating that the molecule is both permeable and binds to the GAS41 protein in cells. Importantly, the IC₅₀ for the negative control DLG-41nc was > 25 µM in this assay. DLG-41 blocked proliferation in a panel of NSCLC cell lines, though DLG-41nc also showed some activity, albeit at higher concentrations. Gene expression studies in one cell line showed that DLG-41 caused changes in hundreds of genes, while DLG-41nc was inactive.

 

This is a nice example of fragment optimization in academia. With both biochemical and cell-based potency around one micromolar, DLG-41 is hovering on the edge of the 2015 suggestions for a chemical probe. But used alongside the negative control, the compound should be useful for further exploring the biology of GAS41.

Fragments vs FEN1: A chemical probe

Synthetic lethality is a relatively new approach to treating cancer by targeting proteins whose inhibition is lethal to cancer cells that have specific mutations. Disruption of flap endonuclease 1 (FEN1), an enzyme important for DNA replication and repair, becomes synthetic lethal combined with BRCA1 and BRCA2 loss of function mutations. However, although a few inhibitors have previously been reported, these had poor cell activity and physicochemical properties. In a new J. Med. Chem. paper, Sam Mann and collaborators at Artios Pharma, Merck KGaA, and several other organizations describe a chemical probe.

 

FEN1 is a member of the RAD2 nuclease family, all of which contain two magnesium atoms in the active site. Thus, the researchers set out to build a metal-chelating library, a strategy we’ve written about previously. Noting that a bivalent metal chelator requires at least three hydrogen bond acceptors, the researchers included fragments that were not strictly rule-of-three compliant. More than 300 fragments were screened in a biochemical assay against FEN1 and three related proteins, EXO1, GEN1, and XPG. Hits were selected based on potency, ligand efficiency, and selectivity. Two related fragments came out on top, one of which was characterized crystallographically bound to FEN1, confirming engagement with the catalytic magnesium ions

 

 

Compound 6 was trimmed back to the chelating core compound 7 before attempts were made to grow the molecule in several directions, leading eventually to compound 21, the first molecule to show target engagement in cells. Modeling suggesting the presence of a high-energy water that might be displaced, which was attempted by expanding the core to include a morpholine moiety. Further modulation of properties ultimately led to MSC778, with modest oral bioavailability in mice, rats, and dogs. The paper describes some nice medicinal chemistry that goes beyond the scope of this post. For example, there was a correlation between cellular target engagement and off-rates as determined by surface plasmon resonance (SPR). One wonders if a covalent inhibitor, with essentially no off-rate, could be even more effective.

 

MSC778 is at least 65-fold selective for FEN2 over related RAD2 family members. It is also clean in a panel of off-target safety assays. The molecule is cytotoxic to a cancer cell line in which the BRCA2 gene had been knocked out but less so to the same cell line carrying wild-type BRCA2. Surprisingly though, no tumor growth inhibition was seen in a mouse xenograft model using the mutant BRCA2 cell line at the highest tolerable dose. However, tumor stasis was seen when the compound was dosed in combination with niraparib, a PARP inhibitor, consistent with earlier cell experiments suggesting that PARP inhibitors could synergize with FEN1 inhibitors.

 

The lack of single agent activity seen with MSC778 was undoubtedly disappointing, though the researchers note that it is unclear whether this is “due to insufficient target coverage, or an unexpected disconnect between the phenotypic consequences of FEN1 inhibition in vitro and in vivo.” Nonetheless, MSC778 looks to be a useful chemical probe for further understanding the biology of FEN1. This paper is also a nice application of building and screening a metal-chelating fragment library, which could be useful for targeting additional metalloproteins.

Exploiting avidity for finding fragments

As our poll last year demonstrated, there is no shortage of methods to find fragments. But that doesn’t mean new approaches aren’t welcome, particularly when they also apply to fragment growing. This is the promise of a recent paper in J. Med. Chem by Thomas Kodadek and collaborators at University of Florida Scripps and Deluge Biotechnologies. (Tom and first author Isuru Jayalath also presented this at the DDC meeting earlier this year.)

 

The researchers were inspired by the concept of avidity, the observation that multiple copies of a ligand bound to a multiprotein assembly can form a more stable complex than monomeric ligands bound to monomeric proteins. Could this phenomenon be exploited to find weak fragments?

 

A previous DNA-encoded library screen on streptavidin had identified 28 macrocycles, all of which contained one of two closely related fragments. The affinity of the more potent fragment came in at 706 µM using SPR. The researchers coupled this fragment to TentaGel beads, 10 µm wide polystyrene spheres covered in polyethylene glycol (PEG) chains terminated by amine groups. The PEG makes the beads water soluble. The beads were soaked in a solution of fluorescently labeled streptavidin, washed, and analyzed. Importantly, streptavidin exists as a tetramer, so each tetramer could bind up to four bead-bound fragments.

 

Streptavidin bound avidly to the beads, even when incubated at low (50 nM) concentrations. A control protein did not bind, nor did streptavidin bind to beads modified with a negative control fragment. Moreover, a monomeric version of streptavidin did not bind to the beads, illustrating the importance of avidity. Finally, adding the natural ligand biotin kept streptavidin from binding to the beads.

 

TentaGel beads have long been used in combinatorial synthesis, so the researchers built a small library in which the initial fragment was coupled to 48 carboxylic acids. These were then incubated with labeled streptavidin, and some of the beads showed more intense fluorescence, suggesting more protein binding. SPR analysis revealed that these new molecules had improved affinity, with the best coming in at 90 µM as a monomer. Thus, the primary screen can rank order affinities.

 

This is great for oligomeric proteins, but what about the large number of targets that are monomeric? Many recombinant proteins are expressed as fusions with glutathione S-transferase (GST), which facilitates purification. Importantly, GST exists as a homodimer in solution. The researchers screened a GST fusion of the oncology target Rpn13 against a small library of 94 fragment-coupled beads and found five hits. SPR studies confirmed weak (K_D > 2 mM) binding for two hits to pure Rpn13 (ie, without the GST fusion), and this binding could be competed with a known peptide ligand of Rpn13.

 

Screening beads in individual wells is one thing, but to really increase throughput it would be nice to be able to screen mixtures of different beads. To do so, the researchers developed a photocleavable linker between bead and fragment. The linker also contained an alkyne group that could be modified with a brominated imidazopyridinium moiety. This tag is UV active, ionizes well, and the bromine’s unique isotopic signature helps distinguish true hits from noise. Beads containing more than 50 different compounds, including the two fragment hits we mentioned above, were incubated with labeled streptavidin. Beads to which protein bound were separated by fluorescence-activated cell sorting (FACS), clicked with the tag, cleaved from the beads, and analyzed by mass spectrometry. Only the two known binders were identified, demonstrating the specificity of the approach.

 

This is a neat paper well worth reading. I particularly like the fact that the method can be done with minimal equipment. I look forward to seeing how it works against more targets.