Fragments in Brazil

Most of the fragment events we’ve highlighted are in the US, Europe, and Australia, but that does not fully reflect where all the good science is happening. In a recent ACS Med. Chem. Lett. paper, Carolina Horta Andrade, Maria Cristina Nonato, and Flavio da Silva Emery introduce CRAFT: the Center for Research and Advancement in Fragments and molecular Targets.

 

Established in 2021, CRAFT is a collaboration between the University of Saõ Paulo and the Federal University of Goiás. The center is focused on endemic diseases of Brazil. As the researchers note, only one of the 60 or so fragment-derived drugs that have entered the clinic is an anti-infective, so there is clearly significant need. CRAFT also has an educational and training component reminiscent of the European FragNet and the Australian Centre for Fragment-Based Design.

 

One focus of CRAFT is fragment library design, including underexplored heterocyclic systems. Importantly, the researchers are investigating new synthetic methodologies to be able to functionalize different regions of the fragments. They are also exploring fragments similar to or derived from natural products.

 

Targets are of course essential, and CRAFT is investing in protein production and characterization, such as the enzyme DHODH from Leishmania; we’ve written recently about a fragment approach to the mammalian counterpart.

 

Finally, CRAFT is investing in structure-based design, ligand-based design, and phenotypic screening. And in 2024 no venture would be complete without use of machine learning.

 

Academic laboratories often struggle with downstream drug discovery efforts such as drug metabolism and pharmacokinetics. CRAFT recognizes this and has partnered with the Welcome Centre for Anti-Infectives Research to train participants in DMPK.

 

The researchers “invite the global scientific community to collaborate with us in addressing neglected diseases.” I hope they succeed. Five years ago we highlighted the consortium Open Source Antibiotics, but that site seems to be updated infrequently. The COVID Moonshot has been more successful but is arguably less urgent given the billions of dollars of industry money that poured into research on SARS-CoV-2. From an ethical perspective society should invest more on combating tropical diseases. And as the planet warms, these diseases will increasingly move out of the tropics.

Fragments in the clinic: Lirafugratinib

With crystal structures of protein-ligand interactions becoming increasingly accessible, it is easy to forget that proteins do not exist as the static structures seen on page or screen. Indeed, back in 2018 we quoted Karplus quoting Feynman that “everything that living things do can be understood in terms of the jiggling and wiggling of atoms,” and even the smallest proteins have lots of atoms. In an open-access paper published in Proc. Nat. Acad. Sci. USA earlier this year, Heike Schönherr, David Shaw, and collaborators at Relay Therapeutics, D.E Shaw Research, Pharmaron, and Columbia University take advantage of these movements.

 

The researchers were interested in finding selective inhibitors of fibroblast growth factor receptor 2 (FGFR2), which is activated in many cancers. The four members of the FGFR family are so closely related that finding selective inhibitors is difficult. Inhibiting FGFR1 can lead to hyperphosphatemia, while inhibiting FGFR4 can cause diarrhea, side effects seen with the approved fragment-derived drug erdafitinib.

 

Although the structures of FGFR1 and FGFR2 are very similar, extended (25 µs) molecular dynamics simulations revealed that the so-called P-loop of the proteins behaved differently: in FGFR1 it became disordered, while in FGFR2 it remained more rigid. The researchers sought to take advantage of these differences with a covalent inhibitor.

 

The researchers started with a non-selective hinge-binding fragment, compound 1. Adding an acrylamide warhead led to a nanomolar inhibitor with modest selectivity for FGFR2. (All IC₅₀ values are measured after 30 minute incubations.) Growing the molecule into the so-called back pocket of the kinase led to compound 5, with nearly 100-fold selectivity for FGFR2 over FGFR1. 

 

 

The path from compound 5 to lirafugratinib (also called RLY-4008) looks straightforward but was anything but. First, the aryl acrylamide was a metabolic liability, so the researchers attenuated the reactivity by adding a methyl group. Mechanistic studies with this molecule revealed that while it had only a slightly better affinity (K_I) for FGFR2 than FGFR1, it had a k_inact value about 15-fold higher for FGFR2. Molecular dynamics studies suggested that the relevant cysteine in FGFR1 is locked in a position too far from the acrylamide to react, while the corresponding cysteine in FGFR2 may be able to more closely approach the acrylamide warhead.

 

Further optimization, guided by extended molecular dynamics simulations, led eventually to lirafugratinib with ~250-fold selectivity for FGFR2 over FGFR1 and >5000-fold selectivity over FGFR4. Remarkably, the noncovalent version of lirafugratinib, compound 11, shows dramatically lower affinity for both FGFR1 and FGFR2 and very little selectivity between them. The ligand seems to assume a different binding mode after covalent bond formation, which could explain these differences in selectivity.

 

Mouse studies of lirafugratinib showed tumor stasis or regression without increased serum phosphate levels. More importantly, early clinical data has shown “minimal hyperphosphatemia and diarrhea.”

 

This is a lovely example of structure and dynamics-based design (SDBD?). Commonly cited advantages of covalent drugs include improved potency and extended pharmacological effects, but this work shows that they can also achieve remarkable selectivity between closely related proteins, even when both proteins contain cysteine residues in the same location. Moreover, an open-access paper in Cancer Discov. that dives more deeply into the biology shows that lirafugratinib is selective across the kinome, inhibiting just two of 468 kinases other than FGFR2 by >75% at 500 nM.

 

The next time you’re trying to find a selective inhibitor for one member of a protein family, it may be worth taking a covalent approach, and paying close attention to dynamics along the way.

Fragments vs β-glucocerebrosidase

The protein β-glucocerebrosidase, also called GCase and GBA, is a lysosomal enzyme that cleaves glucosylceramide. People with inactivating mutations in both copies of GCase develop Gaucher’s Disease, which can be treated with a recombinant form of GCase. Heterozygous mutations increase risk for Parkinson’s Disease and for dementia with Lewy bodies, and though the mechanism is unclear, stabilizing the enzyme and/or boosting activity of residual GCase might help. This approach is described in a recent J. Med. Chem. paper by Nick Palmer and colleagues at Astex Pharmaceuticals.

 

The researchers started with a crystallographic screen of 440 fragments, resulting in a whopping 91 hits. In parallel, 1800 fragments (including the aforementioned 440) were screened using ligand-observed NMR, SPR, and thermal shift assays, and hits were confirmed crystallographically to yield another 15 structures. Astex has previously reported that multiple ligand binding sites are common in proteins, and GCase is no exception, with the 106 ligands binding to 13 distinct sites.

 

With this embarrassment of riches, prioritization became critical. Sites formed by crystal packing and shallow solvent-exposed sites were deprioritized, along with those near the active site, since ligands binding there might inhibit the enzyme. SPR was not well-suited to measuring ligand affinities due to non-specific binding, and ligand-observed NMR was similarly complicated due to multiple binding sites. However, isothermal titration calorimetry (ITC) proved to be effective, and this technique was used to narrow in on two binding sites.

 

Site A was particularly attractive: it had 31 fragment hits, one of which has a respectable dissociation constant of 12 µM. Screening of analogs did not lead to anything better, but merging this fragment with another Site A fragment led to compound 15. Interestingly, crystallography revealed that this molecule binds not at Site A but at Site B. Although the affinity is low, the ligand efficiency is respectable. The fragment also makes several polar interactions and has multiple vectors for growing the molecule.

 

 

Testing analogs of compound 15 led to compound 16, and growing led to compound 17, with low micromolar affinity. Further structure-based design ultimately led to compound 22, with low nanomolar affinity. The molecule increased GCase activity in a cellular assay, albeit at a fairly high (mid-micromolar) concentration. The molecule was found to be cell permeable with no efflux, so the source of the disconnect between affinity and cell activity is unclear.

 

This lovely example of structure-guided fragment-based ligand design holds several lessons. First, as noted above, finding fragments is often the easy part; selecting among them and figuring out what to do next can be challenging. Second, especially at the earliest stages of optimization, fragments can change not just their binding mode but their binding site entirely.

 

Finally, figuring out which sites will be best for high-affinity allosteric ligands isn’t necessarily straightforward. Of the 105 fragment hits at 13 sites, only four bound in Site B, yet this site turned out to be more fruitful than Site A, which had many more bound fragments. The researchers note that Site B had previously been identified as ligandable by FTMap, supporting the utility of computational approaches.

 

The researchers conclude, “we hope that our findings will be of use to the wider community.” Certainly from a best practices perspective the paper succeeds. And although the most advanced molecules described do not meet all the criteria for robust chemical probes, and it is unclear whether they will work with mutant proteins, they could still be useful to better understand the complicated biology of GCase.

A bright idea for rapid affinity measurements

Finding fragments that bind to a target is important but so is measuring their affinities. NMR methods can find even weak fragments, but accurately assessing affinities takes time. In a recent (open-access) J. Am. Chem. Soc. paper, Felix Torres, Roland Riek, and collaborators at the Institute for Molecular and Physical Science and NexMR provide a new, fast method.

 

The approach is based on photochemically induced dynamic nuclear polarization (photo-CIDNP), which we wrote about here; Felix also spoke about it at the FBDD-DU meeting in June. As the name implies, the technique involves illuminating NMR samples to electronically excite ligands, thus increasing the signal to noise ratio of the NMR signal by as much as 100-fold. Previous work focused on using the method to identify binders, even with cheap, benchtop NMR instruments.

 

The new paper describes how to quantitatively measure dissociation constants using photo-CIDNP. The theory gets a bit hairy, but the basic idea is that the more photochemically excited ligand that binds to the protein, the more the signal decreases. A series of samples are prepared with increasing concentrations of ligand and either no protein or a fixed concentration of protein. After measuring the NMR signals, the data are plugged into equations to derive the K_D values in a method called CIDNP-K_D.

 

As the researchers have previously noted, not every ligand can be photosensitized. However, dissociation constants can still be measured for these using competition experiments with previously characterized reporter ligands that can polarized, akin to using to NMR competition studies with ¹⁹F reporter ligands (see here).

 

So how well does the technique work? The researchers first turned to the PDZ2 domain of a phosphatase called hPTP1E, which is involved in cell proliferation. They measured the affinities of a series of peptides having 4 to 8 amino acid residues and compared these values to those obtained using two dimensional [¹H,¹⁵N]-HSQC chemical shift perturbation, the gold standard NMR technique. Affinities ranged from low micromolar to low millimolar, and there was reasonable agreement (generally within about two-fold) between both techniques. Most of the peptides contained tryptophan, which is suitable for photo-CIDNP, but CIDNP-K_D also worked in competition mode when non-tryptophan containing peptides were competed against peptides containing tryptophan. And the technique was fast, with each datapoint taking only 30 seconds for photo-CIDNP compared to as long as 80 minutes for HSQC NMR.

 

Next the researchers turned to fragments. They had previously conducted a screen against the oncology target PIN1 and identified a number of fragment hits, two of which had been characterized in detail. The affinities of these were measured by CIDNP-K_D, and the low millimolar values agreed with those from HSQC NMR.

 

Another neat application described in the paper is “CIDNP-based epitope mapping,” which is based on the fact that an excited proton on a ligand that is in close proximity to the protein will relax more rapidly than one that is distant from the protein. This phenomenon is similar to STD epitope mapping, and the two methods yielded similar information for the two PIN1 ligands: one region of each molecule was buried in the protein, consistent with crystal structures.

 

One drawback of the technique is that, because measurements require fast protein-ligand exchange, CIDNP-K_D is limited to relatively weak binders (K_D > 10 µM), but this is usually not a problem in the early stages of a fragment program. A full affinity measurement takes about 15 minutes, which compares very favorably to two hours using [¹H,¹⁵N]-HSQC and without the need for isotopically labeled protein. It would be interesting to run head-to-head comparisons with two ligand-based NMR techniques we wrote about last year, imaging STD NMR and R2KD, to see how they compare in terms of speed, accuracy, and generality. Please let us know if you’ve done so.

Fragments vs GPx4 – in reverse micelles

Membrane proteins account for more than half of drug targets, but the fraction is far smaller for fragment-derived drugs. In part this is because biophysical methods, the mainstay of FBLD, have been harder to apply to membrane proteins. A recent (open-access) paper in JACS Au by Courtney Labrecque and Brian Fuglestad at Virginia Commonwealth University tackles this challenge.

 

The researchers use an approach called membrane-mimicking reverse micelles, or mmRM: tiny water-filled bubbles surrounded by lipids and suspended in an organic solvent. We last wrote about reverse micelles back in 2019, where they were being used to study high local concentrations of water-soluble proteins and ligands. Here, the researchers turned to membrane proteins.

 

There are actually two types of membrane proteins: integral membrane proteins and peripheral membrane proteins. The former, as their name implies, have at least part of the protein anchored in the membrane at all times; GPCRs are a prominent class. Peripheral membrane proteins are water soluble but associate with the membrane, and this interaction is often required for folding or function. One example is glutathione peroxidase 4 (GPx4), which reduces oxidized lipids. It is an intriguing but  challenging cancer target, with the only ligands being fairly reactive covalent modifiers. Thus the researchers turned to mmRMs, hoping these could both stabilize the protein in a biologically relevant state and also present binding opportunities unavailable in standard screens.

 

A library of 1911 fragments from Life Chemicals was screened against mmRM-encapsulated GPx4 using ¹⁵N-¹H HSQC protein-detected NMR. Fragments were chosen to have high aqueous solubility (at least 1 mM in PBS) and were screened in mixtures of 10 at 400 µM per fragment. After deconvolution, 14 hits were identified, and dose-response titrations revealed that 9 had apparent dissociation constants < 1 mM, with the most potent having a K_d = 105 µM.

 

Three fragments were studied in greater detail, and these were chosen to have a range of hydrophobicities from clogD = -2.1 (most polar) to clogD = 2.1 (most hydrophobic). Chemical shift perturbation (CSP) analyses suggested that the two more lipophilic fragments bind to the membrane-interacting region of the protein, while the more polar fragment likely binds to a water-exposed site. SAR-by-catalog was applied to find analogs, some of which had increased affinity for the protein, with the best being around K_d = 15 µM.

 

Interestingly, the fragments showed minimal binding to GPx4 under normal aqueous conditions (ie, in the absence of the mmRMs), even at very high fragment concentrations. The researchers suggest this is because the fragments are binding to the membrane-bound state of the protein found in mmRMs, which may adopt a different conformation than that in the absence of membranes. Perhaps. But as prior work shows, it is possible to detect extraordinarily low affinity interactions inside reverse micelles, so maybe these are just very weak binders. Ultimately it remains to be seen whether these fragments will have practical applications. I hope so, and look forward to seeing how they progress.

How to avoid metal artifacts

Back in 2017 we observed with characteristic subtlety that “heavy metals suck.” That post described a hit-finding campaign which foundered when the apparent activity of the fragments turned out to be due to contaminating zinc. A new paper in J. Med. Chem. by Thomas Gerstberger, Peter Ettmayer, and colleagues at Boehringer Ingelheim (BI) describes a similar story, along with suggestions of how to avoid being misled.

 

BI had a collaboration with FORMA Therapeutics that entailed screening roughly 1.7 million compounds against ten targets using biochemical and cell-based assays. The effort resulted in chemical probes against BCL6 and SOS1 and a clinical compound against the latter. Another target was the activated (GTP-loaded) form of KRAS^G12D. Of the 6917 hits from the primary AlphaScreen assay, 1535 gave dose-response curves and passed various counter screens. Of these, 87 representative compounds were tested in STD NMR and thermal shift assays. Only seven confirmed by STD NMR, but these did not confirm by SPR or crystallography.

 

In parallel, the researchers were successfully using FBLD to develop inhibitors of KRAS^G12D, which we wrote about here. Some of the fragment hits were structurally similar to those from the HTS screen, and further searching of the FORMA library led to fairly potent (high nanomolar or low micromolar) hits in the AlphaScreen assay. Two of these even yielded crystal structures, though despite their chemical similarity to one another they bound to the protein in completely different orientations.

 

Unfortunately, follow-up work “revealed erratic structure-activity relationships,” and upon resynthesis the compounds were much less active. At this point the researchers became suspicious, and analyses of the original samples showed they contained >20,000 ppm of palladium contamination. Furthermore, PdCl₂ itself turned out to be a low micromolar inhibitor in the assay.

 

Metals are frequently used as catalysts or reagents in organic synthesis and can be difficult to completely remove during purification. Worse, their presence is often not detectable using standard purity assessments such as HPLC and NMR. Particularly in the case of fragments, which are expected to have low affinities, a small amount of metal contaminant could give a reasonable-looking but misleading signal in an assay.

 

To avoid this problem in the future the researchers developed a Metal Ion Interference Set, or MIIS, consisting of a dozen different metal ions and other salts, all soluble in DMSO so as to be compatible with typical screens. The MIIS is now routinely screened before initiating HTS campaigns, and the results of 74 assays are summarized in the paper. Pd²⁺, Au³⁺, and Ag¹⁺ are particularly nasty, often giving IC₅₀ values < 1 µM, but every metal gave IC₅₀ values < 10 µM in at least two assays. Biochemical assays such as AlphaScreen or TR-FRET were more susceptible to artifacts, with 20.9% showing IC₅₀ < 10 µM, while biophysics assays such as mass spectrometry were better behaved, with only 2.3% showing IC₅₀ < 10 µM. Cellular assays were also surprisingly robust, with 6.3% showing IC₅₀ < 10 µM.

 

This is a nice paper showing that even a massive screen may produce no useful chemical matter. Soberingly, the fact that some of the fragments gave reasonable-looking crystal structures even though the functional activity came from metal contaminants is a salutary reminder that just because you have a crystal structure of a bound ligand doesn’t mean you have a viable starting point.

 

Forewarned is forearmed, and the MIIS appears to be a valuable tool for assessing assay sensitivity to metal ions, which are all too often lurking invisibly in compound samples.

Multiplexing (native) mass spectrometry

Native mass spectrometry (nMS) is one of the less commonly used fragment-finding methods. The approach entails mixing proteins and ligands and gently ionizing them under non-denaturing conditions to look for complexes. As with many other methods, multiple fragments can be screened in a single sample. In a new ACS Med. Chem. paper, Ray Norton and collaborators at Monash University and CSIRO report screening multiple proteins in a single sample.

 

The researchers were interested in fatty acid-binding proteins, or FABPs. As their name suggests, these transporter proteins shuttle lipophilic molecules such as fatty acids around cells. The ten human isoforms are expressed in different tissues and have different functions in metabolic signaling, but their similarity to one another has made finding selective chemical probes difficult. Enter nMS.

 

FABP isoforms 1-5 are the most heavily studied, and these were first assessed individually. They ionized well, though in some cases peaks corresponding to both the native protein and a complex with acetic acid was observed, not surprising given that the buffer contained 50 mM ammonium acetate.

 

Next, all five proteins were mixed together at 10 µM each. All the proteins could still be observed (with or without bound acetate), though some proteins did give stronger signals than others due to differences in ionization efficiency.

 

Adding small molecule WY14643, which the researchers had previously found to bind to FABPs in a fluorescence polarization (FP) assay, led to a more complex spectrum, with peaks corresponding to unbound proteins, proteins bound to WY14643, proteins bound to acetate, and proteins bound to both acetate and WY14643. When WY14643 was added at 10 µM, the selectivity profile was consistent with the FP data. Interestingly though, when ligand was added at the total concentration of all protein isoforms (50 µM), the selectivity profile changed. The researchers suggest this may be due to nonspecific binding at higher ligand concentrations, as has been seen previously for nMS.

 

To explore the generalizability of multiplexing nMS, the researchers turned to more potent (nanomolar) ligands. As with WY14643, these molecules showed good agreement with published selectivity rankings at lower ligand concentrations with some non-specific binding at higher concentrations.

 

When I first wrote about nMS back in 2010, I noted that “the stability of protein-small molecule complexes in native mass spectrometry assays does not necessarily correlate with the (more relevant) solution-phase affinity,” and this fact is investigated in the paper. Careful optimization of the experimental conditions, including ionization voltage and temperature, led to good relative selectivity rankings for a given ligand across the different FABP isoforms but differences in absolute values from those measured by ITC.

 

Another challenge is the fact that the five FABP isoforms tested have similar molecular weights; in one case a ligand complexed with FABP3 was difficult to distinguish from free FABP2. The researchers could solve this by using different protein constructs, such as a hexa-histidine-tagged version of FABP3.

 

Overall this is an interesting approach, and the paper does an excellent job describing the technical details and limitations. Along with protein-observed ¹⁹F NMR, mass spectrometry is a rare experimental technique suitable for screening mixtures of proteins in solution. Indeed, this becomes even easier when screening covalent binders, as seen in this paper from 2003, since there is no need to worry about ligand dissociation during ionization. And with the increasing interest in covalent drugs, the use of MS is only likely to increase.

SAR by TR-HT-SAXS

Well that’s an acronym soup! SAR by NMR was the first practical fragment-finding method, and over the years Practical Fragments has covered lots of other techniques. Small-angle X-ray scattering, or SAXS, has not been among them. As the name suggests, this technique uses X-rays, typically produced at a synchrotron. However, unlike conventional crystallography, it doesn’t require crystalline material. Instead, proteins in solution are analyzed to provide information on their size and shape. The resolution is too low to assess small molecule binding, but suitable for observing dimerization or changes in conformation.

 

Time-resolved SAXS, or TR-SAXS, examines SAXS over time in response to a trigger. For example, you can rapidly add a ligand to a protein and watch for changes in conformation. And HT simply means high throughput. A recent Nature Chemical Biology paper from Chris Brosey, John Tainer, and collaborators at the University of Texas MD Anderson Center, Lawrence Berkeley National Laboratory, University of California Santa Cruz, and University of Arkansas for Medical Sciences Little Rock describes structure-activity relationships by time-resolved high throughput small-angle X-ray scattering (TR-HT-SAXS).

 

The researchers were interested in apoptosis-inducing factor (AIF), a mitochondrial protein with potential implications for cancer and other diseases. AIF normally exists as a monomer in complex with an FAD cofactor. Binding of NADH causes reduction of FAD to FADH^- and concomitant dimerization of the protein. Could fragments do the same, allowing dimerization on demand?

 

A library of 2500 fragments purchased from Life Chemicals was screened at 0.75-1.5 mM against the AIF-FAD complex using differential scanning fluorimetry (DSF), and those that raised or lowered the temperature by more than 1.7 ºC were further characterized by microscale thermophoresis (MST). This led to 32 binders and 7 negative controls, or molecules that did not confirm either by DSF or MST. (Side note: although many people discount compounds that give negative thermal shifts, the natural ligand NADH lowers the melting temperature of AIF by a whopping 10.8 ºC.)

 

Next, the fragment binders and negative controls were screened at 0.5-1 mM by TR-SAXS. Intense X-rays cause reduction of the FAD cofactor, but in the absence of NADH or other ligands the AIF protein remains monomeric. However, some fragments did cause dimerization of the protein during TR-SAXS. Interestingly, these fragments were structurally related to one another. Subsequent crystallography revealed that they bind where NADH normally binds and make some of the same interactions to induce protein dimerization. The paper includes much more detailed characterization, including mutagenesis, spectroscopic, and protein crosslinking experiments to further understand the mechanism.

 

TR-SAXS is an interesting addition to our toolbox of biophysical methods suitable for fragment screening. It does have some disadvantages, such as the need for large amounts of protein at high concentrations: 67 µM in this case. Also, the “HT” may be somewhat aspirational, with a current throughput of 100-200 compounds per synchrotron shift. Finally, the technique is probably best suited to well-characterized proteins where SAXS data can be carefully modeled. With these limitations in mind, it will be fun to see how generally TR-SAXS finds fragments that alter the conformation and multimerization of proteins.

Fragment-based Drug Discovery Down Under (FBDD-DU) 2024

The end of June brought me to Brisbane for the fifth FBDD-DU Conference, which was meeting for the first time outside Melbourne. This was also my first FBDD-DU conference since 2019, and it was nice to see a wide range of talks from around Australia and beyond. As always, I won’t attempt to be comprehensive, so if you attended, please feel free to add your observations.

 

Techniques

Experimental techniques received considerable attention. Félix Torres (NexMR) described using an inexpensive benchtop NMR that doesn’t require liquid helium. Fragments were screened using photochemically induced dynamic nuclear hyperpolarization (photo-CIDNP). The method is so rapid that it is limited more by sample handling than data collection, and the Torres team is speeding things up using flow technology. Right now photo-CIDNP is still very much DIY, but rumor has it that Bruker may soon launch a photochemical module for their benchtop instrument.

 

We’ve written about high-throughput crystallographic screening at the Diamond Light Source, and synchrotrons around the world are building similar platforms. Kate Smith described integrated systems at the Swiss Light Source which automate crystallization, fragment screening, data collection, and data processing. She also described increasing automation of fragment screening using the free-electron laser (FEL), which we wrote about here. Current throughput is around 40 compounds per day and requires large amounts of protein, but these are still early days.

 

Australia is building their own high-throughput crystallography platform, and various components were described by Roxanne Smith (University of Melbourne), Gautham Balaji (Monash Univesrity), and Yogesh Khandokar (ANSTO-Australian Synchrotron). Watch this space!

 

Speaking of Australia, Nyssa Drinkwater described Compounds Australia, a national repository of more than 2.5 million molecules, including several fragment collections. Members, who can be from outside Australia, can store their own libraries within the facility to ease collaborations with other groups, and they can also access public libraries of compounds, including unusual Antipodean natural product extracts. I was fortunate to be able to visit the facility at Griffith University and can attest that it is easily the equal of those in large pharma.

 

Turning to mass spectrometry, Sally-Ann Poulsen (Griffith University) described covalent library screening against PRMT5, a target we’ve written about here. Sally-Ann is also a pioneer of (conventionally non-covalent) native mass spectrometry, and she described applying this methodology to screen small molecules against RNA.

 

But the star of the conference was SPR, appearing in multiple talks. Long-time readers may recall an instrument made by SensiQ, with its gradient injection capability to accelerate data collection. This is now marketed by Sartorius, and Lauren Hartley-Tassell (Griffith University) described using it to screen a glycoprotein. The larger plumbing in the instrument is less prone to clogging, and Lauren said it can even accommodate screening of whole cells.

 

Anything to accelerate the (sometimes painful) process of advancing fragments is always welcome. As Jason Pun (Monash University) noted, eight of nine targets screened in Martin Scanlon’s group started with fragments having affinities worse than 100 µM. Off-rate screening, an SPR technique we wrote about here, can rapidly identify more potent molecules from crude reaction mixtures, but data processing can be tedious. Jason described new software tools to automate this process, and hopefully he will publish the methodology and code. (An aside: over coffee Yun Shi of Griffith University noted that off-rate screening, or ORS, should really be called off-rate constant screening, which would give the more amusing acronym ORCS.)

 

Targets

Turning to targets, Ben Davis (Vernalis) described a collaboration with Servier to advance oncology target USP7 inhibitors from a literature fragment to a preclinical candidate. Crude reaction mixture screening was used extensively, not just by SPR but even in microsome stability studies. Unfortunately the project ended when on-target toxicology effects emerged, which were perversely more severe in higher animal species than they were in mice.

 

Yun Shi described finding tiny heterocyclic fragments that react with the NAD⁺ cofactor of neurodegenerative target SARM1 in situ to generate a potent inhibitor, as we wrote about here. Yun is using ¹⁹F NMR to follow the base-exchange reaction to identify inhibitors to other glycohydrolases too.

 

Deaths due to E. coli are – somewhat surprisingly – more common than those caused by any other pathogen, and Christina Spry described her work at the National Australian University to discover inhibitors of the essential dephosphocoenzyme A kinase (GPCK) enzyme, which catalyzes the final step in the synthesis of Coenzyme A (CoA). Fragment screening by DSF and NMR identified a weak (K_D=380 µM) binder, and fragment growing has led to a low nanomolar inhibitor that is selective against the human form of the enzyme.

 

Continuing the E. coli theme, several talks discussed efforts against the challenging bacterial virulence target DsbA, a twenty-year campaign in Martin Scanlon’s group at Monash as noted by Yildiz Tasdan. The enzyme has a shallow, hydrophobic active site, but the discovery of fragments binding to a cryptic site and crude-reaction screening by ORS (ORCS?) and affinity-selected mass spectrometry (ASMS) has finally led to molecules with dissociation constants around 1 µM.

 

Finally, in his closing keynote address Alvin Hung, who recently founded NeuroVanda, described a wide range of fragment success stories, many of them covered on Practical Fragments, against targets including pantothenate synthetase, GSK3β, PKC-ι, and MNK1/2. Although structural enablement helped in many cases, Alvin was not rigid about the need for atomic-level details: in response to the question whether he would advance a fragment in the absence of structure, he answered simply, “of course.” Perhaps it's time to redo my poll on this subject.

 

I’ll wrap up here, but if you missed this or earlier events this year there are still a couple more conferences in Boston, and 2025 is already starting to take shape.

Fragment events in 2024 and 2025

The year is half-way done, and we've seen some great events; I'll share my thoughts on FBDD Down Under 2024 next week.

Boston is where it's at in the second half of 2024, and it's not too soon to start planning for 2025.

September 22-25: After a six year hiatus, FBLD 2024 will be held in Boston. This will mark the eighth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2018, FBLD 2016, FBLD 2014, FBLD 2012, FBLD 2010, and FBLD 2009. Early-bird registration ends August 12, so don't delay!

 

September 30 to Oct 3: Autumn is usually a nice time of year in Boston, so stick around to attend CHI’s Twenty-Second Annual Discovery on Target. As the name implies this event is more target-focused than chemistry-focused, but there are always plenty of FBDD-related talks. You can read my impressions of the 2023 meeting here, the 2022 meeting here, the 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.

 

October 21-22 Returning after a four year hiatus, Industrial Biostructures of America will be held in Cambridge, MA and includes a session on FBLD. You can still submit abstracts for talks until September 1. (Updated Aug 21.)

 

Finally, from December 3-5 CHI holds its first-ever Drug Discovery Chemistry Europe in beautiful Barcelona. This will include tracks on lead generation, protein-protein interactions, degraders, and machine learning, with several fragment talks. (Updated July 8.)

 

2025

April 14-17: CHI’s Twentieth Annual Fragment-Based Drug Discovery, the longest-running fragment event, returns as always to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2024 meeting here, the 2023 meeting here, the 2022 event here, the 2021 virtual meeting here, the 2020 virtual meeting here, the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting here, here, 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. 

  

Know of anything else? Please leave a comment or drop me a note.

Fragments vs LTA4H: LipE in action

Three years ago we described the discovery of LYS006, an inhibitor of leukotriene A4 hydrolase (LTA4H) from Novartis currently in phase 2 clinical trials. Companies often pursue multiple chemical series for important targets, and in a recent J. Med. Chem. paper Gebhard Thoma and colleagues describe another fragment-derived lead against LTA4H.

 

A biochemical high-throughput screen yielded compound 2, which is quite potent for a fragment-sized molecule. However, despite good ligand efficiency, the LipE (or LLE) was less impressive due to the high lipophilicity of the fragment. (Note that throughout the paper LipE is calculated based on measured logD rather than logP.) A co-crystal structure revealed that it bound in a similar fashion to other previously characterized LTA4H inhibitors such as compound 1, derived from LYS006 and reported in a J. Med. Chem. paper last year. Adopting elements from these led eventually to compound 12, which though less potent was also much less lipophilic and more soluble while still remaining fragment-sized.

 

 

Continuing to borrow from the rich literature around this target, the researchers added a basic amine group to get to the very potent compound 14. This was metabolically unstable, but further optimization led to compound 3.

 

Compound 3 was profiled extensively in a battery of tests. In addition to good biochemical potency, it showed mid-nanomolar activity in a human whole blood assay and was also active in other assays, including a mouse arthritis model. Other attractive features included a clean profile against a plethora of off-targets, good oral bioavailability in mice, rats, and dogs, and a predicted human oral dose of 40 mg once daily. However, a two week toxicology study in rats and dogs was “slightly less favorable” than compound 1.

 

This is a lovely example of property and structure-guided drug design, and the researchers are refreshingly open about borrowing elements from other molecules, even from outside Novartis. Interestingly, a crystal structure of compound 3 bound to LTA4H revealed that while the overall binding mode was similar to compound 1, which contains the same left-hand portion, the pyrazole and pyridine rings rotated 180º to make different hydrogen-bond interactions. Another reminder that despite our leaps in predictive capability, molecules can still provide many surprises.

Fragments vs MAT2a: a chemical probe

As many of us know all too well, traditional methods to treat cancer often result in severe and even intolerable side effects. An emerging, gentler approach is based on synthetic lethality: targeting a protein that is essential only in certain cancer cells but not in normal cells. One prominent target is MAT2a, one of two human methionine adenosyltransferases. We’ve written previously about AG-270, a fragment-derived MAT2a inhibitor that entered the clinic. AstraZeneca has also pursued this target, as we discussed here. In a new J. Med. Chem. paper, Stephen Atkinson, Sharan Bagal, and their AstraZeneca colleagues describe a new chemical probe.

 

A differential scanning fluorimetry (DSF) screen of about 55,000 compounds at 100 µM, nearly a third of which were fragments, resulted in a healthy 1.5% hit rate. Further DSF as well as biochemical testing ultimately delivered compound 8, which is quite potent for a fragment. A crystal structure of the compound bound to MAT2a demonstrated that it bound in the same allosteric site targeted by other compounds. The methoxy group was pointed towards a couple backbone carbonyl oxygen atoms, and adding a couple fluorine atoms created a weak hydrogen bond donor with a satisfying 50-fold boost in potency.

 

Adding a hydrogen bond acceptor (compound 12) slightly reduced potency but also decreased lipophilicity. Further inspection suggested opportunities for fragment growing, and free energy perturbation (FEP) calculations suggested that adding the methoxyphenyl group of compound 15 would be fruitful. This turned out to be the case, and further optimization led to AZ’9567. The paper provides plenty of meaty medicinal chemistry, with significant efforts focused on reducing lipophilicity and clearance. FEP was used extensively during the design process, and a retrospective analysis found a good correlation between predicted and measured affinity.

 

AZ’9567 was studied in considerable detail. It has excellent oral bioavailability and good pharmacokinetics in both mice and rats. The compound does not significantly inhibit cytochrome P450 enzymes or hERG and is reasonably clean against a panel of 86 off-targets. The main liability is poor solubility, a problem also faced by AG-270. Nonetheless, the AstraZeneca researchers were able to develop a liquid formulation.

 

The paper compares AZ’9567 with AG-270, showing that both compounds are potent in biochemical assays as well as against cell lines in which MAT2a is essential. A mouse xenograft model with AZ’9567 showed considerable and sustained tumor growth reduction.

 

Unfortunately, AG-270 is no longer in clinical development, and there is no mention of a MAT2a inhibitor in the AstraZeneca pipeline. Nonetheless, having a second well-characterized chemical probe will be useful for further characterizing the biology of MAT2a and assessing whether it will be a productive drug target.

Fragments vs CDC14 phosphatases

Practical Fragments has periodically written about protein tyrosine phosphatases (PTPs), which remove phosphate moieties from tyrosine side chains in proteins. Despite decades of attention, progress towards selective inhibitors has been slow due to both the similar active sites and their highly charged nature. A new paper in J. Med. Chem. by Zhong-Yin Zhang and colleagues provides some hope.

 

The researchers were interested in CDC14 phosphatases, so-called dual-specificity phosphatases that can dephosphorylate phosphoserine and phosphothreonine in addition to phosphotyrosine. Two members of this family, hCDC14A and hCDC14B, are widely expressed in humans, but their role in cancer is ambiguous, with some studies suggesting they are oncoproteins while others suggest they may have a protective function. Clearly a chemical probe would be useful.

 

The researchers started by considering non-hydrolyzable phosphotyrosine mimetics, specifically those replacing the central oxygen with a difluoromethyl moiety; we wrote about this bioisostere back in 2013. Eight fragments were made and assessed at 1 mM in aqueous buffer to demonstrate they did not aggregate. They were then tested in functional assays against a panel of ten PTPs, and compound 9 turned out to be quite potent and selective for hCDC14A. Subsequent experiments showed it to have similar activity against hCDC14B, and Lineweaver-Burk plots revealed it to be a competitive inhibitor of both, as expected.

 

Although no hCDC14A structures have been reported, modeling the compound into a published structure of hCDC14B gave some insights into the binding mode and selectivity. In particular, hCDC14B has a larger active site than some other PTPs, thus explaining why the tricyclic compound 9 could fit. Further analysis suggested the possibility of growing the compound towards a hydrophobic pocket, so the researchers synthesized a small set of molecules, of which compound 15 turned out to be the most potent.

 

Compound 15 was tested against 16 PTPs and found to be quite selective against hCDC14A and hCDC14B, with IC₅₀ values 5 µM or worse against the others. Mutagenesis studies in the hydrophobic pocket were consistent with the proposed binding mode. Despite the presence of the highly charged difluorophosphonate moiety, compound 15 showed activity in cells at low micromolar concentration and had some oral bioavailability in mice.

 

Although better cell activity is probably necessary to make a truly useful chemical probe, this is a nice start. Researchers at AbbVie have taken a competitive inhibitor of a different PTP into the clinic, so perhaps we will start to see more successes against these challenging enzymes.

Throwing the kitchen sink at IL-1β

Last year we highlighted a paper out of Novartis describing a fragment-to-lead story for interleukin-1 beta (IL-1β), a pro-inflammatory cytokine implicated in numerous diseases. The approved antibody drug canakinumab targets IL-1β, but a small molecule would provide easier oral dosing as well as better access to tissues such as the central nervous system. A new paper in J. Med. Chem. by Anna Vulpetti, Konstanze Hurth, and their Novartis colleagues describes the multiple approaches they've taken. (Anna also presented this work at Fragments 2024.)

 

The paper starts by summarizing the fragment work we described here. Notably, of nearly 4000 fragments screened, only a single super-sized fragment was validated, and it was quite weak. The researchers were able to optimize this to a molecule that inhibits binding of IL-1β to its receptor with an IC₅₀ = 1.1 µM.

 

Starting from the initial fragment hit, the researchers performed virtual screens to find alternative binders. Of 281 selected for testing by ¹⁹F NMR or TR-FRET, two hits were obtained, one with an affinity of around 230 µM and the other worse than 1 mM. These molecules were similar to each other, and merging them led to a 43 µM binder. All molecules exceeded conventional fragment size, with the smallest containing 24 non-hydrogen atoms. We’ve previously discussed the possible need for larger fragments for difficult targets such as protein-protein interactions.

 

In addition to FBLD, the researchers also performed DNA-encoded library (DEL) screens using 15 libraries containing >1.6 billion molecules. This led to one family of hits, one member of which inhibited binding of IL-1β to its receptor with an IC₅₀ = 8.3 µM. This molecule contains an aldehyde moiety, a reversible covalent electrophile. Subsequent experiments confirmed that the aldehyde reacts with a lysine residue on IL-1β, and the researchers were able to improve the potency to 1.2 µM. This molecule is even larger than the hit derived from fragments, with >50 non-hydrogen atoms. Interestingly, the molecule binds at a different site on the protein from the initial fragment hit.

 

Finally, the researchers screened a library of macrocyclic peptides in an mRNA display system. The macrocycles consisted of 10-14 amino acid residues, and the library was impressively large, containing “<10¹³ unique cyclic peptides.” This effort yielded a 14 µM inhibitor. Strikingly, crystallography revealed that the molecule binds at a site distinct from either the fragment- or DEL-derived hits.

 

This paper is a tour de force addressing a difficult target. Although the researchers conclude that the protein is “ligandable,” the physicochemical properties of all the hits will need to be improved, along with the affinities, in order to make useful chemical probes, let alone drugs. On the other hand, the fact that the ligands bind to different sites and yet can all inhibit the protein-protein interaction is encouraging, offering multiple opportunities for optimization.

Free computational fragment growing with ChemoDOTS

Back in 2018 we highlighted diversity-oriented target-focused synthesis, or DOTS, a combined computational and experimental method for growing fragments. The computational piece of this has now been turned into a free web server, called ChemoDOTS, and is described in Nucleic Acids Research by Xavier Morelli, Philippe Roche, and colleagues at Aix-Marseille University.

 

To get started, the user draws or uploads the structure of a fragment hit they wish to expand. ChemoDOTS identifies potentially reactive functionalities, such as amine groups. For each functionality, the program also provides compatible reactions, derived from a set of 58 commonly used in industry. The user then chooses one or more reactions of interest, at which point the program generates a list of molecules that could be created by linking the fragment to various building blocks using the selected chemistries. The building blocks themselves consist of 501,542 commercially available molecules from MolPort and 988,112 molecules from Enamine having between 4 and 24 non-hydrogen atoms.

 

The program generates molecules quite rapidly, between 1000-1500 per second. All of these can be downloaded at this point, but ChemoDOTS also allows further processing. Histograms showing molecular weight, cLogP, total polar surface area, the number of hydrogen bond donors and acceptors, and Fsp³ for the library are displayed, and the user can adjust sliders to select molecules having, for example, cLogP between 1 and 3 and 0-2 hydrogen bond donors. Finally, ChemoDOTS generates three dimensional conformers in a ready-to-dock format for each compound.

 

As a retrospective example, the researchers return to the BRD4 case study we wrote about here. Starting from the amine-containing fragment and the sulfonamidation reaction, ChemoDOTS generated 5546 molecules in just 5 seconds, including all 17 of those previously identified.

 

This is a nice approach, and I believe the researchers are correct when they say that to the best of their knowledge “ChemoDOTS is the only freely accessible functional and maintained web server to combine the design of medchem-compatible virtual libraries with an integrated graphical postprocessing analysis.” They plan to continue improving it, for example by adding new commercial building blocks from other sources.

 

If I could make one suggestion, it would be to include new types of chemistries beyond the 58, which came from a paper published in 2011. In particular, C-H bond activation methodologies have made impressive strides in recent years. Adding these is all the more important given that, according to a recent analysis, about 80% of successful fragment-growing campaigns involved growth from a carbon atom. But even in its current form, ChemoDOTS looks to be a useful approach for growing focused chemical libraries around fragment hits. Let us know how it works for you!

Screening MiniFrags by NMR

Small is becoming big. Five years ago we highlighted MiniFrags, consisting of just 5-7 non-hydrogen atoms; FragLites and MicroFrags soon followed. Screening these tiniest of fragments at high concentrations can thoroughly explore hot spots on a protein and identify favorable molecular interactions. But because they are so extraordinarily small, experimental methods for screening them have been mostly limited to crystallography. In a new J. Med. Chem. paper, Annagiulia Favaro and Mattia Sturlese (University of Padova) turn to the most venerable of fragment-finding methods, NMR.

 

The researchers started with the 81 reported MiniFrags and removed those with aqueous solubility less than 250 mM or without protons observable by NMR (such as phosphate). The remaining 69 fragments were dissolved directly in phosphate buffer, mostly at 1 M concentration, though lower solubility fragments were dissolved at 250 mM. Importantly, the pH of each sample was carefully adjusted to 7.1 to ensure that any signals correspond to MiniFrag binding and not to changes in experimental conditions.

 

As a test case, the researchers chose the antiapoptotic target BFL1. This protein is related to BCL2, the target of venetoclax, which was discovered using SAR by NMR. BFL1 has a hydrophobic cleft with five subpockets and has been studied by NMR. Like other BCL2 family members it is a difficult target, as we noted earlier this year.

 

The actual screen was done using chemical shift perturbation (CSP) detected by two-dimensional ¹H-¹⁵N HMQC. Fragments were screened at 100 mM, a 5000-fold excess above the protein concentration. Hits were confirmed at 20 mM (more on that below). As with the library preparation, pH was carefully controlled.

 

At such high ligand concentrations, any impurities could become a problem: a 2% contaminant would be present at 2 mM. To weed these out, the researchers performed WaterLOGSY experiments. These only produce a signal at ligand to protein ratios much lower than 1000 to 1, so any hits could only come from impurities.

 

Even at high concentrations, CSPs caused by weak fragments are small, so the researchers developed an analysis method to identify those that shift more than at least one standard deviation from the average. CSPs can shift in any direction on a two-dimensional map, but any one protein-ligand interaction should shift signals in the same direction. Here is where the 20 mM confirmation experiment comes into play: a “cosine similarity” assesses whether two CSPs are in the same direction and thus likely to be real.

 

Screening BFL1 led to 53 hits, a hit rate of 78%, similar to crystallographic screens of MiniFrags against other targets. Forty percent of MiniFrags bound to multiple sites on the protein; only 11 (16%) bound to a single site. The five subpockets were each liganded by 6-17 MiniFrags. In subsequent experiments, the researchers were also able to measure binding of two different fragments to different pockets simultaneously, akin to SAR by NMR.

 

This is an interesting approach, but while fragments with >5 mM dissociation constants have been advanced to drugs, the utility of a 100 mM binder remains to be seen. That said, the technique could be a boon for understanding protein-ligand interactions, and I look forward to seeing it applied more broadly. In particular, screening the same set of MiniFrags on the same protein by NMR, crystallography, and computational methods could be quite informative.

Fragments in cells, writ large

Earlier this year we highlighted work in which a dozen fragments were screened against cells to look for noncovalent binders across the proteome. A new paper in Science by Georg Winter and collaborators at the Austrian Academy of Sciences, Pfizer, and several other organizations ups the game by more than an order of magnitude, and uses machine learning to make predictions about fragments’ cellular destinations and binding partners. (See also Derek Lowe’s post here.)

 

The researchers started with 407 diverse fully functionalized fragments (FFFs), which as we previously discussed consist of a variable fragment coupled to a photoreactive group and an alkyne moiety that can be used to pull down any bound proteins using click chemistry. These were selected from a larger set of ~6000 FFFs available from Enamine. The FFFs were incubated at 50 µM with intact HEK293T cells, followed by ultraviolet crosslinking.

 

Next, cells were lysed and treated with a biotin-azide probe that reacts with the alkyne on the FFFs. Covalently modified proteins were captured on streptavidin resin and proteolytically digested. Tandem mass tag (TMT) proteomics, which we wrote about here, was used to identify captured proteins. Unlike earlier methods, the researchers did not pinpoint the specific fragment binding sites on proteins.

 

In total the researchers found 2667 proteins bound to one or more fragments, of which ~86% had no reported ligands. Both proteins and ligands varied considerably in promiscuity: some proteins bound to more than half of the FFFs, and some fragments bound to hundreds of proteins, while others bound only a few, or none. To look for specific interactions, the researchers focused on proteins bound by fewer than 10 different ligands.

 

Three protein-ligand interactions were analyzed in some detail: the kinase CDK2 (and other CDK family members), the adapter protein DDB1, and the solute carrier protein SLC29A1. In each case the researchers confirmed the results from their chemoproteomic screens. Follow-up studies with related molecules led to more potent derivatives, with a CDK2 inhibitor showing low micromolar activity in a biochemical assay and an SLC29A1 inhibitor showing micromolar activity in a cell-based assay.

 

The researchers also found patterns in their larger data set. Armed with 47,658 protein-ligand interactions, the researchers were able to use machine learning to start to predict which molecular features were associated with binding. They ranked fragments as promiscuous or nonpromiscuous and built a promiscuity model. Molecules with higher lipophilicity and a greater fraction of aromatic carbon atoms tended to be more promiscuous, but the model could correctly categorize compounds as promiscuous even if they had lower ClogP values, or nonpromiscuous even if they had higher ClogP values.

 

Beyond promiscuity, the researchers used machine learning to predict other behavior, such as subcellular localization. A relatively easy case was to predict which molecules would accumulate in lysosomes; these tended to be hydrophobic basic amines. More impressively, the researchers could predict fragments likely to bind to transmembrane transporters, RNA binding proteins, and even intrinsically disordered proteins. And this is just the start: they hope one day to predict “target proteins from an input chemical structure alone.”

 

Perhaps most exciting, all of the data and models are available for free at Ligand Discovery. You can explore the proteins bound across all 407 fragments, input one or more proteins and find ligands, predict whether any given FFF is likely to be promiscuous or not, and even “build a machine learning model on the fly to predict potential interactions.” 

 

Check it out and let us know your experience.

Covalent fragments vs WRN

Last week Practical Fragments highlighted a covalent clinical compound from Vividion and Roche against the oncology target WRN. Another series of inhibitors against this protein are described in a recent Cancer Discov. paper by Gabriele Picco, Mathew Garnett, and collaborators at the Wellcome Sanger Institute, GSK, IDEAYA, and several academic institutes.

 

As we described in more detail last week, WRN is a synthetic lethal target for microsatellite instability (MSI) cancers. In contrast to the Vividion paper, which started by screening covalent fragments against cell lysates, here the researchers incubated purified WRN protein against each member of their covalent library (at 20 µM for 24 hours at 21 ºC) and analyzed the reactions by intact protein mass spectrometry. The fragment library was based around the methyl acrylate warhead, which, as we discussed a decade ago, has a narrower range of reactivities than more common acrylamides.

 

GSK_WRN1 was one of the prominent hits, with 81% modification. Tryptic digestion revealed that it modified C727, the same cysteine found by the Vividion researchers. Medicinal chemistry led to GSK_WRN3, with sub-micromolar activity in MSI SW48 cells. (Unfortunately no other details on the chemistry are provided; the paper states that these will be written up separately.)

 

GSK_WRN3 or a closely related compound were tested in a battery of assays and found to be inactive against three other helicases, which is not surprising given that C727 is unique to WRN. Chemoproteomic studies in cells also revealed the compound to be quite selective towards WRN vs other proteins. The compounds selectively inhibited MSI cancer cell lines and patient-derived organoids while sparing microsatellite stable (MSS) cell lines and organoids. One of the compounds showed activity in a mouse xenograft model.

 

In a useful public service, the researchers tested two previously reported WRN inhibitors, MIRA-1 and NSC617145, in the same set of several dozen cell lines and found that they were not only ineffective, they lacked selectivity for MSI cells over MSS cells. Although Dr. Saysno might object, I nominate these molecules to be added to the “Unsuitables” bestiary at the Chemical Probes Portal.

 

I do wish more details about the molecules were provided, especially the k_inact/K_i values. It is interesting that GSK_WRN3 bears remarkable structural similarities to VVD-109063. IDEAYA recently announced that their collaboration with GSK has resulted in a development candidate targeting WRN, and it will be fun to see the full story emerge.

Covalent fragments in the clinic: VVD-133214

Back in 2016 we highlighted a paper describing chemoproteomic screening of covalent fragments. That technology formed the basis of Vividion, which was acquired by Bayer in 2021. Now, a paper just published in Nature by Matthew Patricelli, Todd Kinsella, and collaborators at Vividion, Roche, and Universitat Autònoma de Barcelona describes one of the fruits to come from this platform.

 

The work stems from another promising recent approach to find oncology targets, synthetic lethality: searching for proteins that are essential in certain types of cancer cells but dispensable for normal cells, which might mean reduced toxicity. WRN is a DNA helicase that can clean up secondary DNA structures caused by expanded TA-dinucleotide repeats found in cancer cells with microsatellite instability (MSI), which is caused by mutations in DNA repair genes. Previous research had shown that knocking out WRN caused double-stranded DNA breaks and cell death in MSI-high (MSI-H) cancer cells but not normal cells, which do not have so many expanded TA-dinucleotide repeats. This has set off an industry-wide search for WRN inhibitors.

 

The researchers screened several thousand fragment electrophiles against cell lysates and found that some, such as VVD-109063, modified C727 of WRN. Although this cysteine is located some distance from the ATP binding site, functional activity studies with the pure protein found that the molecule was an inhibitor.

 

Optimization of VVD-109063 and related molecules found inconsistencies between results in lysates and intact cells. Some engaged C727 better in intact cells than lysates, others worse. Differences in cell permeability were ruled out by the fact that a cysteine on an unrelated protein was liganded to a similar extent in cell lysates and intact cells. The researchers speculated that, because cell lysates are diluted, they have lower ATP concentrations, and sure enough some molecules were less active in the presence of ATP while others were more active.

 

The team decided to focus on the second class. Optimization ultimately resulted in the clinical candidate VVD-133214. (Unfortunately details are not given; the paper does say these will be provided elsewhere).

 

A crystal structure of VVD-133214 confirmed covalent binding to C727, with the molecule in a hydrophobic pocket in a flexible “hinge region” of the protein. This causes a conformational rearrangement into a “closed” form, which presumably affects the catalytic activity of the helicase. Surprisingly, there are no hydrogen bonds between WRN and VVD-133214. This is highly unusual: a paper we discussed in 2021 found >90% of fragment-derived leads had at least one polar contact.

 

The k_inact/K_i value is reported as being 4848 M^-1s^-1, which is on the low side for clinical-stage irreversible inhibitors. Like sotorasib, its potency seems driven by k_inact, with the K_i being greater than > 15 µM. Consistent with this low inherent affinity, the molecule was inactive against the C727A mutant enzyme.

 

Much of the paper focuses on the biology, which is interesting but beyond the scope of this post. Suffice it to say that VVD-133214 is cytotoxic in MSI-H cells, where it causes G2 arrest and DNA damage, but inactive in microsatellite stable (MSS) cells. Oral dosing led to tumor regression in several MSI-H mouse models, including patient-derived xenografts.

 

This is a nice paper, though I look forward to a full account of the medicinal chemistry. In particular, vinyl sulfones are generally considered quite reactive, and I know of only one other clinical-stage molecule with this warhead. Presumably the cyclopropyl substituent was added at least in part to sterically block access to the electrophile.

 

Also, while the paper refers to VVD-133214 as “clinical-stage,” it appears neither on clinicaltrials.gov nor on Vividion’s website. The Roche website lists RG6457 as a phase 1 WRN covalent inhibitor partnered with Vividion, so perhaps this is the same molecule.

 

The paper ends by mentioning another clinical-stage WRN inhibitor from a different company, this one noncovalent. It notes that “this presents a rare opportunity to compare two small molecule oncology drugs targeting the same protein by different mechanisms,” and that using both could be useful in combating resistance. Practical Fragments wishes luck to these – and other drugs targeting WRN – helping patients quickly.