Fragments vs DCAF1: a new tool for targeted protein degradation

Targeted protein degradation (TPD) goes beyond merely inhibiting a protein; it takes a protein out of commission entirely. This is frequently done using a bivalent ligand: one part binds to the protein of interest, while the other part binds to an E3 ligase, which ubiquitinates the protein of interest, targeting it for destruction in the proteasome. Human cells have hundreds of E3 ligase proteins, some of which may work better in certain situations, such as specific cell compartments or tissues. In a recent ACS Med. Chem. Lett. paper, Anna Vulpetti and colleagues at Novartis describe progress against DCAF1.

 

DCAF1 is one component of the Cullin4-RING E3 ubiquitin ligase complex. The C-terminus of the protein contains a WD40 repeat (WDR) domain, which in this case consists of seven “blades” arranged around a central cavity, or “donut hole”. WDR domains are relatively common, and indeed we wrote about a previous Novartis effort that identified chemical probes against another WDR domain in the protein EED. In the new work, the researchers took 21 EED binders and screened them using both protein-detected and ligand-detected NMR against DCAF1, identifying two hits. Crystallography revealed that compound 1 binds in the central cavity, which previous computational screening had suggested would be ligandable. 

 

 

Next, the researchers screened 30 related compounds from within Novartis. Two of them, including compound 4, had improved affinity (as assessed both by NMR and SPR) and could be characterized crystallographically. In addition to binding in the central cavity, these compounds also bound to a site in the blade region, which the researchers wanted to avoid. Adding a piperazine to compound 4 both improved affinity and disrupted binding to the blade region; further optimization and growing to better fill the central cavity led to compound 13, the most potent molecule in the paper.

 

A crystal structure of a closely related molecule reveals that the acetyl group is near the entrance to the donut hole, providing an easy synthetic attachment point to construct bivalent degraders. A separately published preprint revealed this to be successful, with degraders of BRD9, multiple tyrosine kinases, and BTK.

 

There are several takeaways from this nice fragment to lead story. First, despite the fact that compound 1 is clearly fragment-sized (albeit a bit too lipophilic to be fully rule-of-three compliant), the word fragment never appears in the article. FBLD has become so routine that researchers may not even mention it, which does mean that our list of fragment-derived drugs is destined to be incomplete.

 

Second, although DCAF1 and EED share less than 25% sequence similarity, screening EED hits turned out to be successful, which could argue for screening specific subsets of fragments (for example kinase-focused or, in this case, WDR-focused). On the other hand, compound 1 binds in a different manner to DCAF1 than it does to EED. Indeed, compound 1 actually binds in two different orientations to DCAF1, consistent with its low affinity. The researchers mention a paper published earlier this year that reports a successful DEL screen against the target. Perhaps DCAF1 is just very ligandable, and a naïve fragment screen would have worked just as well as the pre-selected set.

 

Finally, the fact that this program yielded bivalent degraders suggests that many E3 ligases might be coopted for drug discovery. The field of targeted protein degradation is just getting started.

Fragments vs hIL-1β: Growing into a cryptic pocket to inhibit a protein-protein interaction

Protein-protein interactions have a well-deserved reputation for being difficult to drug with small molecules. This is particularly true for cytokine-receptor pairs, which are involved in a host of extracellular signaling functions. Human interleukin-1β (hIL-1β) plays a key role in inflammation by binding to its receptor IL-1R1. Biologics such as anakinra and canakinumab have been approved as drugs, but apart from some very low affinity fragments no small molecule inhibitors are known. In a new (open access) Nat. Commun. paper, Frédéric Bornancin, and collaborators at Novartis and University of Leicester report the first.

 

The researchers started by screening the 3452-compound LEF4000 library, which we described here, using ¹⁹F-NMR. After confirmation using protein-observed 2D NMR just a single super-sized fragment hit remained, consistent with the difficulty of the target. The individual enantiomers of this racemic compound were studied, and only (S)-1 was found to be active. Further characterization revealed that, despite weak affinity, this compound had both slow association and dissociation rates. More on that below.

 

Fragment growing in multiple directions led to mid-micromolar compounds such as 11 and 12. Combining elements from these molecules ultimately led to compound (S)-2, with low micromolar affinity as assessed by SPR. 

 

 

Compound (S)-2 specifically blocked the binding of hIL-1β with its receptor IL-1R1, but did not inhibit the binding of the related cytokine hIL-1α to IL-1R1. Even better, the compound blocked IL-1R-mediated signaling in cells at low micromolar concentrations in two different assays. The similar activity in biochemical and cell assays is likely due to the fact that the compound only needs to act at the cell surface, so permeability is not an issue, in contrast to our post last week.

 

A crystal structure of (S)-2 bound to hIL-1β revealed important interactions between the protein and both the phenol and lactam nitrogen, two contacts that were maintained during fragment optimization. The structure explains why only the (S)-enantiomer is active, as maintaining these contacts would cause clashes for the other enantiomer.

 

The structure also explains the mechanism of inhibition. (S)-2 binds to a cryptic pocket that forms in a region of hIL-1β important for interacting with IL-1R1, and formation of the pocket involves a loop movement that would be incompatible with the protein-protein interaction. The researchers argue convincingly that that the compound stabilizes the cryptic pocket, which naturally exists as a minor population within solution. This also explains the slow kinetics, which would be expected if the compound essentially has to wait until the cryptic pocket opens before it can bind.

 

There is still a long way to go to a drug. Not only is the affinity of (S)-2 modest, the two carboxylic acid moieties and the phenol are likely to impede oral bioavailability. Nonetheless, this is a lovely paper, and the researchers point out that cryptic pockets frequently involve “large movements of secondary structural elements” that could block biological function. Indeed, this is the case for approved drugs such as sotorasib. Don’t give up just because your protein of interest appears like a featureless billiard ball: there may well be opportunities hidden just beneath the surface.

Fragments vs malarial DHFR

Malaria continues to be a worldwide scourge, with some quarter billion cases last year. A seventy-year-old drug called pyrimethamine targets the dihydrofolate reductase (DHFR) enzyme from Plasmodium falciparum, but resistance mutations have rendered this molecule mostly useless. An analog called P218 was developed to overcome this resistance and completed a handful of phase 1 clinical trials, but unfortunately the human pharmacokinetics were found lacking. In a new RSC Med. Chem. paper, Marie Hoarau and colleagues at the National Center for Genetic Engineering and Biotechnology in Thailand describe their efforts to improve this molecule.

 

The researchers recognized that the phenyl propanoate moiety of P218 was a metabolic liability and sought a replacement. They screened a library of 1163 fragments (from Key Organics) at 1 mM using a thermal shift assay. This resulted in 64 hits, 52 of which confirmed by SPR. Of these, 22 showed some level of inhibition at 0.5 mM against mutant PfDHFR.

 

Among the hits, five were “bi-aromatic carboxylates,” such as compound 136. These were prioritized because, while reminiscent of the phenyl propanoate in P218, they had fewer rotatable bonds. Some of them also showed slow off-rates by SPR, though in my opinion the sensorgrams look suspicious, perhaps due to excessive protein loading on the chip. (For example, the K_d for compound 136 calculated from the on and off rates comes in at 160 nM, unrealistically potent given that it shows only 20% enzymatic inhibition at 0.5 mM. Note – all values here and in the figure are for the mutant form of the enzyme.)

 

SAR by catalog was used to find additional analogs, such as compound AF10, which showed measurable inhibition of the enzyme. Next, the researchers tested hits in the presence of a pyrimidine fragment (L4) derived from P218, known to bind nearby. Compound AF10 showed greater inhibition than would be expected by simple additivity, perhaps suggesting some preorganization of the binding site, as in a different example discussed here.

 

Molecular modeling was used to link the carboxylate fragments with L4, and eight were made and tested. All inhibited both wild type and mutant PfDHFR, and compound 8 showed good selectivity over human DHFR too. A crystal structure confirmed that it bound as predicted. From a fragment-linking perspective, the sub-nanomolar affinity of compound 8 is impressively better than would be expected given the weak affinities of L4 and AF10.

 

Unfortunately, despite similar in vitro potency against the isolated enzymes, compound 8 and the other molecules tested showed “disappointing” activity against Plasmodium falciparum carrying either wild-type or mutant DHFR, roughly 100- to 1000-fold less potent than P218. The researchers suggest solubility may be a factor.

 

This paper is a useful reminder of the dramatic disconnects often seen between enzymatic and cell activity. Nonetheless, it is another good example of using fragment-based methods to replace one portion of an existing molecule.

Fragment screening on a benchtop NMR

Practical Fragments has been on an NMR theme for the last two weeks, and this post continues that trend. One of the main barriers to entry for NMR methods is the instrument itself: not only are the machines large, requiring a good size room, the price starts at several hundred thousand dollars. Then there is the maintenance, which includes regular refills of liquid helium, which is both costly and often scarce. And if the helium runs out, your precious superconducting magnet “quenches”, which looks like this.

 

Large magnets such as those in 600 MHz instruments are unlikely to change until room temperature superconductors become a reality. Less powerful permanent magnets are available though, and you can purchase a benchtop 80 MHz machine for less than $100,000. But the low sensitivity requires very high concentrations of sample, too high for fragment screening. Unless, that is, you could increase the sensitivity. This has now been described in a new (open-access) Angew. Chem. Int. Ed. paper by Felix Torres, Roland Riek, and collaborators at the Swiss Federal Institute of Technology, Bruker, and NexMR.

 

The somewhat complicated method is called photochemically induced dynamic nuclear polarization (photo-CIDNP), which we wrote about in June. As the name suggests, this involves light excitation of a photosensitizer molecule which can then increase sensitivity for detecting other small molecules, particularly when they are not bound to proteins. Weirdly and fortuitously, photo-CIDNP theory predicts that polarization transfer is actually higher at lower magnetic fields, making it ideal for benchtop NMR.

 

The researchers first tested three fragments, each at 500 µM, using 25 µM fluorescein as the photosensitizer. Just 3 minutes of measurements each gave very clear spectra after light irradiation at 450 nm. In the absence of light it would take between 22 hours and 10 years to achieve comparable signal-to-noise enhancement.

 

Next, the researchers screened 32 fragments from their custom-designed "NMhare1.0 library” we previously described, which contains molecules suitable for photo-CIDNP. As before they used the protein PIN1 (at 10 µM) and collected data for 3 minutes per sample. Six compounds had reduced polarization in the presence of protein, four of which had been previously detected as binders and validated using a 600 MHz NMR. Of the two new hits, one confirmed using protein-detected NMR while the other did not.

 

To explore the limits of sensitivity, the researchers conducted a series of experiments lowering the concentrations of protein and small molecules. One of the compounds could be detected at concentrations as low as 250 nM and quantified at 1 µM in just 3 minutes. At 50 µM this compound clearly showed binding to 5 µM protein, despite having an affinity in the low millimolar range.

 

This is a fun paper, and I particularly like the fact that it expands fragment screening to an instrument previously not thought to be suitable. As we wrote previously, one limitation of photo-CIDNP is that only some molecules are able to be photo-sensitized. A solution would be to find one such ligand and then run a displacement assay to see whether a second ligand could compete with it, akin to what has been done for fluorine NMR. I look forward to seeing how this technique develops.

Affinity measurements in a single NMR tube?

Last week we highlighted a ligand-detected NMR method to measure affinities of protein-ligand interactions. That technique, R2KD, requires preparing multiple NMR samples with the ligand at different concentrations. In a new open-access paper published in J. Am. Chem. Soc., Serena Monaco and collaborators at University of East Anglia and Universidad de Sevilla describe a method that can be done in a single NMR tube.

 

The researchers have actually combined two methods, chemical shift imaging (CSI) and Saturation Transfer Difference (STD) NMR, to create imaging STD NMR. We’ve written previously about STD NMR, which relies on the transfer of magnetization from an irradiated protein to a bound ligand. In CSI, chemical shift information is recorded at multiple slices along the length of an NMR tube. Normally the solution in an NMR tube is homogenous and so the chemical shifts would be identical at the bottom and top of the NMR tube. Here, though, the researchers create concentration gradients by carefully pipetting a solution containing ligand on top of a solution containing protein and allowing the ligand to diffuse the length of the NMR tube.

 

Like all things NMR-related, the mathematics get a bit complicated. One important factor is the rate of diffusion for a given small molecule. This “diffusion coefficient” can be experimentally measured by creating a concentration gradient in the absence of protein and measuring the ligand concentration at various positions in an NMR tube after a given length of time (typically more than 12 hours). Diffusion is dependent on molecular weight, so it is also possible to calculate the diffusion coefficient, and in fact the researchers found that the calculated values matched the experimental values for three different small molecules.

 

Knowing the diffusion coefficient helps establish the maximum ligand concentration to use and the ideal diffusion time. The researchers examined three different protein-ligand pairs, all of which had weak affinities, with K_D values from 0.2 to 2 mM. Measuring STD signals at different slices along the NMR tube effectively yields STD signals at different concentrations of ligand, and fitting this to an equation allows calculation of the dissociation constant. For the three model systems the affinities agreed with literature values, which had been determined using ITC or WAC.

 

One nice feature of imaging STD NMR is that it can identify non-specific binding. This is because STD signals vary depending in part on how close a proton on the ligand is to the protein, resulting in different STD signals for different protons for specific binders. If this “epitope pattern” is lost at higher concentrations, this suggests non-specific binding, where the ligand can bind in random orientations to multiple sites on the protein. The researchers demonstrated this for one of their model systems: tryptophan binds specifically to bovine serum albumin with a dissociation constant of 0.2 mM, but above 1 mM or so the epitope disappears, suggesting non-specific binding.

 

Imaging STD NMR does have some limitations. For one thing, it requires a high initial concentration of ligand: 30 mM in the case of tryptophan, and even higher for the other two ligands. Most small molecules are nowhere near this soluble in water. The researchers suggest that ligands could be dissolved in DMSO and placed on the bottom of the NMR tube, with the protein solution gently layered on top. They show that the concentration gradients develop in a similar manner as a fully aqueous system, but acknowledge that high DMSO concentrations may not play well with most proteins.

 

Also not stated is the sensitivity of the method for higher affinity binders. Last week’s R2KD could measure affinities as tight as 10 µM, but it is unclear how much below 200 µM imaging STD NMR can go.

 

Finally, as we noted in 2019, STD effects are remarkably complex and not well-correlated with affinity. In particular, binding kinetics can play a role in the strength of the signal. It would have been nice to see more than three protein-ligand pairs tested.

 

All that said, this is an intriguing approach. Laudably, the researchers provide extensive supporting information, including mathematical derivation of the fitting equations, a spreadsheet, NMR pulse sequences, and macros. I’ll be curious to see how it works for others.

Ligand-observed NMR – quantitatively

Ligand-observed NMR is one of the most popular fragment-finding methods. Among its strengths is the ability to find extraordinarily weak fragments that most other techniques would miss. However, common ligand-observed NMR methods such as STD are not quantitative: they can tell you that a fragment binds, but not how tightly. In a new open-access J. Med. Chem. paper Manjuan Liu and colleagues at the Institute of Cancer Research provide an easy solution.

 

The approach is based on an NMR phenomenon called transverse relaxation (see here), which describes how atomic nuclei return to their ground state after being excited by a radiofrequency pulse in a magnetic field. The transverse relaxation rate R₂ for a given nucleus depends on the tumbling speed of the molecule in which it is contained: small molecules tumble rapidly and have small R₂ values, while larger molecules tumble slowly and have larger R₂ values. When a small molecule binds to a protein its tumbling speed slows and its R₂ increases. The R₂ values can be measured experimentally using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence.

 

This is all fairly standard for NMR spectroscopists, and in fact CPMG is widely used to find fragments. Liu and colleagues proposed that, by measuring the change in R₂ with changing concentrations of small molecule, they would be able to extract the dissociation constant (K_d). The theory gets a little hairy (14 equations), and the analysis depends on non-linear regression curve fitting, but this is easily done using modern analytical software. The technique is called R2KD.

 

The experiment itself is straightforward. The ligand alone is prepared at two different concentrations; these are used to determine the R₂ values of the free ligand. Another eight samples contain protein and various concentrations of ligand. The R₂ values are measured and fit to an equation to extract the dissociation constant. An initial test case with a known 50 µM ligand for the protein BCL6 was encouraging, giving a K_d of 53 to 78 µM for four different protons on the ligand. The accuracy could be further improved by using a “global fit” with all the data rather than analyzing each NMR peak in isolation.

 

Next, seven ligands against three proteins were analyzed using R2KD and compared with their literature values. Here too the results were in agreement, mostly within a factor of two. The lower limit for sensitivity is dependent on the NMR signal for the ligand; below concentrations of about 20 µM the experiments become impractically long. The upper limit is dictated by the solubility of the ligand. The researchers could reliably measure dissociation constants around 1 mM and suggested that with a sufficiently soluble ligand even weaker ligands could be measured.

 

The R2KD experiment requires that the protein concentration be less than about 20% of the lowest ligand concentration. (That said, protein concentrations up to 35 µM gave reasonable results.) Preserving protein is usually a goal, so lower concentrations (single-digit micromolar) are desirable from both a practical and theoretical standpoint.

 

Finally, the researchers demonstrated the application of R2KD to assess 10 fragment hits from a 1000-compound screen against the E3 ligase complex CRBN/DDB1, one of the most popular targets for PROTACs. The hits had dissociation constants ranging from 70 to 1200 µM, and the R2KD values were similar to those found in a fluorescence polarization (FP) assay, though for the most part the affinities from R2KD were higher. In particular, two compounds with essentially no activity in the biochemical assay came in at sub-millimolar by R2KD, which may speak to the insensitivity of the FP assay.

 

Overall this is a lovely and, as befits this blog, practical paper, and I hope R2KD becomes widely adopted. With a sweet spot for K_d values of 10-1000 µM the technique fills an important niche: biochemical assays are well-suited for tighter binders but less reliable at millimolar ligand concentrations. As crystallography becomes increasingly popular as a primary screen, I could imagine R2KD being used to rank the resulting fragment hits.

Stabilizing protein-protein interactions: part 3 (fragment linking)

Stabilizing protein-protein interactions is becoming increasingly popular, and not just for PROTACs. Nearly three years ago we highlighted the use of crystallographic screening to find fragments that could stabilize interactions between the adapter protein 14-3-3δ and peptides derived from p53, a prominent cancer target. After noting how much work lay ahead, we ended the post with, “expect a part 3!” This has now been published (open access) in Angew. Chem. by Adam Renslo, Luc Brunsveld, Michelle Arkin, Christian Ottmann, and collaborators at UCSF and Eindhoven University of Technology.

 

In addition to crystallographic fragment screening, the researchers had previously performed a disulfide Tethering screen on the 14-3-3δ protein, which we described here. The fragments from the two screens bound next to one another, so the researchers decided to link them. They started by solving the crystal structure of compound 1 disulfide-bonded to 14-3-3δ in the presence of fragments from the crystallographic screen as well as a peptide derived from estrogen receptor alpha (ERα, another anti-cancer target). These co-structures guided the synthesis of new linked molecules, and these were soaked into crystals of 14-3-3δ and the ERα peptide. Compound 6 gave strong electron density and overlayed nicely on the initial fragments.

 

 

To determine whether the linked molecule could stabilize the 14-3-3δ/ERα complex, the researchers developed a fluorescence anisotropy assay with a dye-labeled peptide from ERα. Some of the linked molecules produced an increase in anisotropy, suggesting stabilization of the 14-3-3δ/ERα complex, but when the researchers ran the important control of repeating the experiment in the absence of 14-3-3δ they found that several molecules still increased anisotropy, which could be due to aggregation. (Adam published a nice early paper on aggregation and is thus particularly attuned to the dangers.)

 

Fortunately, some of the molecules passed this control, and with a robust crystallography system the researchers were able to use structure-based design to improve them, ultimately arriving at compound 24, which increased the affinity of the 14-3-3δ/ERα complex by 25-fold. It was also quite specific towards ERα, and did not increase the affinity of nine peptides from from other proteins for 14-3-3δ. The researchers attribute this selectivity to the fact that most other peptides would sterically clash with compound 24. (Not reported was the peptide from p53, which would be interesting.)

 

This is a nice paper on several levels. In addition to selectively stabilizing a therapeutically relevant protein-protein interaction, this is a rare example of starting with a covalent fragment and developing a non-covalent binder. (For another, see here.) Also, this is a good example of fragment linking, which is often challenging.

 

There is still a long way to go. The most potent molecules all contain amidine moieties, whose high polarity is a liability for cell permeability, let alone oral bioavailability. Moreover, the affinity of compound 24 is still quite weak, with a low ligand efficiency.

 

That said, with a wealth of structural and biological understanding I am optimistic further progress can be made, perhaps by rebuilding the covalent linkage to the protein, as was the case of sotorasib or this more recent paper from the UCSF team. I look forward to part 4!

Democratizing computational FBLD with BMaps

Computational approaches to FBLD continue to gain in power. For the most part, they require significant knowledge and installation of expensive, customized software. To remedy this, John Kulp, III and colleagues at Conifer Point Pharmaceuticals have introduced a new web-based application, BMaps, which they describe in a recent J. Chem. Inf. Mod. paper.

 

As the researchers note (and appropriately reference), there are more than a dozen virtual fragment-based design tools and another dozen web-based tools. BMaps (for Boltzmann Maps) aims to provide a full range of functions, from visualizing proteins, finding hot spots, docking fragments, and growing them. It also provides information on the energetics of bound water molecules, which as we’ve written can be crucial players in optimizing protein-ligand interactions.

 

Two key techniques used by BMaps are Grand Canonical Monte Carlo (GCMC) simulations and Simulated Annealing of Chemical Potential (SACP). The first entails comprehensive sampling of different fragment conformations on a protein of interest and assessing binding free energy. The second tool “forcefully inserts fragments into all the binding sites of the protein” and then removes them slowly to evaluate which are most difficult to remove, and thus most tightly bound. Together, GCMC-SACP can be used to evaluate fragment binding to any protein uploaded to the site from the protein data bank, AlphaFold, or any other source.

 

One nice feature of BMaps is a repository of several hundred proteins each with more than 100 fragment and water simulations. BMaps also contains a database of more than 4000 fragments, including MiniFrags. Users can import their own fragments or computationally deconstruct larger ligands. The paper itself is quite short, but the supporting information provides more guidance on how to use the software.

 

The researchers “aim to democratize the availability of accurate fragment and water maps,” a laudable goal. Most computational features are available with a free account, though with restrictions on the number of operations per month.

 

BMaps looks quite powerful and easy to use, but I do wish the researchers had included some full case studies, for example those used by the free FastGrow tool we highlighted last year. Try it out and let the community know what you think!

DNA-encoded fragment growing

When growing fragments into leads, the typical route is making and purifying one compound at a time. In recent years parallel synthesis and screening of crude reaction mixtures has been catching on. However, no physical screening approach can match the throughput of DNA-encoded libraries (DEL). In a new (open access) Chem. Sci. paper, Michael Waring and collaborators at Newcastle University, University of Oxford, and Genentech use DEL to grow fragments.

 

The researchers started by creating a “poised DNA-encoded library.” We’ve previously discussed the concept of poised libraries, which are designed for rapid follow-up chemistry. Typically, the library members are fragments that contain a handle to facilitate growing. Here, the concept is reversed, with the DEL itself poised to react with a pre-chosen fragment. In this first test case, the DEL consisted of just 42 members made by coupling 7 amino acids to 6 aryl halide-containing acids, which could then be used for Suzuki-Miyaura couplings.

 

Bromodomains such as the BD1 domain of BRD4 bind a plethora of published fragments, and the researchers chose the 7-atom 3,5-dimethylisoxazole, one of the first fragments published. A boronic acid version of this was coupled to the DEL library and screened against BD1. One DNA sequence in particular occurred 11-times more frequently than any of the 41 others. The corresponding molecule was synthesized without being attached to DNA and found to have a dissociation constant of 51 nM as assessed by NMR. Three control molecules which used different amino acid or aryl-halide building blocks had affinities considerably lower, the best being 2.5 µM.

 

 

A crystal structure of compound 22 bound to BD1 showed several important contacts that explain why the molecule was selected in the DEL screen. Moreover, a more lipophilic version of the molecule showed some cell-based activity.

 

The “NUDEL” (for Newcastle University DEL) is an interesting approach to rapidly explore regions of chemical space around a fragment hit. By including every possible combination of building blocks in the library it is possible to find synergistic combinations, such as those found in compound 22; molecules derived from alanine but a different aryl halide or pyrazole but a different amino acid were not selected over background levels.

 

Of course, as the researchers acknowledge, 42 compounds is very modest for a DEL. Considerable care was taken to ensure each library member was properly synthesized to facilitate proper analysis (for example, that the selection was not based on differences in concentrations of different library members). This level of care would be more difficult with a million compound library. Also, finding high affinity binders to BRD4 is a rather low bar, particularly when starting with a known fragment. Nonetheless NUDELs look like they could prove quite useful, and I look forward to seeing applications to more novel targets. Perhaps they could even be combined with the DEL-based fragment finding approach we highlighted last year. I predict growing bonds between fragments and DEL.

Fragments vs VE-PTP: biophysics in action

Protein kinases attach a phosphate group onto amino acid side chains in proteins. Phosphorylation regulates myriad aspects of cell signaling, and thus kinases are common drug targets. Indeed, roughly one third of fragment-derived clinical compounds target kinases. Protein phosphatases remove phosphate groups and thus also make potentially valuable drug targets. Unfortunately, they are very difficult to selectively inhibit, and indeed no fragment-based drugs have entered the clinic. A new paper in Biochemistry from Wataru Asano, Yoshiji Hantani, and colleagues at Japan Tobacco takes the first steps towards rectifying this.

 

Phosphatases are so difficult to drug because most of them have small, highly charged active sites that have evolved to bind phosphate. This moiety and strongly anionic analogs are not very cell permeable or orally bioavailable. Moreover, the small size of the active site makes selectivity challenging, and the fact that many phosphatases contain an active-site cysteine makes them particularly susceptible to assay artifacts.

 

The researchers were interested in vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays a role in vascular homeostasis and angiogenesis. They chose 25,000 fragment-sized molecules (with < 20 heavy atoms) from their HTS collection, all with aqueous solubility > 300 µM, and screened these at 250 µM in a mass-spectrometry-based functional assay. Those that inhibited enzyme activity by at least 40% were retested in dose-response format and also characterized by SPR. Many highly acidic compounds such as sulfonic acids were found, but the researchers were particularly intrigued by Cpd-1, which is only modestly acidic with a calculated pKa of 3.9.

 

Cpd-1 inhibited VE-PTP, but although SPR showed binding, this was not saturable. Thus, the researchers turned to NMR, using multiple protein-observed as well as ligand-observed methods to demonstrate that the molecule binds to the active site of the enzyme. This was confirmed with a crystal structure, which also revealed an “unhappy” water molecule nearby, leading to Cpd-2. This molecule was characterized by crystallography, SPR, and ITC. The molecule proved to be unexpectedly selective for VE-PTP over four other PTPs. The researchers hypothesize that binding to PTPs is often dominated by conserved electrostatic contacts, and because Cpd-2 is less highly charged it relies on other, more specific interactions.

 

This is a nice example of using a variety of biophysical techniques to find and advance fragments. The researchers do a good job of describing the strengths and weaknesses; for example, it was impossible to determine the dissociation constant of Cpd-1 by SPR due to non-specific binding with the protein, reminiscent of a Pin1 story from several years ago.

 

There is still a long way to go, with no cell activity or permeability described for Cpd-2. Still, the paper ends boldly: “we believe that this compound will be developed as a potential drug for VE-PTP-related diseases.” Here’s wishing them success.

A rule of two for using chemical probes?

Earlier this year we highlighted the growth of the Chemical Probes Portal, a free website that profiles more than 500 small molecules targeting more than 400 proteins. Each chemical probe is evaluated by experts based on published literature and then scored for use in cells or in vivo. More than 300 chemical probes have received three or four stars and are thus recommended. But even a good probe can be misused, and this is the subject of a recent (open-access) Nat. Comm. paper from Adam McCluskey, Lenka Munoz, and colleagues at the University of Sydney and the University of Newcastle. (The paper has also been discussed by Paul Workman and Derek Lowe.)

 

The researchers chose eight probes targeting histone methyltransferases, a histone demethylase, a histone acetyltransferase, and several kinases. All but one of these probes had first been disclosed before 2015. A literature search revealed 662 papers that used these probes in cellular studies, ranging from 21 to 134 publications per probe.

 

Centuries ago the alchemist Paracelsus noted that everything is poisonous at high enough doses, and indeed even the best probes might hit dozens or hundreds of protein targets. For this reason the Chemical Probes Portal recommends maximum concentrations for cellular assays. The researchers examined whether papers exceeded these concentrations. The overall results were encouraging, with just 22% of papers exceeding recommended limits. However, there was considerable variation: for one chemical probe, 70% of papers exceeded the limit. (For this particular case, the maximum recommended cellular concentration was just 250 nM.)

 

Because chemical probes can have off-target activity even at recommended concentrations, best practices are to include a related but inactive control compound plus a second chemically differentiated probe. All but one of the eight probes chosen for analysis had orthogonal probes available, and five had inactive controls. So how frequently were these used? Unfortunately, 58% of papers did not use an orthogonal probe, and a whopping 92% of papers did not use available inactive control compounds. In fact, just 4% of the papers “used chemical probes within the recommended concentration range and included inactive compounds as well as orthogonal chemical probes.”

 

A wider analysis of nearly 15,000 papers that cited the 662 publications produced similar results, with 17% exceeding recommended concentrations, 59% not using differentiated chemical probes, and 83% not using inactive controls.

 

The researchers propose a “'rule of two': At least two chemical probes (either orthogonal target-engaging probes, and/or a pair of a chemical probe and matched target-inactive compound) to be employed at recommended concentrations in every study.” To encourage best practices, the paper provides a simple “Researchers’ Flowchart” to help investigators select probes and controls. And because science is self-regulated, they provide a five-item “Reviewers’ Checklist.” The paper also includes a nice list of links to other resources, including webinars and slide decks.

 

Overall I think following these guidelines would be beneficial, and the Reviewers’ Checklist in particular could be usefully incorporated into journal publication requirements.

 

Of course, the vast majority of protein targets don’t have even a single good chemical probe, let alone two or more. Which means that there are plenty of opportunities to identify new probes and make better use of those that already exist.

Fragment events in 2023 and 2024

We're at the midpoint of the year but there is still at least one more good conference in 2023, and 2024 is shaping up to be even better.

September 25-28: CHI’s Twenty-first Annual Discovery on Target will be held in beautiful Boston, as usual. 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 2022 meeting here, the 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.

 

2024

March 4-5: RSC-BMCS Ninth Fragment-based Drug Discovery Meeting will be held in Cambridge, UK. This venerable biannual event will be particularly focused on case studies "that have delivered compounds to late stage medicinal chemistry, preclinical, or clinical programmes." You can read my impressions of the 2013 meeting here and the 2009 event here.

 

April 1-4: CHI’s Nineteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, returns as always (pandemics aside) to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of 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. 

 
September: FBLD 2020 was sadly canceled due to COVID-19, but after a six year hiatus FBLD 2024 is scheduled to be held in Boston (exact dates TBD). 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.

 

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

Fragment merging in silico, two ways

Roughly speaking there are three ways to advance fragments to leads: growing, linking, or merging. Growing is the most common, but as the number of crystal structures of bound fragments continues to increase so too does the opportunity for fragment merging, in which elements of two fragments are combined into a new molecule. In a new (open access) J. Chem. Inf. Mod. paper, Charlotte Deane and collaborators at University of Oxford, Informatics Matters, Vernalis, and LifeArc compare two in silico methods.

 

Fragment merging, as described in the paper, “is used for fragments that bind in partially overlapping space by designing compounds that incorporate substructural features from each.” Each fragment may have extraneous bits that are not kept in the merged molecule. Indeed, sometimes only a small portion of one of the fragments is incorporated into the final molecule. (Both these concepts are shown in an example we recently wrote about here.)

 

When chemists merge two fragments, they consider the synthetic tractability of the merged molecules. Computers, on the other hand, sometimes propose compounds that are either unreasonable or would require a doctoral-thesis worth of effort to make. One solution is to invert the problem: rather than trying to assess whether specific in-silico-generated molecules can be made, possible merged molecules can be searched against a large virtual library of synthetically accessible molecules for similar compounds (see for example here).

 

A key question for this approach is the definition of similar. The most common method for finding similar molecules is by reducing them to molecular “fingerprints,” such as the presence or absence of a chlorine atom. The more fingerprints two molecules have in common, the more similar they are; this is the approach used for Tanimoto similarity.

 

An alternative approach is to use a “graph database” in which molecules are represented as nodes and edges, with nodes being substructures and edges being connections between the substructures. This approach was described by researchers from Astex as the Fragment Network.

 

Which of these approaches works better?

 

In the new paper, the researchers built a virtual library of more than 120 million commercially available molecules. They then selected four proteins with published crystallographic fragment hits. Between 9 and 19 individual fragments were chosen for merging for each protein, forming 55 to 134 potentially mergeable pairs. These were then computationally merged and queried against the database using either similarity searching or the Fragment Network.

 

Both search methods yielded comparable numbers of possible hits, ranging from just under 23,000 to nearly 169,000 per protein. These were then computationally filtered to find those molecules most likely to bind to the proteins, resulting in 56 to 952. Interestingly though, the specific molecules varied considerably depending on which search method was used. In fact, molecules from the Fragment Network mostly occupied different regions of chemical space than those found using similarity searching. Moreover, in many cases fragment pairs yielded merged compounds from one method but not the other. The number of predicted interactions with the protein targets also differed, and these differences extended not just to specific interactions but also to functional diversity.

 

The researchers did not purchase and test compounds themselves, but they did run the analysis against two published examples of fragment merging (one of which we wrote about here) and found that both the Fragment Network and similarity searching could find molecules related to experimentally validated binders.

 

The question of which approach works better remains open, so the researchers suggest using both. They do note that running a Fragment Network search is computationally less demanding, in this case taking an average of 2 to 14 minutes of CPU time vs 40 minutes for the similarity search. These differences become even more significant when searching billion-compound libraries.

 

Importantly, the researchers provide the code to generate your own Fragment Network, so you can try this at home. I look forward to seeing how the two techniques perform prospectively.

Nucleophilic covalent fragments against cysteine (!)

Covalent fragment-based drug discovery continues to gain momentum, as evidenced by the number of talks at the CHI Drug Discovery Chemistry meeting in April. All of those involved nucleophilic residues on proteins, especially cysteine, reacting with electrophilic fragments. However, as we noted last year, it is possible to do the reverse. This is the topic of a new paper in Nat. Chem. Biol. by Jing Yang (Beijing Institute of Lifeomics), Kate Carroll (UF Scripps), and collaborators.

 

The reason most covalent fragments are not nucleophiles is that none of the twenty standard amino acids are particularly electrophilic. The work we mentioned last year focused on post-translational modifications introducing aldehydes or ketones into proteins. But it turns out that the thiol group of cysteine, which is normally nucleophilic, can be oxidized to a sulfenic acid, which is electrophilic. The Carroll group has been studying this “cysteine redoxome” for years and found that S-sulfenation can serve a regulatory function akin to phosphorylation.

 

To assess the reactivity of sulfenic acids across the proteome, the researchers synthesized cyanoacetamide and nitroacetamide derivatives of 3,5-bis(trifluoromethyl)aniline. The chloroacetamide and acrylamide derivatives of this fragment have previously been used in chemoproteomics experiments to probe for reactive cysteines. Cell lysates were treated with one of these four fragments, followed by treatment with a generic probe for sulfenic acids or thiols. If a cysteine residue is modified with the fragment, it will be unavailable to react with the second probe, and this loss in signal can be quantifiably detected using mass spectrometry.

 

For the thiol-reactive chloroacetamide and acrylamide, the researchers found that 25.2% and 11.0% of quantifiable cysteines in the proteome could form adducts. But only 24 cysteine residues formed adducts with the nitroacetamide (1.3% of the total sulfenic acids quantified), and none formed adducts with the cyanoacetamide.

 

Despite this lower hit rate, the researchers constructed a library of 65 cyanoacetamide- and nitroacetamide-containing fragments, which was similarly screened in cell lysates. Adding hydrogen peroxide to cell lysates to mimic oxidative stress increased the number of sulfenic acid sites. In total the researchers found 524 liganded sites across 441 proteins. As expected from the earlier experiments, nitroacetamides tended to bind to more sites than cyanoacetamides.

 

The researchers studied the functional effect of covalent modification for several proteins. The enzymes GAPDH, GSTO1, and ACAT1 all have active-site cysteine residues that can be reversibly modified by oxidation to the sulfenic acid. Reaction of this form of the recombinant proteins with one of the covalent fragments led to irreversible inhibition.

 

Similarly, the researchers demonstrated that a fragment which hits the enzyme PRXL2A activated MAPK signaling in cells, as expected. Importantly, this effect was not seen in cells containing PRXL2A with a cysteine to serine mutation. The non-enzyme proteins HDGF and BCCIP could also be functionally inhibited in cells with covalent fragments.

 

This “umpolung” approach to covalent ligand discovery is scientifically interesting, but how useful will it be? In most cases sulfenic acid formation is already deactivating, so targeting this form of the protein will simply keep it in the off-state. However, the researchers do note that sulfenic acid formation can be activating.

 

A second challenge is that sulfenic acid formation tends to be substoichiometric, with only a small percentage of cysteines existing predominantly in the sulfenic acid form. Thus, it will be difficult to achieve the near quantitative level of modification often required for biological effects. That said, there are cases where you want to tweak a pathway rather than shut it down entirely. Protein activation or the development of new PROTACs could also benefit from limited target protein engagement.

 

As for the covalent fragments themselves, nitroacetamides may be too reactive, but the cyanoacetamide moiety is actually found in a few approved drugs, such as the anti-inflammatory tofacitinib. And if compelling sulfenic acid targets are identified, chemists will likely develop additional nucleophilic probes suitable for dosing in humans.

A rule of 1 (hydrogen bond donor) library

Hydrogen bond donors (HBDs) in ligands are troublemakers. Having more than a couple tends to decrease permeability, bioavailability, and even solubility. HBDs can also lead to efflux, which is particularly problematic for drugs that must cross the blood-brain barrier. While this is true in general, the problems become even more acute for heterobifunctional drugs such as PROTACs, which contain two moieties that each recognize a separate protein. To minimize the number of HBDs at the outset of a project, Benjamin Whitehurst and colleagues at AstraZeneca have built a “Low HBD” fragment set, which has just been described in J. Med. Chem. Soc.

 

The researchers started by examining roughly 205,000 compounds in their collection having between 11 and 19 non-hydrogen atoms and no more than one HBD, defined as “a proton bonded to an oxygen or nitrogen atom in its neutral form.” An extensive series of filters was used to winnow the molecules based on physicochemical properties, diversity, and absence of reactive groups. Consistent with our recent poll, synthetic tractability was considered explicitly. The researchers also made use of a multiparameter optimization score (see here). After quality control, which included solubility and compatibility with SPR, they ended up with a set of 551 fragments.

 

AstraZeneca has recently revamped their general purpose “Biophysics” fragment library, which consists of 2741 compounds. They also have a set of 402 “Kinase Hinge” fragments, which contain hydrogen bond donors near hydrogen bond acceptors.  Comparing the Low HBD set with the other two revealed that it was as diverse as the Biophysics set and more diverse than the Kinase Hinge set. Other parameters such as the number of hydrogen bond acceptors (HBAs), polar surface area, and molecular weight were similar between the Low HBD and Biophysics libraries. Happily, and perhaps defying expectations, lipophilicity was not higher in the Low HBD set.

 

So how does the library perform? The researchers describe five screens against an E3 ligase, a protein-protein interaction, a kinase, a histone methyltransferase, and a transcription factor. Confirmed hits (defined as having K_d < 1 mM by SPR) were obtained for all targets. Hit rates for two targets were comparable to hit rates for the Biophysics set, as were the dissociation constants and ligand efficiencies. Not surprisingly the Kinase Hinge set produced a higher hit rate for the kinase. (Two targets were only screened with the Low HBD set.)

 

The percentage of hits from the other fragment libraries having 0 or 1 HBD was 44%, 46%, and 80%, so the Low HBD set does seem to be fulfilling its role of enriching these types of compounds. Interestingly, when the researchers analyzed successful fragment-to-lead studies published between 2015 and 2021, they found that 53% of them had just 0 or 1 HBD.

 

All these results suggest that sharply curtailing the number of hydrogen bond donors in a fragment library doesn’t have negative consequences. Perhaps this isn’t surprising: an analysis we highlighted in 2021 based on 131 fragment-to-lead success stories noted that most of them only retained one or two polar interactions from the initial hit. That paper also noted that while 35% of the polar interactions were from N-H hydrogen bond donors on the ligands, an even higher percentage came from hydrogen bond acceptors. That paper and the AstraZeneca researchers also note the potential of other types of interactions, such as polarized C-H hydrogen bond donors and halogen bonds. It will be fun to watch hits from this library progress.

Fragment screening by photo-CIDNP

Last year we highlighted a talk by Félix Torres in which he described photochemically induced dynamic nuclear polarization (photo-CIDNP) as a rapid, sensitive method for fragment screening. He, Roland Riek, and collaborators at the Swiss Federal Institute of Technology and the Latvian Institute of Organic Synthesis have just published details (open access) in J. Am. Chem. Soc.

 

The discovery of photo-CIDNP dates back to 1967, which is a useful reminder that technology advancement does not necessarily happen rapidly. The physics and mathematics are a bit complicated, but in essence the process requires a photosensitizer molecule that is excited by light and can also form a radical pair with a given ligand molecule. This “hyperpolarized” ligand is easily detectable by NMR. If the ligand is bound to a protein, the ligand is less able to be hyperpolarized, and thus conducting experiments in the presence and absence of protein reveals whether a small molecule binds to a protein of interest.

 

In practice, the researchers used fluorescein as the photosensitizer. To prevent quenching of the excited state by dissolved oxygen, the samples also included glucose (at 2.5 mM) and the enzymes glucose oxidase and catalase. Samples were illuminated with a 450 nm laser whose light was fed into a 600 MHz NMR instrument via an optical fiber.

 

So, what do you get for all this elaborate setup? Speed and sensitivity. The hyperpolarization allows ligands to be detected with a single scan taking just 2 seconds, as opposed to a typical STD NMR experiment which can take tens of minutes. Moreover, compound and protein concentration can be reduced, which both saves on precious materials and reduces the risk of aggregation.

 

But to realize these benefits, the researchers needed to construct a fragment library suitable for photo-CIDNP. Only about 30 molecules had been reported to be suitable for photo-CIDNP, but these included aromatic moieties frequently found in drugs such as indole, phenol, and imidazole rings. The researchers tested over 1300 fragments and selected a set of 212 that were rule-of-three compliant and showed at least five-fold signal-to-noise enhancement in photo-CIDNP.

 

This “NMhare” library was screened against the enzyme PIN1, which has been implicated in cancer and other diseases. Each fragment was screened individually at 50 µM with or without 25 µM PIN1. Although each experiment took only 2 seconds, changing the samples took longer, and the entire set of 424 experiments took 11 hours. The researchers described a flow-based system that could potentially screen 5000 compounds per day.

 

After visual inspection and quality control, twenty hits were identified. Remarkably, all twenty of these confirmed as binders using protein-detected (¹⁵N,¹H-HSQC) NMR, with fragments at 200 µM and isotopically labeled protein at 50 µM. Two of the fragments had been previously reported as binders, and the researchers were able to determine dissociation constants for these in the low millimolar range. Moreover, they were able to demonstrate that photo-CIDNP could detect one of these fragments at just 5 µM in the presence of 2 µM PIN1.

 

Overall this is neat technology, though as it requires some engineering I’m not sure where it falls under the “practical” descriptor of this blog. That said, if it proves sufficiently useful I’m sure vendors will supply off-the-shelf solutions. I look forward to hearing what NMR aficionados have to say.

Poll results: fragment libraries in 2023

Our latest poll on fragment libraries suggests the field is settling into some standard practices. The poll ran from April 9 through May 26. Of the 59 participants, all but one answered all the questions (there was one skip for the last question). This is slightly down from previous years; perhaps people are sick of internet polls? We also don’t know how many organizations the respondents represent; it is possible several people voted from one company or university, which might skew the results. Nonetheless, we think this survey gives a reasonable snapshot of how people construct and maintain fragment libraries.

 

Our first question asked about library size, and the results are similar to when we last asked this question in 2018, with the average library having between 1001 and 2000 fragments.

 

 

Next, we asked about the size of fragments themselves, specifically the minimum and maximum number of non-hydrogen atoms allowed in a fragment. The minimum hasn’t really changed from 2018, averaging 7-8 heavy atoms. However, the fraction of respondents who include the tiniest fragments has doubled (albeit from a low number), perhaps due to increasing interest in MiniFrags and MicroFrags.

 

Unlike in 2018, the maximum size of fragments seems to be bimodal, with some folks drawing the line at 15-16 heavy atoms (consistent with this analysis from Astex) while others allow larger fragments. It will be interesting to see whether this bifurcation represents a true shift, though even fragments with 22 heavy atoms are likely to be under 300 Da, consistent with the rule of three, which is twenty years old this year.

 

 

We then asked about the presence of chiral molecules. There was little change from 2017, with most respondents stating that they have racemic compounds in their library, though there was a slight increase in the number of respondents excluding chiral fragments.

 

 

A new question for this poll asked whether synthetic tractability was considered at the outset of library design. This was a consideration for 85% of people who took the poll; more than a third said they considered progressability for every fragment in the library.

 

 

 

 

 

 

 

 

 

Finally, we asked about library storage conditions. As was the case when we asked this question nine years ago, more than two-fifths of respondents said they store their library at -20 ˚C. However, the fraction of respondents who store their library at room temperature dropped, while those who store their libraries at -80 ˚C increased.

 

 

 

 

 

Although some changes are noticeable over the years, it seems that best practices have been established and widely adopted in fragment library design. What do you think – does anything surprise you?