25 September 2023

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.

18 September 2023

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.

11 September 2023

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 Kd 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.

04 September 2023

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.