From noncovalent fragment to covalent KRASG12C inhibitor

Last week we highlighted Steve Fesik’s presentation at the Discovery on Target meeting in which he discussed the discovery of a covalent inhibitor of the oncology target KRAS^G12C. The paper describing this work, by Joachim Bröker, Alex Waterson, and collaborators at Boehringer Ingelheim and Vanderbilt University, has just appeared (open access) in J. Med. Chem.

 

A decade ago we described how the Fesik lab reported finding millimolar fragments that bind to the so-called switch I/II pocket on KRAS. In collaboration with researchers at Boehringer Ingelheim, these were optimized to sub-micromolar ligands that block nucleotide exchange (see here). However, these molecules hit all RAS isoforms and show only modest cell activity. In contrast, the approved drug sotorasib binds in a different pocket, called switch II, and forms a covalent bond with an oncogenic cysteine mutation, G12C.

 

To find molecules that would bind in the switch II pocket, the researchers first needed to block the switch I/II pocket, which seems to be a hot spot for fragment binding. They did so by introducing a cysteine mutation near the pocket and linking this via a disulfide to a small fragment. All this was done in the context of KRAS^G12V, a mutant that is more common in cancer than KRAS^G12C. The modified protein was then screened using two-dimensional protein-observed (HSQC) NMR against 13,000 fragments. This process identified 20 fragments that bind outside of the switch I/II pocket, including compound 1, which bound to the modified protein with mid-micromolar affinity.

 

 

A combination of SAR-by-catalog and synthesis suggested the importance of both the amino group and the nitrile, and these observations were confirmed by a crystal structure of compound 1 bound to the protein deep in the switch II pocket, as predicted from the NMR data. The crystal structure also revealed a vector to grow the molecule, leading to compound 12. This molecule had sufficiently high affinity to bind to KRAS^G12V without the introduction of the switch I/II blocking fragment. Further growing to compound 19 and addition of a phenyl group (compound 20b) led to low micromolar binders. Installation of an acrylamide warhead and further decoration led to BI-0474, which rapidly reacted with the mutant cysteine in KRAS^G12C. Interestingly, the initial fragment is carried through unchanged.

 

In addition to potent biochemical activity, BI-0474 showed low nanomolar cell activity. The “bioavailability was not yet optimized,” but intraperitoneal administration led to anti-turmor activity in mouse xenograft models. The paper also notes that “a more advanced orally available analogue from this series has recently entered phase I clinical trials.” As we noted earlier this year, this is BI 1823911.

 

It is worth contrasting this work with the discovery of sotorasib, which we discussed in 2020. Sotorasib traces its origins to covalent fragment screens, and an electrophile was maintained throughout the optimization process. In contrast, the new paper starts with a non-covalent fragment that was optimized before an electrophilic warhead was introduced. This is probably more typical of how covalent drugs are discovered, as exemplified last year for the BTK inhibitor TAK-020. However, it is not necessarily easy; Steve mentioned in his presentation earlier this month that achieving the optimal configuration of the warhead took some effort.

 

KRAS has become a poster child for the power of fragment-based approaches to deliver drugs against previously intractable targets. The fact that the new molecules have good non-covalent affinity broadens the range of ligandable oncogenic mutants beyond KRAS^G12C. Indeed, the researchers end by noting that their approach has led to “molecules that are highly attractive for further use in the discovery of inhibitors against other KRAS mutants.”

 

Let’s hope they – and others – succeed.

Twentieth Annual Discovery on Target Meeting

Cambridge Healthtech Institute held its annual Discovery on Target meeting in Boston last week. Although it was technically a hybrid event, about 90% of attendees were physically present, so it really felt like a return to normalcy. Still, the online option was useful: just as with the spring DDC meeting, at least one speaker tested positive for COVID-19 and had to give his presentation remote. Also, with up to eight concurrent tracks, the fact that sessions were recorded for future viewing reduces FOMO, though likely at a cost of spontaneity (see our poll). Panel discussions were in-person only and not recorded, allowing for more candid conversations.

 

Fragments made appearances throughout the event. In a keynote talk, Steve Fesik (Vanderbilt) described work on several targets, most notably KRAS. Long-time readers will recall how NMR screens a decade ago identified molecules that bind to what has become known as the switch I/II pocket. Heroic efforts in collaboration with Boehringer Ingelheim have led to a chemical probe, but the biology around this particular site is complicated.

 

Also, the switch I/II pocket seems to be a magnet for fragments: all 25 crystallographically-characterized fragments from an NMR screen bound here. To look for new sites, the researchers introduced a cysteine residue to covalently block the switch I/II pocket with a known fragment, and then ran an NMR screen to find noncovalent binders at other sites. This identified fragments binding at the switch II pocket used by sotorasib. Extensive optimization and addition of a covalent warhead to target the G12C mutation led to clinical-stage BI 1823911. Steve emphasized the importance of diverse vectors for fragment growing and linking, and not being seduced by potency alone.

 

According to Christopher Davies of Genentech, one of the reasons KRAS has been so hard to drug is that it is very dynamic; in particular, the switch I and switch II loops can adopt multiple conformations. To constrain the protein, the researchers generated antibodies against the G12C mutant covalently bound to a small molecule inhibitor. One of these CLAMPs (Conformational Locking Antibody for Molecular Probe discovery) could stabilize the “open” form of the switch II pocket, thereby improving the affinity of ligands for this pocket and increasing the hit rate from an SPR-based fragment screen. (This work was published in Nat. Biotech. earlier this year.)

 

KRAS is a small GTPase. Samy Meroueh (Indiana University) discussed screening electrophilic fragments against Rgl2, which activates RAL, another small GTPase. We recently wrote about some of this work, and he mentioned that future publications are on the way.

 

Continuing the theme of difficult targets, Brad Shotwell described various hit-finding approaches used at AbbVie against the “cytokinome,” including IL-36γ, TNFα, and two sites on IL-17. We covered some of their TNFα work last year, and the IL-17 work will be the subject of a future post. In line with observations on other proteins, fragment hit rates predicted target ligandability.

 

The protein-protein interaction between NRF2 and KEAP1 is also a challenging target, and David Norton (Astex) discussed how a fragment-inspired virtual screen of the GlaxoSmithKline library ultimately led to low nanomolar inhibitors distinct from an earlier series. He emphasized the importance of growing fragments deliberately rather than attempting dramatic changes.

 

The pocket on KEAP1 is difficult because it is highly polar, but Marianne Schimpl (AstraZeneca) faced the opposite problem with the lipophilic allosteric site on MAT2a (work we highlighted last year). She mentioned the role of synthetic tractability: one fragment hit with higher LLE and Fsp³ was deprioritized in favor of a less shapely molecule that was more readily derivatized.

 

I spent much of the conference in PROTACs and molecular glues talks. Despite my arguments in 2018, FBLD is still not prominent here, but hopefully this will change. For PROTACs especially, which consist of two separate binding elements and a linker, minimizing the overall size is important. Indeed, Yue Xiong (Cullgen) described finding E3 ligands with molecular weights less than 300 Da. Despite only having micromolar affinity, they could be used to make highly effective PROTACs.

 

A broad view of drug discovery was provided by plenary keynote speaker Anabella Villalobos, who described the multiple therapeutic modalities used at Biogen to tackle neuroscience diseases. She mentioned that there are around 15 FDA-approved oligonucleotide-based drugs, but that this did not happen overnight: the first was approved more than a quarter century ago. This long “induction period” reminds me of my post last year comparing the rise of therapeutic antibodies with FBDD.

 

There is plenty more of interest; for those of you who attended, what talks would you recommend watching? And mark your calendars for September 25-28 next year, when DoT returns to Boston!

Inter-ligand STD NMR: Better than ILOE?

Although our poll in 2019 suggested that crystallography has surpassed NMR in FBLD, not all proteins can be crystallized. Ligand-detected methods such as saturation transfer difference (STD) NMR can be particularly useful for quickly identifying individual fragment binders and getting some sense of how they bind. A new variation published (open access) in Pharmaceuticals by Jesus Angulo and collaborators at University of East Anglia and Universidad de Sevilla provides information on the relative binding modes of two ligands.

 

Long-time readers may remember the inter-ligand NOE method (ILOE) we wrote about in 2010, in which proximity of two ligands is assessed by measuring NOE signals between them. However, despite being described more than 20 years ago, the technique seems to be rarely used, with fewer than a dozen papers in Pubmed, perhaps because ILOE requires large amounts of both protein and NMR time.

 

The new method is called inter-ligand STD NMR (IL-STD NMR), and it was discovered serendipitously while studying the binding of the drug naproxen to bovine serum albumin (BSA). As Teddy discussed several years ago, STD NMR normally involves irradiating specific protons in a protein (for example, the hydrogen atoms on buried methyl groups) and then measuring the “transfer” of this magnetization to bound ligands. When the researchers instead irradiated protons on one end of naproxen, they found that while the STD effect fell along the length of the molecule as expected based on distance, the signal suddenly increased at the other end of the molecule. Naproxen is known to bind to three sites on BSA, and this increased signal was attributed to the proximity of two adjacently bound naproxen molecules.

 

Inspired by this observation, the researchers developed IL-STD NMR. The experiment requires two samples, with two NMR experiments on each. One sample contains the protein and ligand of interest, while the other sample also contains a “reporter ligand” with a known binding mode. For each sample, one NMR experiment is run with selective irradiation of protons on the protein, while the other is run using irradiation of the reporter ligand. Comparison of the spectra reveals which regions of the ligand of interest are near the reporter ligand. The researchers demonstrated that the method works using a model system they had previously studied, the cholera toxin subunit B (CTB), which binds two ligands at nearby sites.

 

Importantly, the time and amount of protein is considerably less than required for ILOE: in this case 2 hours of NMR time and 0.3 mg of protein compared with 88 hours (!) and 1.8 mg. Moreover, the experiment could be run on a 500 MHz NMR, which is a relatively common instrument.

 

IL-STD NMR does have limitations. The researchers note that it is important to avoid irradiating the ligand of interest while irradiating the reporter ligand. Also, the approach obviously only applies to proteins with two nearby pockets (or one larger pocket). Still, it does look interesting, and I could imagine it being used as part of a screening cascade to find candidate fragments for merging or linking. What do the NMR aficionados think?

Crystallographic covalent fragment screening – but why?

Once considered a time-intensive method justified only by the most-promising molecules, crystallography is fast becoming a routine technique for general fragment screening. Just last month we highlighted an example where crystallographic hits were successfully used in virtual screens. In a new J. Med. Chem. paper, Jeffrey St. Denis, Benjamin Cons, and colleagues at Astex describe a crystallographic screen with a covalent fragment library.

 

Driven in part by the rapid development of KRAS^G12C inhibitors such as sotorasib, covalent fragments are continuing to increase in popularity. To screen, you first need a library, and here the researchers assembled a set of 114 molecules, most of which were commercially available from Enamine. The majority (101) contained an acrylamide moiety, the most common warhead in approved covalent drugs. The library is rule of three compliant and fairly shapely, as assessed by deviation from planarity. Laudably, the structures of all 114 library members are disclosed in the supporting information.

 

In addition to physicochemical properties, the library was also characterized experimentally. Importantly, it seemed to be quite stable when stored frozen at -80 °C in deuterated DMSO for up to a year. Most (83%) of the compounds were soluble to at least 3 mM in aqueous buffer. And just over half of the molecules withstood a test for reactivity with 5 mM glutathione, the main nucleophile found in cells, assessed as >90% remaining after 30 minutes at 37 °C.

 

As a test case, the researchers screened the kinase ERK2, an oncology target for which Astex previously developed the non-covalent clinical compound ASTX029. Soaking ERK2 crystals in 50 mM of each fragment led to 29 bound ligands, a whopping 25% hit rate. Of these, 16 compounds bound to C166, a cysteine near the hinge region in the active site. Unsurprisingly, less reactive fragments made hydrogen bonding interactions with the protein, while the more reactive fragments did not and also had poor electron density, suggesting non-specific alkylation. As we noted in 2014, distinguishing specific from non-specific binding is a general challenge when screening irreversible fragments.

 

Among fragments making specific interactions, compound 9 was chosen for further study due to its low inherent reactivity and interesting structure. The molecule showed no inhibition of the protein after 1 hour at 1 mM, but scaffold hopping and fragment growing led to compound 22, with low micromolar inhibition after 1 hour. (A back of the envelope calculation suggests k_inact/K_i ~ 25 M^-1s^-1, though I wish the researchers had reported this.)

 

This is an interesting paper, and it is useful to get structural information up-front, but I can’t help thinking that it would be more efficient to use an alternative method for the initial screen. As the researchers acknowledge, many covalent fragment screens use intact-protein mass spectrometry. For example, in a paper we highlighted in 2019, nearly 1000 fragments were screened against ten different proteins. Moreover, as we noted last year, the ability of crystallography to find such very weak hits can be a distraction as much as a blessing.

 

The current paper promises to “report on the further developments and observations pertaining to our electrophilic library in due course.” I look forward to seeing more.

Metal-binding fragments vs glutaminyl cyclases

Metal-binding fragments have a long history in FBLD; the first mention on Practical Fragments was back in 2010. The idea is to use the strong interaction between a fragment and a protein-bound metal as an affinity anchor for further optimization. The latest example, by Jie-Young Song, Soosung Kang and collaborators at Korea Institute of Radiological & Medical Sciences, Ewha Womans University, and elsewhere was published in ACS Med. Chem. Lett.

 

Glutaminyl cyclases such as glutaminyl-peptide cyclotransferase (QC) and glutaminyl-peptide cyclotransferase-like protein (isoQC) convert N-terminal glutamine or glutamate residues on proteins to pyroglutamates. This modification tends to stabilize proteins, and it has been implicated in several diseases. In particular, modification of CD47 by isoQC seems to be important for the ability of cancer cells to evade the immune system.

 

QC and isoQC are closely related enzymes with a zinc-containing active site. Capitalizing on this, the researchers tested a library of 36 potential metal-binding fragments in a functional assay against QC. Most of the compounds tested were inactive, though 11 had IC₅₀ values less than 0.8 mM. A few of these, including compound ab, were used to generate a second library of just half a dozen larger fragments, and compound 9 turned out to quite potent.

 

The researchers recognized that compound 9 has two potential zinc-binding moieties, and docking suggested the newly added amino-thiadiazole was likely responsible for the increased activity. Structure-based design ultimately led to compound 22b, with low nanomolar activity against QC and isoQC. The molecule did not seem to be generally cytotoxic, but it did increase phagocytosis of cancer cells in vitro, consistent with an effect on the “don’t eat me” function of CD47.

 

Unfortunately, no information is provided on the selectivity of compound 22b against other zinc-dependent enzymes. Moreover, unlike an earlier example of starting with metallophilic fragments, no ADME data are provided. But whether or not this particular series advances, it is nice to see metallophilic fragments being explored.