Covalent fragments vs Rgl2

Just over a year ago the FDA granted accelerated approval to sotorasib, the first marketed inhibitor of KRAS and the first approved fragment-derived covalent drug. In a recent ChemMedChem paper, Samy Meroueh and colleagues at Indiana University School of Medicine describe their efforts against a protein in a related pathway.

 

KRAS is a GTPase which cycles between an “on” state, where GTP is bound, and an “off” state, where GTP is hydrolyzed to GDP. KRAS is just one member of a superfamily of GTPases. Two other members also associated with cancer include RalA and RalB. Sotorasib acts by binding to a mutant form of KRAS in which a glycine is replaced by a cysteine, but this mutation does not occur in RalA or RalB. An alternative approach to targeting GTPases is to prevent them from becoming activated by guanine exchange factors (GEFs), which help exchange GDP to GTP. We’ve previously written about how fragments have led to noncovalent inhibitors of the GEF SOS1, which activates RAS proteins.

 

To sum up, there’s more than one way to block GTPase activity: directly, or by preventing activation by an associated GEF. The new paper focuses on Rgl2, a GEF that serves RalA and RalB.

 

Rgl2 sports four surface-exposed cysteine residues, so the researchers screened the protein against a library of 1260 electrophilic fragments at 75 µM for 24 hours at 4 °C and then assessed whether it could still activate RalB. 50 fragments inhibited guanine nucleotide exchange by at least 30%, and a dozen were studied in detail. All were time-dependent inhibitors and had EC₅₀ values from 2.6 to 120 µM at 24 hours.

 

Next, the researchers mutated each of the four surface-exposed cysteine residues to serine. The twelve fragments still inhibited all the mutants except C284S. SOS1 does not contain a cysteine at the position corresponding to C284, and indeed none of the twelve fragments significantly inhibited SOS1 activation of KRAS. All this suggests the fragments act via modification of C284.

 

The easiest and most direct measurement of covalent binding is with intact protein mass spectrometry, and the researchers confirmed that 10 of the 12 fragments did in fact form adducts. Interestingly, Rgl2 was modified two or three times by each fragment, which is perhaps not surprising given that they had relatively reactive warheads (chloroacetamides or propiolamides). Mass-spec studies with the mutants revealed that most of the modifications were at C284 and C508.

 

Whether or not these fragments are advanceable, the discovery that modification of C284 inhibits Rgl2 is useful. Interestingly, C284 is near but not at the Ral binding interface, and the researchers suggest that their fragments block protein activity allosterically. I believe such allosteric sites are common throughout the proteome, and readily addressable using covalent approaches. Watch this space!

A fragment-sized chemical probe for Notum

Practical Fragments has written previously (here and here) about the enzyme Notum, which shuts down Wnt signaling by removing a palmitoyl group. Aberrant Wnt signaling is implicated in maladies from cancer to osteoporosis, but Paul Fish has been particularly focused on neurological conditions such as Alzheimer’s disease. In a paper just published in J. Med. Chem., Fish and collaborators at University College London, University of Oxford, and The Francis Crick Institute describe their discovery of a chemical probe for this target.

 

As we discussed last year, the researchers conducted a crystallographic screen of the 768-member Diamond-SGC Poised Library, which resulted in 59 hits. Biochemical confirmation studies revealed that fragment 6b, a close analog of a fragment described earlier, is remarkably potent. The substituted phenyl ring nicely fills the lipophilic active site, and the triazole forms a hydrogen bond with a backbone amide of the protein. Structure-based design subsequently led to compound 7y, with low nanomolar potency.

 

The previous fragment-based efforts against Notum also yielded potent molecules, but they had poor brain-penetration. In contrast, compound 7y has a high brain-to-plasma ratio, though the compound also has high clearance, which was attributed to phase 2 metabolism at the hydroxyl. The researchers explored a variety of replacements and substitutions, all of which led to loss in potency, but interestingly removing the hydroxymethyl substituent altogether was tolerated.

 

The resulting molecule, ARUK3001185, is a potent inhibitor of Notum both in biochemical and cell assays. It has good oral bioavailability and pharmacokinetics in mouse and rat. Importantly, it also has excellent brain penetration in both species. The molecule showed virtually no inhibition of 39 other serine hydrolases or 485 kinases and was fairly clean in a safety panel of some four-dozen human targets, including hERG. In other words, ARUK3001185 appears to be an excellent chemical probe.

 

This is a nice example of how a fragment-sized molecule can nonetheless achieve high affinity and selectivity. As we’ve seen repeatedly, potency is not enough; one often needs to spend considerable effort to optimize other properties such as brain penetration. It will be fun to see what this new probe can teach us about Wnt signaling in the brain.

SAMPL7: Epic computational fail or just no solution?

Every few years computational chemists are invited to compete in the Statistical Assessment of Proteins and Ligands (SAMPL) challenges. Researchers are asked to solve a problem for which the solution is known but not yet published; this blinded format allows a more rigorous test of methods than the typical retrospective studies. SAMPL7 focused on fragments binding to proteins, and the results have been published (open access) in J. Comp. Aided Mol. Des. by Philip Biggin and collaborators at University of Oxford and elsewhere.

 

The subject of this challenge was PHIP, a multidomain protein implicated in insulin signaling and tumor metastasis, though the biology is a bit complicated. PHIP contains two bromodomains, small modules that act as epigenetic readers by binding to acetylated lysine residues (Kac), and the researchers chose to focus on the second bromodomain (PHIP2). Bromodomains have proven to be highly ligandable, though this one is unusual in having a threonine in place of a conserved asparagine.

 

The experimental results that contestants were challenged to predict came from fragment screening using high-throughput crystallography at Diamond Light Source’s XChem. PHIP2 crystals diffracted to high resolution (1.2 Å) and were soaked with 20 mM fragment for 2 hours at 5 °C. In total 799 fragments were screened: 768 from the DSI-poised library (see here) and 31 FragLites (see here). The team took great pains to gather high-quality data, screening the FragLites twice and re-soaking 202 fragments that produced poor R factors or resolution worse than 2 Å. This resulted in 52 hits, a hit rate of 6.5%, consistent with the 2-15% typically seen at XChem. Most (47) of these were in the Kac-binding site, and these were the focus of the SAMPL7 challenge.

 

The first task was for modelers to simply predict which of the 799 fragments bound and which did not. Full experimental details were provided, including pH and the crystallization conditions. Entrants were given 1 month. There were eight submissions plus a control, which randomly selected compounds as binders or non-binders. Most of the contestants used some sort of docking strategy; details are provided in the paper.

 

Shockingly, none of the submissions scored better than random. Three of the entrants failed to correctly identify a single binder, and four identified between 1 and 5 of the 47.

 

The second task was to predict the binding modes of the crystallographically identified ligands. Contestants were provided with the 47 hits and asked to submit up to five poses for each. Perhaps stung from their performance on the first task, or perhaps put off by the two-week requested turnaround time, only five groups submitted entries.

 

Performance was assessed by calculating the root mean square deviation (RMSD) between the experimental and docked structure(s), with RMSD ≤ 2 Å considered successful. Despite this fairly lenient cutoff, “the performance of the methods was disappointing.” The best scored 24%, while two methods scored 2% and 0%. I’ll leave it to chemists to opine whether even a 24% success rate for docking would give confidence to embark on analog synthesis.

 

The third task was to select follow-up molecules from a large database for experimental validation, but alas “the COVID-19 pandemic resulted in a diversion of funds before this follow-up study could be done.” Nonetheless, four intrepid groups submitted entries, and these are discussed in the paper.

 

Taken at face value, this is downright damning for computational chemists. It is also at odds with many nice success stories, for example those described at last month’s DDC conference. So what’s going on?

 

For one thing, not everyone paid attention to the information provided. The crystals were at pH 5.6, but some of the entrants nonetheless assumed pH 7.4.

 

This raises a second and more important point. As the researchers acknowledge, “there is the possibility that our fragments do not necessarily bind in solution, whereas scoring functions are almost always calibrated and validated against solution and structural data.” In other words, perhaps the fragments were not identified computationally because they only bind extremely weakly to a crystalline protein soaking in dilute acid.

 

This highlights perhaps the biggest drawback of fragment screening by crystallography: no matter how beautiful the structure may appear, you get no measure of affinity. Indeed, a paper we highlighted last year was able to confirm binding by NMR for only a minority of crystallographically identified fragments against the SARS-CoV-2 main protease. This does not mean that the crystal structures are “wrong,” but the ligands may be so weak as to be unadvanceable.

 

A picture can be worth a thousand words, but it can also be misleading. Advancing fragments is best done with the help of multiple orthogonal methods.

Fragments vs TLR7/8, starting from HTS

The toll-like receptors TLR7 and TLR8 are closely related proteins that respond to single-stranded RNA, often associated with viral infection, to activate the immune system. While this is useful to ward off disease, when the proteins become overactivated they can lead to autoimmune disorders such as lupus (see here for a recent discussion by Derek Lowe). In a recent ACS Med. Chem. Lett. paper Claudia Betschart and colleagues at Novartis describe advancing a fragment to a potent inhibitor of both proteins.

 

The researchers built a biochemical (specifically, a TR-FRET competition) assay in which they screened 50,000 molecules, each at 20 µM. The campaign yielded some 1500 hits, and this 2020 paper describes the optimization of one of these.

 

The new paper describes the optimization of a completely different molecule, compound 2. This rule-of-three compliant fragment was not only potent in the biochemical assay, it also showed low micromolar cell activity. A crystal structure of the compound bound to TLR8 revealed that it binds at the interface of a homodimer, making hydrogen bonds to both monomers and stabilizing an inactive conformation of the receptor. 

 

A carbon atom in compound 2 was replaced with a nitrogen in compound 3 in the hopes of picking up an additional hydrogen bond, and this led to a ten-fold increase in potency. TLR8 is located in acidic endosomes, and adding a basic piperidine moiety to try to optimize the subcellular localization did in fact improve cellular potency for compound 5. However, basic amines are often associated with hERG binding, which can cause cardiac problems, and this turned out to be the case for this series. This liability was addressed by adding a fluorine to lower the pKa of the amine. Further addition of small moieties to complement the protein led to additional increases in potency, ultimately yielding compound 15.

 

In addition to low nanomolar and even picomolar cellular activity against TLR7 and TLR8, respectively, compound 15 is selective against other TLRs as well as a panel of 100 off-targets. The compound has good DMPK properties in mice and reduced TLR7-dependent interferon-α release in a mouse model.

 

This is a nice medicinal chemistry story focusing on all aspects of optimization, not just potency. Like last month’s Notum and SARM1 posts, it is also another example of a fragment rising to the top of a high-throughput screen. Fragments don't have to be weak.

Fragment events in 2022 and 2023

The first third of 2022 has already been graced with two major fragment conferences. Two more have recently been added, and 2023 is starting to take shape.

May 9-11:  While not exclusively fragment-focused, the Eighth NovAliX Conference on Biophysics in Drug Discovery will have several relevant talks, and for the first time will use a hybrid model, both online and in Munich. You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

 

May 24-25:  BioSolveIT is holding a DrugSpace Symposium, with a heavy emphasis on fragments. It's both virtual and free, with an impressive lineup of speakers.

September 28-30: FBDD Down Under 2022 will take place in beautiful Melbourne. If you've been longing to travel, Australia has recently opened its borders. This is the fourth major FBDD event in the country, and given the success of the first and third, it should be excellent.

 

October 17-20: CHI’s Twentieth Annual Discovery on Target will be held both virtually and in Boston, as it was last year. 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 2020 virtual event here, the 2019 event here, and the 2018 event here.

 

 

2023

April 10-13: CHI’s Eighteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, has already been scheduled for 2023 in San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of 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 FBLD 2023 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!