Malicious metals muddy fragment-to-lead optimization

Despite the effectiveness of vaccines against SARS-CoV-2, COVID-19 continues to plague us. The handful of approved small molecule drugs target only two proteins and have much room for improvement. One interesting but underexplored target is the nonstructural protein 14 (NSP14), a 3’ to 5’ RNA exonuclease, which is important both for viral replication as well as immune escape. In a new open-access ACS Chem. Biol. paper, Jae Jung, Shaun Stauffer, and colleagues at the Cleveland Clinic describe how their efforts against NSP14 were thwarted.

 

The researchers started with the crystal structures of two fragments that had been identified in a high-throughput crystallographic screen at XChem. They reproduced these in-house, confirming the published structures, and also made and characterized a few analogs. Crystallography demonstrated these bind in a similar manner. Encouragingly, they also showed activity in a biochemical assay.

 

The two published fragments bind next to one another, presenting a good opportunity for fragment merging or linking. The researchers used the computational tool Fragmentstein, which we wrote about here, to design new molecules. Some of these molecules were active in the biochemical assay, and a crystal structure of a merged compound revealed that it bound as expected. Importantly, none of the molecules inhibited an unrelated endonuclease.

 

So far, so good, but the researchers were suspicious about the SAR. For example, changing an isopropyl to a cyclopropyl group weakened the activity  from 2.4 to 150 µM, despite the fact that the moiety is largely solvent exposed. After resynthesizing and more carefully purifying the molecules, the researchers found them to be completely inactive in the biochemical assay.

 

NSP14 contains two catalytic magnesium ions and three structural zinc ions, and the researchers considered the possibility that metal contaminants might have been responsible for the activity. Sure enough, when they screened the Metal Ion Interferences Set (MIIS), which we wrote about here, they found that half a dozen metal ions potently inhibited the assay. They tested whether any of the spuriously active compounds contained palladium and ruled this out, but did not test for other metals. Indeed, metals may not even be to blame: the active molecules all contain thiazoles, and as we discussed in 2022 these can sometimes interfere with assays. What is clear is that the exciting initial activity results were artifacts, and the researchers were sufficiently diligent to figure it out for themselves.

 

One of the most disturbing findings is that the crystal structures looked fine, despite the compounds having no measurable activity. As we’ve written previously, the lack of affinity information is the biggest drawback of fragment screening by crystallography. Perhaps NMR would have been able to invalidate the false-positives, though as we have written both protein-detected and ligand-detected methods can be fooled. As our 2024 poll emphasized, using multiple methods to validate fragment binding is important. And resynthesizing and carefully purifying compounds helps too.

 

These sorts of cautionary tales are not published as often as they should be. Kudos to this team for both warranted skepticism and providing a warning for others.

Selectivity in cells may vary

Last year we celebrated the ten-year anniversary of the Chemical Probes Portal. One of the key requirements for a chemical probe is selectivity, which was set to >30-fold vs related targets when the Portal launched in 2015. For enzymes such as kinases, selectivity is often measured in cell-free assays. A new open-access J. Med. Chem. paper by Matthew Robers, Alison Axtman, and collaborators at Promega and University of North Carolina at Chapel Hill suggests that such data don’t necessarily translate to cellular assays.

 

Kinases are one of the most heavily mined classes of targets this century; five of the eight FBLD-derived approved drugs target kinases. With more than 500 in the human proteome, selectivity has long been a focus. One common method for assessing selectivity in cell-free assays is the Eurofins DiscoverX panel, which currently includes more than 450 kinases. Each kinase has a DNA tag and is paired with a promiscuous high affinity binder attached to a solid support. Test compounds are added, and qPCR is used to assess and quantify which kinases are displaced. The competition assay allows determination of dissociation constants.

 

To measure the binding of compounds to kinases in living cells, the researchers turned to the NanoLuc-bioluminescence resonance energy transfer (NanoBRET) assay. This is also a displacement assay that relies on a bivalent molecule containing a kinase ligand and a fluorophore. Kinases are tagged with NanoLuc, which causes luminescence of the fluorophore when it is in close proximity (ie, bound to the kinase). Ligands that bind to the kinase displace the bivalent molecule, decreasing luminescence.

 

The researchers started with four promiscuous kinase inhibitors, two of which (dasatinib and sorafenib) are approved drugs. They ran these against 240 or 300 kinases in the NanoBRET assay and compared the values with published DiscoverX dissociation constants. Most of the compounds were more potent in the DiscoverX assay than in the cell-based assay, and the researchers suggest several possible reasons. First, the DiscoverX assay is run in the absence of the cofactor ATP, which can compete with ligands that bind to the active site. Second, cell (im)permeability could decrease cellular potency. Finally, most of the DiscoverX kinases are truncated, whereas the NanoBRET kinases are full length.

 

For these and other reasons, it is common for compounds to be less active in cell assays than biochemical or biophysical assays. Surprisingly though, for a few kinases the compounds were actually more potent in the cellular assay than they were in the DiscoverX assay.

 

To extend these findings, the researchers tested additional kinase inhibitors and found that three kinases were particularly susceptible to inhibition in cells. One of these kinases, PIP4K2C, was engaged at mid nanomolar potency by cabozantinib, and the researchers suggest this could be useful for immuno-oncology. More worrisome, several approved drugs bind to the tumor suppressor STK11 in cells, raising the potential that these compounds could inhibit exactly the wrong pathway.

 

On the bright side, the researchers find that some molecules that look moderately selective in the DiscoverX assay are actually quite selective in cell assays, and they propose new chemical probes for the little-studied kinases BRSK1/2 as well the kinases DDR1/2.

 

Kinases are certainly not the only class of targets for which compounds’ performance differs outside vs inside cells; we wrote about covalent WRN inhibitors here. This paper is a good reminder that as useful as cell-free assays are, things can go weird once you go into cells – for better or for worse.

Best practices for applying HDX-MS to FBLD

Among the many biophysical techniques presented at the recent Novalix meeting, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was mentioned only a few times. Practical Fragments last covered it nearly four years ago in the context of a paper that described theoretical challenges and improvements to the method for assessing fragment binding modes. A new open-access Comm. Chem. paper from Tiago Bandeiras, Alessio Bortoluzzi, and collaborators at iBET-Instituto de Biologia Experimental e Tecnológica and Merck KGaA implements some of these suggestions.

 

The researchers find only two examples where HDX-MS had been applied to fragments, one of which we covered here. The new paper focuses mostly on Cyclophilin D (CypD), a mitochondrial protein implicated in several diseases. A screen we wrote about in 2020 described several fragment hits, some of which were crystallographically found to bind in three overlapping sites designated the proline pocket, the aniline pocket, and the pyrazolo pocket.  The researchers chose a binder from each pocket to study as well as two more fragments whose binding sites were not known. All five fragments are extremely weak hits, with at best 7 mM (yes, millimolar) affinity as assessed by SPR, making this a particularly challenging test case.

 

As a reminder, HDX-MS examines the exchange of deuterium from D₂O to protein backbone amides. Nearby ligands can slow this exchange, and mapping the locations and magnitude of these changes reveals where the ligand binds. Optimization experiments were initially run on a compound with a K_D of 44 mM for the aniline pocket. Three protein concentrations were tested. Since the highest (10 µM) yielded the highest number of detected peptides, it was used for subsequent experiments.

 

As suggested in the 2022 paper, compound was added both to the initial protein solution as well as to the deuterium exchange buffer. The fragment was tested at 2.5, 5, and 10 mM, and changes in deuterium uptake were found in all cases, which is remarkable given that the theoretical occupancies range from 5 to 18%. An experiment using substoichiometric concentrations of a high affinity ligand confirmed that 18% protein occupancy is sufficient to generate a reliable signal.

 

Still, given the low occupancy, the researchers used statistical methods to ensure that changes in signals were significant. Mapping those that were onto the structure of the protein confirmed that the fragment bound to the aniline pocket.

 

A second fragment was tested by HDX-MS, and the results confirmed that it binds in the pyrazolo pocket, as previously shown by crystallography. However, for a third fragment that had been shown to bind in the proline pocket, the results suggested instead that it binds in the aniline pocket. The proline pocket exhibited very low levels of deuterium exchange, but when the researchers increased the pH from 7.4 to 9 they did find evidence that the fragment binds here as well.

 

Next, the researchers turned to the two fragments with unknown binding sites. HDX-MS revealed that these bind to the aniline binding site, though with subtly different protection patterns suggesting that they bind in slightly different regions of the pocket.

 

Finally, the researchers used their optimized HDX-MS conditions on a previously identified fragment that binds the kinase FAK with low millimolar affinity. This showed that it binds in the so-called hinge region, in agreement with crystallography. This fragment was also used as a negative control for CypD, showing that it does not bind.

 

This paper is a nice resource for those hoping to apply HDX-MS to fragments. The fact that the binding sites of such weak binders can be determined is quite remarkable. That said, the resolution is not as good as crystallography or protein-detected NMR; for FAK in particular, the ligand reduces deuterium update across a large fraction of the protein surface. And the fact that a proline-pocket binder was initially mapped to the aniline pocket also gives one pause. Perhaps the binding mode in solution really is different from that found crystallographically. It would be interesting to see whether NMR can resolve the conundrum.