26 April 2021

STD NMR on putative SARS-CoV-2 main protease ligands

Over the past sixteen months SARS-CoV-2 has infected more than 146 million people worldwide and killed over 3 million. Highly effective vaccines are now available, but not everywhere, and how long the vaccinations will last as new variants arise remains unknown. COVID-19 will likely be with us indefinitely, necessitating drugs as well as vaccines.
More than a year ago we highlighted the COVID Moonshot effort, which began with a crystallographic screen against the essential main protease (Mpro) to find starting points for drug discovery. In a new open-access J. Biomol. NMR paper, Ioannis Vakonakis and collaborators at University of Oxford and University of Patras have attempted to characterize some of the hits using saturation transfer difference NMR (STD NMR, see here for a brief description).
The researchers had access to a 950 MHz NMR (jealous much?). Samples were screened at 10 µM protein using irradiation of a Mpro methyl group with a chemical shift at 0.5 ppm. To try to minimize differences in relaxation parameters among different ligands, only the strongest STD signals in aromatic moieties were examined.
Of 39 non-covalent ligands discovered in the crystallographic screen, five either did not produce an NMR signal or the spectra were inconsistent with the expected structures, suggesting the ligands may be insoluble or unstable in aqueous buffer. The remaining 34 compounds were nominally screened at 0.8 mM each, but the reference spectra differed in intensity by as much as 15-fold, suggesting dramatic differences in concentration. Since the strength of an STD signal is related to both affinity and concentration, this could obviously complicate interpretation of results. As we’ve written previously, careful curation of your library is essential.
Of thirteen active site ligands, only four showed strong STD signals. Dose-response titrations between 0.05 and 4 mM revealed dissociation constants of 1.6-1.7 mM for two of them, with the other two being too weak to accurately measure. Molecular dynamics simulations starting with the known structures were consistent with these results, with the tighter binders tending to maintain their positions more than the weaker binders.
The researchers also characterized 650 elaborated molecules from the COVID Moonshot, some of which had been reported to be nanomolar inhibitors. Disturbingly, 35 gave no NMR signal and another 86 yielded weak signals. Among those remaining there was a weak correlation (R2=30%) between IC50 in an enzymatic assay and the STDratio (integrated signal intensity of peaks in the STD spectrum over reference spectrum). STD NMR is not appropriate for molecules with Kd < 10 µM, so the researchers also used a competition experiment in which four putative high-affinity molecules would compete a weaker “spy” fragment. This exercise confirmed two ligands but not two others, calling into question their mechanism.
STD NMR is often used as part of an assay cascade prior to attempting crystallography, but as crystallography throughput increases there is a case for starting with crystallography, as we argued five years ago. Results from the 39 crystallographic hits perhaps gives pause to that notion, or at least emphasize the need for confirmatory assays. It is easy to be seduced by a high-resolution structure, but because of its sensitivity crystallography may identify ligands so weak as to be unadvanceable. As for the 650 elaborated molecules, it’s too early to draw conclusions, though it’s good to always be on the lookout for false positives.
Hopefully the COVID Moonshot will ultimately lead to drugs against SARS-CoV-2. But even if it doesn’t, the intensive focus of multiple techniques on a few proteins is providing useful guidance and best practices that will be applicable to other targets.

19 April 2021

Fragments vs KEAP1: deconstruction and merging

One of the more challenging protein-protein interactions targeted by drug hunters is the interface between the transcription factor NRF2 and its repressor KEAP1. This is part of the cellular defense against reactive oxygen species; increasing NRF2 activity may be useful for treating a variety of diseases. Unfortunately, the binding site on KEAP1 that interacts with NRF2 is large and has a predilection for carboxylic acids. Thus, many of the molecules reported as inhibitors tend not to be druglike. Anders Bach (University of Copenhagen) and a multinational team of collaborators sought to do better, and have just published some of their journey in J. Med. Chem.
The researchers had previously tested 19 reported small-molecule KEAP1 inhibitors, of which only nine confirmed. (This is a salutary reminder to take any individual publication with a large grain of salt.) The nine fell into six chemical series (two shown below), and the researchers decided to fragment some of these molecules into 77 fragments. The fragments were then tested in four assays: fluorescence polarization (FP), a thermal shift assay (TSA), saturation transfer difference (STD) NMR, and surface plasmon resonance (SPR).
Primary hit rates were generally high, from 25%-64%, but long-time readers will not be surprised that the overlap was not great: no fragments hit in all four assays, and only eight hit in three. As the researchers point out, this could reflect differences in sensitivity, conditions (from 3-8% DMSO and from 0.5 to 8 mM fragment), and different types of false positives and false negatives. Interestingly, and in contrast to previous work, overlap was good between STD NMR and SPR.
Crystal structures of seven hits were solved bound to the protein, and compounds 4c and 1m (from different precursor molecules) were merged to provide compound 8, with low micromolar affinity. Compound 8 was the subject of considerable medicinal chemistry, with five different vectors chosen for growing. Despite being structurally enabled, the researchers struggled; changes that improved affinity in one context did not do so in another. After considerable effort, the researchers obtained compound 77o, with mid-nanomolar activity.

Compound 77o is stable in human plasma and mouse liver microsomes. Unfortunately, and unsurprisingly given the two carboxylic acids, it has poor permeability. Indeed, a fragment-derived KEAP1 inhibitor we described previously has only a single carboxylic acid, as does precursor compound 7. As the researchers themselves acknowledge, “the physicochemical properties of our compounds are not favorable for membrane permeability.”
Nonetheless, this paper is a lovely example of fragment-based deconstruction reconstruction (FBDR) and is well worth studying for the thorough descriptions of fragment screening in orthogonal assays and structure-based design. Another lesson may be that despite considerable effort, the final molecule is far from a chemical probe, let alone a drug. Perhaps some targets truly are undruggable. Or maybe – as for other seemingly undruggable targets – a change in strategy is needed.

12 April 2021

Fragment merging on c-MET

Fragment-based inhibitors of kinases are legion, particularly those that bind in the so-called hinge region where the adenine of ATP normally sits. However, even among these there are many different flavors of inhibitors. In particular, about 10 kinases can adopt a “folded P-loop” conformation, in which the phosphate-binding loop collapses into the ATP binding site. This was the focus of a recent open-access paper in ACS Med. Chem. Lett. by Gavin Collie and colleagues at AstraZeneca.
The researchers were interested in the oncology target c-MET. A ligand-based NMR screen of 1150 fragments (in pools of 6 at 200 µM each) yielded a 6% hit rate, of which 20 confirmed by SPR. Crystallography was attempted unsuccessfully on most of these, but compound 1 was found to snuggle into the active site with the protein in the folded P-loop conformation.
A computational similarity search of AstraZeneca’s internal library identified compound 2, which crystallography revealed to bind in a similar manner, with two hydrogen bonds to the hinge region and the benzyl group buried in a hydrophobic pocket. A second similarity search of the library – this time based on compound 2 – identified compound 3. Crystallography confirmed that the core azaindole moieties of compounds 2 and 3 overlay, and thus fragment merging was attempted.

The resulting compound 5 bound as expected. This prompted yet another computational search of the internal library, and after a bit of medicinal chemistry compound 7 was identified as a mid-nanomolar inhibitor with low micromolar cell-based activity. Crystallography revealed that it too binds to the folded P-loop conformation of c-MET.
Because the folded P-loop conformation is rare among kinases, the researchers hoped that the resulting molecule would be selective. Unfortunately, when profiled against a panel of 140 kinases at the low concentration of 100 nM, 27 of them were inhibited by at least 60%. This is perhaps not surprising given the 7-azaindole core, which has been found to bind to more than 90 kinases, though some compounds containing this moiety are selective.
Nonetheless, this paper is a nice example of structure-guided fragment merging. A cynic could point out that had the researchers screened the entire AstraZeneca compound collection they likely would have identified molecules very similar to compound 7 anyway, but this may have cost more and would not be an option at smaller organizations without million-compound libraries. And the approach is useful for more difficult targets for which high-affinity molecules may not exist – yet.

05 April 2021

A general fragment-based approach to… targeting RNA?

This is taken from the title of a recent open-access paper by Matthew Disney and collaborators at Scripps Research Institute Jupiter and Florida Atlantic University in Proc. Nat. Acad. Sci. USA. RNA has long been a target of FBLD: Practical Fragments first blogged about it in 2009, and a 2002 paper reported using fragment linking to obtain a low micromolar binder. So how general is the new approach?
The researchers describe chemical cross-linking and isolation by pull-down fragment mapping (Chem-CLIP-Frag-Map). This involves using photoaffinity probes that can crosslink to biomolecules such as RNA. The probes also have an alkyne tag that can be used to isolate bound molecules using click chemistry. We’ve written previously about such “fully functionalized fragments” (FFFs).
Earlier work had resulted in the identification of compound 1, which binds to a specific site on pre-miR-21, the precursor to a non-coding microRNA linked to cancer. An FFF version of compound 1 was shown to crosslink to pre-miR-21 after irradiation with UV light, and the site of modification could be mapped using a reverse-transcriptase-mediated primer extension, which stalled at the modified bases.
Next, the researchers screened 460 FFFs at 100 µM and found 21 that crosslinked to pre-miR-21. They were ultimately looking to link fragments with compound 1, and thus competition studies were used to eliminate fragments that bound at the same site. This left three fragments, and primer extension studies confirmed that these bound near but not at the binding site of compound 1.
Next, the researchers attached these three fragments to compound 1, with or without various linkers. Some of the resulting molecules had improved affinity, and compound 9 showed the tightest binding according to microscale thermophoresis (MST). Mutational and competition studies confirmed that the molecule binds to the expected site. Importantly, compound 9 not only bound to pre-miR-21, it also blocked processing by the enzyme Dicer. Moreover, it showed activity in cell models consistent with inhibition of pre-miR-21.

This is a nice paper, but there are several limitations. First, compound 9 is still a fairly modest binder with lackluster ligand efficiency. Indeed, while potency can be overrated, I would love to see a fully synthetic low nanomolar RNA binder. Second, while the approach may be general, it is not necessarily easy, and it requires specialized fragments. And as we noted last year, there is no relation between crosslinking efficiency and affinity. I wish the researchers had tried linking some of the non-selected fragments to see whether these were false negatives. Indeed, given the complexity of the approach, I wonder if the researchers would have been better off simply making and testing an anchor library around compound 1, in a similar fashion as described here.
But whether or not Chem-CLIP-Frag-Map turns out to be the solution to targeting RNA, I wholeheartedly agree with the conclusion: “It may be time to describe biomolecules that are perceived to be challenging small molecule targets as ‘not yet drugged’ rather than ‘undruggable.’”

01 April 2021

Fragments from Mars

Four years ago today we highlighted work invalidating ligand efficiency on Venus. Recently though, the planet of war has been getting all the love. You've probably seen the stunning pictures from the Perseverance rover on Mars. One of its missions is to collect samples for later return to Earth. Many scientists are eagerly awaiting their allotment, among them Herbert George Wells of Bromley University.
There are those who believe that life here began out there - maybe even on Mars. According to this theory, Martian meteorites, or even Martians themselves, seeded life on Earth. If so, any organic fragments on Mars could be ancestral to all life and may generate particularly high hit rates, perhaps approaching those of the "universal fragments" we profiled here and here. And any molecules that can remain intact near the harsh surface of Mars for a few billion years may also have good metabolic stability.
Prof. Wells plans to separate and identify all the organic fragment-like molecules in his sample. While this will undoubtedly make an interesting publication, Prof. Wells is also dreaming of cosmic riches and has teamed up with Dr. Lyttle, Jr. to sell a custom collection of Martian Fragments.
Some people are concerned that a Martian sample may harbor dangerous organisms, but Prof. Wells is not worried. "Frankly, with so many viruses running around Earth these days, it's the Martians who should be afraid!"