25 October 2021

Fragments vs TIM-3

In order to thrive, cancer cells need to evade the immune system. Preventing them from doing so is the goal of cancer immunotherapy. Although it has not entirely lived up to its initial hopes, this promising approach has generated multiple new targets, such as T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), whose upregulation correlates with tumor progression. Several antibodies targeting this protein are working their way through the clinic, but small molecules may have advantages in terms of oral dosing and improved tumor penetration. The discovery of one small molecule binder is reported in a new J. Med. Chem. paper by Stephen Fesik and colleagues at Vanderbilt University.
 
As is customary for this group, the project began with a two-dimensional (1H/15N HMQC) NMR screen of 13,824 fragments, each at 0.8 mM in pools of 12. This yielded 101 hits, a respectable 0.7% hit rate, and higher than might be expected for this immunoglobulin-like protein. The hits belonged to 11 chemotypes, and 18 had dissociation constants better than 1 mM and ligand efficiencies (LE) better than 0.25 kcal mol-1 per heavy atom. All of the fragments caused similar resonance perturbations, suggesting a common binding pocket, though as specific backbone resonance assignments were not known the exact location was unclear. Compound 1 was pursued due to its (relatively) high affinity, LE, and chemical tractability.
 
Substitutions off two vectors of the molecule improved affinity, and combining these substituents led to compound 22. This molecule bound sufficiently tightly that NMR could no longer be used to measure the dissociation constant. At this point the researchers were able to solve a crystal structure of the compound bound to TIM-3, revealing that it binds to a protein loop with the tricyclic core sandwiched between two tryptophan residues. The structure also revealed a portion of the molecule that extended toward solvent, and this insight was used to construct a fluorescent probe for use in a fluorescence polarization anisotropy (FPA) competition assay to accurately measure binding of more potent molecules.
 
 
With the probe results and crystal structure in hand, the researchers continued to optimize the molecule by growing towards a couple arginine and aspartic acid residues. This led to compound 34, which again started bottoming out the FPA assay and necessitated constructing yet another fluorescent probe. Further optimization using structure-based design ultimately led to compound 38, the most potent molecule in the series. NMR experiments revealed that compound 38 causes a rigidification of the TIM-3 loop where it binds.
 
And that’s where things stand. Unfortunately no data are presented as to whether compound 38 blocks binding of TIM-3 to its biological partners. The binding site is actually somewhat distant from where natural ligands bind, suggesting that the compounds would likely need to act allosterically. Moreover, the researchers note that many of the compounds are not particularly soluble. Still, whether the compounds move forward or not, this is a nice example of finding fragments that bind to a novel target and using diverse insights to improve them by several orders of magnitude.

18 October 2021

Fragments vs the SARS-CoV-2 main protease – this time by NMR

Last week we highlighted NMR screens against RNA from SARS-CoV-2. As we noted then, much of the drug discovery action has focused on the virus’s main protease, called Mpro or 3CLp. These efforts have included two separate screens by crystallography and/or mass spectrometry. A new (open-access) paper in Angew. Chem. Int. Ed. by Benoit Deprez, Xavier Hanoulle, and collaborators at CNRS and University Lille describes an NMR screen against this same protein.
 
The main protease is 306 amino acid residues long and forms a 67.6 kDa dimer in solution. Proteins this large are challenging for protein-detected NMR, both because the number of potentially overlapping resonances increases and because of line broadening. Nonetheless, the researchers used several sophisticated NMR techniques to assign more than 60% of backbone resonances as well as quite a few main-chain and side-chain hydrogens to gain information on binding locations.
 
A library of 960 fragments was purchased from Life Chemicals and Maybridge. These were pooled in groups of five, with each fragment at 377 µM, and screened by Water-LOGSY and, for 427 fluorinated fragments, 19F line broadening and chemical shift perturbation. This exercise yielded 159 hits.
 
These hits were validated in a protein-detected 1H, 15N TROSY-HSQC experiment, which confirmed 38 fragments, giving an overall hit rate of around 4%, comparable to that seen in the crystallographic fragment screen against Mpro. Also in common with the previous screen is the fact that most of the fragments (32) bind somewhere in the active site, while a few (5) bind at the dimerization interface. Fragment hits tended to be both larger and more lipophilic than those in the overall library.
 
Earlier this year we highlighted an NMR study of non-covalent fragment hits from the crystallographic fragment screen, which found that only two of them had measurable affinities, and both were weak (KD = 1.6 mM at best). In contrast, one of the new fragments, F01, has a dissociation constant of 73 µM. With a molecular weight of 287 Da and 20 non-hydrogen atoms this is a somewhat portly fragment, but it does have a ligand efficiency of 0.3 kcal mol-1 per heavy atom. It also shows functional activity with an IC50 = 54 µM in a biochemical assay and EC50 = 150 µM in an antiviral assay. The researchers further characterized their molecule crystallographically, which confirmed that it binds to the active site; it makes three hydrogen bond interactions and multiple hydrophobic contacts with the protein.
 
Although crystallography has been receiving increasing attention among fragment-screening techniques, this paper is a reminder than NMR remains highly relevant, even for larger proteins that crystallize easily. And at the end of the day, it’s not how you screen but what you find and what you do next. Hopefully folks will follow up on F01. While PF-07321332 is making rapid progress in the clinic against this enzyme and the COVID Moonshot effort is moving molecules into animal studies, HIV has taught us that we’ll need multiple small molecule drugs.

11 October 2021

Fragments vs SARS-CoV-2 RNA

The first mention of SARS-CoV-2 on Practical Fragments in early March of last year highlighted a crystallographic fragment screen against the main viral protease. As discussed last week this effort has now led to compounds with nanomolar activity in cells. We’ve also highlighted a separate crystallographic screen against this target as well as a screen against the Nsp3 macrodomain. But proteins are not the only potential viral targets.
 
A recent (open access) paper in Angew. Chem. Int. Ed. by Harald Schwalbe and a large group of collaborators mostly at Johann Wolfgang Goethe-University focuses not on proteins but on RNA. Harald also presented this work at Discovery on Target last week, where he noted that the effort is part of the COVID19-NMR project, a collaboration of 240 people in 18 countries.
 
The researchers investigated 15 RNA regulatory elements that are conserved between SARS-CoV-2 and SARS-CoV, ranging from 29-90 nucleotides, as well as 5 larger multielement RNAs (118-472 nucleotides). These were screened against the DSI-poised library (discussed here): 768 fragments designed for rapid follow-up chemistry.
 
Three different ligand-detected NMR methods were used for screening: chemical shift perturbation (CSP) or line-broadening, waterLOGSY, and T2-relaxation. Fragments were screened at 200 µM in pools of 12 against 10 µM RNA. Compounds that hit in at least two assays were investigated individually.
 
In total 40 fragments bound to one or more of the 15 shorter RNAs, and an additional 29 fragments bound to the five longer RNAs. Between 5 and 49 hits were found for all but two of the RNAs. Selectivity varied: some fragments bound to just one RNA while one fragment bound to 18 of 20.
 
Given the negatively-charged phosphate backbone of RNA, it is not surprising that many of the fragment hits are positively charged at physiological pH. Nearly one-third of the 40 hits against the shorter RNAs contain a basic amine; pyrimidine and benzimidazole moieties are enriched, and not one of the hits contain a carboxylic acid. All the hits have at least one aromatic ring and most have two or three, perhaps suggesting intercalation. Moreover, as seen in a previous ambitious RNA screen from the same group, hits tend to have fewer sp3-carbons than non-hits.
 
The highest affinity fragment had a dissociation constant of just 64 µM but an impressive ligand efficiency of 0.38 kcal/mol/atom. A search of commercial analogs yielded a compound with low micromolar affinity against two RNA targets. In his presentation Harald noted that this series has since been optimized to a 200 nM binder.
 
This paper is a tour de force, but as I have noted, there remains a dearth of high-affinity specific RNA binders. The researchers also note another potential problem: viral RNA accounts for roughly two-thirds of total RNA in cells infected with SARS-CoV-2. Would this necessitate high concentrations of drug for effective antiviral activity?
 
Whether or not the work leads to drugs, it should further basic research. Laudably, structures of all the hits and non-hits are provided in the paper, and the extensive supporting information provides more details. Hopefully we will soon see whether fragments poised for ready elaboration really will enable rapid progress against RNA.

04 October 2021

Nineteenth Annual Discovery on Target Meeting

Cambridge Healthtech Institute held its annual Discovery on Target meeting last week. For the first time the event was hybrid, with slightly fewer than half the attendees in Boston and the rest online, and I’m happy to report that it was quite successful. In-person attendees were required to show proof of vaccination against COVID-19, and masks and social distancing guidelines were observed. Ten of the individual tracks were hybrid, while four were virtual only. However, even in these cases it was valuable to attend in person; after one vendor presentation I immediately went from my hotel room to the exhibit hall to find out more.
 
For many of us this was the first in-person conference we had attended in nearly two years, and the return to some semblance of normalcy. At the same time, the fact that in-person talks were broadcast opened the conference to people unable to travel. One of the most active Q&A participants in one track was in Singapore, despite the 12 hour time difference.
 
Another nice feature of the virtual or hybrid model is reduction in FOMO; if you find it difficult to choose between the seven concurrent talks you can watch some later. But, as our 2020 poll showed, speakers may be less forthcoming with newer, more speculative results in a recorded format.
 
With the heavy focus on biology there seemed to be fewer “conventional” fragment stories, though Lars Neumann (Proteros) did discuss the identification and optimization of a kinase inhibitor that does not interact with the hinge region. Novel targets were represented in work from Harald Schwalbe (Johann Wolfgang Goethe University), who described fragment screens against RNA; I’ll post more on this later this month.
 
We’ve previously discussed the COVID Moonshot Consortium to rapidly discover drugs for SARS-CoV-2. Annette von Delft (Oxford University) provided an update, noting that fragments from a crystallographic screen have been advanced to compounds with mid-nanomolar biochemical and cellular activity. DMPK properties are reasonable, though this is an area of continued optimization. Annette mentioned the goal is to enter clinical development in 2023. Progress has been accelerated by the crowd-sourced nature of the initiative, with nearly 40 groups and 150 individuals working together. She also noted that many of the molecules are active against other coronaviruses.
 
The main series being advanced by the COVID Moonshot are noncovalent inhibitors of the SARS-CoV-2 main protease MPro. However, covalent molecules against this target are also moving forward. Matthew Reese described Pfizer’s oral PF-07321332, which is currently in several phase 3 trials. The program began on March 16 of last year and the clinical compound was first synthesized just four months later. Clinical trials began in February of this year, a mere 11 months after the program began. This is astonishingly rapid, though the researchers did benefit from previous work on SARS-CoV-1 and even earlier work from the 1990s on rhinovirus inhibitors. It is worth re-reading Glyn Williams’ 2020 discussion of HIV protease inhibitors for more historical context and insights.
 
Although PF-07321332 did not come from FBLD, fragments capable of forming covalent bonds were well represented. We’ve previously discussed fully-functionalized fragments (FFFs, or PhABits), which in addition to having a photoreactive group also contain an alkyne handle so that any target they bind can be captured and identified. Aarti Kawatkar and Jenna Bradley described using these at AstraZeneca to identify new targets. They’ve constructed a library of just under 500 FFFs and are using these to do phenotypic screening, particularly in hard-to-get cells such as primary tissue samples. They are also making the FFF library available through their open innovation initiative.
 
Fully functionalized fragments are just one flavor of covalent fragments. Indeed, unlike the light-activated warhead of FFFs, most covalent fragments have a moiety that reacts selectively with amino acid residues such as cysteines. Steve Gygi (Harvard) and Dan Nomura (UC Berkeley) both described covalent screening in cells to identify starting points against challenging targets. The approach is also gaining traction in industry; Heather Murrey described how Scorpion is using covalent fragments, and noted that Vividion (mentioned here) was recently acquired by Bayer for up to $2 billion.
 
A prominent recent success story from covalent fragments is sotorasib, which was approved earlier this year to treat certain non-small cell lung cancer patients whose tumors carry the G12C mutant form of KRAS. Sotorasib binds to a mostly cryptic pocket, and the protein itself has low ligandability. To improve the odds of finding new fragments, Mela Mulvihill described how she and her colleagues at Genentech have developed antibodies that stabilize the so-called Switch II loop in an “open” conformation more accessible to small molecules. An SPR-based fragment screen in the presence of the antibody led to more than twice as many hits, many of which could bind more tightly than without the antibody. Darryl McConnell (Boehringer-Ingelheim) also described using fragment-based methods to pursue KRAS, including mutants other than G12C.
 
In addition to inhibitors, Darryl also described bifunctional molecules that selectively cause degradation of KRAS by bringing it to the proteasome via E3 ligases. In his opinion PROTACs are “the best thing since sliced bread.” PROTACs and targeted protein degradation were in fact the subject of two tracks that spread across all three days of the conference, and were also covered in a pre-conference short course taught by Stewart Fisher (C4 Therapeutics) and Alexander Statsyuk (University of Houston). Here too fragments are playing an increasing role; in a second talk Dan Nomura described how he has been using chemoproteomic fragment approaches to identify ligands for E3 ligases.
 
The recent excitement around PROTACs is probably justified, but as our post last week noted, new technologies are not necessarily fast or inevitable. PROTACs were first described in 2001; Adam Gilbert (Pfizer) puckishly described them as a “20-year overnight success story.” But by the end of this year there will be roughly a dozen PROTACs in the clinic, with more likely to join them soon.
 
I’ll end on this positive note, but welcome your thoughts on science or experience with hybrid conferences. I look forward to seeing you at one in the near future!