A library of covalent fragments vs a library of kinases

Protein kinases have proven to be a fruitful class of targets, as evidenced by more than 80 FDA-approved drugs, five of which came from fragments. Because all protein kinases bind ATP, selectively inhibiting just one of the more than 500 family members can be challenging. This is a bit easier for the 215 protein kinases that contain a cysteine within the ATP-binding pocket capable of reacting with covalent ligands. In a recent (open access) Angew. Chem. Int. Ed. paper, Matthias Gehringer, Stefan Knapp, and collaborators at Johann Wolfgang Goethe-University and Eberhard Karls University Tübingen provide such starting points for dozens of kinases.

 

The researchers built a small library of 47 fragments consisting of six classic hinge-binding moieties such as pyrazole and azaindole coupled through nine aryl linkers at varying positions to an electrophilic acrylamide warhead. Although most of the compounds are rule-of-three compliant, the researchers note they “reside at the upper end of fragments space,” similar to what we discussed last week. Chemical reactivity towards the abundant cellular thiol glutathione was tested and found to be lower than the approved drug afatinib, meaning the fragments might be good starting points for optimization.

 

Each member of the fragment library was screened against 47 different protein kinases chosen to present cysteine residues at a variety of positions around the ATP binding site. Two types of screens were conducted: intact protein mass spectrometry to assess covalent binding and differential scanning fluorimetry (DSF) to assess protein stabilization. Screens were run at fairly high concentrations, 50 µM protein and 100 µM fragment.

 

The results, plotted as a two-dimensional figure with kinases on one axis and compounds on the other, provide a wealth of information. Some compounds hit multiple kinases while others hit few or none. Similarly, some kinases are hit by multiple compounds while others are recalcitrant.

 

A couple more general observations emerged. First, there was little if any correlation between the inherent reactivity of a given fragment (as assessed by reactivity with glutathione) and the number of kinases hit, suggesting that covalent modification was driven by specific interactions rather than nonspecific reactivity. Second, there was also no clear correlation between the ability of a fragment to stabilize a given kinase and the ability of the same fragment to covalently bind to that kinase. This latter observation isn’t surprising, since one could imagine a fragment binding noncovalently to a kinase and stabilizing it without forming a covalent bond.

 

Most proteins contain multiple cysteine residues, and the researchers confirmed that the fragments were covalently modifying the cysteines in the ATP-binding pocket using mutagenesis, trypsin digestion, or, for MAP2K6, RIOK2, MELK, and ULK1, crystallography. The crystal structures were particularly informative in showing hydrogen bond interactions between the covalently-bound fragments and the hinge region.

 

As we’ve noted, the best metric for characterizing irreversible covalent inhibitors is k_inact/K_I, and the researchers determined these for covalent inhibitors of PLK1, PLK3, RIOK2, CHEK2, and CSNK1G2. The values ranged from 2 to 8 M^-1s^-1, comparable to other early covalent fragments.

 

This is a lovely, systematic paper that is in some ways an irreversible complement to a study we wrote about in 2013 focused on reversible covalent kinase inhibitors. The fact that hit rates are relatively high likely reflects the fact that all the fragments contain privileged hinge-binding pharmacophores.

 

Perhaps most importantly, all the data are available in the supporting information. If you’re interested in pursuing any of these 47 kinases, you may find good starting points here.

Do covalent fragments need to be larger?

A few months ago we highlighted work out of AstraZeneca detailing how to build a covalent fragment library. One of the design features was including larger molecules beyond the traditional rule of three (Ro3) criteria. A new open-access paper in J. Med. Chem. by György Keserű and collaborators at the HUN-REN- Research Centre for Natural Sciences and the Weizmann Institute of Science explores “size-dependent target engagement of covalent probes.”

 

The paper starts with a theoretical discussion of covalent inhibitors, focusing on the classic two-step mechanism in which binding of a ligand to a protein is followed by covalent bond formation. These steps are characterized by the inhibition constant (K_I) and the inactivation rate constant (k_inact). The most appropriate way to assess an irreversible covalent inhibitor is by the ratio k_inact/K_I, as we discussed last year.

 

A two-step mechanism is not the only possibility: the researchers also consider a three-step model in which binding of the ligand is followed by a second step, deprotonation of the amino acid nucleophile, before the final bond-forming step.

 

Fragments typically have lower affinities than lead-size or drug-size molecules, and thus k_inact will usually need to be higher for smaller molecules in order to see significant protein labeling. Simulations in which K_I is held constant show that at the high micromolar affinities often seen for fragments, protein modification requires either long incubation times or high reactivities. In addition to these simulations, the researchers also reanalyze publications we’ve previously covered such as this and this to argue that “reactivity contributes to labeling when the effects of other factors cancel out.”

 

Next, the researchers examine the kinase BTK and the oncology target KRAS, both of which have been successfully drugged with covalent molecules, ibrutinib and adagrasib, respectively. They trimmed back these molecules to smaller lead-like and fragment-like molecules and found that while some lead-sized molecules could still label the proteins, this was not the case for the fragment-sized molecules. From this they conclude that “fragment-sized covalent agents do not offer smooth optimization and are not ideal starting points.”

 

Two examples do not a trend make, but the researchers point to other examples in the literature. In 2020 we noted the larger size of covalent KRAS hits, and Vividion’s WRN inhibitor also started from a molecule with a molecular weight of 337 Da, while GSK’s starting point weighs in at 312 Da. The AstraZeneca library we mentioned at the start of this post yielded a hit against BFL1 that also just missed the Ro3 cutoff, coming in at 302 Da.

 

That said, there are counterexamples. Just last month we highlighted a covalent fragment hit that fits comfortably within the rule of three. Fragment-sized covalent hits can be found, but don’t expect them to be common. The alternative approach, screening lead-like compounds, will also likely require screening more compounds due to lower coverage of chemical space. Either way, libraries containing more molecules are likely to be beneficial for finding covalent starting points.

How to build a covalent fragment library

Covalent fragment-based lead discovery is becoming ever more popular, driven by success against difficult targets such as KRAS^G12C. These efforts require the design of new libraries, and in a recent J. Med. Chem. paper Simon Lucas and colleagues at AstraZeneca describe their design philosophy. (Co-author Henry Blackwell presented some of this work at the CHI FBLD meeting earlier this year.)

 

AstraZeneca has taken great care in building their fragment libraries; we discussed the revamp of their general fragment library as well as a “low HBD” (hydrogen bond donor) library here and here. For their covalent library, they considered several design features. First, given that any warhead will add molecular weight (four non-hydrogen atoms and a hydrogen-bond acceptor for an acrylamide), larger molecules are necessary, which requires relaxing the rule of three. Indeed, the researchers refer to their library as “lead-like.”

 

Because larger fragments are more complex, more are needed to explore chemical space. The researchers have built their library to 12,000 compounds, larger than the typical respondent from our poll last year. They have also chosen compounds to be maximally diverse rather than including near neighbors.

 

Attractive covalent hits make specific interactions with a protein target; warheads that are too “hot” can react non-specifically, as is the case with certain PAINS. Thus, the researchers chose molecules having moderate reactivity with the biologically relevant nucleophile glutathione (GSH).

 

The design principles are summarized as:

• Molecular weight 250-400 Da
• cLogD 0-4
• GSH t_1/2 > 100 minutes
• Propensity for molecular interactions (such as hydrogen bond donors and acceptors)
• Diversity
• No diastereomeric mixtures (racemates are OK)
• Synthetically tractable
• Purity > 85% (and stable)
  

 

These criteria were used to select ~700 historical compounds from within AstraZeneca’s collection. Next, the researchers chose amines from their internal collection and capped these with an acrylamide moiety, leading to an additional 1200 molecules. They then turned to custom synthesis of scaffolds that were under-represented, commercial compounds, and covalent warheads besides acrylamides, such as cyclic sulfones. The final library consists of 88% acrylamides. Molecular weights range from 150 to 420 Da, and compounds contain 1-6 HBAs, 0-3 HBDs, and 1-3 rings.

 

The paper briefly describes a screen against Bfl-1 (or BFL1), a difficult oncology target we wrote about earlier this year. The protein contains a cysteine residue in the biologically important BH3 binding site, and previous research by others had identified covalent binders.

 

The AstraZeneca researchers tested Bfl-1 against an early version of the library having just 1400 compounds, which were incubated at 20 or 200 µM for 24 hours at 4 °C before analyzing by intact protein mass spectrometry. Hits were defined as giving >50% single labeling and that could be competed with a peptide derived from the binding partner BIM. Six hits are shown in the paper, with k_inact/K_I values ranging from 0.7 to 9.5 M^-1s^-1, comparable to some of the early KRAS^G12C hits. Further development of these molecules is described in a pair of papers that will be the subject of my next post.

 

Including Bfl-1, the library has been screened against 15 targets using mass spectrometry, typically yielding 1-2% hit rates defined as at least 20% labeling of a single site. Given this record of success, if you’re contemplating building a covalent library, this paper is well worth studying.

Poll results: fragment libraries in 2023

Our latest poll on fragment libraries suggests the field is settling into some standard practices. The poll ran from April 9 through May 26. Of the 59 participants, all but one answered all the questions (there was one skip for the last question). This is slightly down from previous years; perhaps people are sick of internet polls? We also don’t know how many organizations the respondents represent; it is possible several people voted from one company or university, which might skew the results. Nonetheless, we think this survey gives a reasonable snapshot of how people construct and maintain fragment libraries.

 

Our first question asked about library size, and the results are similar to when we last asked this question in 2018, with the average library having between 1001 and 2000 fragments.

 

 

Next, we asked about the size of fragments themselves, specifically the minimum and maximum number of non-hydrogen atoms allowed in a fragment. The minimum hasn’t really changed from 2018, averaging 7-8 heavy atoms. However, the fraction of respondents who include the tiniest fragments has doubled (albeit from a low number), perhaps due to increasing interest in MiniFrags and MicroFrags.

 

Unlike in 2018, the maximum size of fragments seems to be bimodal, with some folks drawing the line at 15-16 heavy atoms (consistent with this analysis from Astex) while others allow larger fragments. It will be interesting to see whether this bifurcation represents a true shift, though even fragments with 22 heavy atoms are likely to be under 300 Da, consistent with the rule of three, which is twenty years old this year.

 

 

We then asked about the presence of chiral molecules. There was little change from 2017, with most respondents stating that they have racemic compounds in their library, though there was a slight increase in the number of respondents excluding chiral fragments.

 

 

A new question for this poll asked whether synthetic tractability was considered at the outset of library design. This was a consideration for 85% of people who took the poll; more than a third said they considered progressability for every fragment in the library.

 

 

 

 

 

 

 

 

 

Finally, we asked about library storage conditions. As was the case when we asked this question nine years ago, more than two-fifths of respondents said they store their library at -20 ˚C. However, the fraction of respondents who store their library at room temperature dropped, while those who store their libraries at -80 ˚C increased.

 

 

 

 

 

Although some changes are noticeable over the years, it seems that best practices have been established and widely adopted in fragment library design. What do you think – does anything surprise you?