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.