Covalent fragments vs BFL1: a selective chemical probe

Last week we highlighted the construction of a covalent fragment library at AstraZeneca. The first fruits of this library have recently been published as a pair of papers.

 

The protein BFL1 (or Bfl-1) is a member of the BCL2 family and blocks apoptosis by binding to pro-apoptotic proteins such as BIM, BID, and Noxa. Blocking these types of protein-protein interactions should increase apoptosis in cancer cells. Indeed, BCL2 itself is the target of the approved fragment-derived drug venetoclax, which took heroic measures to discover.

 

Finding noncovalent inhibitors of BFL1 was also expected to be difficult, but fortunately the protein contains a unique cysteine (C55) in the protein-protein binding site, facilitating both covalent attachment and selectivity. As we mentioned last week, the protein was screened against the emerging AstraZeneca covalent library, resulting in the discovery of several hits, including compound 8. Its optimization is described by Simon Lucas and colleagues in the first J. Med. Chem. paper.

Compound 8 showed promising k_inact/K_I for BFL1 as well as micromolar inhibition in a TR-FRET assay using a BIM-derived peptide. Crystallography was initially unsuccessful, but synthesis of close analogs led to compound 13, which is slightly more potent and could be co-crystallized with the protein. The structure confirmed covalent binding and revealed that one of the phenyl rings binds in a lipophilic pocket created by movement of a phenylalanine side chain.

To explore more regions of the protein-protein binding site, the researchers performed a high-concentration crystallographic screen with 384 non-covalent fragments. This yielded nine hits, four of which made hydrogen bonds with a glutamic acid side chain (E78) that had previously been targeted by others. To try to engage with this residue, the researchers modeled and synthesized a series of amine-containing molecules. Happily, one of the highest priority compounds gave a ten-fold boost in potency. Adding a methyl to the benzylic position and tweaking substituents around one of the phenyl rings ultimately led to compound (R,R,S)-26, the best molecule in this paper.

 

Because C55 is unique to BFL1, the hope was that compounds would be selective against other BCL2 family members, and indeed (R,R,S)-26 showed no activity against BCL-xl, BCL2, or MCL1. In vitro ADME parameters were encouraging, and the molecule also showed moderate bioavailability in mice. (R,R,S)-26 showed some cellular activity, though a mass-spectrometry assay showed only ~50% target engagement in cells after treatment at 10 µM for five hours.

 

The second J. Med. Chem. paper, by Adeline Palisse and colleagues, describes further optimization. Structure-based design was supported by “multiple X-ray cocrystal structures,” and as in the first paper the researchers consistently measured the half-life of new molecules against the cellularly abundant thiol glutathione to ensure they were not simply optimizing non-specific reactivity. The paper is an excellent blow-by-blow account of some of the challenges of medicinal chemistry: improving activity at the expense of stability or permeability, for example. The most potent compound has k_inact/K_I = 120,000 M^-1s^-1, but the hepatocyte stability data suggested it would be rapidly cleared.

 

In the end, compound 20 was chosen as the best overall molecule, with a k_inact/K_I comparable to that of the approved drug sotorasib. As with (R,R,S)-26, it showed no activity against BCL-xl, BCL2, or MCL1, and it was also clean against a panel of 48 kinases and fairly clean against a panel of other potential off-target proteins.

 

Among the several BCL2 family members, the protein MCL1 can also bind to BIM, thereby blunting the effects of inhibiting BFL1. Thus, the researchers performed cell assays in the presence of the MCL1 inhibitor AZD5991, whose discovery we wrote about here. In the presence of 0.5 µM AZD5991, compound 20 had an EC₅₀ = 350 nM in a cell viability assay and also activated caspase 3, as expected in apoptosis. A similar effect is also seen in combination with venetoclax.

 

Pharmacokinetic studies in mice revealed that compound 20 is 55% orally bioavailable, and this combined with the other properties suggest this molecule will be a useful chemical probe for exploring the biology of BIM.