Identification of a covalent BFL-1 inhibitor

Evading apoptosis is one of hallmarks of cancer and highly associated with chemotherapy resistance. The BCL-2 family of proteins govern both intrinsic apoptosis and are frequently dysregulated in various cancers. So far, small molecules have been developed to effectively block BCL-2, BCL-XL, and MCL-1. A selective BCL-2 inhibitor, Venetoclax, is the first FDA approved for the treatment of CLL, and other BCL-XL and MCL-1 inhibitors are currently in clinical trials. Among six anti-apoptotic BCL-2 family proteins, BFL-1 is considered undruggable, being the least studied, however, a growing body of evidence suggests its value as a therapeutic target acting as resistant factors to other BCL-2 family proteins inhibitors in lymphoma and to MAPK inhibitors in melanoma. Thus, selective BFL-1 inhibitors held clinical promise. Recently, a small molecular covalent BFL-1 inhibitor targeting cysteine has been described in a paper in “Cell Chemical Biology”.

The unique cysteine55 in the BH3 binding groove of BFL-1 provides an opportunity to develop selective covalent inhibitors. Using a disulfide tethering screen, the group identified 31 hits capable of disulfide formation with BFL-1 cysteine55 (Figure 1A, B). The subsequent fluorescence polarization (FP) secondary screen revealed hit 4E14 as the most effective competitor (Figure 1C). And intact mass spectrometry detected complete conjugation of 4E14 to BFL-1DC (M + 304) (Figure 1D). Moreover, 4E14 conjugated with neither cysteine 55 mutant (BFL-1DC C55S) nor other BCL-2 family proteins BCL-xL and MCL-1 (Figure 1E, 1F). The above evidence demonstrated 4E14 selectively modified cysteine55 of BFL-1 protein.

Figure 1: A Disulfide Tethering Screen Identifies Covalent BFL-1 Inhibitor Molecules that Disrupt BH3-Binding Activity

Then they performed hydrogen/deuterium exchange mass spectrometry (HXMS) analyses to assess the impact of disulfide bond formation between 4E14 and cysteine55 on the structural dynamics of BFL-1DC C4S/C19S. The result realved that 4E14 strongly protected the distal region of a2 and proximal portion of a3 from deuterium exchange, which comprise the upper portion of the canonical groove including cysteine 55 (Figure 2).

Figure 2. Conformational Consequences of 4E14 Derivatization of BFL-1

At last, they evaluated the effect of 4E14 and its analogs on mitochondrial cytochrome c release to determine if the covalent interaction with BFL-1 had functional consequences. They purified BAX/BAK-deficient mitochondria and treated them with monomeric full-length BAX, tBID, or the BAX/tBID combination in the presence or absence of BFL-1DC C4S/C19S. Treatment with monomeric BAX or tBID alone had little to no effect on the mitochondria, however, the combination induced robust cytochrome c release, which was significantly suppressed by the addition of BFL-1. 4E14 and its analogues effectively blocked the anti-apoptotic functionality of BFL-1. Consistent with previous FP assay, 4E14 exhibited the most potent inhibitory activity (Figure 3).

Figure 3. Covalent Targeting by 4E14 Blocks BFL-1 Suppression of BAX-Mediated Mitochondrial Apoptosis

Herein, this work explored whether incorporating a covalent reaction with the unique cysteine55 of the BFL-1 groove could enable smaller molecules to effectively block anti-apoptotic activity. They identified a series of small molecules that effectively conjugated with cysteine55 of BFL-1 groove as well as blocked BFL-1 activity via a disulfide tethering screen. The inspired dataset suggests that developing a cysteine55 targeting covalent inhibitor based on a matured non-covalent inhibitor could yield potent and selective small molecular inhibitors of BFL-1.

Reference:

Harvey EP, et al. Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering. Cell Chem Biol. 2020 Apr 21. pii: S2451-9456(20)30118-5.