MLKL inhibitor | BI-8925
Highlights
BI-8925 is an inhibitor of the MLKL protein, belonging to the xanthine class. It is the first covalent tool compound for MLKL, with a structurally understood mode of action. It works by stabilizing the inactive state of MLKL by an essential π–π stacking interaction. The molecule is found to inhibit necroptosis in Jurkat and U937 cells with an IC50 of 541 and 271 nM, respectively.
Background information
Target Information
Mixed lineage kinase domain-like protein (MLKL) is the effector protein in the signal pathway leading to Necroptosis. MLKL is comprised of two domains. The C-terminal pseudokinase domain is connected via two brace helices to the N-terminal four helix bundle domain. The N-terminal executioner domain is locked in its inactive state by the auto-inhibitory first brace helix (α-helix 6). Upon TNF signalling in the absence of caspase activity MLKL becomes activated via the kinases RIPK1 and RIPK3 through phosphorylation in its pseudokinase domain. Upon activation the auto-inhibitory brace helix unfolds and the N-terminal executioner domain of MLKL multimerizes and integrates into the membrane, which then leads to membrane rupture and necroptosis.
Complex of BI-8925 with MLKL, as solved by X-ray crystallography (PDB code: 6ZZ1). The structure was also solved by NMR spectroscopy (PDB code: 6ZPR). Both structures show a very high level of agreement6
In vitro activity
BI-8925 displays nM activity in two different cellular necroptosis assays.
| Probe name / negative control | BI-8925 | BI-8762 |
| MW [Da] | 346.36 | 240.28 |
| Assay A (IC50) [nM]a | 541 | >100000 |
| Assay B (IC50) [nM]a | 271 | >100000 |
a see reference 6
Assay A: Stimulation of Jurkat FADD-/- cells was achieved with huTNFα and subsequent cell viability analysis was carried out with Cell Titer Glo® Luminescent Cell Viability Assay
Assay B: U937 cells were treated with the caspase inhibitor zVAD-fmk and stimulated with huTNFα; cell viability analysis was performed with Cell Titer Glo® Luminescent Cell Viability Assay.
In vitro DMPK and CMC parameters
| Probe name / negative control | BI-8925 | BI-8762 |
| logD pH2/pH11 | 1/0.5 | - |
| Solubility @ pH 6.8 [µg/ml] | 75 | 59 |
| CACO permeability @ pH 7.4 [*10-6 cm/s] | 17 | 53 |
| CACO efflux ratio | 0.7 | 0.8 |
Negative control
BI-8762 which serves as a negative control
Selectivity
| SELECTIVITY DATA AVILABLE | BI-8925 | BI-8762 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | No | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI-8925_selectivityData.xlsx
BI-8762_selectivityData.xlsx
Co-crystal structure of the BI probe compound and the target protein
The Xray crystal structure of the target without ligand is available (PDB code: 6ZVO)
The Xray crystal structure of the target in complex with BI-8925 is available (PDB code: 6ZZ1)
The NMR structure of the target without ligand is available (PDB code: 6ZLE)
The NMR structure of the target in complex with BI-8925 is available (PDB code: 6ZPR)
Summary
BI-8925 is the first covalent tool compound for the necroptosis effector protein MLKL with a structurally understood mode of action.
Supplementary data
2 D structure formats available
MLKL inhibitor | BI-8925.smiles
Negative control | BI-8762.png
References
Direct Activation of Human, MLKL by a Select Repertoire of Inositol Phosphate Metabolites.
McNamara D. E., Dovey C. M., Hale A. T., Quarato G., Grace C. R., Guibao C. D., Diep J., Nourse A., Cai C. R., Wu H., Kalathur R. C., Green D. R., York J. D., Carette J. E.
Cell Chem Biol 2019, 26, 863-877.e7.
Necrosulfonamide inhibits necroptosis by selectively targeting the mixed lineage kinase domain-like protein.
Liao D., Sun L., Liu W., He S., Wang X., Lei X.
MedChemComm 2014, 5.
Locking mixed lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.
Rübbelke M., Fiegen D., Bauer M., Binder F., Hamilton J., King J., Thamm S., Nar H., Zeeb M.
PNAS (2020) accepted.
How to cite opnMe?
When you plan a publication, please use the following acknowledgement:
BI-8925 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://opnme.com.
Fast response and process with opnMe