Apoptolidin

Apoptolidin : Inhibitor of ATP5F1B

Structure

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Information

ATP5F1B

Inhibitor

Probe Availability:

MedChemExpress

In Vitro Validations

Uniprot ID: P06576

Target Class: Enzyme

Target SubClass: --

Potency: IC50

Potency Value: 100 nM

Potency Assay: Enzyme-coupled ATPase assay. The assay buffer consisted of 60 mM Tris–acetate pH 7.8, 1 mM MgCl2, 2.5 mM phosphoenolpyruvate and 1 mM KCN. The working solution was prepared at 2× for a final concertation of 0.4 mM NADH; 3 U ml−1 of pyruvate kinase + 4.5 U ml−1 lactate dehydrogenase (Sigma P0294); ± 10 μM FCCP. Unless otherwise specified, ATP was added as a 4× solution at 200 μM. Absorbance was monitored at 340 nm using a SpectraMax Plus 384 plate reader (Molecular Devices) set at 25 °C using Softmax Pro v.4.3 acquisition software. Fifty microliters of enzyme was added to 100 μl of working solution and monitored for 2 min, 50 μl of ATP was then added for a final volume of 200 μl and absorbance was monitored over 10 min.

PDB ID for probe-target interaction (3D structure): 7MD3

Target aliases:

ATP synthase subunit beta, mitochondrial, ATPSB, A ...

DOI Reference: 10.1038/s41589-021-00900-9

In Cell Validations

In Vivo Data

No in Vivo Validations

Off-Target Selectivity Assesments

Probe Selectivity in Cell:

Apoptolidin A is a selective inhibitor of the F1 subcomplex of ATP synthase. Selectivity was assessed by generating diazirine-alkyne photoaffinity probes to identify binding partners which revealed ATP5B as the sole specific [competively displaced by the parent compound] target. On target activity was established by deep mutational scanning to identify resistance mutations. CRISPR-Cas9 knock-in of identified resistance mutation eliminated both binding and cytotoxicity, establishing that the ATP synthase binding was required for cytotoxicity and providing a model for investigating potential off-target effects.

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(last updated: 1 Feb 2022 )