Drp1 GTPase activity

The GTPase activity of Drp1, which is required for the latter to mediate mitochondrial fission, was examined using the colorimetric GTPase assay kit (Innova Biosciences, Cambridge, UK) according to the manufacturer’s instructions²³. Compounds were diluted in DMSO (all with a final concentration of 0.1% DMSO). Recombinant human Drp1 protein isoform-3 (final concentration 0.25 ng/µL) was diluted in assay buffer containing 50 mM Tris HCl (pH 7.5) and 2.5 mM MgCl₂, followed by incubation with compounds for 15 min at 37 °C in a 96 well plate. 0.5 mM GTP, diluted in assay buffer, was then added into the mixture followed by incubation at 37 °C for 30 min.

Upon binding to Drp1, GTP is hydrolysed into GDP and free phosphate (Pi)²⁴. Therefore, the degree of GTPase inhibition was assessed by measuring the concentration of Pi released into the assay buffer, which is detected by a malachite green-based dye. The intensity of Pi-dye complex was assessed by measuring the light absorbance at 635 nm wavelength using a microplate reader (BenchMark Plus, MS, USA). Absorbance of each experimental group was normalised to the vehicle control (DMSO).