Table 1.

Main features of the 3 OMA1 assays side by side.

	FRET peptide	PINK1C125G-EYFP reporter	Luke-S1 reporter
Assay type:	In vitro assay	Cellular reporter assay	Target-based cellular reporter assay
Principle:	FRET pair serves as	Fluorescent protein	Luciferase targeted to the inner
	OMA1 substrate	targeted to the inner membrane serves as OMA1 and PARL substrate	membrane serves as OMA1 substrate
Correlation with OMA1 activity:	Direct*	No	Indirect
Advantages:	+ Straightforward assay set up + Plate reader compatibility + Direct correlation of emitted signal and OMA1 activity	+ Additional readout of cell permeability and cell toxicity + Direct correlation of OMA1 inhibition and emitted signal	+ High specificity though spatial confinement of the reporter to the inner membrane + High sensitivity through enzymatic signal amplification + Additional readout of cell permeability and cell toxicity + Plate reader compatibility + Indirect correlation of emitted signal and OMA1 activity
Disadvantages:	− Requires purified and functional OMA1 protease for specificity − Potentially confounded by autofluorescence of test molecules	− Requires high content imaging system − OMA1-independent events can also generate signal − Limited to cell permeable and non-toxic compounds − Potentially confounded by autofluorescence of test molecules	− Limited to cell permeable and non-toxic compounds − Potentially confounded by luciferase modulators
Reference:	Tobacyk et al. 2019 [46]	Houston et al. 2021 [49]	Alavi 2021 [27]

^*The FRET peptide is presumably recognized by other proteases as well.