Recent advances in, and challenges of, designing OMA1 drug screens

Abstract

The proteases of the mitochondrial inner membrane are challenging yet highly desirable drug targets for complex, multifactorial diseases prevalent mainly in the elderly. Among them, OMA1 with its substrates OPA1 and DELE1 safeguards mitochondrial homeostasis at the intersection of energy metabolism and apoptosis, which may have relevance for neurodegeneration, malignancy and heart failure, among other diseases. Little is known about OMA1. Its structure has not been solved and we are just beginning to understand the enzyme's context-dependent regulation. OMA1 appears dormant under physiological conditions as judged by OPA1's processing pattern. The protease is rapidly activated, however, when cells experience stress or undergo apoptosis. Intriguingly, genetic OMA1 ablation can delay or even prevent apoptosis in animal models for diseases that can be broadly categorized as ischemia-reperfusion related disorders. Three groups have reported their efforts implementing OMA1 drug screens. This article reviews some of the technical challenges encountered in these assays and highlights what can be learned for future screening campaigns, and about the OMA1 protease more broadly. OMA1 does not exists in a vacuum and potent OMA1 inhibitors are needed to tease apart OMA1's intricate interactions with the other mitochondrial proteases and enzymes. Furthermore, OMA1 inhibitors hold the promise of becoming a new class of cytoprotective medicines for disorders influenced by dysfunctional mitochondria, such as heart failure or Alzheimer's Disease.

Keywords: AZD1080 (PubChem CID: 135564570); CCCP (PubChem CID: 2603); Cancer; Ceritinib (PubChem CID: 57379345); Drug discovery; MG132 (PubChem CID: 462382); Membrane proteases; Mitochondria; Neurodegeneration; Protease inhibitors; SB216763 (PubChem CID: 176158); Sorafenib (PubChem CID: 216239); Tamoxifen (PubChem CID: 2733526); Valinomycin (PubChem CID: 3000706).

Figure 1:. OMA1, OPA1 and Luke-S1 reporter protein turnover in Western blots.

OMA1 is activated within minutes upon exposure of HEK293T cells to 2 μM CCCP (A) or 100 nM Valinomycin (B) as evident by cleavage of the large OPA1 isoforms. OMA1 hydrolysis by contrast follows a slower kinetics. PHB2 is not an OMA1 substrate and remained stable even after 9 h of CCCP or valinomycin treatment. Luke-S1 reporter protein levels were significantly higher in HEK293T reporter cells cultured for 24 h in serum-free medium with 100 μM sorbitol than in cells cultured with 100 μM galactose (C). This effect was less pronounced after 48 h (D). OMA1 and OPA1 protein levels were about the same after 24 h, but OMA1 protein levels were significantly higher in cells maintained for 48 h in galactose medium. Panels E & F show the densitometric quantification of Western blots; n = 2 independent replicates; 2-way ANOVA: p<0.05 (*).

Figure 2:. Influence of the energy-metabolic state on the Luke-S1 OMA1 reporter.

Luke-S1 reporter cells exhibit a sorbitol-dose dependent increase in bioluminescence compared to Luke cells, which express just the native luciferase (A). No signal changes were observed in cells cultured for 24 h with galactose (B). The GSK3 inhibitor AZD1080 mitigated sorbitol’s effect (C) and had no other effects on cells cultured with galactose (D). All cells were cultured for 24 h in 96-well plates in serum-free RPMI 1640 medium without glucose, supplemented with sorbitol or galactose before luciferase substrate was added and bioluminescence measured.

Figure 3:. OMA1 substrate levels and OMA1 protein levels can modulate the protease’s response to CCCP and valinomycin.

Luke-S1 reporter cells showed a higher CCCP (A) and valinomycin (B) sensitivity in OPA1 Western blots than native HEK293T cells. OPA1 knockdown lowered valinomycin’s EC₅₀ in luciferase assays. OPA1 protein levels were significantly reduced in Luke-S1 cells treated for 36 h with OPA1 siRNA (C); these cells showed a higher valinomycin sensitivity in luciferase assays (D). Ectopic OMA1-Flag expression reduced the baseline fluorescence, which diminished the valinomycin response but did not change valinomycin’s EC₅₀. OMA1 protein levels were significantly increased and more than doubled in Luke-S1 cells 24 h post transfection with an OMA1-Flag plasmid (E). The OMA1-Flag expressing reporter cells showed about 40% reduced bioluminescence at low valinomycin concentrations but still responded to higher valinomycin concentrations with the same EC₅₀ as non-transfected cells (F). All cells were treated for 30 minutes with CCCP or valinomycin in serum-free DMEM/F12 media before the analysis. Panels C & E show the densitometric quantification of Western blots; n = 3 independent replicates; 2-way ANOVA: p<0.001 (***).