Science

We are developing drugs that target the proteases of the mitochondrial inner membrane. These enzymes partake in quality control and stress response pathways with implications for fatal diseases, including heart failure, cancer and dementia. Mitochondria form dynamic networks of interconnected tubules, whose morphology is determined by balanced fission and fusion events. These events are intimately linked to the energy metabolism as well as quality control measures, such as mitochondrial autophagy, aka mitophagy, and apoptotic cell death signaling.

Illustration of the impact of the dynamic mitochondrial network on different cellular processes, such as ATP synthesis, autophagy and apoptosis.

Figure 1. The dynamic mitochondrial network is tied to the energy metabolic function and the integrity of the cell. Shifts in the equilibrium of mitochondrial fission and fusion can influence and at the same time reflect the denoted cellular functions. (Alavi 2019 Int J Cancer.)

Mitochondria constantly merge and divide, whereby damaged mitochondria are sorted out and recycled by mitophagy. Severely harmed cells on the other hand are recycled by apoptosis, which involves mitochondria breakdown. The processes behind the judgment call between recycling of single mitochondria and entire cells are unknown. One potential pathway may involve the mitochondrial proteins OPA1 and OMA1, more precisely cleavage of the OPA1 protein by the OMA1 protease, which is a signature event of both mitophagy and apoptosis.

Illustration of OPA1's regulation by the two mitochondrial membrane proteases OMA1 and YME1L1.

Figure 2. Context-dependent OPA1 cleavage by the i-AAA protease YME1L1 and OMA1. Healthy cells usually maintain stoichiometric OPA1 ratios through YME1L1 (a), which are completely shifted by OMA1 to the cleaved S-OPA1 isoforms in stressed cells (b).

OPA1 belongs to the dynamin-family of GTPases and can coat membranes like the other members of this family. A subset of OPA1 (dubbed ‘L-OPA1’) carries for this reason a membrane anchor. OMA1 is a membrane protease, which cleaves L-OPA1 in challenged cells, thereby removing OPA1’s anchor and releasing ‘S-OPA1’ from the inner membrane. Blocking OMA1 as well as ectopic OPA1 expression can prevent apoptosis, which suggests OPA1 contributes to apoptosis beyond its structural support of the inner membrane (Alavi & Fuhrmann 2013 Mol Neurodegen.).

Illustration depicting OMA1 with its substrates PGAM, PINK1, OPA1, and DELE1 and the functional overlap with PARL and the i-AAA protease.

Figure 3. The proteases of the inner mitochondrial membrane orchestrate complex functions. OMA1 together with PARL integrates the mitochondrial fission machinery via PGAM5 and clears misrouted PINK1 from the inner membrane. OMA1 can communicate with the integrated stress response via DELE1. Reciprocal hydrolysis of OMA1 and the i-AAA protease can adjust mitochondrial capacity and promote apoptosis by processing OPA1. (Alavi 2021 BBA – Proteins Proteom.)

OMA1 has only few other substrates besides OPA1. For example, OMA1 can cleave DELE1, which activates the integrated stress response thereby reducing protein synthesis and initiating other countermeasures, such as the expression of chaperons. OMA1 together with the rhomboid protease PARL also processes PGAM5, which synchronizes the mitochondrial fission and fusion machineries. Interestingly, OMA1 cleaves OPA1 in mammals, while PARL cleaves OPA1 in yeast and flies. This suggests OMA1 has taken over some of PARL’s function during evolution.

Illustration of the Luke-S1 reporter assay, which emits light in healthy cells but is cleaved and inactivated by OMA1 in stressed cells.

Figure 4. The Luke-S1 reporter inversely correlates with OMA1 activity. An engineered luciferase dubbed Luke-S1 creates an artificial OMA1 substrate in the mitochondrial inner membrane. Luke-S1 emits light in healthy cells (a). OMA1 cleaves Luke-S1 in stressed cells, which kills its bioluminescence (b). (Alavi 2021 ACS Chem Biol.)

712 North’s core technology builds around the proprietary Luke-S1 assays, which is a scalable, cellular OMA1 protease assay for high-throughput drug screening. Luke-S1 is a suitable reporter to screen for both OMA1 inhibitors and activators. (Alavi 2021 ACS Chem Biol.) 712 North has a growing portfolio of patents and patent applications protecting our core technology as well as novel and not so novel chemical entities emerging from our screening campaigns. A key value driver is to advance these molecules towards clinical studies.

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