Mitochondrial proteases as drug target

Introduction

 

Mitochondria are essential organelles present in most eukaryotic cells and typically form discrete structures or elaborate tubular networks in the cytoplasm (1). They were first cytologially characterized as bioblasts by Richard Altmann in 1894. Subsequently, they were renamed into mitochondria by Carl Brenda in 1898. Insight into their structure was provided by the first detailed electron microscopy pictures of these organelles in 1952 (2). It has now been well established that mitochondria fulfill a crucial role in a plethora of biological processes (figure 1 adapted from 3). For example, mitochondria are the main source of cellular ATP production, while they also serve as an important cellular storage for calcium, a key signaling molecule. In this way, mitochondria control calcium levels and thereby regulate calcium-dependent signaling pathways. Additionally, mitochondria participate in maintaining redox homeostasis through their production of reactive oxygen species. These drive crucial cellular processes such as differentiation and proliferation. Furthermore, mitochondria are key regulators of apoptosis or programmed cell death (1). Based on their crucial metabolic role, it is not surprising that mitochondrial dysfunction is closely associated with ageing and different neurodegenerative diseases (4). Moreover, accumulating evidence shows that mitochondria are important mediators of tumorigenesis owing to their ability to influence cancer initiation, growth, survival and metastasis (5). It is commonly accepted that mitochondria originate from a bacterium that was taken up by a primordial eukaryotic cell about 2 billion years ago. During their subsequent evolution, mitochondria retained two phospholipid bilayers that segregate two aqueous compartments – the intermembrane space and matrix (1). Although the vast majority of the mitochondrial genes was transferred to the nucleus, a small genome is still present in most mitochondria, encoding 13 proteins in humans (4). Consequently, most mitochondrial proteins are nuclear-encoded and imported into the organelle following their synthesis in the cytoplasm. Thus, the mitochondrial proteome is of dual genetic origin and comprises about 1500 different proteins in humans (4). Maintaining a functional proteome is of paramount importance to ensure mitochondrial activity. To this end, mitochondria are equipped with molecular chaperones and proteases that jointly monitor the quality of the proteome and degrade misfolded, unassembled or otherwise damaged proteins (6). Human mitochondria probably contain about 25 different proteases distributed over the different mitochondrial compartments (7). These include ClpP, a conserved serine protease with homologs in bacteria and chloroplasts, that is responsible for the removal of non-functional proteins in the matrix such as enzymes of the electron transport chain (7). To this end, ClpP cooperates with ClpX, a molecular chaperone of the AAA+ superfamily. Structurally, ClpP is assembled into a cylindrical-shaped structure made up of two rings comprising seven subunits each with a central hollow chamber that contains the proteolytic sites. ClpX is located at both ends of the ClpP cylinder and is probably required for substrate recognition, unfolding and translocation into the ClpP proteolytic chamber as has been established for its bacterial counterpart (8). Interestingly, disruption of ClpP affects the infectivity and virulence of several bacterial pathogens, while in humans ClpP seems to play an important role in different tumors (9), pointing towards ClpP as potential drug target. Therefore, small molecule drugs were designed that inhibit or hyperactivate bacterial ClpP and display potent cytotoxic activity against human bacterial pathogens (9). Likewise, small molecule hyperactivators of human ClpP have been designed exhibiting strong anti-tumor activity with successful results in clinical trials (10). Recently, the first detailed structures of human ClpP in complex with hyperactivators were reported, providing important insight into their mechanism (11). Here, I will discuss these structures as well as the mode of action of ClpP hyperactivators.  

ClpP as potential drug target

In bacteria, ClpP and Lon proteases are a key component of the cellular quality control system that constantly surveys the proteome, thereby removing damaged and misfolded proteins. These are subsequently replaced by newly synthesized copies. In fact, it has been estimated that bacterial ClpP and Lon are jointly responsible for 80% of the cellular proteolysis, demonstrating that ClpP activity severely impacts the overall bacterial proteome (9). It is therefore not surprising that ClpP is critical for virulence and infectivity in some bacterial pathogens (9). Similar to its bacterial homolog, mitochondrial ClpP is a crucial component of the matrix protein quality control system (6). Moreover, several lines of evidence indicate that human ClpP plays an important role in different tumors. For example, ClpP is overexpressed in some carcinomas, including carcinomas in breast, prostate, colon, lung and liver (9). Likewise, ClpP is upregulated in metastatic non-small-cell lung cancer cells relative to nonmetastatic cells, while knockdown of ClpP reduced the viability of acute myeloid leukemia cells (9,12). These findings, therefore, point towards a role of ClpP in proliferation, viability and metastasis of cancer cells. In sum, ClpP has emerged as an attractive target for the potential treatment of bacterial infections as well as novel therapeutic approaches for cancer. 

In furtherance of this aim, small molecule drugs were initially designed that inhibit the activity of bacterial ClpP but despite their success in in vitro experiments, the in vivo applicability is limited mainly because of poor selectivity and stability. With regards to the latter, it should be noted that novel acyldepsipeptide (ADEP) antibiotics were isolated from Streptomyces hawaiiensis in 1985 that dysregulate or activate ClpP (top panel of figure 2 adapted from 9). Mechanistically, this is achieved by ADEP antibiotics through displacing the regulatory ClpX chaperone, while subsequent binding to ClpP keeps it in active state, enabling the unspecific degradation of proteins. ADEP antibiotics are based on a common structure made up of a peptidolactone macrocyclic core that is linked to an N-acylphenylalanine moiety through an exocyclic amide bond. ADEP1 and 2 represent compounds that were originally isolated from S hawaiiensis but display poor efficacy against Gram-positive and negative bacteria. Therefore, the ADEP core was chemically modified to obtain more potent variants, including ADEP4, ADEP1g and ADEP26. These display improved stability and bactericidal activity mainly against Gram-positive pathogens. However, ADEP antibiotics are currently not clinically evaluated, despite their potential to treat infections caused by multi-drug resistant Gram-positive superbugs (13).

 

 

To identify small molecules with potential anti-tumor activity, a large compound library was initially screened. This yielded one molecule, ONC201, exhibiting tumor-specific cytotoxic activity (14). Subsequent assessment of its chemical structure revealed that ONC201 is an imidazopyridopyrimidone derivative (bottom panel of figure 2) (15), which is currently evaluated in a number of clinical trials targeting a range of different tumors (10,16-19). Moreover, a more potent variant of ONC201 was obtained through fluorination of its impiridone core (20). This novel ONC201 derivative was termed ONC212 (bottom panel of figure 2) and displays a broad-spectrum anti-tumor activity relative to ONC201. Pre-clinical evaluation of ONC212 established that impiridones are promising anti-cancer agents (21). To obtain more insight into the potential cellular target of ONC201 and ONC212, they were included in a library of more than 700 compounds with established anti-tumor activity. This was screened using an assay with ClpP as target to isolate compounds able to activate the protease, yielding ONC201 and ONC212 as possible activators of ClpP (11). In sum, impiridone compounds have emerged as powerful anti-tumor agents that are currently evaluated in different clinical trial stages. Similar to ADEP antibiotics, impiridones serve as activators of ClpP. 

Biochemical and structural features of ClpP

Structural features of bacterial ClpP

Within each cell a specific set of energy-dependent proteases ensure the maintenance of a functional proteome through selectively removing dysfunctional proteins. These proteases include members of the FtsH, Lon, Clp and proteasome families, which display both ATPase and proteolytic activity (22). ClpP is a conserved serine peptidase that is found in bacteria as well as in most mitochondria and chloroplasts of eukaryotic cells, although it is probably absent in archaea. ClpP degrades a number of model proteins in vitro, including casein hence its name caseinolytic protease (8). Crystal structures of ClpP homologs from different organisms have been solved and collectively they established that the protease is organized into a cylindrical-shaped structure made up of two rings comprising seven subunits each with a central hollow chamber that contains the proteolytic sites (ser-his-asp) (8). E. coli ClpP represents biochemically the best characterized variant. It is initially synthesized as a proenzyme of 207 residues that is autoproteolytically processed to remove an N-terminal propeptide, yielding the mature monomer of 193 residues (8). Insight into the final makeup of the E. coli ClpXP complex was obtained by electron microscopy, confirming that ClpX, a molecular chaperone of the AAA+ superfamily, is located at both ends of the ClpP cylinder (top left panel figure 3 adapted from 23). In addition to ClpX, ClpP also cooperates in bacteria with ClpA, another AAA chaperone. Although ClpP is able to degrade small peptides that are able to access the proteolytic chamber through its central pore, proteolysis of larger proteins is enabled by association with ClpX or ClpA. These are required for substrate recognition, unfolding and translocation into the ClpP proteolytic chamber. Therefore, ClpX and ClpA determine the substrate specificity of the ClpXAP system. Similar to other AAA chaperones, ClpX and ClpA are assembled into a hexameric ring with a central pore and use ATP hydrolysis to power substrate unfolding and translocation (22). Structurally, the assembly of ClpXAP resembles the eukaryotic proteasome complex. Different crystal structures of E. coli ClpP have been solved. For example, figure 3 shows its structure at 1.9 Å (24) in surface (top center and right panel) and ribbon representation (bottom left panel). This reveals that the E. coli protease is a homomeric complex assembled into the typical barrel-shaped structure that is composed of two heptameric rings (top center panel) with a central pore (top right panel) that provides access to the proteolytic chamber (8). The structure of the monomer, a 21 kDa protein, is presented in the bottom right panel and comprises three subdomains, namely: an N-terminal loop segment, the head domain and the handle region (8). The handle domains form the interface which links the two heptameric rings in the fully assembled complex. The flexible N terminal regions of each subunit are located around the axial pore of both heptamers and make up a gate that controls access into the pore. Binding of ClpA or ClpX to ClpP causes opening of the channel. Moreover, the first 20 N-terminal residues of ClpP are crucial for stabilizing the ClpAP and ClpXP complexes (24).  

 

Hyperactivation of human ClpP by impirodones

 

Accumulating evidence demonstrates that impiridones are efficient anticancer agents that are effective against a variety of different tumors and are probably directed against human ClpP. To analyze whether ONC201 directly interacts with ClpP, the structure of human ClpP bound to ONC201 was solved at 2 Å (REF). This structure is shown in figure 4 and reveals that all subunits bind one ONC201 molecule (shown as red spheres in the top panel). In the heptameric ring, the ONC201 molecules are located at the interface with ClpX. The bottom panel shows a close-up of the ONC201 binding site, unveiling that ONC201 is bound non-covalently to a single subunit through interactions with Tyr118, Gln107, Leu104 and Tyr138. A structural comparison of human ClpP without ONC201 (REF) and the complex bound to this impiridone (shown as yellow spheres) is presented in figure 5 and shows that in the absence of ONC201 the axial pore is closed, while in its presence the pore is opened. Therefore, binding of ONC201 induces opening of the axial pore, while additional changes are induced around the active site region. These affect the orientation of the catalytic residues (Ser153, His178 and Asp227). Binding of ONC201 also triggers opening of channel-like pores in the wall of the proteolytic chamber that are probably used for the exit of peptide products. 

Extensive analysis of the anticancer properties of ONC201 and ONC212 established that both impiridones induce cell death in a variety of human tumor cells through hyperactivation of ClpP. These include hard-to-treat tumors such as leukemias and lymphomas. Importantly, these compounds are much less toxic to non-malignant cells. To assess the effect of ClpP hyperactivation and thereby determining the underlying cause of ONC201s cytotoxicity, ClpP substrates were mapped by analyzing its interactome following ONC201 treatment of cancer cells. This showed that under these conditions ClpP specifically degrades respiratory chain enzymes as well as mitochondrial translation factors. This is accompanied by structural damage of mitochondria. Based on these promising in vitro results, the efficacy of ONC212 was investigated in a tumor mouse model, revealing that impirdones reduce the tumor burden in this experimental set up. This confirms that ONC212 is also effective in vivo, thereby validating its antitumor activity. The structural data presented above demonstrate that ONC201 and ONC212 are directed towards ClpP and exert their cytotoxic effects through hyperactivation of ClpP, resulting in increased degradation of respiratory chain enzymes as well as destruction of mitochondrial structure. This ultimately induces mitochondrial dysfunction and triggers apoptosis. Collectively, these data emphasize that ClpP is an effective therapeutic target for a wide spectrum of tumors.

Conclusion

 

It is well established that mitochondria play a crucial role in a variety of biochemical processes, ranging from bulk ATP production to calcium ion storage, fatty acid oxidation and iron-sulfur cluster biogenesis. It is, therefore, not surprising that mitochondrial dysfunction is closely associated with aging and different neurodegenerative diseases (4). Moreover, several lines of evidence emphasize that mitochondria are important mediators of tumorigenesis owing to their ability to influence cancer initiation, growth, survival and metastasis (5). Mitochondria contain a specific set of energy-dependent proteases that are essential for maintaining mitochondrial activity trough maintaining a functional proteome. These proteases include members of the FtsH, Lon, Clp and proteasome families, which display both ATPase and proteolytic activity (22). In humans ClpP seems to play an important role in different tumors because it was found that it is overexpressed in some carcinomas, including carcinomas in breast, prostate, colon, lung and liver (9). Likewise, ClpP is upregulated in metastatic non-small-cell lung cancer cells relative to nonmetastatic cells, while knockdown of ClpP reduced the viability of acute myeloid leukemia cells (9,12). These findings, therefore, point towards a role of ClpP in proliferation, viability and metastasis of cancer cells and suggest that it is a potential target for anticancer drugs. To identify small molecules that are directed against ClpP, a library of more than 700 compounds with established anti-tumor activity was screened with human ClpP as target to isolate compounds able to activate the protease, yielding ONC201 and ONC212 as possible activators of ClpP (11). These compounds are small molecules of the impiridone family that have emerged as powerful anti-tumor agents that are currently evaluated in different clinical trial stages. To assess whether ONC201 directly interacts with ClpP, the structure of human ClpP bound to ONC201 was solved (11), showing that this impirodone interacts non-covalently with each subunit of the heptameric ClpP ring. Importantly, ONC201 is located at the interface with ClpX and binding of ONC201 induces opening of the axial pore as well as additional conformational changes that affect the orientation of the catalytic residues and trigger opening of channel-like pores in the wall of the proteolytic chamber. These are probably used for the exit of peptide products. Thus, hyperactivation of ClpP by ONC201 and ONC212 results in increased degradation of respiratory chain enzymes as well as destruction of mitochondrial structure. This ultimately induces mitochondrial dysfunction and triggers apoptosis. It can be expected that the improved mechanistic understanding of ONC201 and ONC212 as hyperactivators of ClpP will contribute to the design of novel impiridone anticancer drugs, thereby further exploiting ClpP as an effective therapeutic target for a wide spectrum of tumors.

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