Protein Hyperactivation Could Kill Cancer Cells and Bacteria

Protease enzymes break down old or misfolded proteins as part of the quality control process that ensures proper protein form and function within cells. When hyperactivated, however, proteases could have the ability to destroy cells from within by removing essential proteins, not just those that are faulty.

A research team led by Walid A. Houry, professor of biochemistry, University of Toronto, is researching compounds that can induce protease hyperactivation to kill cancer cells. Technology Networks had the pleasure of speaking with Houry to find out more about the discovery of such compounds, how they work and their applications as anticancers and antibacterial.

Kate Robinson (KR): What led you to study proteases as a possible anticancer target?

Walid Houry (WH): The mitochondrion is a vital organelle in the cell with diverse functions, including energy metabolism and biosynthesis of important metabolites. Many diseases are, therefore, associated with mitochondrial dysfunction, including cancer. The cell has evolved various mechanisms to preserve mitochondrial integrity and functionality, an important part of which is the maintenance of the mitochondrial proteome via molecular chaperones to aid in protein folding and proteases to remove misfolded and damaged proteins. Among the different proteases utilized is the ClpXP complex (ClpXP) found in the mitochondrial matrix, consisting of a ring-shaped ClpX ATPase and ClpP serine protease that forms a cylindrical structure. Multiple studies have shown that ClpP is upregulated in both primary and metastatic cancers. ClpP is now considered to be a novel anticancer drug target.

My group is interested in the general area of protein homeostasis, and we have been studying the structure and mechanism of function of ClpP for a few years. We recently became interested in developing novel compounds with a novel mode of action that can specifically target human ClpP and induce cell death or cell growth inhibition. These compounds are promising novel anticancers.

KR: What effect does protease hyperactivation have and how is this state achieved?

WH: Human ClpXP consists of the AAA+ ATPase ClpX and the serine protease ClpP. Assembly of ClpXP involves the capping of the cylindrical ClpP tetradecamer by up to two ClpX hexamers. In this configuration, ClpX serves as a gatekeeper that recognizes and targets only specific substrate proteins. A substrate for the protease initially interacts with ClpX and is then actively unfolded by the ATPase using energy derived from ATP binding and hydrolysis. The unfolded substrate is then threaded through ClpX’s axial pore and into the lumen of ClpP for proteolysis. The peptidyl remnants are subsequently ejected through either the side pores or one of two open ends of the ClpP cylinder to complete the proteolysis cycle.

As part of our work, we identified new ClpP-targeting compounds of high potency and specificity. These compounds bind at the top of the ClpP cylinder at the same hydrophobic pockets where the loops of ClpX would engage ClpP and, hence, the compounds prohibit the formation of the ClpXP complex. Furthermore, these compounds also enhance ClpP protease activity by opening the axial entrance pool, which then causes ClpP to degrade proteins in a dysregulated and unspecific manner, resulting eventually in cell death or growth arrest. This represents a novel mechanism to target cells in which a protease is activated rather than inhibited. We call these compounds "ClpP activators". Data were collected at the Canadian Light Source to obtain the structures of compound-bound ClpP. My group was the first to show that activation of ClpP by specific compounds can lead to cancer cell death.

KR: Could these protease dysregulation compounds be a viable treatment option for difficult-to-treat cancers?

WH: ClpP overexpression has been reported in acute myeloid leukemia (AML), multiple myeloma and various lymphomas. Increased ClpP expression is also universally observed in type I endometrial cancer, triple-negative breast cancer and multiple other types of solid tumors from various tissues. Importantly, high ClpP expression is shown to correlate with poor prognosis in patients. In many cases, ClpP overexpression is required to regulate mitochondrial function and maintain cell viability in face of increased reactive oxygen species accumulation, particularly in cancers that are more dependent on oxidative phosphorylation such as AML and prostate adenocarcinoma PC3. We demonstrated that ClpP activators can inhibit growth in a screen of 60 cancer cell lines. Several ClpP activators have already been shown to have efficacy in mouse xenograft models. Given the novel mode of action of these compounds, we think that they can be a viable treatment option for difficult-to-treat cancers, especially in a combination therapy with other established drugs.

KR: How can your research translate to antibacterial development?

WH: Our initial effort prior to our most recent work was directed towards targeting the ClpP protease in bacteria. The activation of bacterial ClpP to develop novel antibiotics was first described by a group at Bayer. My group had carried out a large screen to identify compounds that activate the bacterial Escherichia coli ClpP. These compounds have the same mechanism of action as the compounds targeting human ClpP. However, the bacterial ClpP-targeting compounds that we work on have a different chemical scaffold than the human ClpP-targeting compounds that we describe in our recent work. In the high-throughput screen, we identified five structurally diverse bacterial ClpP activators, which we termed Activators of self-Compartmentalizing Proteases (or ACPs). ACP1 was further optimized yielding several potent analogs that showed good antibacterial properties for both Gram-positive and Gram-negative bacteria. We found that Neisserial species are especially susceptible to such compounds, and most of our efforts are currently concentrated on targeting Neisseria meningitidis.

KR: Do you have any plans to build upon this research?

WH: We continue to optimize the various classes of compounds targeting human ClpP and bacterial ClpP to develop novel anticancers and antibiotics, respectively. We are also interested in gaining a more detailed understanding of ClpP function and the consequences of its activation. We ultimately hope that these compounds will move from the bench and into the clinic to provide another line of treatment against these diseases.

Professor Walid A. Houry was speaking to Kate Robinson, Assistant Editor for Technology Networks.