Tuberculosis is a deadly bacterial disease that has been infecting civilizations around the world for thousands of years. The disease is caused by the Mycobacterium tuberculosis bacterium and is the most infectious bacterial disease. Before the discovery of antibiotics, a tuberculosis infection most commonly lead to death. Although antibiotics have helped treat many bacterial infections the development of drug resistant bacterial strains makes it more and more difficult to cope with certain infections. Multi drug resistant (MDR) and extensively drug resistant (XDR) cases off M. tuberculosis have been identified and become more and more prevalent, leading to an increased need for the development of new drugs and drug targets to combat this evolving public health concern. Caseinolytic protease proteolytic subunit protease (ClpP) has emerged as a novel drug target as it is essential in many bacterial species for maintaining cell proteostasis and aiding in virulence and survival. Most commonly used antibiotics have similar structures, and target similar pathways in the cell, like cell wall synthesis, DNA/RNA synthesis, and protein synthesis. ClpP is a novel target as it is responsible for protein degradation and maintaining proteostasis. The peptidase has also been largely conserved through evolution among different bacterial species, as well as eukaryotic organelles (chloroplasts and mitochondria). In cells ClpP interacts with an unfoldase partner that recognizes and degrades damaged, misfolded, and unwanted proteins in a controlled and regulated fashion. The essentiality of this peptidase in some bacteria, including M. tuberculosis, makes it a promising drug target. Acyldepsipeptides (ADEPS) are a unique class of non-ribosomally encoded peptides that were discovered in and isolated from Streptomyces hawaiiensi, a soil dwelling bacterium that produces ADEPs as a byproduct of aerobic fermentation. In nature ADEPs likely serve as a tool against other soil inhabiting organisms, like Bacillus subtilis, in competition for nutrients. ADEPs work by targeting ClpP and mimicking the unfoldase binding partner, to compete for the unfoldase docking sites on ClpP. The interaction between ADEP and ClpP prevents the assembly of the complete protease preventing the degradation of large and structure proteins, while stabilizing the ClpP peptidase in an active conformation and allowing small proteins and peptides to freely diffuse into the degradation chamber and be destroyed in an unregulated fashion. However, there are certain limitations of using ADEPs as drugs to target ClpP in cells, ranging from poor bioavailability to poor species specificity. The ADEP macrocycle is not necessary for ADEP binding and activity. Here we investigate the binding and activity of ADEP fragments to bacterial ClpPs, and how ADEP macrocycle modifications can improve species specificity.

In Chapter 2, we determine the position of ADEP fragment binding to the ClpP of a model organism B. subtilis, and identify a secondary binding site within the degradation chamber of the peptidase that is located near the active site residues (Ser97, His122, Asp175). Through peptidase degradation assays, we identify ClpP peptidase inhibition at higher ADEP concentrations, something that is not observed with full ADEPs. Suggesting that ADEP fragments have both an activating effect (similar to ADEPs) and an inhibitory effect on the ClpP peptidase activity. This opens up potential for developing new ADEP fragment derivatives that could help to stabilize binding in the active site of the peptidase and enhance peptidase inhibition.

In Chapter 3, we seek to determine if ADEP fragments have an inhibitory effect on other bacterial Clp peptidases, like the MtbClpP1P2. In crystal structures, and by cryo-EM, we see that ADEP fragments are also able to bind in the active site of MtbClpP1P2 and that this is not a phenomenon limited to just BsuClpP. We observe an inhibitory effect of ADEP fragments with MtbClpP1P2 just as we did with BsuClpP, showing that this likely occurs across multiple Clp species. By comparing crystal structures, we see that ADEP fragment binds in the active site of BsuClpP similarly to how an N-terminally blocked peptide substrates binds to EcoClpP or how agonist binds to MtbClpP1P2. In vitro MtbClpP1P2 requires the presence of ADEP and agonist to stabilize the active 14-mer. However, we show that ADEP fragment alone in the absence of agonist and ADEP is sufficient to stabilize the MtbClpP1P2 peptidase and induce peptidase activity.

In Chapter 4, we investigate the potential for improving the species specificity of ADEPs by modifying the two proline positions of the macrocycle with the addition of a fluorine, di-fluorine or fluorine and methyl. Fluorine helps to stabilize ligand binding by interacting with hydrophobic residues, like those found lining the ADEP binding site of the ClpP peptidase. Through peptidase assays we show that the addition of fluorine at certain positions and orientations helps to stimulate activity and binding of ADEPs to some of the tested ClpP species, particularly MtbClpP1P1 and EcoClpP. Indicating promise for the improvement of ADEP species specificity by modifying the ADEP macrocycle.