Structural and Functional Aspects of the Paradoxical ClpP Modulation by Peptidomimetic Boronate Compounds

Abstract

The low availability of novel antibiotics and the spread of multi-resistant organisms create a severe situation worldwide. Each year, many individuals perish or develop serious disabilities due to persistent infections that conventional antimicrobial chemotherapies cannot treat. This reality also places economic pressure on governments, as strategies to cope with this burden in the public health system demand high investments. Efforts to promote better management of this scenario involve preventive measures to promote the rational use of antimicrobials in daily life, from hospitals to agriculture. Data from several international agencies show that adopted measures and policies have been effective in recent years, helping to control the rise of new resistant strains, especially in developed countries. Unfortunately, in undeveloped and developing regions, the dramatic reality serves as a reminder that much work remains to be done. Similarly, plans for producing new drugs are needed. Over the last decades, only a restricted portfolio of antibiotics has been available for hard-to-treat microbial diseases. For some of these drugs, like vancomycin, resistance has already been detected in certain environments. Some Staphylococci are found among resistant organisms. Staphylococcus aureus (Sa) and Staphylococcus epidermidis (Se) strains, commensal organisms found on skin and mucosa, can affect immunocompromised and long-term hospitalized patients with persistent nosocomial infections. Besides that, some Staphylococci also have the potential to build biofilms that shield them against antibiotics effective upon metabolically active cells in the bloodstream. However, studies involving caseinolytic protease P subunit (ClpP) unveil that this enzyme is a promising drug-target macromolecule for eliminating multi-resistant and dormant Staphylococci. ClpP is an endopeptidase that forms a proteolytic machine with chaperones from the AAA+ (ATPases associated with diverse cellular activities) superfamily. This machine plays an important role in bacterial protein homeostasis, keeping a controlled proteolysis in the cytosol. This control is performed by the chaperones since they make the ClpP’s catalytic chamber accessible to selected substrates– unfolded, misfolded, and regulatory proteins – to be degraded. Nevertheless, in recent years, it has been discovered that natural and synthetic molecules mimic the ATPases, dysregulating the ClpP function and unleashing extensive and promiscuous proteolysis in the intracellular milieu, which leads bacteria to death by self-digestion. On the contrary, other compounds bind to the ClpP’s catalytic sites, inhibiting the enzymatic activity, affecting the organism’s virulence, and hampering microbial dissemination in the host. In both ways, activation or inhibition, there are still open questions about their mechanism. In this regard, some boron-based compounds pose as intriguing ClpP modulators. Commonly classified as canonical proteasome inhibitor, the peptidomimetic boronate known as bortezomib is linked to a paradoxical allosteric activation of a bacterial ClpP, leading to an increase of proteolysis in a linear way.While some biophysical and biochemical approaches have given insights into structural and functional aspects of the ClpP-bortezomib complex, a detailed explanation at the atomic level about how the ligand modulates the enzymatic activity is still lacking. Considering this gap, in this work, another peptidomimetic boronate, ixazomib, was selected to study its effect on the ClpP from Staphylococcus epidermidis. In the first two parts, the crystal structure of native SeClpP and SeClpP-ligand complex obtained at PETRA III/DESY and EMBL (Hamburg, Germany) are explored and compared, aiming the discovery of structural differences with the assistance of low-resolution techniques like Batch-SAXS and SEC-SAXS available at EMBL (Hamburg, Germany). This comparative investigation revealed the reorientation of a key amino acid residue related to the extension of the protein’s axial pores, a process that allows the degradation of globular proteins inside the ClpP’s catalytic chamber. In the latter part, assays with SeClpP and ixazomib, under distinct conditions, confirmed the degradation of beta-casein from bovine milk. Peptidolytic assays in the presence of peptidomimetic boronates were taken into consideration as well, indicating a change of modulation dependent on the ligand concentration. Additionally, SEC-SAXS measurements demonstrated that ixazomib induces the assembly of the tetradecameric SeClpP from its heptamers. For the first time, experiments confirm that a ligand bound to the catalytic sites plays a role in the protein’s oligomerization. Undoubtedly, the contributions displayed in this thesis are of great relevance for the understanding of modulation and conformational shifts of ClpP. The results show that the ligand used can be a scaffold for the development of novel antibiotics against nosocomial infections caused by opportunistic pathogens.