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

The low availability of novel antibiotics and the spread of multi-resistant organismscreate a severe situation worldwide. Each year, many individuals perish ordevelop serious disabilities due to persistent infections that conventionalantimicrobial chemotherapies cannot treat. This reality also places economicpressure on governments, as strategies to cope with this burden in the publichealth system demand high investments.Efforts to promote better management of this scenario involve preventivemeasures to promote the rational use of antimicrobials in daily life, from hospitalsto agriculture. Data from several international agencies show that adoptedmeasures and policies have been effective in recent years, helping to control therise of new resistant strains, especially in developed countries. Unfortunately, inundeveloped and developing regions, the dramatic reality serves as a reminderthat much work remains to be done.Similarly, plans for producing new drugs are needed. Over the last decades, onlya restricted portfolio of antibiotics has been available for hard-to-treat microbialdiseases. For some of these drugs, like vancomycin, resistance has already beendetected in certain environments.Some Staphylococci are found among resistant organisms. Staphylococcusaureus (Sa) and Staphylococcus epidermidis (Se) strains, commensal organismsfound on skin and mucosa, can affect immunocompromised and long-termhospitalized patients with persistent nosocomial infections. Besides that, someStaphylococci also have the potential to build biofilms that shield them againstantibiotics effective upon metabolically active cells in the bloodstream. However,studies involving caseinolytic protease P subunit (ClpP) unveil that this enzyme isa promising drug-target macromolecule for eliminating multi-resistant anddormant Staphylococci.ClpP is an endopeptidase that forms a proteolytic machine with chaperones fromthe AAA+ (ATPases associated with diverse cellular activities) superfamily. Thismachine plays an important role in bacterial protein homeostasis, keeping acontrolled proteolysis in the cytosol. This control is performed by the chaperonessince they make the ClpP’s catalytic chamber accessible to selected substrates–unfolded, misfolded, and regulatory proteins – to be degraded. Nevertheless, in12recent years, it has been discovered that natural and synthetic molecules mimicthe ATPases, dysregulating the ClpP function and unleashing extensive andpromiscuous proteolysis in the intracellular milieu, which leads bacteria to deathby self-digestion. On the contrary, other compounds bind to the ClpP’s catalyticsites, inhibiting the enzymatic activity, affecting the organism’s virulence, andhampering microbial dissemination in the host.In both ways, activation or inhibition, there are still open questions about theirmechanism. In this regard, some boron-based compounds pose as intriguingClpP modulators. Commonly classified as canonical proteasome inhibitor, thepeptidomimetic boronate known as bortezomib is linked to a paradoxical allostericactivation of a bacterial ClpP, leading to an increase of proteolysis in a linearway.While some biophysical and biochemical approaches have given insights intostructural and functional aspects of the ClpP-bortezomib complex, a detailedexplanation at the atomic level about how the ligand modulates the enzymaticactivity is still lacking. Considering this gap, in this work, another peptidomimeticboronate, ixazomib, was selected to study its effect on the ClpP fromStaphylococcus epidermidis. In the first two parts, the crystal structure of nativeSeClpP and SeClpP-ligand complex obtained at PETRA III/DESY and EMBL(Hamburg, Germany) are explored and compared, aiming the discovery ofstructural differences with the assistance of low-resolution techniques like Batch-SAXS and SEC-SAXS available at EMBL (Hamburg, Germany). This comparativeinvestigation revealed the reorientation of a key amino acid residue related to theextension of the protein’s axial pores, a process that allows the degradation ofglobular proteins inside the ClpP’s catalytic chamber.In the latter part, assays with SeClpP and ixazomib, under distinct conditions,confirmed the degradation of -casein from bovine milk. Peptidolytic assays in thepresence 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 theassembly 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 theprotein’s oligomerization.13Undoubtedly, the contributions displayed in this thesis are of great relevance forthe understanding of modulation and conformational shifts of ClpP. The resultsshow that the ligand used can be a scaffold for the development of novelantibiotics against nosocomial infections caused by opportunistic pathogens.