Objective

Acyldepsipeptides (ADEPs) are potential drugs against bacterial infections. They act in a way distinct from all other antibiotic mechanisms known so far. ADEPs target the caseinolytic peptidase P (ClpP), the proteolytic core of bacterial ATP-dependent proteases. ClpP on its own can only act as a peptidase, for its proteolytic activity an ATPase is needed. This ATPase binds into hydrophobic pockets at the surface of ClpP and regulates proteolysis. ADEPs bind to the same binding pockets, mimic the binding of a Clp ATPase and as a consequence open the pore for larger polypeptides. This and an additional stimulating effect results in uncontrolled proteolysis and thus leads to cell death.

Due to its therapeutic potential it is important to identify any off-targets of ADEPs in bacterial and human cells. In order to investigate these, an ADEP-photoprobe is designed and used in an affinity-based protein profiling (AfBPP) approach. Since the synthesis of ADEPs is sophisticated, an ADEP-fragment-photoprobe is synthesized first and tested in the shown experiments in S. aureus.

In a tailored assay, the synthesized photoprobes showed activity in SaClpP stimulation and were able to label ClpP in an analytical AfBPP experiment with fluorescent readout on a SDS-gel. In a preparative AfBPP experiment the labeling will be confirmed by gel-free proteomics.