Ecumicin and Cyclomarin A (CymA) are known antimicrobial compounds that target the NTD of ClpC1. In proteomics analysis of compound treated M. smegmatis, two novel protein, ClpC2 and ClpC3, have been identified as strong interactors of those compounds in addition to ClpC1. In this study we used PRM to monitor the levels of ClpC1, ClpC2 and ClpC3 over time after treatment with CymA or ecumicin or CymA-based BacPROTACs.

Experiment 1: To analyze the impact of ecumicin and CymA, cells were treated with compound and levels of ClpC1, ClpC2 and ClpC3 were measured over time, in addition to several housekeeping proteins. In addition, Hetero BacPROTACs (DOI:10.1016/j.cell.2022.05.009) were tested, as well as their respective head groups. BacPROTAC treatment were carried out in a M. smegmatis strain overexpressing BRDT-BD1. Sample key A: BP-4 (WT strain) B: THP + D9 (WT strain) C: BP-5 (BRDT strain) D: JQ1 + D9 (BRDT strain) E: Ph-9-90 (WT strain) F: dCymA (WT strain) G: DMSO (WT strain) H: DMSO (BRDT strain) 1-4: timepoint 0 to 2h Experiment 2: In a second experiment, Homo BacPROTACs, carrying 2 equal Cym headgroups were used to induce selective degradation of ClpC1 and ClpC2. Levels of ClpC1, ClpC2 and ClpC3 were measured over time. For additional absolute quantification, peptide standards of ClpC1 and ClpC2 were used. Sample key: EuEu: active dimer Eumono: active monomer DisDis: inactive dimer Dismono: inactive monomer 6: exit vector 6 7: exit vector 7

Experiment 1: M. smegmatis mc2-155 were treated with 10 µM compound or DMSO (final concentration of 1%) and timepoints of 5, 10, 30 or 120 min were analyzed. Samples were lzyed and processed according to the standard SP3 sample protocol (https://doi.org/10.1038/s41596-018-0082-x). Experiment 2: M. smegmatis 607 were treated with 12 µM compound or DMSO (final concentration of 1%) and timepoints of 2, 6 and 24 hours were analyzed. Samples were lzyed and processed according to the standard SP3 sample protocol (https://doi.org/10.1038/s41596-018-0082-x).