Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria

Abstract

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.

Fig. 1. Elements and mode of action of Homo-BacPROTACs.

a Naturally occurring cyclomarins and simplified synthetic dCym derivatives. The numbering system for amino acid positions used in this work is based on their introduction in the chemical synthesis and is indicated in the structure. b Concept for the Homo-BacPROTAC-induced degradation of ClpC1.

Fig. 2. Chemical synthesis of Homo-BacPROTACs.

a Chemical synthesis of exit vector 6 Homo-BacPROTACs via CuAAC. b Synthesis of exit vector 7 Homo-BacPROTACs via CuAAC. c Synthesis of exit vector 6 and 3 Homo-BacPROTACs via olefin metathesis. BEP: 2-Bromo-1-ethylpyridinium tetrafluoroborate; DMBA: N,N’-Dimethylbarbituric acid; EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt: 1-Hydroxybenzotriazole; NMM: N-methylmorpholine; TPPTS: 3,3’,3”-Phosphanetriyltris(benzenesulfonic acid) trisodium salt; HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate.

Fig. 3. Further modifications of exit vector 7 Homo-BacPROTACs.

Chemical synthesis of exit vector 7 Homo-BacPROTACs bearing an additional methyl group (shown in green) attached to tryptophan-N^α. Pra Propargylglycine. NMI N-methylimidazole. TREN Tris(2-aminoethyl)amine.

Fig. 4. Activity of Homo-BacPROTACs in cell-free degradation assays.

a Degradation curves of ClpC1-NTD in the cell-free degradation assay (quantified by capillary Western (WES)) induced by Homo-BacPROTAC 8 (UdSBI-0545) compared to its enantiomer 8a (UdSBI-0966), matching monomer 5 and dCymC (3 independent experiments done in triplicates). b WES visualization of ClpC1-NTD degradation after titration of Homo-BacPROTACs, developed with anti-His antibody recognizing His6-tagged ClpC1-NTD and processed, His4-tagged ClpP1P2. Concentration-dependent degradation of ClpC1-NTD can be observed for 8 (UdSBI-0545) (lanes 10–13) but not for 8a (UdSBI-0966) (lanes 2–8). c Analogous to a, except that exit vector 7-based Homo-BacPROTAC 12 (UdSBI-4377), enantiomer 12a (UdSBI-0117) and monomer 10 were used. d WES-derived gel picture visualizing ClpC1-NTD degradation (lane 2–5) from representative experiment summarized in c. e SYPRO^TM Ruby-stained SDS-PAGE gel from exemplary cell-free degradation assay depicting efficient degradation (lane 3,4) of ClpC1-NTD by Homo-BacPROTAC 8 (UdSBI-0545), while full length ClpC1 is not significantly degraded. Green vertical lines indicate the DC₅₀ (for 8, 12). Error bars represent mean ± SD of n = 3 independent experiments in triplicates. The actual mean DC₅₀ values for all cell-free degradation experiments conducted for this study are summarized in Supplementary Table 8. Source data are provided as a Source Data file.

Fig. 5. Homo-BacPROTACs degrade intracellular ClpC1.

a Intracellular degradation of ClpC1 in Msm #607 cells following a 24 h incubation with Homo-BacPROTACs 8 (UdSBI-0545), or 12 (UdSBI-4377). Green vertical lines indicate the DC₅₀ values obtained in that particular experiment. b ClpC1 levels in Msm #607 following a 24 h incubation with distomers 8a (UdSBI-0966), or 12a (UdSBI-0117). c ClpC1 levels in Msm #607 following a 24 h incubation with monomers 5 (green), 10 (blue), or dCymC (red). The curves show representative experiments performed in well triplicates. Experiments for each compound were performed independently n = 3 (compounds 8, 8a, 12a, dCymC, 5, 10) or n = 4 (compound 12) times with similar results. Error bars indicate mean ± SD of n = 3 well replicates for that given experiment. The actual mean DC₅₀ values reported in the text for compound 8 or 12 are calculated from the respective individual DC₅₀ values obtained for the independent cellular degradation experiments conducted in this study and are summarized in the Source Data file for Fig. 5a. Source data are provided as a Source Data file.

Fig. 6. Intracellular MIC assay in THP-1 macrophages.

a, b Exit vector 6-based Homo-BacPROTACs 7 and 8 (UdSBI-0545), as well as exit vector 7-based Homo-BacPROTACs 11 and 12 (UdSBI-4377) were assessed in a 4- and 7-day incubation on THP-1 macrophages following infection with Mtb H37Rv. The Homo-BacPROTACs were compared to their corresponding monomers (5 for exit vector 6 and 10 for exit vector 7) and the reference antibiotics rifampicin and moxifloxacin, which are known to inhibit the intracellular propagation of Mtb H37Rv. Exit vector 6 Homo-BacPROTACs showed a more efficient concentration-dependent reduction of cfu/ml over time with an E_max of 1.22 − 1.27 at 50 µM, as compared to exit vector 7 based Homo-BacPROTACs or matched monomers. The various compound concentrations are indicated in different colours, matching across the panels. CC means Mtb culture control, where no drug treatment was given. For further details see text. Error bars indicate mean ± SD of n = 2 well replicates. The CC, rifampicin and moxifloxacin values were taken as common reference points in the various graphs. Source data are provided as a Source Data file.