BacPROTACs mediate targeted protein degradation in bacteria

Summary Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.

. Amino acid sequences of fusion proteins, Related to STAR Methods    2) Solid phase amino acid assembly The peptide was built up by application of generic solid phase peptide synthesis procedures: Fmoc deprotection: A solution of DMF:piperidine (3:2, 5 mL) was added to the resin.
The resulting suspension was shaken for 20 minutes at room temperature. The solution was removed and the cleavage step was repeated once more. The remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.
This solution was added to the resin and the resulting suspension was shaken for 2 hours at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.

3) N-terminal acylation
Acetic anhydride (30 µL) and DIPEA (100 µL) were dissolved in NMP (5 mL). This solution was then added to the resin and the resulting suspension was shaken for 1 h at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× DMF.

4) Acidic cleavage and purification of the Tce-protected peptide
A solution of TFA:DCM (3:1, 5 mL) was added to the resin and the resulting suspension was shaken for 1 h at room temperature. The resin was filtered off and ice-cold diethyl ether (35 mL) was added to the filtrate and stored over night at -20 °C.
The resulting suspension was centrifuged and the filtrate was decanted off. The residue was taken up in water:acetonitrile (4:1,15 mL) and purified by RP-HPLC with 0.1% TFA in water and 0.1% TFA in acetonitrile as the eluent system. Pooling and lyophilization of product containing fractions resulted in 121 mg (0.087 mmol, 73% yield based on initial resin loading) of the desired product as a white powder. [M+H] + .

5) Hydrogenation and purification
The Tce-protected peptide (66 mg, 0.0475 mmol) was dissolved in a mixture of 100 mM NH4HCO3 buffer (pH 9, 10 mL) and EtOH (5 mL). Argon was bubbled through the solution for 15 min, followed by an addition of the Pd/C catalyst (25 mg). Next, hydrogen gas was bubbled through the resulting suspension for 4 h at room temperature. The catalyst was filtered off and the remaining filtrate was removed under reduced pressure. The residue was taken up in water:acetonitrile (19:1,10 mL) and purified by RP-HPLC using 0.1% ammonia in water and 0.1% ammonia in acetonitrile as the eluent system. Pooling and lyophilization of product containing fractions

Synthesis of BacPROTAC-1a
BacPROTAC-1a was synthesized analogously to the previously described solid phase peptide synthesis approach for BacPROTAC-1.
55 mg (0.046 mmol, 56% yield based on initial resin loading) of the protected peptide were obtained as a white powder.

Synthesis of BacPROTAC-1b
BacPROTAC-1b was synthesized analogously to the previously described solid phase peptide synthesis approach for BacPROTAC-1.
45 mg (0.042 mmol, 53% yield based on initial resin loading) of the protected peptide were obtained as a white powder.

Synthesis of BacPROTAC-1c
BacPROTAC-1c was synthesized via a solid phase peptide synthesis approach.

1) Loading of Rink amide resin
Commercially available Fmoc-protected Rink amide resin (50 mg, 0.8 mmol g -1 initial loading) was suspended in a solution of DMF:piperidine (4:1, 10 mL) and shaken for 40 min at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× with NMP. Fmoc-L-Lys(Biotin)-OH (48 mg, 0.08 mmol) and DIPEA (28 µL, 0.16 mmol) were dissolved in NMP (5 mL). This solution was added to the resin and the resulting suspension was shaken for 16 h at room temperature. After removal of the solution, the remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF. For capping, acetic anhydride (15 µL, 0.16 mmol) and DIPEA (100 µL) were dissolved in NMP (5 mL). This solution was added to the resin and the resulting suspension was shaken for 30 min at room temperature.
After removal of the solution, the remained resin was washed 2× with DMF, 2× with DCM and again 2× with DMF.
2) Solid phase amino acid assembly The peptide was built up by application of generic solid phase peptide synthesis procedures: The resulting suspension was shaken for 20 minutes at room temperature. The solution was removed and the cleavage step was repeated once more. The remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.
This solution was added to the resin and the resulting suspension was shaken for 2 hours at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.

3) N-terminal acylation
Acetic anhydride (15 µL) and DIPEA (100 µL) were dissolved in NMP (5 mL). This solution was then added to the resin and the resulting suspension was shaken for 1 h at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× DMF.

4) Acidic cleavage and purification of the crude peptide
A solution of TFA:DCM (3:1, 5 mL) was added to the resin and the resulting suspension was shaken for 1 h at room temperature. The resin was filtered off and ice-cold diethyl ether (35 mL) was added to the filtrate and stored over night at -20 °C.
The resulting suspension was centrifuged and the filtrate was decanted off. The residue was taken up in water:acetonitrile (4:1,5 mL) and purified by RP-HPLC with 0.1% TFA in water and 0.1% TFA in acetonitrile as the eluent system. Pooling and lyophilization of product containing fractions resulted in 10 mg (0.009 mmol, 24% yield based on initial resin loading) of the desired product as a white powder. BacPROTAC-2 was synthesized via a solid phase peptide synthesis approach. Prior to SPPS, two non-commercial amino acid building blocks had to be synthesized.
Proton sponge (11 g, 51.4 mmol) and Me3OBF4 (7.6 g, 51.4 mmol) were added and the resulting solution was stirred for 4 days at room temperature. The solvent was evaporated under reduced pressure to dryness and the residue was purified with silica gel column chromatography using 50-100% EtOAc in cyclohexane as the eluent system. Product containing fractions were pooled and evaporated to dryness, yielding 2) Solid phase amino acid assembly The peptide was built up by application of generic solid phase peptide synthesis procedures: The resulting suspension was shaken for 20 minutes at room temperature. The solution was removed and the cleavage step was repeated once more. The remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.
Coupling of Fmoc-protected amino acids: The corresponding Fmoc-protected amino acid (3 eq.), HOBt monohydrate (3 eq.), HBTU (3 eq.) and DIPEA (3 eq.) were dissolved in DMF (5 mL). This solution was added to the resin and the resulting suspension was shaken for 2 hours at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.

5) Protecting group cleavage and purification
This cyclized peptide ( hours. The reaction mixture was diluted by addition saturated aq. NaHCO3 (15 mL).
The resulting phases were separated and the organic phase was washed with brine, followed by drying over MgSO4. The solvent was removed under reduced pressure, yielding a residue that was used in the next step without further purification.
This residue was suspended in 4 M HCl in 1,4-dioxane (10 mL) and the resulting suspension was stirred for 1 hour. The solvent was removed by evaporation to dryness and the resulting residue was dissolved in DCM (10 mL), followed by addition of Et3N 2) Solid phase amino acid assembly and resin cleavage: The peptide was built up by application of generic solid phase peptide synthesis procedures: Fmoc deprotection: A solution of DMF:piperidine (3:2, 5 mL) was added to the resin.
The resulting suspension was shaken for 20 minutes at room temperature. The solution was removed and the cleavage step was repeated once more. The remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.
Coupling of Fmoc-protected amino acids: The corresponding Fmoc-protected amino acid (3 eq.), HOBt monohydrate (3 eq.), HBTU (3 eq.) and DIPEA (3 eq.) were dissolved in DMF (5 mL). This solution was added to the resin and the resulting suspension was shaken for 2 hours at room temperature. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.
Modified coupling procedure for couplings on N-methyl amino acids: The corresponding Fmoc-protected amino acid (3 eq.), 2-Bromo-1-ethyl-pyridinium tetrafluoroborate (3 eq.) and DIPEA (6 eq.) were dissolved in DMF (5 mL). This solution was added to the resin and the resulting suspension was shaken at room temperature for 4 hours. The solution was removed and the remained resin was washed 2× with DMF, 2× with DCM and 2× with DMF.
Cleavage from resin: A mixture of DCM:hexafluoroisopropanol (3:1, 5 mL) was added to the resin. The resulting suspension was shaken at room temperature for 20 min.
The cleavage solution was removed from the resin and transferred into a flask. The resin was washed 3× with DCM (2 mL) and the washing solutions were combined with the cleavage solution. The resulting solution was evaporated under reduced pressure to dryness. The residue was dried at high vacuum to obtain 229 mg (0.137 mmol, 55% based on initial resin loading) of a linear peptide that was used in the next step without further purification.

4) Final deprotection and purification
This cyclic peptide (146 mg, 0.088 mmol) was dissolved in TFA/DCM (3:1, 10 mL) and the resulting mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure and the residue was re-dissolved in DCM (25 mL).
The organic layer was washed with saturated aq. NaHCO3 and brine and dried over

Synthesis of BacPROTAC-3a
BacPROTAC-3a was synthesized analogously to the previously described solid phase peptide synthesis approach. Prior to SPPS, one further non-commercial amino acid building blocks was synthesized.
The resulting solution was stirred for 16 hours at room temperature. The solvent was evaporated under reduced pressure and the resulting residue was used in the next step without further purification.
The obtained residue was dissolved in DCM (10 mL). Et3N (174 µL, 2.75 mmol), HOBt monohydrate (148 mg, 1.1 mmol), DIC (78 µL, 1.1 mmol) and Boc-TOTA-NH2 (122 mg, 0.82 mmol) were added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted by addition saturated aq. NaHCO3 (15 mL). The resulting phases were separated and the organic phase was washed with brine, followed by drying over MgSO4. The solvent was removed under reduced pressure, yielding a residue that was used in the next step without further purification.
This residue was suspended in 4 M HCl in 1,4-dioxane (10 mL) and stirred for 1 hour.
The solvent was removed by evaporation to dryness and the resulting residue was dissolved in DCM (10 mL and added to 2-chlorotrityl resin (100 mg, 1.6 mmol g -1 initial loading 2) Solid phase amino acid assembly and resin cleavage The solid phase peptide synthesis and resin cleavage was performed as described for BacPROTAC-3, yielding 89 mg (0.050 mmol, 38% based on initial resin loading) that was used in the next step without further purification.

4) Final deprotection and purification
This cyclic peptide (61 mg, 0.035 mmol) was dissolved in DCM/TFA (1:3, 10 mL) and the resulting mixture was stirred for 16 hours at room temperature. The solvent was removed under reduced pressure and the residue was re-dissolved in DCM (25 mL).
The organic layer was washed with saturated aq. NaHCO3 and brine and dried over

Synthesis of sCym-1
sCym-1 was synthesized via the previously described solid phase peptide synthesis approach used for BacPROTAC-2 and BacPROTAC-3. Therefore, only reagent quantities and product characterizations are provided.
Resin loading: Fmoc-L-Phe(3R-MeO)-OH (SI-5, 209 mg, 0.5 mmol) and DIPEA (170 µL, 1 mmol) were dissolved in DMF (5 mL). This solution was added to 2-chlorotrityl resin (150 mg, 1.6 mmol g -1 initial loading). The resulting suspension was shaken at room temperature for 4 hours. Washing of the resin and the capping step were performed as described for BacPROTAC-2. The subsequent solid phase peptide synthesis was performed as described before.

Chemical Synthesis of bacPROTAC-4a
SI-17 was prepared by solution phase peptide synthesis in analogy to a previously published procedure using the enantiomeric starting materials and Fmoc-Dpropargylglycine as the 7 th amino acid .