Induced degradation of target proteins is a new concept in drug discovery. For eukaryotes, the ubiquitin-proteasome system is the main pathway of intracellular protein degradation, which participates in more than 80% of intracellular protein degradation. Ubiquitin is tantamount to “death” for proteins, and once targeted, they will eventually be destroyed.
The most prominent synthetic “degrader” is proteolysis targeted chimera (PROTAC)—bifunctional small molecules, including the E3 ubiquitin ligase binding part and the target protein (POI) binding chemical part. PROTAC promotes POI ubiquitination and subsequent proteasome degradation by bringing E3 ligase close to POI.
Compared with classical inhibitors, protein degradants have many advantages. They demonstrate better degradation efficiency, for example, based on this catalytic mode. In addition, PROTAC can target almost any cellular protein, and both of its ports can be modularized, allowing protein ligands to be modified to design “degrader” for specific target proteins.
Although it is hoped that PROTAC technology will be used for the bacterial degradation pathway, now it is only applicable to the ubiquitin labeling system of eukaryotes. The realization of target protein degradation in bacteria will present a compelling approach for the design of protein functional regulators and the development of new antimicrobial agents.
A study titled “BacPROTACs to mediate targeted protein degradation in bacteria” was recently published in the journal Cell by the Tim Clausen Laboratory of the Institute of Molecular Pathology in Vienna, Austria, in cooperation with the Markus Kaiser Laboratory of the University of Duisburg-Essen in Germany.
In this study, a new type of small-molecule degrader—BacPROTAC—was developed to realize the degradation of target protein in bacteria based on the ClpCP protease degradation system in bacteria, providing a new strategy for the development antimicrobial agents.
Although ubiquitin is unique to eukaryotic cells, there is also a protein degradation system similar to ubiquitin-protease in bacteria—ClpC: ClpP (ClpCP) protease system 3. Compared with eukaryotic proteasomes that recognize complex polyubiquitin signals, the recognition mechanism of ClpCP protease is much simpler—a phosphate group is connected to the target protein’s arginine residue and is recognized as a degradation signal by ClpCP protease, achieving the degradation of the target protein. ClpCP protease existing in Gram-positive bacteria and mycobacteria has the potential to be used in bacterial protein degradation agents as a protein degradation element.
Based on the ClpCP protease degradation system, the researchers redesigned the BacPROTAC which is composed of a POI ligand, chemical linker, and ClpCNTD ligand, using Bacillus subtilis ClpCP as the protein degradation original.
Theoretically, every protein in bacteria can be degraded by this modular design. As a proof of concept, the researchers formed BacPROTAC-1 compounds by combining pArg (ClpCNTD ligand) with biotin (high affinity mSA ligand) in vitro using monomeric streptavidin (mSA) as a model protein. BacPROTAC-1 can bind mSA and ClpCNTD to form an active ternary complex, which can effectively degrade the target protein at a dosage of 100μM.
In addition, it has been reported that ClpC exists in the form of decamer in which the AAA ring is destroyed in the resting state. When the binding protein MecA interacts with ClpC, it will promote the reassembly of a functional hexamer and activates ClpCP protease. However, it is not clear how the majority of pArg-labeled ClpC substrates trigger the remodeling of ClpC decimers. In order to clarify the activation mechanism based on pArg recognition, the researchers analyzed the structure of ClpC compounded with BacPROTAC-1 and mSA-Kre fusion proteins by freeze electron microscopy.
It is surprising to learn that the four ClpC hexamers interact with each other through their coiled coils and form an almost perfect tetrahedral symmetry. In addition, the researchers also performed a focused cryogenic electron microscopic analysis of a single hexamer to analyze the functional units of the activated unfolding enzyme at a higher resolution, providing a snapshot of ClpC in the process of unfolding the BacPROTAC binding substrate. The structure of ClpC 24-mer indicates that pArg markers are not only used as degradation signals, but also mediate the formation of high-order oligomers and the activation of ClpC. BacPROTAC with pArg part can trigger this remodeling mechanism, so the bifunctional small molecule can be used as both a chemical junction and an activator of ClpCP protease.
However, the pharmacokinetics of BacPROTAC based on pArg design is poor and the guanidine phosphate group is unstable. To overcome these limitations, the researchers looked for some ClpCNTD ligands that could replace pArg. Among them, the most characteristic ClpCNTD-directed antibiotic is CymA, whose binding site is highly conserved in ClpC1 unfolding enzyme, but does not exist in ClpC protein of Gram-positive bacteria, so it can selectively target mycobacterial protease. In addition, CymA can also be encapsulated by mycobacteria. And because cytobiotin competes to bind to mSA and hinders BacPROTAC activity, the researchers further modified the POI ligand of BacPROTAC to JQ1, where JQ1 is a widely used BRDTBD1 ligand in PROTAC. These data confirm that BacPROTAC is a multi-functional molecular tool that can be used to replace different target protein ligands and degradation induction module ligands with various protein substrates via modular design.
Evaluation of BacPROTAC activity at the cellular level is required following adequate protein level characterization. The researchers created a mycobacterium that could produce the BRDTBD1 protein permanently because it was not expressed in the bacteria. Studies showed that BacPROTAC-3 decreased the level of BRDTBD1 in bacteria in a dose-dependent manner, and proteomic tests indicated that only BRDTBD1 was degraded considerably, demonstrating that the medication had strong specificity.
In general, this study developed a new bifunctional small molecule BacPROTAC, which extends the targeted protein degradation technology to bacteria. BacPROTAC can open up a new field of vision and develop antimicrobial agents with high selectivity and species specificity due to the benefits of the bifunctional small molecule modular design. BacPROTAC also offers the ability to study the function of bacterial proteins and discover new drug targets in addition to producing new antimicrobial agents. However, it is worth mentioning that there are still some limitations in this study, such as the low degradation efficiency of target proteins in vivo and the limited cell permeability of BacPROTAC. It is necessary to further optimize the design and prove that endogenous bacterial proteins can be degraded in the same way.