BacPROTACs: Novel PROTACs Targeting the ClpCP
Bacterial infections kill hundreds of thousands of people every year, especially in low - and middle-income countries. Considering that only a small number of antibiotics have been approved in the last 50 years, the development of new antibiotics also faces the challenge of low permeability of the bacterial envelope and few specific targets. The difficulty of finding effective antimicrobials is further compounded by the rate at which pathogens develop resistance to existing drugs. Given this uneven development, the resurgence of bacterial epidemics is a big threat, and innovative strategies to fight infections are urgently needed.
PROTAC (proteolysis targeting chimera) is a targeted protein degradation technique that uses small molecules to regulate protein levels. After 20 years of development, PROTAC technology has been continuously improved and is now widely used in the development of a variety of tumor therapeutics. However, ubiquitin proteasome system is unique to eukaryotic cells, and prokaryotic cells such as bacteria cannot utilize proteasome in eukaryotic cells, so PROTAC based on ubiquitin proteasome system cannot be applied to bacteria. In 2016, Tim Clausen's team found that the ClpC:ClpP (ClpCP) protease of Bacillus subtilis recognizes and degrades proteins with phosphorylated arginine. In 2022, the team used the bacteria's ClpCP system to develop a functional molecule that could be applied to bacterial cells: BacPROTACs. BacPROTACs uses the ClpCP in bacteria to target the target protein with high specificity and achieve selective degradation of the bacterial protein, resulting in the death of the bacteria.
Design of BacPROTACs
Although bacteria do not have the ubiquitin proteasome system of eukaryotic cells, the ClpCP system within bacterial cells has a similar function of protein degradation. In gram-positive bacteria and mycobacteria, ClpCP is a folding enzyme driven by ATP. The arginine of the target protein is phosphorylated to form phosphorylated arginine residues (pArg), after which the ClpCP protease recognizes the target protein labeled by pArg, the target protein is bound to the ClpCP protease receptor domain, and the ClpCP protease degrades the target protein. It can be seen that the recognition and mechanism of ClpCP proteasome is much simpler than that of eukaryotic proteasome.
The researchers used mSA (monomer streptavidin) as a model protein. BacPROTAC-1 was designed to bind pArg to biotin (a high-affinity mSA ligand) to form an active ternary complex structure. BacPROTAC-1 makes mSA and ClpCNTD "meet", and 100 μM BacPROTAC-1 can selectively degrade mSA.
mSA-Kre is considered to be the most specific binding substrate and can be degraded by 1 μM BacPROTAC-1. Kre has an unstructured C-terminal chain with a length of 28 amino acids, and its effective degradation may be related to the extended tail of this C-terminal, indicating that the structural characteristics of the substrate significantly affect the degradation efficiency. Parg-containing BacPROTAC can recruit POI to the ClpCNTD domain and be degraded by ClpCP proteases. In addition to the binding properties of the chemical joint, the intrinsic properties of the target protein play an equally important role in the degradation efficiency. The specific degradation of BacPROTAC was confirmed.
Fig. 1 In vitro reprogramming of B. subtilis ClpCP by BacPROTAC-1. (Morreale, F. E., 2022)
Activation mechanism based on pArg recognition
ClpC exists in the resting state as a decamer with a broken AAA ring. The interaction with the adaptor MecA destabilizes the decamer and promotes the assembly of the functional hexamer with the active arrangement of the ATPase unit and activates the ClpC protease.
Parg-labeled ClpC substrates trigger remodeling of ClpC decamers. The four ClpC hexamers stabilize the resting state of the ClpC by interacting to coil the helical M-domain, and when the labeled substrate binds to the ClpC, the resting decamers are reshaped into active higher-order complexes. The structure of ClpC 24-mer indicates that pArg labeling not only acts as a degradation signal, but also mediates the formation of higher-order oligomers and the activation of ClpC. BacPROTAC, which contains parts of pArg, triggers this remodeling mechanism and thus acts not only as a chemical connector but also as an activator of ClpCP proteases.
BacPROTAC in mycobacterium
The major disadvantages of BacPROTAC are poor pharmacokinetics and chemical instability of guanidine phosphate group. Therefore, it is necessary to find ClpCNTD ligands similar to pArg. To further demonstrate the universality of the system, and to avoid biotin interventions that could affect BacPROTAC activity by competing mSA binding, the investigators turned to the cyclopeptide antibiotic CymA, a ClpCP protease system inhibitor designed to synthesize BacPROTAC-3. sCym-1 (CymA cyclic peptide) is a high-affinity ClpCNTD ligand. BRDT bromodomain-1(BD1) is a new model substrate, and its ligand JQ1 has been widely used in various PROTACs due to its high affinity. To target BRDTBD1, BacPROTAC-3 connects sCym-1 and JQ1. The data suggest that BacPROTACs represents a versatile molecular tool suitable for a variety of POI substrates and for integrating different degradation-inducing ligands.
Fig. 2 Chemical structure of BacPROTACs-1, -2 and -3. (Bonjorno, A. F., 2024)
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Induces the degradation of mycobacterium intracellular target protein
D-alanine synthase (DdlA) is an important part of peptidoglycan synthesis pathway, and is also the target of broad-spectrum antibiotic D-cycloserine (DCS). However, overexpression of DdlA can desensitize M. smegmatis to DCS. After inducing M. smegmatis to produce an excess of the fusion protein DdlA-BRDTBD1 and then treating bacteria with BacPROTACs, M. smegmatis with the fusion protein DdlA-BRDTBD1 regained their DCS sensitivity.
Threonine synthetase ThrC is an essential enzyme to catalyze the last step of threonine biosynthesis, and the lack of threonine leads to cell growth. The fusion protein BRDTBD1-THRC was expressed by integrating the BRDTBD1 coding region into the start codon of the ThrC gene of M. smegmatis. BRDTBD1-ThrC itself did not interfere with cell growth, but bacterial cell growth was inhibited after adding BacPROTACs, while bacterial cell growth was restored after adding L-threonine. The above experiments indicate that BacPROTACs can target fusion proteins in bacterial cells and mediate the degradation of target fusion proteins by ClpCP protease.
Treatment of tuberculosis with BacPROTACs strategy
TB is the leading cause of death from bacterial infections worldwide, and TB infections are increasing especially during the COVID-19 pandemic, making it urgent to develop new and further anti-TB strategies. Currently, one of the most promising drug targets for mycobacterium tuberculosis is the ClpC1:ClpP1P2 protease, which is a core component of the mycobacterium protein stabilization system and plays an important role in maintaining protein homeostasis and combating host-induced stress.
In order to improve the efficacy of anti-TB drugs that target Clp proteases, in 2023 the team developed a new BacPROTACs that can induce the self-degradation of essential Clp components in the mycobacterium tuberculosis protein stabilization system while overcoming the protein's own protective system. Thus, a new and effective antibiotic treatment strategy against Mycobacterium tuberculosis was introduced.
Fig. 3 BacPROTACs mechanism of action with Clp targeting. (Hoi, D. M., 2023)
The most studied natural antibiotics targeting ClpC1 are cyclomarin A and ecumicin. Therefore, the authors used quantitative proteomics to investigate the effects of cyclomarin A and ecumicin on the proteome of binding mycobacterium. Quantitative proteomics showed that antibiotics caused a large number of proteomic imbalances and induced the enrichment of two Clp proteins, both of which contain the ClpC1NTD domain, two unlabeled but conserved stress response factors, which the authors named ClpC2 and ClpC3, and speculated that they are important regulatory components of the Clp degradation system in mycobacteria.
Through phylogenetic analysis combined with sequence alignment and crystal structure comparison, the authors determined that ClpC1, ClpC2, and ClpC3 share a common receptor domain. Clpc1-directed antibiotics stimulate the Clp protease by binding to conserved hydrophobic points in the Clp repeat domain, while also blocking misfolded proteins. cyclomarin A and ecumicin hijacks the ClpC1P1P2 protease by mimicking the damaged protein. As a result, CLPC1-directed antibiotics can cause a significant imbalance in the proteome.
ClpC2 produces competitive inhibition of the specific degradation identified by ClpC1, chelates the potential substrate in the form of a competitive inhibitor and prevents its degradation, thus, ClpC2 exerts a safe protective role against ClpC1P1P2 protease. ClpC2 exerts a protective effect by reducing the effective concentration of desoxycyclomarin C in cells. Because of its protective function, security systems from ClpC2 must be considered when developing antibiotics that target the stress response mechanisms of mycobacteria.
In order to overcome the security system inherent in Clp, the author developed homo-BacPROTACs (HBPs) on the basis of BacPROTAC. In vitro degradation experiments showed that HBP could target the Clp CRD motif in vitro. While inducing ClpC1 to be degraded by ClpC1P1P2 protease, this compound can also target ClpC2 through its desoxycyclomarin C binding site, eliminating the protective effect of ClpC2. Together, this compound is highly effective in killing pathogenic mycobacterium tuberculosis. It is 115 times more potent than cyclomarin A, a traditional natural antibiotic. This excellent efficacy is due to PROTAC's unique mode of action: inducing degradation of the housekeeping proteins ClpC1 and ClpC2, rather than inhibiting them. BacPROTACs antibiotics overcome the safety system of bacterial protein quality control, are also active in bacteria with a dormance-like phenotype, and simultaneously target multiple components of the mycobacterial stress response.
Perhaps the most threatening bacterial pathogen in terms of antibiotic resistance and infection severity is Mycobacterium tuberculosis, which is the cause of tuberculosis. The study concluded that the BacPROTAC approach is an effective strategy for the development of anti-mycobacterium drugs that degrade the essential Clp protein while also overcoming Clp's built-in protective system. The mechanistic advantages of small molecule degraders over classical drugs seem to be reflected in BacPROTACs. The advancement of this technology provides an attractive technology platform for the development of the next generation of potent antibiotics.
References:
- Morreale, F. E., et al. BacPROTACs mediate targeted protein degradation in bacteria. Cell. 2022, 185(13): 2338-2353.
- Bonjorno, A. F., et al. BacPROTACs targeting Clp protease: a promising strategy for anti-mycobacterial drug discovery. Frontiers in Chemistry. 2024, 12: 1358539.
- Hoi, D. M., et al. Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival. Cell. 2023, 186(10): 2176-2192.
