Bertozzi et al. have demonstrated that a degradation technology targeting extracellular proteins called Lysosome-targeting chimaeras (LYTAC) can successfully degrade epidermal growth factor receptor (EGFR), programmed death ligands 1 (PD-L1) and other membrane proteins^[2]. Autophagy-targeting chimera (AUTAC) that degrade target proteins through the lysosomal pathway has also been reported. Moreover, Yuhua Fu et al. developed ATTEC that can target pathogenic proteins for autophagic degradation. Recently, this group used lipid droplets (LDs) as exemplar targets and demonstrated that ATTEC can degrade non-protein biomolecules^[3]. Additionally, an RNA degrader RIBOTAC was constructed with the idea of PROTAC protein degrader.

However, PROTAC and the other approaches mentioned above are limited to targeted degradation of substrates in eukaryotes and are not yet applicable in bacteria and other prokaryotes.

Most recently, Morreale et al. developed Clpcp-dependent BacPROTACs, expanding the technology of targeted protein degradation to bacteria with potential antibacterial applications.

Previously, Tim Clausen et al. found that the ClpC–ClpP (ClpCP) protease, constituted by the AAA unfoldase ClpC and the protease ClpP, is an important proteolytic machine for the clearance of unfolded and aggregated proteins in B. subtilis and other gram-positive bacteria^[4]. The docking site for phosphoarginine (pArg) is located in the amino-terminal domain of the ClpC ATPase, therefore, pArg can function as a bona fide degradation tag for the ClpC–ClpP protease.

BacPROTACs, composed of a POI ligand, a chemical linker and a ClpCNTD anchor, can induce in vitro and in vivo degradation of non-eukaryotic proteins in bacteria without the ubiquitin proteasome system^[5]. As shown in Figure 2A, Morreale et al. first used monomer streptavidin (mSA) as a model protein. BacPROTAC-1 was synthesized by linking pArg (ClpCNTD ligand) with biotin (mSA ligand). BacPROTAC-1 binds to mSA and ClpCNTD with K_Ds of 3.9 and 2.8 μM (Figure 2C)^[5]. The formed ternary complex of BacPROTAC-1, ClpCNTD, and mSA leads to the effective degradation of the target protein.

Due to the chemical instability of the phosphoguanidinium group and poor PK properties for pArg-based PROTACs, the endogenous pArg was replaced with the highly selective sCym-1. sCym-1 can not only bind to ClpCP of Bacillus subtilis, but also to ClpC1P1P2 of Mycobacterium^[5].

Endogenous biotin could compete for binding to mSA and interfere with the formation of the ternary complex, thus hindering the activity of biotin based BacPROTACs. In the novel designed BacPROTACs, sCym-1 or its cyclomarin analogs are linked to JQ1. Bromodomain-1 (BD-1) is the model POI and the substrate of JQ1 . The new BacPROTACs with natural cyclomarin derivative dCycmM that binds to ClpCNTD, promote the degradation of BRDT_BD1 in a concentration-dependent manner.

To sum up, BacPROTACs direct bacterial ClpCP proteases to substrates in a highly specific manner. These BacPROTACs not only induce proximity between substrate and protease, but also promote the reassembly of inactive ClpCP decamers into active hexamers, directly triggering the hydrolysis of target proteins by ClpCP^[5].

Molecular glue, another TPD (targeted protein degradation) approach, is progressing in a remarkable pace. Molecular glues refer to a class of molecules without linker that can induce protein proximity and degradation, such as Thalidomide, CC-92480 , CC-90009 , etc. Molecular glue is closer to traditional small molecules in structure and has more advantages in membrane permeability and bioavailability, which can provide a new avenue to modify and improve PROTAC-type degraders.

The ultimate goal of degradation agents is to degrade the associated proteins, whether it is PROTAC dependent on the ubiquitinase pathway, LYTAC dependent on the proteasome pathway, or BacPROTAC dependent on the ClpCP protease. Distinct from K_d determination, western blots are more intuitive to evaluate the levels of the relevant proteins degraded by UPS with PROTACs, lysosome with LYTAC, or ClpCP with BacPROTAC. Changes in protein levels indicate the efficacy of these TPD agents.

PROTAC has derived a class of molecules with the POI ligand-Linker-degradation system guide as the model. Such molecules complement the deficiencies of PROTAC as a degradation agent in the degradation of certain protein and non-protein molecules, giving the concept of a degradation agent more possibilities. In addition to the difference in activity test results with conventional molecules caused by the molecule's own mechanism of action, the appropriate degradation pathway has a particularly great influence on the activity.

MCE is the world's leading supplier of chemicals including bioactive compounds for translational science and biological research. We can supply a wide variety of TPD products such as PROTACs, AUTACs, ATTECs, molecular glues and their building linkers. We also provide integrated synthesis services for PROTAC products.