Targeted Protein Degradation of β-Lactamases and DHX36: Developing Bioconjugate Strategies Using BacPROTACs and rG4-PROTACs

Abstract

Targeted protein degradation (TPD) has been introduced to induce the elimination of target proteins via hijacking the cellular protein degradation system. TPD has shown great promise for medical chemistry and drug discovery, with molecules being developed against well-established cancer targets and even explored for “undruggable” targets, particularly proteolysis-targeting chimeras (PROTACs). PROTACs exhibit greater efficacy than traditional inhibitors due to their catalytic mode of action. Although TPD has been adopted and widely utilized in clinical trials, few PROTACs have been developed to tackle infectious and RNA-binding protein-associated diseases.

β-lactam antibiotics are the most frequently used antibacterial agents, but they are losing effectiveness due to the rapid spread of antimicrobial resistance (AMR) genes. β-lactam resistance genes, encoding β-lactamase enzymes that hydrolyze the core β-lactam ring, are the most common resistance mechanism in Enterobacterales. β-lactamase inhibitors have been introduced to counteract this issue. However, resistance to some inhibitors restricts their further applications. Novel approaches that counter AMR are urgently needed. Recently, bacterial proteolysis-targeting chimeras (BacPROTACs) have been expanded to bacteria with potential antibacterial applications. They redirect the ClpC-ClpP protease, present in Gram-positive bacteria and mycobacteria, to target drug-resistance proteins in a highly specific manner. Nevertheless, no BacPROTACs have been reported in Gram-negative bacteria involved in most bacterial infections. In this study, we designed a novel Gram-negative specific BacPROTACs strategy to report the first AMR ligand-peptide conjugate, NacssrA-1. This conjugate can specifically recognize the β-lactamase CTX-M-14 and unfoldase ClpX with high affinity. Notably, we determined the efficacy of NacssrA-1 in interacting with β-lactamase CTX-M-14 and demonstrated this conjugate could induce CTX-M-14 degradation by redirecting protease complex ClpXP in vitro and bacterial cells. Moreover, this conjugate can potentially re-sensitize the cefotaxime-resistant E. coli. Overall, we have generated a novel strategy for developing an adjuvant to promote the utilization of antibiotics by eliminating the key AMR proteins in Gram-negative bacteria. The innovative bioconjugation strategy reported here is not limited to ligands and could be extended to other previously disregarded compounds, peptides, or nucleic acids. To the best of our knowledge, this study is the first to design an AMR ligand-peptide conjugate and highlight its importance in targeting AMR protein degradation in Gram-negative bacteria.

In addition to the above application of TPD in bacteria, we have introduced it to RNA-binding protein degradation. RNA G-quadruplexes (rG4s) are non-canonical secondary nucleic acid structures in the transcriptome. They play a crucial role in gene regulation by interacting with G4-binding protein (G4BP) in cells. rG4-G4BP complexes have been associated with human diseases, making them an essential target for drug development. Creating innovative molecular tools to disrupt rG4-G4BP interactions will provide a unique opportunity to explore new biological mechanisms and potentially treat related diseases. DEAH-BOX helicase 36 (DHX36) regulates gene expression by binding and unwinding rG4 structures in mRNAs and non-coding RNAs. DHX36 is a key regulator for the translation of rG4-containing transcripts. As such, developing DHX36 targeting tools is a promising avenue to control the rG4-mediated gene activity. However, there are currently limited DHX36 targeting tools in the field of rG4, so developing novel tools to characterize and interfere with rG4-DHX36 interactions becomes timely and essential. Here, we rationally designed and developed a robust click-chemistry-based ligation strategy to report the first rG4-based proteolytic targeting chimeras, rG4-PROTACs, aiming at degrading G4-binding proteins, such as DHX36. Systematic mapping reveals that rG4-PROTACs predominantly and selectively degrade DHX36 through a proteosome-dependent mechanism, which promotes the formation of the rG4 structure in mRNA, leading to the translation inhibition of rG4-containing transcripts. Notably, rG4-PROTACs inhibit 3'UTR rG4-mediated native APP protein expression and impact the proliferative capacity of skeletal muscle stem cells by negatively regulating Gnai2 protein expression. Overall, we introduced a new and important DHX36 targeting tool for studying rG4-DHX36 interaction and biology in cells. The innovative bioconjugation strategy reported here is not limited to targeting and degrading DHX36 and could likely be extended to other G4BPs. Furthermore, the novel rG4-PROTACs tool can promote the development of PROTACs technology not only based on G-quadruplexes, but also on other non-canonical structures of nucleic acids.

Taken together, the first work exemplified reprogramming Gram-Negative bacterial protease for targeted protein degradation. The methodology used in this work could apply to other antimicrobial resistance proteins and inspire further development of new antimicrobial agents. In the second work, we developed rG4-based PROTACs, which provide a new avenue to understand rG4-G4BP interactions and the biological implications of dysregulated G4BP, promoting the development of PROTACs technology based on the non-canonical structure of nucleic acids.

Date of Award	25 Jul 2025
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Chi Kong Terrence LAU (Supervisor) & Liang ZHANG (Co-supervisor)