Scientists Build Screening Toolkit to Find Bait Ligands for Antibacterial Protein Degradation Drugs

Antimicrobial resistance has quietly become one of the defining medical crises of the decade. According to the World Health Organization, one in six bacterial infections in 2023 was caused by a strain resistant to antibiotics, a figure that underscores how quickly the pharmaceutical arsenal is eroding. Now, a team of researchers at the University of Nebraska Medical Center and Iowa State University has developed a set of high-throughput laboratory methods designed to accelerate a fundamentally new kind of antibiotic discovery: drugs that work not by blocking a bacterial enzyme, but by deliberately marking bacterial proteins for destruction.

The strategy the Nebraska and Iowa State teams pursued rests on a piece of bacterial machinery that has, until recently, attracted more attention from microbiologists than from drug developers. The caseinolytic protease system, or Clp system, is a two-part molecular machine found across a broad range of bacterial species. One component, ClpP, is the protease that actually chops proteins into fragments. The other component is a family of ATP-powered unfoldases, including ClpC, that grab onto proteins destined for destruction, unwind their three-dimensional structures, and feed the unfolded chains into the ClpP chamber for degradation. Because the cell relies on this system to clear damaged or misregulated proteins, the pathway is essential for bacterial survival, and both overstimulating and inhibiting it can be lethal to the cell. That dual vulnerability is precisely what makes the system attractive as a drug target, since it offers two distinct pharmacological angles of attack.

What distinguishes ClpC from many other bacterial proteins is the way it selects its substrates. In Gram-positive bacteria and several other pathogens, a kinase enzyme called McsB decorates target proteins with a phosphate group attached to an arginine residue, a modification known as phospho-arginine. The N-terminal domain of ClpC, roughly the first 145 amino acids of the protein, acts as the reading head for this tag, recognizing the phospho-arginine mark through conserved binding pockets. Once a tagged substrate is captured, adaptor proteins help assemble ClpC into its active multimeric form, ATP hydrolysis drives the unfolding of the cargo, and ClpP completes the destruction. In effect, the phospho-arginine receptor on ClpC functions as a molecular docking port for the cell’s own protein-destruction labels.

This recognition mechanism is the conceptual foundation for the emerging field of antibacterial targeted protein degradation. In cancer research, proteolysis-targeting chimeras, or PROTACs, have shown that bifunctional molecules can drag disease-relevant proteins toward cellular degradation machinery, eliminating targets that resist conventional inhibition. Bacterial analogs, sometimes called BacPROTACs, apply the same logic: one end of the molecule binds a bacterial protein of interest, while the other end engages a degradation signal receptor such as the phospho-arginine binding site on ClpC. Proof-of-concept work has already demonstrated that such molecules can drive degradation of recombinant proteins in vitro and can modulate protein turnover inside living mycobacteria. The practical bottleneck has been finding suitable small-molecule ligands for the degradation machinery itself, ligands that can serve as the anchoring end of these bifunctional compounds.

The new study addresses that bottleneck by building screening infrastructure around the ClpC N-terminal domain. The researchers first established a differential scanning fluorometry workflow, a technique that monitors how the thermal stability of a protein changes when small molecules bind to it. A ligand that stabilizes a protein typically raises the temperature at which the protein unfolds, producing a detectable shift in a melting curve. This method is inexpensive, miniaturizable, and well suited to scanning large fragment libraries, but it does not by itself reveal whether a binder competes with the natural phospho-arginine substrate or binds somewhere else entirely. It therefore served as a first-pass filter to nominate candidate ligands for more rigorous characterization.

The second and more decisive tool was a fluorescence polarization displacement assay built on a custom-synthesized fluorescent probe. The probe is derived from phospho-arginine itself, the natural degradation tag, and carries a fluorescent label. When the probe is bound to the ClpC N-terminal domain, its rotational freedom is restricted, and polarized light emitted after excitation remains highly polarized. When a competing ligand displaces the probe, the freed fluorophore tumbles rapidly in solution and the polarization signal drops. By titrating candidate ligands and measuring how effectively they strip the probe from the protein, researchers can quantify binding affinity with precision. The reported dissociation constants for the probe across multiple ClpC homologs ranged from 0.1 to 0.8 micromolar, a range tight enough to support robust high-throughput screening while remaining loose enough that competitive ligands can realistically displace it.

A notable strength of the work is its cross-species design. Rather than optimizing an assay for a single organism, the team characterized ClpC N-terminal domains from an assortment of pathogenic bacteria, including Staphylococcus aureus and Chlamydia trachomatis, both of which yielded new X-ray crystal structures of the domain. ClpC is among the most widely conserved AAA+ unfoldases in bacteria, present in the ESKAPE pathogens that account for many hospital-acquired resistant infections, as well as in Mycobacterium tuberculosis, Listeria monocytogenes, Clostridioides difficile, and Escherichia coli. A screening platform that functions across homologs allows medicinal chemists to compare how a given ligand behaves against the ClpC of different species, opening the door to either broad-spectrum antibacterials or pathogen-selective compounds designed to spare commensal bacteria.

The crystal structures themselves carry practical significance. By revealing the geometry of the phospho-arginine binding pockets and the surrounding surface features, they provide templates for computational docking, which the researchers performed against three homologs using a panel of confirmed binders. Docking results suggested possible allosteric interactions, meaning some ligands may influence substrate recognition not by occupying the phospho-arginine pocket directly but by reshaping the domain from a nearby site. Allosteric ligands are often prized in drug discovery because they can offer selectivity that active-site competitors lack, and because resistance mutations that block an active site may not affect a distant allosteric pocket.

The study also situates the phospho-arginine receptor within a broader landscape of known ClpC-directed chemistry. Natural product antibiotics, including cyclomarin A, ecumicin, rufomycin, and lassomycin, bind a conserved surface on the ClpC N-terminal domain and exert antimicrobial effects by dysregulating the unfoldase, activating its ATPase activity while interfering with productive ClpC-ClpP complex formation. Cyclomarin A, for instance, shows activity against both replicating and non-replicating Mycobacterium tuberculosis. However, that conserved binding surface has been shown to be vulnerable to mutational resistance, a reminder that any single-site ligand faces evolutionary pressure. Combining multiple binding sites, or pairing a degradation-anchor ligand with a payload targeting an essential protein, may complicate the emergence of resistance.

Earlier Clp-directed drug discovery has largely centered on ClpP itself. Acyldepsipeptide antibiotics bind ClpP and force it into unregulated proteolysis of loosely folded proteins, while simultaneously blocking productive interaction with unfoldases. These compounds have validated the Clp system as a druggable axis but have required optimization for stability, specificity, and bioavailability. The current work shifts attention upstream, to the substrate-recognition step controlled by the ClpC N-terminal domain, which offers a complementary and potentially more controllable point of intervention.

For the targeted protein degradation field, the practical value of these assays lies in what comes next. A validated fluorescence polarization displacement assay is compatible with the multiwell plate formats used in high-throughput screening facilities, meaning thousands to hundreds of thousands of compounds can be evaluated for phospho-arginine-competitive binding in a matter of days. Hits from such screens can then be triaged by thermal shift data, structural models, and cross-homolog profiling before any synthesis of bifunctional molecules begins. This tiered workflow, from fragment screening through displacement confirmation to structural validation, mirrors the pipelines that matured in oncology PROTAC discovery and could compress the timeline for antibacterial degrader programs.

The broader context is sobering. Antimicrobial resistance has been attributed in part to overconsumption of antibiotics, and the pipeline of genuinely new drug classes has thinned over decades. Approaches that hijack bacterial protein degradation rather than simply inhibiting enzymatic activity represent a mechanistically distinct modality, one that can in principle eliminate proteins previously considered undruggable. By supplying the screening tools, probe reagents, and structural maps needed to identify anchor ligands for the ClpC N-terminal domain, this study lowers a concrete barrier to that modality. Whether such anchor ligands can be optimized into compounds with the potency, permeability, and selectivity required of real drugs remains an open question, but the infrastructure to begin answering it now exists.

Subject of Research: High-throughput identification of ClpC N-terminal domain bait ligands for antibacterial targeted protein degradation drug discovery

Article Title: High-throughput methods for identification of ClpC N-terminal domain bait ligands for antibacterial targeted protein degradation drug discovery

Article References: Erickson, H. E., Jayasinghe, Y. P., Sheth, Z., Wolfe, D. B., Roche, J., & Ronning, D. R. (2026). High-throughput methods for identification of ClpC N-terminal domain bait ligands for antibacterial targeted protein degradation drug discovery. Frontiers in Chemical Biology, 5, Article 1922299. https://doi.org/10.3389/fchbi.2026.1922299

Image Credits: AI Generated

DOI: 10.3389/fchbi.2026.1922299

Keywords: antimicrobial resistance, ClpC, targeted protein degradation, BacPROTAC, fluorescence polarization, thermal shift assay, phospho-arginine, drug discovery, Staphylococcus aureus, Mycobacterium tuberculosis, X-ray crystallography, molecular docking