Protease-targeted antimicrobial therapy is an example of a novel strategy in the therapeutics of infectious diseases. We at Protheragen focus on the development of protease-targeted antimicrobial therapies owing to our strength in molecular biology, biochemistry, and drug development. We offer complete solutions in drug development, starting from target identification and validation through preclinical evaluation.
Overview of Protease-targeted Antimicrobial Therapy
Antimicrobial therapy that targets protease is an innovative and unique approach to combating multi drug-resistant bacteria. The older antibiotics acted on the bacterial cell walls, membranes, or central metabolic pathways of the cells. With the use of vaccines and antibiotics, these mechanisms have been rendered useless due to the development of antibiotic resistance. There is now a need to find alternative ways of dealing with the problem. Protease would be able to help because they are enzymes that cleave peptide bonds of the proteins constituting the bacteria. This therapy can be done by engineering a protease that specifically targets to cleave vital bacterial proteins so as to disable the pathogen easily and without employing traditional antibiotics. Because of the targeted nature of this strategy, the therapy is likely to have fewer chances of resistance and offers a more controlled way of addressing infections.
Fig.1 Protease-targeted sensors. (Naga N. G., et al., 2023)
Development of Protease-targeted Antimicrobial Therapy
The development of protease-targeted antimicrobial therapies has gained significant attention due to the increasing antibiotic-resistant bacteria. Traditional antibiotics target bacterial cell wall synthesis, protein synthesis, or DNA replication. However, multidrug-resistant pathogens have led to the exploration of alternative targets like bacterial proteases. Notably, the AAA+ family of proteolytic complexes, involved in essential cellular functions, has shown potential as antimicrobial targets. Additionally, antimicrobial peptides (AMPs) resistant to protease degradation have been developed to enhance stability and efficacy. These peptides can target specific bacterial proteases or evade host protease degradation. The current landscape of protease-based antimicrobial therapy development is marked by advancements in understanding bacterial protease structure and function, as well as the identification of novel inhibitors and peptide-targeted agents.
Table 1. Quick overview of the most often studied protease inhibitors and their target. (Sandri A., et al., 2025)
| Target |
Inhibitor |
Note |
| Bacterial proteases |
N-mercaptoacetyl-Phe-Tyr-amid |
Potent LasB inhibitor, capable of blocking virulence processes, reducing biofilm growth |
| Sixteen compounds |
Optimization of LasB inhibitors suppressing IL-1β activation in macrophages and mouse lung infection models. |
| ANT3273 |
LasB inhibitor, able to suppress IL-1β activation and reduce bacterial burden in both cellular and mouse infection models. |
| Aprotinin |
A serine protease inhibitor has shown promise as an antimicrobial agent against Gram-positive and Gram-negative bacteria. |
| Marimastat, ilomastat |
They also demonstrated cross-reactivity with bacterial proteases, showing a reduction of lung inflammation in CF animal models. |
| MMP-2, MMP-9, and NE. |
Epigallocatechin gallate (EGCG) |
EGCG has been shown to inhibit MMP-2 and MMP-9, and it is a potent inhibitor of NE. It suppresses ROS activity, inhibits apoptosis in activated neutrophils, and acts on pulmonary inflammation in in vivo studies. |
| NE and bacterial proteases |
Marimastat and ilomastat |
Cross-reactivity with NE and bacterial proteases. |
| Dipeptidyl peptidase 1 (DPP-1) |
Brensocatib (INS1007) |
An oral reversible inhibitor of DPP-1 (responsible for the activation of neutrophil serine proteases) showed good results in CF patients. |
| DPP-1 |
BI 1291583 |
DPP-1 inhibitor showed good results and safety. A phase 2 trial on CF bronchiectasis is ongoing. |
| Serine proteases and a broad group of other protease |
Five alpha 1-antitrypsin constructs |
Synthetic oxidation-resistant inhibitors, showing also improved inhibitory activity. |
| NE |
VH-Fc 1D1.43 and IgG1 1C10 |
Two antibodies, developed against recombinant NE, show specificity against NE and potent inhibitory effects on its activity. |
| Serine proteases |
Nanobody (Nb4) |
Engineered nanobody derived from camelid antibodies explored for targeting serine proteases. |
| Serine proteases |
Liposome-encapsulated aprotinin |
Have been shown to accumulate efficiently in the lungs. |
Disclaimer: Protheragen focuses on providing preclinical research services. This table is for information exchange purposes only. This table is not a treatment plan recommendation. For guidance on treatment options, please visit a regular hospital.
Our Services
Protheragen focuses on protease-targeted antimicrobial therapy with specialization in bioinformatics, protein design, and in vitro and in vivo validations. Our extensive service offering enables us to assist our clients from the beginning of the target discovery to the preclinical evaluation stage.
Our Solutions
Target Identification and Validation
Protheragen's services begin with the identification and validation of protease targets. We employ a combination of bioinformatics, structural biology, and functional assays to pinpoint essential proteases in bacterial pathogens. Our team of experts utilizes advanced techniques such as proteomics and gene knockout studies to confirm the role of these proteases in bacterial survival and virulence. This rigorous validation process ensures that the selected targets are both biologically relevant and druggable.
Therapeutic Development
After validating a target, high-throughput screening (HTS) is used to identify potential inhibitors with antimicrobial activity from large compound libraries. In addition to small molecule drug development, we also provide therapeutic antibody and mRNA development services. Protheragen offers comprehensive therapeutic development services, providing clients with optimized candidates for preclinical evaluation.
Preclinical Research
Protheragen's preclinical research services include rigorous in vitro and in vivo testing. In vitro assays assess the antimicrobial efficacy of the compounds against various bacterial strains, providing data on the minimum inhibitory concentration (MIC) and bactericidal activity. Cytotoxicity assays are also conducted to ensure the compounds are safe for human cells. In vivo studies are performed in relevant animal models to evaluate the therapeutic efficacy and pharmacokinetics of the lead compounds.
Types of Protease Targets
AAA+ Proteases
AAA+ proteases, such as ClpP, are multimeric complexes that play crucial roles in protein homeostasis, stress response, and virulence. ClpP is regulated by associated AAA+ ATPases, which control substrate selection and unfolding. Inhibitors and activators of ClpP have shown promise in reducing bacterial virulence and enhancing antibiotic efficacy. Protheragen's expertise in this area includes the development of novel scaffolds and optimization of existing compounds to improve their potency, stability, and pharmacokinetic properties.
Signal Peptidases
Signal peptidases (SPs) are critical enzymes that function in the secretion of proteins by cutting the targeting peptides from the secreted proteins. Chemical inhibitors like β-lactam antibiotics and arylomycin lipopeptides have been used to successfully block both type I and type II SPs. These inhibitors bind to the active sites of SPs and consequently halt protein secretion, thus interfering with bacterial virulence. Protheragen’s services in this field aim at designing and refining SP-targeting inhibitors that could broaden the scope of available therapeutics for bacterial infections.
Other Protease Targets
Besides the AAA+ proteases and signal peptidases, other bacterial proteases also present intriguing possibilities as targets for antimicrobial interventions. For example, sortases are enzymes that anchor proteins to the bacterial cell wall and are essential for virulence in many Gram-positive pathogens. Inhibitors of sortases have shown efficacy in reducing bacterial colonization and virulence. Protheragen’s integrated strategy for developing therapies focuses on these and other protease targets, applying our knowledge of biochemistry and drug development to discover and refine new inhibitors and activators.
Types of Protease-targeted Antimicrobial Therapies
Protheragen is committed to providing custom solutions tailored to the unique needs of each client. We offer flexible collaboration models, from standalone services to comprehensive co-development partnerships. Our team works closely with clients to understand their goals and deliver innovative solutions that meet their specific requirements. If you are interested in our services, please feel free to contact us.
References
- Fink, Tina, and Roman Jerala. "Designed Protease-targeted signaling networks." Current opinion in chemical biology 68 (2022): 102146.
- Sandri, Angela, and Federico Boschi. "Exploring Proteases as Alternative Molecular Targets to Tackle Inflammation in Cystic Fibrosis Respiratory Infections." International Journal of Molecular Sciences 26.5 (2025): 1871.
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