Biology subjects

Blazkova, K.

Publications and source records attributed to Blazkova, K..

4 recordsLinked to original sources

bioRxiv2025

Macrocyclic phage display for identification of selective protease substrates

Traditional methods for identifying selective protease substrates have primarily relied on synthetic libraries of linear peptides, which offer limited sequence and structural diversity. Here, we present an approach that leverages phage display technology to screen large libraries of chemically modified cyclic peptides, enabling the identification of highly selective substrates for a protease of interest. Our method uses a reactive chemical linker to cyclize peptides on the phage surface, while simultaneously incorporating an affinity tag and a fluorescent reporter. The affinity tag enables capture of the phage library and subsequent release of phages expressing optimal substrates upon incubation with a protease of interest. The addition of a turn-on fluorescent reporter allows direct quantification of cleavage efficiency throughout each selection round. The resulting identified substrates can then be chemically synthesized, optimized and validated using recombinant enzymes and cells. We demonstrate the utility of this approach using Fibroblast Activation Protein alpha (FAP) and the related proline-specific protease, dipeptidyl peptidase-4 (DPP4), as targets. Phage selection and subsequent optimization identified substrates with selectivity for each target that have the potential to serve as valuable tools for applications in basic biology and fluorescence image-guided surgery (FIGS). Overall, our strategy provides a rapid and unbiased platform for effectively discovering highly selective, non-natural protease substrates, overcoming key limitations of existing methods.

biochemistry↗

bioRxiv2025

Identification of a secreted protease from Bacteroides fragilis that induces intestinal pain and inflammation by cleavage of PAR2

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

microbiology↗

bioRxiv2024

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

bioengineering↗

bioRxiv2024

Mitochondrially targeted deferasirox kills cancer cells via simultaneous iron deprivation and ferroptosis induction.

In principle, two separate ways to target cancer cells exist, the first one using iron deprivation and subsequent dysfunction of iron-dependent enzymes, and the second one inducing iron overload that leads to cell death known as ferroptosis. In this study, we introduce a compound that uniquely employs both pathways at once - mitochondrially targeted deferasirox (mitoDFX). MitoDFX deprives cells of biologically active iron while simultaneously depleting the primary cellular antioxidant glutathione (GSH) and inducing lipid peroxidation, both of which are hallmarks of ferroptosis. The role of the GSH is further supported by enhanced cell death in glutathione peroxidase 4 KO cells (GPX4 KO) induced by mitoDFX. This response is further exacerbated by simultaneous inhibition of glutathione metabolism or the pentose phosphate pathway. MitoDFX strongly affects the structure and function of mitochondria, leading to its fragmentation, ROS production and induction of mitophagy. Moreover, we found that mitoDFX treatment markedly reduces mitochondrial translation and downregulates levels of enzymatic subunits coupled with the ETC/TCA cycle as well as DNA replication and transcription, which could explain its strong cytostatic effects. In summary, mitoDFX achieves high activity and selectivity against cancer cells that seems unattainable for the conventional untargeted iron chelators, which have adverse effects on systemic iron metabolism. Our novel, targeted chelator has also shown enhanced efficacy in animal models, surpassing other mitochondrially targeted drugs and making mitoDFX a promising candidate for the development of future selective and highly effective cancer therapies.

cancer biology↗