Kristyna Blazkova

All Publications

• A Bacteroides fragilis protease activates host PAR2 to induce intestinal pain and inflammation. Cell host & microbe Lakemeyer, M., Latorre, R., Blazkova, K., Wood, H. M., Jensen, D. D., Shakil, N., Thomas, S. C., Saxena, D., Mulpuri, Y., Poolman, D., Duran, P., Keller, L. J., Reed, D. E., Schmidt, B. L., Jiménez-Vargas, N. N., Xu, F., Lomax, A. E., Bunnett, N. W., Bogyo, M. 2025

  Abstract

  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), conditions often associated with gut microbiome alterations. To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing to screen a library of diverse gut microbes. We identify multiple bacteria that secrete proteases capable of cleaving host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we uncovered a previously uncharacterized Bacteroides fragilis serine protease 1 (Bfp1) and show 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 an axis of host-commensal interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

  View details for DOI 10.1016/j.chom.2025.09.010

  View details for PubMedID 41015045

• Macrocyclic Phage Display for Identification of Selective Protease Substrates. Journal of the American Chemical Society Faucher, F. F., Blazkova, K., Lovell, S., Bertolini, M., Herrero-Bourdieu, J., Cosco, E. D., Bogyo, M., Barniol-Xicota, M. 2025

  Abstract

  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 (FAPalpha) 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.

  View details for DOI 10.1021/jacs.5c04424

  View details for PubMedID 40679920

• Identification of 6-Aryl-7-Deazapurine Ribonucleoside Phosphonates as Inhibitors of Ecto-5′-Nucleotidase (CD73) ACS PHARMACOLOGY & TRANSLATIONAL SCIENCE Simova, M., Ormsby, T., Sinkeviciute, U., Sirotova Veselovska, L., Cermakova, K., Hadzima, M., Barton, L., Stanurova, J., Kramna, A., Sacha, P., Tichy, M., Hocek, M., Konvalinka, J., Blazkova, K. 2025

  View details for DOI 10.1021/acsptsci.5c00180

  View details for Web of Science ID 001531635100001

• Polymer-Tethered Quenched Fluorescent Probes for Enhanced Imaging of Tumor-Associated Proteases. ACS sensors Hadzima, M., Faucher, F. F., Blažková, K., Yim, J. J., Guerra, M., Chen, S., Woods, E. C., Park, K. W., Šácha, P., Šubr, V., Kostka, L., Etrych, T., Majer, P., Konvalinka, J., Bogyo, M. 2024

  Abstract

  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 the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents 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 the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall 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.

  View details for DOI 10.1021/acssensors.4c00912

  View details for PubMedID 38941307

• Polymer-based antibody mimetics (iBodies) target human PD-L1 and function as a potent immune checkpoint blocker JOURNAL OF BIOLOGICAL CHEMISTRY Zamani, M., Hadzima, M., Blazkova, K., Subr, V., Ormsby, T., Celis-Gutierrez, J., Malissen, B., Kostka, L., Etrych, T., Sacha, P., Konvalinka, J. 2024; 300 (6): 107325

  Abstract

  Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.

  View details for DOI 10.1016/j.jbc.2024.107325

  View details for Web of Science ID 001345352100001

  View details for PubMedID 38685532

  View details for PubMedCentralID PMC11154707

• The development of a high-affinity conformation-sensitive antibody mimetic using a biocompatible copolymer carrier (iBody) JOURNAL OF BIOLOGICAL CHEMISTRY Blazkova, K., Beranova, J., Hradilek, M., Kostka, L., Subr, V., Etrych, T., Sacha, P., Konvalinka, J. 2021; 297 (5): 101342

  Abstract

  Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design.

  View details for DOI 10.1016/j.jbc.2021.101342

  View details for Web of Science ID 000723119000013

  View details for PubMedID 34710374

  View details for PubMedCentralID PMC8600089

• Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes ACS CHEMICAL BIOLOGY Simon, P., Knedlik, T., Blazkova, K., Dvorakova, P., Brezinova, A., Kostka, L., Subr, V., Konvalinka, J., Sacha, P. 2018; 13 (12): 3333-3342

  Abstract

  Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.

  View details for DOI 10.1021/acschembio.8b00791

  View details for Web of Science ID 000454568000015

  View details for PubMedID 30489064

• Polymers as tools for studying the internalization of membrane protein glutamate carboxypeptidase II Kostka, L., Sedlak, F., Blazkova, K., Etrych, T., Sacha, P., Subr, V., Konvalinka, J. ELSEVIER SCIENCE BV. 2017: E81-E82

  View details for DOI 10.1016/j.jconrel.2017.03.180

  View details for Web of Science ID 000407591400139

• In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control PLOS ONE Felker, A., Nieuwenhuize, S., Dolbois, A., Blazkova, K., Hess, C., Low, L. L., Burger, S., Samson, N., Carney, T. J., Bartunek, P., Nevado, C., Mosimann, C. 2016; 11 (4): e0152989

  Abstract

  Mutant Estrogen Receptor (ERT2) ligand-binding domain fusions with Cre recombinase are a key tool for spatio-temporally controlled genetic recombination with the Cre/lox system. CreERT2 is efficiently activated in a concentration-dependent manner by the Tamoxifen metabolite trans-4-OH-Tamoxifen (trans-4-OHT). Reproducible and efficient Cre/lox experimentation is hindered by the gradual loss of CreERT2 induction potency upon prolonged storage of dissolved trans-4-OHT, which potentially results from gradual trans-to-cis isomerization or degradation. Here, we combined zebrafish CreERT2 recombination experiments and cell culture assays to document the gradual activity loss of trans-4-OHT and describe the alternative Tamoxifen metabolite Endoxifen as more stable alternative compound. Endoxifen retains potent activation upon prolonged storage (3 months), yet consistently induces half the ERT2 domain fusion activity compared to fresh trans-4-OHT. Using 1H-NMR analysis, we reveal that trans-4-OHT isomerization is undetectable upon prolonged storage in either DMSO or Ethanol, ruling out isomer transformation as cause for the gradual loss of trans-4-OHT activity. We further establish that both trans-4-OHT and Endoxifen are insensitive to light exposure under regular laboratory handling conditions. We attribute the gradual loss of trans-4-OHT potency to precipitation over time, and show that heating of aged trans-4-OHT aliquots reinstates their CreERT2 induction potential. Our data establish Endoxifen as potent and reproducible complementary compound to 4-OHT to control ERT2 domain fusion proteins in vivo, and provide a framework for efficient chemically controlled recombination experiments.

  View details for DOI 10.1371/journal.pone.0152989

  View details for Web of Science ID 000374131700016

  View details for PubMedID 27077909

  View details for PubMedCentralID PMC4831813