Mass cytometry-compatible chemical probes for the investigation of proteolytic enzymes

Mass cytometry is one of the newest and most high-throughput technologies that allows for the investigation of complex biological systems at single cell level. It relies on the use of stable metal isotopes as labels of specific cell markers and therefore, allows for simultaneous analysis of more than 40 parameters at single cell level. In order to fully explore the potential of mass cytometry, researchers are trying to develop new experimental setups based on the application of pure metal isotopes in biological studies. The incorporation of antibodies into mass cytometry setups, while extremely selective and well-validated, limits the analysis as it shows the whole protein pool present in the cell. In our group, we developed new technology that allows for the identification of active forms of proteins-the ones that actively participate in cell signaling pathways. Activity-based probes are the most valuable tools for enzyme activity profiling and for years now they have been in the center of the method called Activity-Based Protein Profiling. Classic activity-based probes consist of three parts: a warhead (electrophilic binding group that covalently modifies enzyme active site), linker (specific peptide sequence or non-specific carbon chain) and the fluorescent tag that allows for enzyme detection and localization inside the cell. Spectral properties of commercially available fluorophores allow for the detection of up to dozen different cell parameters, with the use of various techniques such as confocal microscopy or flow cytometry. To increase the number of analyzed parameters, we designed activity-based probes that possess DOTA chelating moiety that is able to trap one metal atom per one probe. The combination of mass cytometry with highly selective activity-based probes allowed for the development of new technology that grants the possibility of multiparametric analysis of complex biological samples such as blood or cancer tissue. The new type of activity-based probes (so-called TOF-probes) incorporate various inhibitor scaffolds designed with HyCoSuL technology (Hybrid Combinatorial Substrate Libraries). These compounds possess a variety of unnatural amino acids in their structures, which significantly increases their selectivity toward proteases of interest

Wizualizacja enzymów proteolitycznych za pomocą sond chemicznych oraz cytometrii masowej

Proteolytic enzymes, also known as peptidases or proteases, are protein catalysts that are primarily responsible for the hydrolysis of a peptide (amide) bond in peptide and protein substrates. By selective hydrolysis of selected substrates, these enzymes control many physiologically important processes including programmed cell death, blood coagulation cascade, protein maturation, fibrinolysis and many others. On the other hand, however, the imbalance in proteases activity leads to the development of diseases, including cancer, neurodegenerative diseases and coronary diseases etc.. In recent decades there has been great progress in studying the biological functions of many proteolytic enzymes. These observations were made possible through the use of various research techniques including genomics, epigenomics and proteomics. However, a major limitation of these techniques is the lack of information about the exact catalytic activity of the enzymes. For this reason, chemical probes are the most convenient toll for functional investigation of proteolytic enzymes. According to the generally accepted convention, chemical probes are compounds that can detect the catalytic activity of proteolytic enzymes. In general, chemical-based probes (activity-based probes, ABPs) consist of three main components: (1) a reactive binding group that binds permanently to the enzyme active site, (2) a recognition sequence (usually a peptide), which is responsible for the selective binding of a given probe to an individual enzyme or group of enzymes, and (3) a tag, mainly a fluorophore, enabling for detection of the probe-enzyme complex. However, the current limitation of ABPs is that only up to four enzymes can be detected and visualized in parallel, which significantly impedes their application for multi-parametric analysis. To date, the detection of proteases with the use of ABPs was limited to individual enzymes being investigated one by one, thus the obtained picture was far from being complete. In this review we describe the development of a new type of enzyme ABPs, so called TOF-probes that are compatible with mass cytometry format. The application of metal isotopes instead of fluorophores, makes possible to significantly increase the number of enzymes, which can be simultaneously visualized using chemical probes. Mass cytometry is a revolutionary technology that adopts atomic mass spectrometry into flow cytometry applications. The excellence of this method is that each metal isotope (mostly from lanthanides) has its own peak on mass spectrum, which eliminates the problem of signal overlap, thus allows for monitoring of more than 40 parameters at single cell level

Enzymatic Synthesis of Lipophilic Esters of Phenolic Compounds, Evaluation of Their Antioxidant Activity and Effect on the Oxidative Stability of Selected Oils

The aim of the study was to compare the effect of the substituent and its position in the aromatic ring on the antioxidant activity of hexanoic acid esters obtained in reactions catalyzed by immobilized lipase B from Candida antarctica. 4-Hydroxybenzyl hexanoate, 2-hydroxybenzyl hexanoate, 4-methoxybenzyl hexanoate, and vanillyl hexanoate were obtained with conversion yields of 50 to 80%. The antioxidant activity of synthesized esters, their alcohol precursors and BHT (Butylated HydroxyToluene) was compared with DPPH (2,2-diphenyl-1-picrylhydrazyl), CUPRAC (cupric ion reducing antioxidant capacity), and CBA (crocin bleaching assay) methods. Furthermore, it was investigated whether the presence of vanillyl hexanoate in a concentration of 0.01 and 0.1% affected the oxidative stability of sunflower and rapeseed oils in the Rancimat test. It was observed that the antioxidant activity of hexanoic acid esters depends on the presence and position of the hydroxyl group in the aromatic ring. The highest activities were found for vanillyl alcohol, vanillyl hexanoate, and BHT. The addition of the ester and BHT significantly extended the induction times of the tested oils, and these compounds exhibited similar activity. Vanillyl hexanoate increased the induction time from 4.49 to 5.28 h and from 2.73 to 3.12 h in the case of rapeseed and sunflower oils, respectively

Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors

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Structural determinants of substrate specificity of SplF protease from Staphylococcus aureus

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system

SARS-CoV-2 Mpro^{pro} inhibitors and activity-based probes for patient-sample imaging

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-dTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 M$^{pro}$. We visualized active SARS-CoV-2 M$^{pro}$ in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests