Mapping the substrate-binding subsite specificity of a Porphyromonas gingivalis Tpr peptidase

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections

Unnatural amino acids as achemical tool for the development of protease substrates, inhibitors and activity - baseproblems

Proteolytic enzymes are molecular scissors that are responsible for the amide bond breakdown in peptide and protein substrates. Over the years, the view on proteases has been considerably changed from non-specific digestive enzymes to sophisticated biocatalysts, which by performing limited proteolysis control virtually all biological processes. In order to better understand how proteases work and what are their biologically relevant target substrates, it is indispensable to determine their catalytic preferences. This knowledge can be further utilized to develop selective substrates, inhibitors and activity-based probes (ABPs) enabling the monitoring of proteases activity in various settings, from in vitro analysis on recombinant enzymes or cell lysates to ex vivo and in vivo imaging at the single cell level. Among many chemical-based approaches that have been developed and applied over the years, the Hybrid Combinatorial Substrate Library (HyCoSuL) technology has emerged as one of the most powerful one. HyCoSuL is a combinatorial peptide-based library of fluorogenic substrates, that comprise natural and unnatural amino acids, that can deeply explore the chemical space in proteases active site, providing a structural framework for the development of highly-selective chemical tools. In this review we present the most prominent examples of proteolytic enzymes that have been profiled with HyCoSuL approach yielding selective substrates, potent inhibitors, and very sensitive activity-based probes

Fluorescent Activity-Based Probe for the Selective Detection of Factor VII Activating Protease (FSAP) in Human Plasma

The zymogen form of circulating Factor VII activating protease (FSAP) is activated by histones that are released as a consequence of tissue damage or excessive inflammation. This is likely to have consequences in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To investigate the existence, as well as the concentration of active FSAP (FSAPa) in complex biological systems an active site probe is needed. We used Hybrid Combinatorial Substrate Library (HyCoSuL) to screen for natural and unnatural amino acids that specifically bind to P4-P2 pockets of FSAPa. This information was used to designing a fluorogenic substrate (Ac-Pro-DTyr-Lys-Arg-ACC) as well as an irreversible, fluorogenic activity-based probe Cy5-6-Ahx-Pro-DTyr-Lys-ArgP(OPh)2. In normal human plasma the probe showed very low non-specific reactivity with some plasma proteins but upon activation of pro-FSAP with histones, strong labelling of FSAPa was observed. This labelling could be inhibited by aprotinin and was not found in the plasma of a subject that was homozygous for a polymorphism, which leads to loss of activity, or in plasma that was depleted of FSAP by antibodies. This 2nd generation substrate exhibited 6-fold higher catalytic efficiency than the 1st generation substrate and a much higher selectivity for FSAPa over other plasma proteases. This substrate and probe can be useful to detect and localize FSAPa in normal and pathological tissue and plasma to gain more insight into its functions

Perceived changes in the university students' health behavior after participating in the study on wearing high-heeled shoes

Wearing high-heeled shoes (HH) is a wide-spread practice among Western women, maintaining popularity despite its harmful potential. We examined the main motivation behind wearing HH in female students, as well as the possible change to wearing HH among the research participants. Methods: Thirty university students (N=30 females, age 21.8±2.09 years; weight: 55.7±4.05 kg, height: 1.66±0.03 m, BMI: 20.34±1.41 kg.m-2, shoe size: EU 36-38), who rarely wore HH. The participants wore HH for no longer than 6 hours per month during the period two past years. The SonoSens Monitor Analyzer system (Gefremed, Chemnitz, Germany) was used to observe participants' posture when walking and movements in individual sections of their spines. In a comparative experiment, the correlation between variables recorded when walking in two types of shoes was identified. The first pair of shoes (HH) had 7 cm heels whereas the second were flat sport shoes (FS). Results: The evaluation of participants' spines and posture when walking in HH and FS revealed significant differences (p < 0.05). The evaluation of the initial and follow-up surveys indicates that the main motivation for wearing HH among the test group was to increase their attractiveness and respond to social expectations. After participating in the research and becoming aware of its outcomes and issues, the test group's attitude to wearing HH has changed. Conclusions: In regards to good posture and spinal health, this study has raised some awareness amongst its participants, and has therefore proved to be a significant positive influence

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