Enzyme activity as a method for soil biochemistry and microbiology research has a long history of more than 100 years that is not widely acknowledged in terms of adherence to strict assay protocols ...and the interpretation of results. However, in the recent past, there is a growing lack of recognition of the historic advancements among researchers that use soil enzymology. Today, many papers are being published that use methods that either do not follow exact protocols as originally vetted in the research literature or individual labs use their own method that has not been optimized for pH, co-factors, substrate concentrations, or other conditions. This is of particular concern for fluorogenic substrates and microplate methods. Furthermore, there is a lack of understanding of the origin and location of a given enzyme being studied. Notably, regardless of the enzyme, it is too often assumed that enzyme activity equals microbial activity—which is not the case for most hydrolytic enzyme assays. Because as established by Douglas McLaren in the 1950s, a considerable amount of activity can come from catalytic enzymes stabilized in the soil matrix but that are no longer associated with viable cells (known as abiontic enzymes). In summary, today, many papers are using imperfect methods and/or misinterpret enzyme activity data that at a minimum confounds cross paper studies and meta-analysis. However, most importantly, lack of historical perspectives and ignoring strict protocols cause redundancy and fundamentally undermine the discipline and understanding of soil microbiology/biochemistry when enzymology methods are used.
The development of a versatile approach for the rapid and sensitive detection of relevant pathogenic bacteria and autonomous signaling of the detection events in reporter hydrogel film coatings is ...reported. Exploiting chitosan hydrogel films equipped with chromogenic or fluorogenic reporter moieties, the presence of the Gram‐negative bacterium Pseudomonas aeruginosa and the Gram‐positive bacterium Staphylococcus aureus is sensed within 1 h by detecting the characteristic enzymes α‐glucosidase and elastase with limits of detection (LOD) <45 × 10−9m and <20 × 10−9m, respectively, for this observation time. The values for the LOD are two to three orders of magnitude smaller than the concentrations of the enzymes detected in the corresponding bacterial supernatants. The results show that the covalently conjugated reporter moieties are exclusively and efficiently reacted by the associated enzyme, allowing in principle for discrimination among different types of bacteria. Since high enzyme concentrations are a result of proliferating bacteria, e.g., in wounds or food, and since the selectivity of the reporting function is easily adapted to bacteria of choice, these reporter hydrogels comprise an interesting platform for the rapid detection of bacteria.
A versatile approach for the rapid and selective detection of enzymes of pathogenic bacteria with a signaling hydrogel film coating is developed. Cleavage of reporter dyes from chitosan functionalized with chromogenic or fluorogenic reporter moieties reveals selectively the presence of P. aeruginosa versus S. aureus with low limits of detection.
The main protease of SARS‐CoV‐2 (Mpro), the causative agent of COVID‐19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched ...fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed Mpro. Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in‐depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of Mpro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards Mpro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (kinac/Ki=37 500 m−1 s−1, Ki=24.0 nm) and pyridyl ester 17 (kinac/Ki=29 100 m−1 s−1, Ki=10.0 nm), promising drug candidates for further development have been discovered.
The main protease (Mpro) of SARS‐CoV‐2 has been recognized as a significant drug target to treat COVID‐19. The design of a new fluorogenic substrate, the recombinant expression of Mpro and the development of an HTS assay were combined with a structure‐based rational approach leading to the discovery of tailored azanitriles and indole pyridyl esters as novel, outstandingly potent Mpro inhibitors.
It is often said that pnictogen‐bonding catalysis, and σ‐hole catalysis in general, would not work in aqueous systems because the solvent would interfere as an overcompetitive pnictogen‐bond ...acceptor. In this study, we show that the transfer of pnictogen‐bonding catalysis from hydrophobic solvents to aqueous systems is possible by replacing only hydrophobic with hydrophilic substrates, without changing catalyst or reaction. This differs from conventional covalent Lewis acid catalysts, which are instantaneously destroyed by ligand exchange. With their water‐proof substituents in place of exchangeable ligands, pnictogen‐bonding catalysts, the supramolecular counterpart of Lewis acid catalysts, are evinced to catalyze transfer hydrogenation of quinolines in neutral aqueous systems. To secure these results, we introduce a water‐soluble fluorogenic substrate that releases a coumarin upon the reduction of quinolines instead of activated quinolidiniums, and stiborane catalysts with deepened σ holes. They demonstrate that pnictogen‐bonding catalysts can operate in higher‐order architectures for supramolecular systems catalysis under biologically relevant conditions, and provide an operational assay for high‐throughput catalyst screening by fluorescence imaging, in situ under relevant aqueous conditions.
Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless ...of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.
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•Identification of proprotein convertase substrates improving kinetic measurements.•Elongation of substrates with basic residues improves their catalytic efficiency.•Detection of slow-binding behavior of a furin inhibitor for the first time.•Frequent observation of kcat-KM compensation.
A titrant for the SARS-CoV-2 main protease (Mpro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active Mpro by active-site ...titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex. Four fluorogenic substrates of Mpro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the Mpro-catalyzed cleavage of these substrates were determined.
The exact concentration of enzymatically active SARS-CoV-2 main protease (Mpro) was determined by means of a newly developed active-site titrant and employed for the kinetic characterization of Mpro substrates. Display omitted
A real‐time, sensitive, and selective detection device to monitor the healing status of chronic wounds at the point of care is urgently required to render the management of this disease more ...effective. The photonic properties of porous silicon resonant microcavity (pSiRM) afford an excellent opportunity to be developed as a highly sensitive optical biosensor to monitor the presence of specific biomarkers found in the wound exudate, such as matrix metalloproteinases (MMPs). In this study, the pSiRM is designed, fabricated, and functionalized using a fluorogenic MMP peptide substrate featuring both a fluorophore and a quencher. The peptide‐functionalized pSiRM is then employed as a fluorescence‐based optical biosensor for MMPs. Active MMPs recognize and cleave the peptide sequence of the substrate, producing an immobilized peptide fragment carrying the fluorophore. The fluorescence intensity of the fluorophore embedded within the pSiRM matrix is enhanced by the photonic structure of the pSiRM compared to other pSi photonic structures. This fluorescence enhancement translates into high sensitivity, enabling detection of MMP‐1 at a limit of detection as low as 7.5 × 10−19 m after only 15 min incubation time. Finally, the biosensor also allows the detection and quantification of the concentration of MMPs in human wound fluid.
An optical biosensor based on functionalized porous silicon resonance microcavity (pSiRM) using matrix metalloproteinase (MMP) substrate containing a fluorophor and a quencher is demonstrated. The presence of MMP cleaves the substrate leaving the fluorophor on the pSiRM matrix which is now emitting the fluorescence. This biosensor sensitively and selectively detects the MMP in buffer solution and human wound fluid.
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A series of fluorogenic heterocyclic azides were prepared and assessed as reductase substrates across a selection of Gram-negative and Gram-positive microorganisms. The majority of ...these azides showed similar activity profiles to nitroreductase substrates. Microorganisms that do not produce hydrogen sulfide reduced the azides, indicating reductase activity was not linked to hydrogen sulfide production.
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