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Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases. [ABSTRACT FROM AUTHOR]

Abstract Trypsin-like serine proteases (TLSPs), are members of the serine protease family and play important roles in innate immune responses. There are no previous studies regarding the role of TLSPs in immune responses to Spiroplasma eriocheiris in Eriocheir sinensis. In this paper, two TLSPs were identified from E. sinensis , namely EsTLSP-I and EsTLSP-II. Phylogenetic analysis showed that EsTLSP-I and EsTLSP-II clustered into two different groups that the crustaceans within each group were specifically. Tissue distribution analysis indicated that both EsTLSP-I and EsTLSP-II were mostly expressed in hemocytes, in the gill and in the intestine. The EsTLSP-I and EsTLSP-II transcription levels in hemocytes significantly increased after infection with S. eriocheiris. After dsRNA interference, the expression of both EsTLSP-I and EsTLSP-II distinctively declined from 48 h to 96 h. The expression of proPO and beta-1,3-glucan binding protein (LGBP) were also notably reduced after silencing. Compared to the control group, the copy number of S. eriocheiris increased significantly in interfering EsTLSP-I and EsTLSP-II groups during S. eriocheiris infection. Meanwhile, the mortality of crabs in the silenced EsTLSP-I and EsTLSP-II groups was higher than in the control group after the S. eriocheiris challenge. The results indicate that EsTLSP-I and EsTLSP-II play important roles in the innate immune system of Eriocheir sinensis, in combating infections of S. eriocheiris. Highlights • EsTLSP-I and EsTLSP-II were involved in the immune responses of E. sinensis to S. eriocheiris challenge. • EsTLSP-I and EsTLSP-II are part of the innate immnue system activating the proPO system. • The copies of S. eriocheiris and crabs' death rate were notably increased in EsTLSP-I and EsTLSP-II RNAi assay. [ABSTRACT FROM AUTHOR]

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl- l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death. [ABSTRACT FROM AUTHOR]

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases. [Display omitted] • TMPRSS2 inhibition is crucial for the antiviral effects of Phe(3-Am) compounds. • The applied Phe(3-Am)-derived substances are potent inhibitors of CYP3A4. • Phe(3-Am)-type inhibitors bind to albumin with low affinity. • Phe(3-Am) derivatives did not inhibit CYP1A2, CYP2C9, and CYP2C19. • Depletion rates of these compounds remained below 30% in human microsomes. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a gel free chemiproteomic method based on chromatography was developed and applied for the biological fingerprinting analysis of complex biological system. p-Aminobenzamidine (ABA), an inhibitor of trypsin-like serine proteases, was immobilized for characterizing their interacting proteins in human plasma. By the proteomic analysis method, 214 proteins were identified with obvious affinity to the immobilized ABA. By searching the sequences of above proteins with consensus patterns of the two active sites, seven proteins belong to trypsin-like serine protease group were found. Based on the Gene Ontology annotation, the identified trypsin-like serine proteases have the function of catalytic activity and calcium ion binding, and are mainly involved in the biological process of blood coagulation. Eight more other proteins related to calcium ion binding and blood coagulation were found. Nearly all of these proteins cannot be identified by directly analyzing the plasma sample demonstrating the chemiproteomics a useful approach to characterize interacting proteins in the low abundance range. [Copyright &y& Elsevier]