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Effect of Mucosal Inflammation on Colonic Smooth Muscle Contraction
Published in William J. Snape, Stephen M. Collins, Effects of Immune Cells and Inflammation on Smooth Muscle and Enteric Nerves, 2020
Yining Xie, William T. Gerthoffer, S. Narasimha Reddy, Viktor E. Eysselein, Fabio Cominelli, William J. Snape
A calcium dependent protease, which degrades membrane plaques, cytoplasmic dense bodies, and intermediate filaments18, could reduce isometric force and isotonic shortening velocity of the colitis muscle. However, there was no obvious electron microscopic difference in the muscle (unpublished observation). It is probable that leupeptin would block the effect of this protease.
Structural Determination of the Polycystin-2 Channel by Electron Cryo-Microscopy
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Protease inhibitors: Leupeptin 5 mg/mL in ethanol (1000× stock)Pepstatin 2.8 mg/mL in ethanol (2000× stock)PMSF 17.4 mg/mL in DMSO (1000× stock)Aprotinin 2 mg/mL (1000× stock)Hypotonic buffer: 10 mM HEPES, pH 8.0; 0.5 mM TCEP or DTT + 4 protease inhibitorsResuspension buffer: 50 mM HEPES, 150 mM NaCl, 10% glycerol, 0.5 mM TCEP, pH 7.8 + 4 protease inhibitors
Chikungunya Virus Infection
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
D. Velmurugan, K. Manish, D. Gayathri
The nsP2 protease domain belongs to the papain super-family of cysteine proteases (Lulla et al. 2006, Sourisseau et al. 2007). Among the CHIKV-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. nsP2 is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while papain-like protease activity resides in the C-terminal portion. The nsP2 protease is an essential enzyme whose proteolytic activity is critical for virus replication. Biochemical characterization of nsP2 cysteine protease of CHIKV has been reported (Pastorino et al. 2008). As an insight into the protease catalytic mechanism, they have studied the effect of different protease inhibitors on the enzymatic activity of CHIKV-nsP2 protease by using the cost-effective BOC-AGG-MCA peptide as substrate. The enzyme was found to be completely resistant to the inhibitors of serine protease (aprotinin), aspartic proteases (pepstatin) and metallo-proteases (EDTA). It has also proven resistance to the cysteine protease inhibitor leupeptin. Based on these findings, nsP2 can be classified as a thiol protease of the papain super family. Pastorino also confirmed that the viral activity of CHIKV-nsP2 protease is contributed by the amino acids in the range 422 to 799 in the C-terminal region of protease-helicase complex. The amino acids in the N-terminal segment (166 to 630) show NTPase or helicase activity (Karpe and Lole 2010).
Chemical tools to monitor bladder cancer progression
Published in Biomarkers, 2022
Natalia Gruba, Lech Stachurski, Adam Lesner
Next, we decided to screen mixed urine samples with inhibitors of certain classes of proteolytic enzymes (Table 4). Such an experiment allowed us to pre-determinate which class of enzymes is responsible for the observed activity. The result of this experiment is shown in Figure 4. Incubation of G1 urine with leupeptin resulted in a decrease in the hydrolysis efficiency, which could suggest the presence of serine or cysteine proteinases. The proteolytic activity was also reduced to some extent after the use of carfilzomib and epoxomicin which are proteasome inhibitors. Such a result could suggest the presence of threonine proteinases. Similar situation was observed in G2 urine. Proteolytic activity was inhibited by about 50% after the use of leupeptin and to some extent after incubation with PMSF. The situation is different in the case of G3 mixed urine. Here, we observed a 60% decrease in activity after incubation with AHX-VYDnVP (C6H2Cl)2 described as the most potent peptidyl irreversible inactivators of neutrophil elastase (HNE) (33). The use of inhibitors of different classes of enzymes did not result in a decrease in the hydrolysis efficiency. The results obtained for each stage of cancer development are not unequivocal. On the one hand, we observed a certain decrease in activity after the use of inhibitors of various classes of enzyme, on the other hand, we did not observe complete enzyme inhibition in any case. Such a result may suggest that the activity we are monitoring correlates with a different type of enzyme than we used.
An update on the therapeutic potential of calpain inhibitors: a patent review
Published in Expert Opinion on Therapeutic Patents, 2020
Gabadur is composed of the poorly membrane permeable calpain inhibitor leupeptin linked to pregabalin which serves as a carrier [55] (Figure 6). Pregabalin is an anti-epileptic drug that is actively transported into the brain by the L-type amino acid transporter [56]. Thus, in Gabadur, pregabalin serves as a trojan horse to carry the poorly BBB permeable leupeptin into the brain where the calpain inhibitor (leupeptin) targets injured tissue [57]. Due to the involvement of calpain in the pathophysiology of traumatic brain injury (TBI) [15,58], Dugue et al. [59] studied Gabadur as treatment for TBI. They showed that a single 80 mg/kg dose of Gabadur significantly decrease cortical neurodegeneration at 48 h post-TBI compared to the pregabalin carrier or the PBS vehicle administered alone. Additionally, active calpain-2 levels were found to be significantly lower in the injured hemispheres of Gabadur treated rats suggesting that the calpain inhibitory activity of Gabadur accounted for its neuroprotective effects. However, it should be noted that leupeptin is not a specific inhibitor of calpain. It inhibits proteases such as cathepsin B, cathepsin S, trypsin, kallikrein, plasmin, and thrombin all of which have been implicated in TBI [60] hence the observed results may not be due exclusively to inhibition of calpain.
A Dual Zinc plus Arginine formulation attenuates the pathogenic properties of Porphyromonas gingivalis and protects gingival keratinocyte barrier function in an in vitro model
Published in Journal of Oral Microbiology, 2020
Amel Ben Lagha, Ying Yang, Harsh M. Trivedi, James G. Masters, Daniel Grenier
To determine the effects of the Dual Zinc plus Arginine aqueous solution and dentifrice on the proteinase activity of P. gingivalis, a 48-h culture was centrifuged at 10 000 x g for 10 min and the supernatant was collected. Assay mixtures containing equal volumes of P. gingivalis culture supernatant, the fluorescent substrate collagen DQTM (100 µg/ml; Molecular Probes, Eugene, OR, USA), and the Dual Zinc plus Arginine aqueous solution or dentifrice were prepared and incubated for 2 h at 37°C. The fluorescence corresponding to collagen degradation was monitored at time 0, 30, 60, 90, and 120 min using a Synergy 2 microplate reader (BioTek Instruments, Winooski, VT, USA), with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Test compounds or the fluorescent substrate alone were used as controls. Leupeptin (1 µM) was used as a positive inhibitory control. Assays were performed in triplicate in two independent experiments and the means ± SD were calculated.