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Journal of Environmental Pathology,... 2024Protease is the enzyme accountable for the breakdown of proteins i.e., proteolysis. Proteases are reportedly involved in the events of growth, development, progression... (Review)
Review
Protease is the enzyme accountable for the breakdown of proteins i.e., proteolysis. Proteases are reportedly involved in the events of growth, development, progression and metastasis of cancers. If any agent could inhibit/retard the protease enzyme, i.e., protease inhibitor, it would arrest the cancer; thus indicating the significance of exploring protease inhibitors for latest anti-malignant drug discovery. Higher plants are the rich sources of different protease inhibitors that are effective against several types of malignancies both at preclinical and clinical stages. Natural protease inhibitors of herbal origin have both cancer chemopreventive and chemotherapeutic properties together with inhibitory activity against different types of pertinent proteases. Clinically, these herbal agents are found to be safe unlike the synthetic antineoplastic agents. Further studies in this direction are necessary in pursuit of newer generation drugs without adverse reactions for the prevention and treatment of malignancies.
Topics: Humans; Protease Inhibitors; Neoplasms; Peptide Hydrolases; Antiviral Agents
PubMed: 38608142
DOI: 10.1615/JEnvironPatholToxicolOncol.2024052872 -
Biomaterials Jan 2015With the advent of the Highly Active Antiretroviral Therapy, the morbidity and the mortality associated to HIV have been considerably reduced. However, 35-40 million...
With the advent of the Highly Active Antiretroviral Therapy, the morbidity and the mortality associated to HIV have been considerably reduced. However, 35-40 million people bear the infection worldwide. One of the main causes of therapeutic failure is the frequent administration of several antiretrovirals that results in low patient compliance and treatment cessation. In this work, we have developed an innovative Nanoparticle-in-Microparticle Delivery System (NiMDS) comprised of pure drug nanocrystals of the potent protease inhibitor indinavir free base (used as poorly water-soluble model protease inhibitor) produced by nanoprecipitation that were encapsulated within mucoadhesive polymeric microparticles. Pure drug nanoparticles and microparticles were thoroughly characterized by diverse complementary techniques. NiMDSs displayed an encapsulation efficiency of approximately 100% and a drug loading capacity of up to 43% w/w. In addition, mucoadhesiveness assays ex vivo conducted with bovine gut showed that film-coated microparticles were retained for more than 6 h. Finally, pharmacokinetics studies in mongrel dogs showed a dramatic 47- and 95-fold increase of the drug oral bioavailability and half-life, respectively, with respect to the free unprocessed drug. These results support the outstanding performance of this platform to reduce the dose and the frequency of administration of protease inhibitors, a crucial step to overcome the current patient-incompliant therapy.
Topics: Adhesiveness; Administration, Oral; Alginates; Animals; Cattle; Chitosan; Dogs; Dose-Response Relationship, Drug; Drug Delivery Systems; Glucuronic Acid; Hexuronic Acids; Indinavir; Nanoparticles; Protease Inhibitors; Spectroscopy, Fourier Transform Infrared; Time Factors; X-Ray Diffraction
PubMed: 25453966
DOI: 10.1016/j.biomaterials.2014.10.026 -
International Journal of Molecular... Mar 2022Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome 2 (SARS-CoV-2), has been one of the most devastating pandemics of recent times. The... (Review)
Review
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome 2 (SARS-CoV-2), has been one of the most devastating pandemics of recent times. The lack of potent novel antivirals had led to global health crises; however, emergence and approval of potent inhibitors of the viral main protease (Mpro), such as Pfizer's newly approved nirmatrelvir, offers hope not only in the therapeutic front but also in the context of prophylaxis against the infection. By their nature, RNA viruses including human immunodeficiency virus (HIV) have inherently high mutation rates, and lessons learnt from previous and currently ongoing pandemics have taught us that these viruses can easily escape selection pressure through mutation of vital target amino acid residues in monotherapeutic settings. In this paper, we review nirmatrelvir and its binding to SARS-CoV-2 Mpro and draw a comparison to inhibitors of HIV protease that were rendered obsolete by emergence of resistance mutations, emphasizing potential pitfalls in the design of inhibitors that may be of important relevance to the long-term use of novel inhibitors against SARS-CoV-2.
Topics: Antiviral Agents; Coronavirus 3C Proteases; HIV Protease; Humans; Molecular Docking Simulation; Peptide Hydrolases; Protease Inhibitors; SARS-CoV-2; COVID-19 Drug Treatment
PubMed: 35408866
DOI: 10.3390/ijms23073507 -
Journal of Biomolecular Structure &... Sep 2022Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin...
Effect of variations in the conserved residues E371 and S359 on the structural dynamics of protein Z dependent protease inhibitor (ZPI): a molecular dynamic simulation study.
Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin superfamily, it possesses unique structural features such as activation by PZ, regulating its inhibitory function. In order to understand the Reactive Centre Loop (RCL) dynamics of ZPI, which is absolutely critical for its activity, we performed Molecular Dynamics (MD) simulation on ZPI and its E371 and S359 variants located at important conserved functional sites. Unexpectedly, the RCL of E371 variants, (E371K, E371R, and E371Q), were shown to be very stable due to compensatory interactions at the proximal end of RCL. Interestingly, RCL flexibility was shown to be enhanced in the double mutant K318E-E371K due to the repulsive effect of increased negative charge on top of the breach region. Principal component analysis (PCA) coupled with residue wise interaction network analysis(RIN) revealed correlated motion between the RCL and the PZ binding regions in the WT. However, a loss of regulation in correlated motion between RCL and PZ binding hotspot Tyr240 in the double mutant was also observed. Additionally, the S359F and S359I mutations resulted in increased RCL flexibility owing to the disruption of stabilizing hydrogen bonding interaction at the distal end of strand S5A. Thus, the current study proposes that the overall stabilizing interactions of S5A is a major regulator of proper loop movement of ZPI for its activity. The results would be beneficial to engineer activity compromised ZPI as a prophylactic agent for the treatment of hemophilia.Communicated by Ramaswamy H. Sarma.
Topics: Blood Proteins; Factor Xa; Kinetics; Molecular Dynamics Simulation; Protease Inhibitors; Protein Binding; Serpins
PubMed: 33554754
DOI: 10.1080/07391102.2021.1883114 -
Journal of the American Academy of... Jun 1988Recently we reported that a kind of serine protease, SH protease, and collagenase might be involved in blister formation and, furthermore, that the cooperative action of...
Recently we reported that a kind of serine protease, SH protease, and collagenase might be involved in blister formation and, furthermore, that the cooperative action of these three proteases was essential for blister formation in recessive dystrophic epidermolysis bullosa. In this study we examined the inhibitory effect of clinically usable serine protease inhibitors for blister formation in organ culture and in clinical trials of recessive dystrophic epidermolysis bullosa patients. Camostat mesylate, a synthetic serine protease inhibitor that is available for the treatment of chronic pancreatitis, demonstrated a striking effect of inhibiting blistering in organ culture of normal human skin with recessive dystrophic epidermolysis bullosa blister fluids. Subsequently we administered camostat mesylate by topical application to four patients with recessive dystrophic epidermolysis bullosa to assess its ability to reduce blistering. Therapeutic response was favorable; a significant effect in decreasing the number of blisters was observed in three of four patients. These findings actually supported the hypothesis that a kind of serine protease had a close relationship with blistering in recessive dystrophic epidermolysis bullosa and that therapy with a clinically usable protease inhibitor was useful for the treatment of this disease.
Topics: Administration, Topical; Adolescent; Adult; Blister; Child; Epidermolysis Bullosa; Esters; Female; Gabexate; Genes, Recessive; Guanidines; Humans; Male; Organ Culture Techniques; Protease Inhibitors; Skin
PubMed: 3385039
DOI: 10.1016/s0190-9622(88)70130-9 -
Journal of Molecular Medicine (Berlin,... Apr 2024Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation...
Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.
Topics: Animals; Chlorocebus aethiops; Humans; Solanum tuberosum; Peptide Hydrolases; Vero Cells; Angiotensin-Converting Enzyme 2; Protease Inhibitors; Enzyme Inhibitors; Inflammation; Antiviral Agents; COVID-19; Elastin
PubMed: 38381158
DOI: 10.1007/s00109-024-02423-x -
Methods in Molecular Biology (Clifton,... 2022Many proteins are regulated post-translationally by proteolytic processing. This includes plant signaling peptides that are proteolytically released from larger...
Many proteins are regulated post-translationally by proteolytic processing. This includes plant signaling peptides that are proteolytically released from larger precursor proteins. The proteases involved in the biogenesis of signaling peptides and in regulation of other proteins by limited proteolysis are largely unknown. Here we describe how protease inhibitors that are specific for a certain class of proteases can be employed for the identification of proteases that are responsible for the processing of a given target protein. After having identified the protease family to which the processing enzyme belongs, candidate proteases and the GFP-tagged target protein are agro-infiltrated for transient expression in N. benthamiana leaves. Cleavage products are analyzed on immuno-blots and specificity of cleavage is confirmed by co-expression of class-specific inhibitors. For the identification of processing sites within the target protein, cleavage product(s) are purified by immunoprecipitation followed by polyacrylamide gel electrophoresis and analyzed by mass spectrometry.
Topics: Endopeptidases; Peptide Hydrolases; Peptides; Protease Inhibitors; Proteolysis; Substrate Specificity
PubMed: 35583773
DOI: 10.1007/978-1-0716-2079-3_6 -
Analytical Biochemistry Mar 1999A new type of solid-phase assay for proteases and protease inhibitors has been developed using biotinylated casein. The assay involves coating of titer plate wells with...
A new type of solid-phase assay for proteases and protease inhibitors has been developed using biotinylated casein. The assay involves coating of titer plate wells with biotinylated casein, hydrolysis of this substrate with a protease such as trypsin, reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and finally quantification of the amount of casein remaining on the plate using alkaline phosphatase activity as the indicator. The activity of the bound indicator enzyme is oppositely related to the protease activity of the sample. In addition, the assay can be modified for quantitating the corresponding amount of protease inhibitor in the sample. The assay is simple, sensitive, accurate, inexpensive, and amenable to automation.
Topics: Biotinylation; Caseins; Chemistry Techniques, Analytical; Endopeptidases; Enzymes, Immobilized; Protease Inhibitors
PubMed: 10036175
DOI: 10.1006/abio.1998.3053 -
The Protein Journal Mar 2013Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in...
Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.
Topics: Enzyme Assays; Enzyme Precursors; Kinetics; Peptide Hydrolases; Protease Inhibitors; Proteolysis
PubMed: 23417772
DOI: 10.1007/s10930-013-9470-9 -
Bioconjugate Chemistry Jul 2019Protease inhibitors are used as both research tools and therapeutics. Many of these inhibitors consist of substrate amino acid sequence-derived structure with a...
Protease inhibitors are used as both research tools and therapeutics. Many of these inhibitors consist of substrate amino acid sequence-derived structure with a transition state mimic to interact with the active site of the protease, suppressing enzymatic activity. However, once they bind, macrodilution or protein denaturation is required to remove them, limiting their usage. In this study, we describe a removable protease inhibitor, which is a directly biotinylated analogue to control the activities of HIV-1 protease and human cathepsin D. In the substrate cleavage assay, we observed that the nanomolar inhibitory activities were lost upon the addition of streptavidin, while the enzymatic activities sufficiently recovered. HIV-1 protease mixed with the removable inhibitor, avoiding autolysis, was still active to be detected by adding streptavidin after one year at room temperature. We also observed that the inhibitor was an effective eluent for the simple detection of the activity of proteases purified from human serum and cells. These results demonstrate that direct biotinylation of protease inhibitors could be a novel method for controlling the enzymatic activity from OFF to ON. We proposed the phenomenon that binding equilibrium of inhibitor was shifted from protease to streptavidin with higher affinity, named "inhibitor stripping action by affinity competition", or ISAAC. We anticipate that ISAAC could be applicable for preservatives of proteases and activity-based diagnosis of protease related diseases. Furthermore, removable inhibitor to be designed for targeted proteases changing the inhibitor structure may elucidate enzymatic activity in intrinsic form with natural modifications from various biological samples.
Topics: Biotinylation; Cathepsin D; Drug Design; HIV Protease; HIV-1; Humans; Models, Molecular; Protease Inhibitors
PubMed: 30990716
DOI: 10.1021/acs.bioconjchem.9b00195