Host Protease Inhibitors Research Articles

Infectious parvovirus B19 circulates in the blood coated with active host protease inhibitors

The lack of a permissive cell culture system has limited high-resolution structures of parvovirus B19 (B19V) to virus-like particles (VLPs). In this study, we present the atomic resolution structure (2.2 Å) of authentic B19V purified from a patient blood sample. There are significant differences compared to non-infectious VLPs. Most strikingly, two host protease inhibitors (PIs), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and serpinA3, were identified in complex with the capsids in all patient samples tested. The ITIH4 binds specifically to the icosahedral fivefold axis and serpinA3 occupies the twofold axis. The protein-coated virions remain infectious, and the capsid-associated PIs retain activity; however, upon virion interaction with target cells, the PIs dissociate from the capsid prior to viral entry. Our finding of an infectious virion shielded by bound host serum proteins suggests an evolutionarily favored phenomenon to evade immune surveillance and escape host protease activity.

Cas13d knockdown of lung protease Ctsl prevents and treats SARS-CoV-2 infection.

SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR-Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.

Insight into the human pathodegradome of the V8 protease from Staphylococcus aureus

SUMMARYStaphylococcus aureus possesses ten extracellular proteases with mostly unknown targets in the human proteome. To assist with bacterial protease target discovery, we have applied and compared two N-terminomics methods to investigate cleavage of human serum proteins by S. aureus V8 protease, discovering 85 host-protein targets. Among these are virulence-relevant complement, iron sequestration, clotting cascade, and host protease inhibitor proteins. Protein cleavage sites have been identified, providing insight into the disruption of host protein function by V8. Complement proteins are cleaved within peptidase and sushi domains, and host protease inhibitors are cleaved outside their protease-trapping motifs. Our data highlight the potential for further application of N-terminomics in discovery of bacterial protease substrates in other host niches and provide omics-scale insight into the role of the V8 protease in S. aureus pathogenesis.

Therapeutic Modalities for Sars-Cov-2 (Covid-19): Current Status and Role of Protease Inhibitors to Block Viral Entry Into Host Cells

An acute respiratory disease (SARS-CoV-2, also recognized as COVID-19/2019-nCoV), caused by nCoV created a worldwide emergency. The World Health Organization declared the SARS-CoV-2 as epidemic of international concern on January 2020. After SARS-CoV in 2002 and MERS-CoV in 2012, the emergence of SARS-CoV-2 is marked as third highly pathogenic coronavirus of 21st century. Till now, various researches have been conducted, highlighting SARS-CoV-2 as β-coronavirus with high phylogenetic and genomic similarity with bat-CoV, indicating bats as natural reservoir of coronaviruses. It has also been confirmed that SARS-CoV-2 uses the same (ACE2) receptor for host cellular entry as of SARS-CoV, and primarily spread through respiratory pathway. Evidences shows continuous human-to-human viral transfer, with numerous worldwide exported cases. Currently, there is no specific approved drug available for the treatment of SARS-CoV-2, but various anti-parasitic and anti-viral drugs are being investigated. In this review, we have described several possible therapeutic modalities for SARS-CoV-2 infection based on (i) host protease inhibitors to block viral entry into the cell; (ii) gene silencing using siRNA-based RNAi and (iii) type I interferons (IFN1)-based therapeutics have been discussed in detail. Background knowledge on these strategies highlight them as potential therapeutic targets, which could be evaluated on urgent basis to combat COVID-19 epidemic.

Coronavirus Antiviral Research Database (CoV-RDB): An Online Database Designed to Facilitate Comparisons between Candidate Anti-Coronavirus Compounds.

Background: To prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. Methods: We reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. Results: As of August 2020, the Coronavirus Antiviral Research Database (CoV-RDB; covdb.stanford.edu) contained over 2800 cell culture, entry assay, and biochemical experiments, 259 animal model studies, and 73 clinical studies from over 400 published papers. SARS-CoV-2, SARS-CoV, and MERS-CoV account for 85% of the data. Approximately 75% of experiments involved compounds with known or likely mechanisms of action, including monoclonal antibodies and receptor binding inhibitors (21%), viral protease inhibitors (17%), miscellaneous host-acting inhibitors (10%), polymerase inhibitors (9%), interferons (7%), fusion inhibitors (5%), and host protease inhibitors (5%). Of 975 compounds with known or likely mechanism, 135 (14%) are licensed in the U.S. for other indications, 197 (20%) are licensed outside the U.S. or are in human trials, and 595 (61%) are pre-clinical investigational compounds. Conclusion: CoV-RDB facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development.

Angiotensin-converting enzyme 2 (ACE2) receptor and SARS-CoV-2: Potential therapeutic targeting

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta coronavirus that uses the human angiotensin-converting enzyme 2 (ACE2) receptor as a point of entry. The present review discusses the origin and structure of the virus and its mechanism of cell entry followed by the therapeutic potentials of strategies directed towards SARS-CoV2-ACE2 binding, the renin-angiotensin system, and the kinin-kallikrein system. SARS-CoV2-ACE2 binding-directed approaches mainly consist of targeting receptor binding domain, ACE2 blockers, soluble ACE2, and host protease inhibitors. In conclusion, blocking or manipulating the SARS-CoV2-ACE2 binding interface perhaps offers the best tactic against the virus that should be treated as a fundamental subject of future research.

Insight into the Human Pathodegradome of the V8 Protease From  <i>Staphylococcus aureus</i>

Staphylococcus aureus possesses ten extracellular proteases, with mostly unknown targets in the human proteome. To assist with bacterial protease target discovery, we appliedand compared two N-terminomics methods to investigate cleavage of human serum proteins by S. aureus V8 protease, discovering 85 host-protein targets. Amongst these were virulence-relevant complement, iron sequestration, clotting cascade, and host protease inhibitor proteins. Protein cleavage sites were identified, providing insight into the disruption of host protein function by V8. Complement proteins were cleaved within peptidase and sushi domains, and host protease inhibitors were cleaved outside their protease-trapping motifs. Our data highlight the potential for further application of N-terminomics in discovery of bacterial protease substrates in other host niches, and provide omics-scale insight into the role of the V8 protease in S. aureus pathogenesis.

HOST PROTEASE INHIBITION BY SPECIFIC PATHOGENS IN PERIODONTAL DISEASE

Proteolytic tissue degradation is a typical phenomenon in chronic inflammatory periodontal disease with the uncontrolled release of host and bacterial derived proteases causing self-digestion and tissue destruction. Antimicrobial proteins and peptides constitute a diverse class of host defense molecules that act early to combat invasion and infection with bacteria and other microorganisms and protease inhibitors forms one of the functional classes of antimicrobial peptides. Plasma protease inhibitors present in gingival crevicular fluid as well as tissues may play a critical role in the protection of periodontal tissues by modulating protease activity, more particularly during active phases. This literature review attempts to highlight the role of host protease inhibitors and their interaction with specific periodontal pathogens in the pathogenesis of periodontal disease.

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.

Differential effects of periopathogens on host protease inhibitors SLPI, elafin, SCCA1, and SCCA2

Objective: Secretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum.Design: The gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 and chemokine (C-C motif) ligand 20 (CCL20).Results: We found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect.Conclusion: Our results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.

Bait region cleavage and complex formation of human alpha2M with a Porphyromonas gingivalis W50 protease is not accompanied by enzyme inhibition.

Three isoforms of extracellular Arg-specific proteases of P. gingivalis, W50, HRgpA, RgpAcat and mt-RgpAcat, which are all products of the same gene, show identical enzymatic properties toward small chromogenic substrates but have different subunit organisation and molecular size. In order to examine the potential inhibition of these proteases in vivo by host protease inhibitors, the interaction of HRgpA (approximately 110 kDa) and RgpAcat (approximately 55 kDa) with human (alpha2M and their cytotoxicity toward cultured fibroblasts were investigated. Both enzymes formed complexes with (alpha2M as shown by gel filtration chromatography and both cleaved the 'bait' region at Arg696-Leu697. However, whereas (alpha2M-RgpAcat) complex was unable to hydrolyse large substrates such as hide powder azure, (alpha2M-HRgpA) complex hydrolysed both small and large substrates. HRgpA was able to bind to alpha2M saturated with trypsin and also to methylamine-treated alpha2M. This suggested that HRgpA is able to bind to both 'slow' and 'fast' forms of alpha2M and formation of (alpha2M:HRgpA) complex does not trap HRgpA and cause inhibition of activity toward hide powder azure. However, the (alpha2M-HRgpA) complex is not able to cleave other alpha2M molecules, which suggests that the active site of HRgpA in the complex is constrained probably due to steric reasons. The (alpha2M-HRgpA) complex was cytotoxic to 3T3 cells, causing them to round up and detach from the surface with a reduction in metabolic activity.

Degradation of host protease inhibitors and activation of plasminogen by proteolytic enzymes from Porphyromonas gingivalis and Treponema denticola.

Bacterial proteases may participate in the pathogenesis of periodontal diseases through their action on host proteins. In the present study, the ability of selected periodontopathogens, as well as two proteases isolated from Porphyromonas gingivalis and Treponema denticola, to degrade host protease inhibitors was evaluated. The activation of human plasminogen by the two bacterial proteases was also investigated. Proteolytic breakdown of host protease inhibitors (alpha-1-antitrypsin, antichymotrypsin, alpha 2-macroglobulin, antithrombin III, antiplasmin and cystatin C) was evaluated by SDS-PAGE. The 80 kDa trypsin-like protease of P. gingivalis completely digested the six protease inhibitors under investigation, whereas the 95 kDa chymotrypsin-like protease of T. denticola was slightly less active, more particularly on alpha 2-macroglobulin and cystatin C. When whole cells from a number of oral bacterial species were tested, the most significant degradation was obtained with P. gingivalis, T. denticola, Prevotella intermedia, Prevotella nigrescens and Capnocytophaga spp. Peptostreptococcus micros and Propionibacterium acnes had only some degradative activity on selected inhibitors, whereas three bacterial species, Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Fusobacterium nucleatum, had no effect on the protease inhibitors. The 80 kDa protease of P. gingivalis demonstrated strong plasminogen activation, whereas no such activity was associated with the 95 kDa protease of T. denticola. This study indicates the high potential of some periodontal pathogens to destroy protease inhibitors and activate plasminogen. This may result in an uncontrolled degradation of periodontal tissues and a rapid progression of the disease.

Trypanosoma cruzi: Enhanced alpha-macroglobulin levels correlate with the resistance of BALB/cj mice to acute infection

Trypanosoma cruzi proteinases are very likely involved in host-cell invasion. Physiological plasma-proteinase inhibitors from the macroglobulin (MG) family, among them alpha-2-macroglobulin (A2M), are found in tissues and in the plasma of mammals. By complexing to all classes of proteinases, MGs inhibit their action on high-molecular-weight substrates. In vitro studies have shown that A2M impairs T. cruzi proteases and, consequently, the parasite's ability to invade host cells and enhances the phagocytic and microbicidal actions of resident macrophages against T. cruzi. To test the hypothesis of a putative "protective" effect for MG, we quantified it in BALB/cj mice during the course of an experimental T. cruzi infection, comparing a posteriori the levels in mice that died with those in animals that survived, which were considered as being susceptible and resistant to the infection, respectively. The results showed that surviving mice showed an increase in plasma concentrations of MG during the first few weeks after the infection, whereas the levels in mice that died during the acute phase did not differ significantly from those in non-infected mice. These findings and the previous in vitro data indicate a role for physiological proteinase inhibitors, particularly alpha-macroglobulins, in resistance to T. cruzi infection, whereby a balance between parasite proteases and host protease inhibitors may be crucial. MG may thus participate in the complex network of reactions involved in the early acute phase of the disease and contribute by conferring to the host an ability to survive the infection.

Proteases in the schistosome life cycle: a paradigm for tumour metastasis

Cancers and parasites have a number of properties in common, particularly those that relate to their respective capacities to evade host defence mechanisms. This review highlights the similarities between metastatic tumours and schistosomes in particular, and describes the role that proteases may have in the migration, growth, survival and transmission of the different stages of the schistosome life-cycle in the vertebrate host. An elastase-like serine protease of schistosome larvae has been particularly well characterized, and its substrate profile and other properties are indicative of a role in facilitating migration of the parasite through skin tissue early after infection. The primary structures of a cathepsin B-like enzyme, and a putative 'haemoglobinase' found in adult worms have also recently been derived, these enzymes being responsible for degradation of haemoglobin in erythrocytes upon which the adults feed. Adult schistosome worms reside and produce eggs intravascularly, and the processes that mediate the extravasation and subsequent migration of the egg through host tissue are dependent on both blood platelets and the immune response. Fibrino(geno)lytic enzymatic activity that is present in the egg could modulate the thrombogenic potential that eggs might have as a result of their capacity to cause platelet aggregation in vitro and in vivo. The roles of other proteases and peptidases that have been found in schistosome larvae, worms and eggs are less clear. Some of these enzymes may modulate immunological and haemostatic defence mechanisms and thus prolong survival of the parasite, and the consequences of the interactions between schistosomes and host protease inhibitors could also be immune modulatory.