Trypsin-like Serine Protease Research Articles

Insights into substrate specificity of proteases for screening efficient dehairing enzymes

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 μM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 μM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 μM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.

FAM111A induces nuclear dysfunction in disease and viral restriction.

Mutations in the nuclear trypsin-like serine protease FAM111A cause Kenny-Caffey syndrome (KCS2) with hypoparathyroidism and skeletal dysplasia or perinatally lethal osteocraniostenosis (OCS). In addition, FAM111A was identified as a restriction factor for certain host range mutants of the SV40 polyomavirus and VACV orthopoxvirus. However, because FAM111A function is poorly characterized, its roles in restricting viral replication and the etiology of KCS2 and OCS remain undefined. We find that FAM111A KCS2 and OCS patient mutants are hyperactive and cytotoxic, inducing apoptosis-like phenotypes such as disruption of nuclear structure and pore distribution, in a protease-dependent manner. Moreover, wild-type FAM111A activity causes similar nuclear phenotypes, including the loss of nuclear barrier function, when SV40 host range mutants attempt to replicate in restrictive cells. Interestingly, pan-caspase inhibitors do not block these FAM111A-induced phenotypes, implying it acts independently or upstream of caspases. In this regard, we identify nucleoporins and the associated GANP transcription/replication factor as FAM111A interactors and candidate targets. Overall, we reveal a potentially unifying mechanism through which deregulated FAM111A activity restricts viral replication and causes KCS2 and OCS.

A trypsin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth

Masquerade (Mas) is a secreted trypsin-like serine protease (SPs) and involved in immune response in some arthropods. However, according to previous studies, Mas presents different functional activities. In the present study, the functional mechanisms of Mas in crayfish Procambarus clarkii immune defense were studied. A fragment cDNA sequence of PcMas was identified and characterized. From the structural analysis, it contains a trypsin-like serine protease domain. The highest expression level of PcMas was detected in hepatopancreas. The infection of A. hydrophila could induce the expression of PcMas, while the WSSV infection did not cause changes in the expression of PcMas. Through the prokaryotic expression system, the PcMas protein was expressed in E. coli. It was verified that PcMas can bind to bacteria in vitro and inhibit the growth of the bacteria. By dsRNA interference with the expression of PcMas, the decrease expression of PcMas led to a decrease in the activity of phenoloxidase in hemolymph and an increase of mortality caused by A. hydrophila infection. The injection of recombinant protein can enhance the activity of phenoloxidase and reduce mortality caused by A. hydrophila infections. Therefore, the present study confirmed that PcMas could improve the body's immune response to eliminate bacterial pathogens by binding with bacteria and activating the prophenoloxidase system. The results will enrich the molecular mechanisms of crustaceans immune defense.

Bivalent antibody pliers inhibit \u03b2-tryptase by an allosteric mechanism dependent on the IgG hinge

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering.

A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.

Development of a specific immunoassay to selectively measure active tryptase in airway samples.

Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.

Thrombin Inhibition by Argatroban: Potential Therapeutic Benefits in COVID-19.

Thrombin is a trypsin-like serine protease with multiple physiological functions. Its role in coagulation and thrombosis is well-established. Nevertheless, thrombin also plays a major role in inflammation by activating protease-activated receptors. In addition, thrombin is also involved in angiogenesis, fibrosis, and viral infections. Considering the pathogenesis of COVID-19 pandemic, thrombin inhibitors may exert multiple potential therapeutic benefits including antithrombotic, anti-inflammatory, and antiviral activities. In this review, we describe the clinical features of COVID-19, the thrombin’s roles in various pathologies, and the potential of argatroban in COVID-19 patients. Argatroban is a synthetic, small molecule, direct, competitive, and selective inhibitor of thrombin. It is approved to parenterally prevent and/or treat heparin-induced thrombocytopenia in addition to other thrombotic conditions. Argatroban also possesses anti-inflammatory and antiviral activities and has a well-established pharmacokinetics profile. It also appears to lack a significant risk of drug–drug interactions with therapeutics currently being evaluated for COVID-19. Thus, argatroban presents a substantial promise in treating severe cases of COVID-19; however, this promise is yet to be established in randomized, controlled clinical trials.

Plant cell culture extract of Cirsium eriophorum with skin pore refiner activity by modulating sebum production and inflammatory response.

Facial pore enlargement is considered a significant esthetic and health concern in skincare cosmetics. The pores fulfill the critical function of keeping the skin surface hydrated and protected against microbial infections. The hyperseborrhea, the stress factors, and the hormonal triggers can cause pore size enlargement, causing higher susceptibility of the skin to microbe aggressions and inflammatory reactions. Thus, reducing excessive sebum production and keeping functional pores are two of the most requested activities in skincare cosmetics. A Cirsium eriophorum cell culture extract was investigated for its role in sebum regulation, stratum corneum desquamation, and anti-inflammation. The extract was able to regulate essential markers associated with sebum secretion and pore enlargements, such as the enzyme 5α-reductase, which plays a central role in sebum production, and the trypsin-like serine protease Kallikrein 5, which promotes skin exfoliation and antimicrobial response. Moreover, the extract showed a sebum-normalizing and pore refining activity in individuals having seborrheic or acne-prone skins, suggesting a role of the C. eriophorum extract in rebalancing altered skin conditions responsible for pore enlargement.

Inhibitory effects of tripeptides to enzymatic activity and life cycle parameters of Anticarsia gemmatalis

Chemical pesticides are the main tool used to handle lepidopteran insects in agriculture. However, in addition to causing environmental problems, the insects have acquired resistance to these molecules. Thus, the development of environmentally friendly biopesticides with higher specificity, such as organic protease inhibitors, has been encouraged. This work evaluated the inhibitory effects of two tripeptides, Pep1 and Pep2, obtained by removing the cleavage site of tripeptidyl substrates for trypsin-like proteases. Anticarsia gemmatalis were used as biochemical model and the trypsin-like activity of larvae exposed to different dosages of the tripeptides and protease inhibitors merged in an artificial diet was evaluated. Further, we selected one dose of each tripeptide to set a biological assay, where the total-proteolytic, cysteine-proteases, chymotrypsin-like and trypsin-like activities were determined. The tripeptides shown to have potential to inhibit trypsin-like serine proteases of A. gemmatalis midgut, being the lowest activity detected at doses of 170.4 μM and 251.2 μM for Pep1 and Pep2, respectively. The survival curves obtained using Kaplan-Meier estimators indicated that Pep2 and Pep1 affected survival when comparing to the control group. However, the effect of Pep1 on survival was more pronounced indicating the lowest percent of survival at the end of the larval phase (20%). In addition, larvae exposed to Pep1 maintained normal total proteolytic activity at the expense of the activities of chymotrypsin-like and cysteine proteases. Collectively, our findings revealed that the tripeptides, especially Pep1, exhibit toxic effects on A. gemmatalis and should be tested against other lepidopteran insects.

Structure-Based Macrocyclization of Substrate Analogue NS2B-NS3 Protease Inhibitors of Zika, West Nile and Dengue viruses.

A series of cyclic active‐site‐directed inhibitors of the NS2B‐NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue‐4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d‐lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α‐amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with K i values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin‐like serine proteases and furin‐like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure‐activity relationships were observed for these enzymes, thus suggesting their potential application as pan‐flaviviral protease inhibitors.

High-Throughput Docking and Molecular Dynamics Simulations towards the Identification of Potential Inhibitors against Human Coagulation Factor XIIa.

Human coagulation factor XIIa (FXIIa) is a trypsin-like serine protease that is involved in pathologic thrombosis. As a potential target for designing safe anticoagulants, FXIIa has received a great deal of interest in recent years. In the present study, we employed virtual high-throughput screening of 500,064 compounds within Enamine database to acquire the most potential inhibitors of FXIIa. Subsequently, 18 compounds with significant binding energy (from -65.195 to -15.726 kcal/mol) were selected, and their ADMET properties were predicted to select representative inhibitors. Three compounds (Z1225120358, Z432246974, and Z146790068) exhibited excellent binding affinity and druggability. MD simulation for FXIIa-ligand complexes was carried out to reveal the stability and inhibition mechanism of these three compounds. Through the inhibition of activated factor XIIa assay, we tested the activity of five compounds Z1225120358, Z432246974, Z45287215, Z30974175, and Z146790068, with pIC50 values of 9.3∗10−7, 3.0∗10−5, 7.8∗10−7, 8.7∗10−7, and 1.3∗10−6 M, respectively; the AMDET properties of Z45287215 and Z30974175 show not well but have better inhibition activity. We also found that compounds Z1225120358, Z45287215, Z30974175, and Z146790068 could be more inhibition of FXIIa than Z432246974. Collectively, compounds Z1225120358, Z45287215, Z30974175, and Z146790068 were anticipated to be promising drug candidates for inhibition of FXIIa.

A Rational Designed PslG With Normal Biofilm Hydrolysis and Enhanced Resistance to Trypsin-Like Protease Digestion.

A glycosyl hydrolase produced by Pseudomonas aeruginosa, PslG, has become a promising candidate for biofilm treatment because of its ability to inhibit and disperse biofilms by disrupting exopolysaccharide matrix at nanomolar concentrations. However, as a protein, PslG used for treatment may be degraded by the ubiquitous proteases (of which trypsin-like serine proteases are a major group) secreted by human cells. This would lead to an insufficient effective concentration of PslG. Here, based on the result of liquid chromatography–tandem mass spectrometry (LC-MS/MS) and structural analysis, we generate a PslG mutant (K286A/K433S) with greatly enhanced trypsin resistance. This measure raises IC50 (the concentration of trypsin that can degrade 50% of protein in 30 min at 37°C) from 0.028 mg mL–1 of the wild-type PslG to 0.283 mg mL–1 of PslGK286A/K433S. In addition, biofilm inhibition assay shows that PslGK286A/K433S is much more efficient than wild-type PslG in the presence of trypsin. This indicates that PslGK286A/K433S is a better biofilm inhibitor than wild-type PslG in clinical use where trypsin-like proteases widely exist.

Protective immunity in mice vaccinated with a novel elastase-1 significantly decreases Trichinella spiralis fecundity and infection

Trichinella spiralis is an important foodborne parasitic nematode that represents an enormous threat to the food safety of pork meat. The development of a preventive vaccine is valuable for the prevention and control of Trichinella infection in domestic pigs to ensure pork safety. Elastase is a trypsin-like serine protease that hydrolyzes the host’s diverse tissue components and participates in parasite penetration, and it might be a novel vaccine target molecule. The aim of this study was to assess the protective immunity produced by vaccination with a novel Trichinella spiralis elastase-1 (TsE) in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsE elicited a systemic humoral response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and significant local enteral mucosal sIgA responses. Anti-rTsE IgG recognized the native TsE at the cuticle, stichosome of intestinal infective larvae and adult worm (AW), and intrauterine embryos of female AW. The rTsE vaccination also produced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) after spleen, mesenteric lymph node and Peyer’s patch cells from immunized mice were stimulated with rTsE. The immunized mice exhibited a 52.19% reduction in enteral AW and a 64.06% reduction in muscle larvae after challenge infection. The immune response triggered by rTsE vaccination protected enteral mucosa from larval intrusion, suppressed larval development and reduced female fecundity. The results indicate that TsE may represent a novel target molecule for anti-T. spiralis vaccines.

The association between synovial fluid serine proteinase activity and response to intra-articular corticosteroid injection in psoriatic arthritis.

Intra-articular corticosteroid (IAS) injections are often used for the immediate relief of pain and inflammation in the joint of psoriatic arthritis (PsA) patients. However, studies identifying factors that predict response to the IAS injections are lacking. We aimed to assess the usefulness of serine proteinase activity measurements in PsA synovial fluid (SF) samples obtained at the time of injection in predicting clinical response. The PsA patients with available SF samples from the knee joint were identified from the University of Toronto PsA cohort. Clinical response was defined as an absence of tenderness or swelling in the injected joint at the first post-injection visit, at either 3 or 6months. SF proteinase activity was determined by measuring cleavage of fluorogenic tri-peptide substrates for trypsin-like (VPR-AMC and VLK-AMC) and chymotrypsin-like (AAPF-AMC) serine proteinases. Generalized estimating equation (GEE) models were used to investigate factors associated with response. A total of 32 patients with 60 injected joints and data available for follow-up at 3 or 6months were included in the analysis, with 25 (41.7%) injected joints resulting in clinical response. Age, sex, active joint count, systemic medications and SF serine proteinase activity at the time of injection were included as covariates. Only treatment with biologics was significantly associated with response at 3 or 6months in the multivariate reduced model (OR 3.02, p = 0.027). We could not demonstrate an association between SF serine proteinase activity and response to IAS injection. Biologic agents significantly improve the likelihood of achieving clinical response.

Functional and structural characterization of an ecotin-like serine protease inhibitor from Trypanosoma cruzi

Ecotin, a serine peptidase inhibitor (ISP), discovered in Escherichia coli, inhibit a wide range of trypsin-like serine peptidases, protecting microorganisms from the host's immune response. In eukaryotes, ISPs encoding genes were found only in Trypanosomatidae protozoa, including the genus Trypanosoma, which harbors Trypanosoma cruzi, the ethiological agent of Chagas' disease. T. cruzi encodes the ISP2 Trypanosomatidae orthologous, which in Leishmania species present inhibitory activity on mammalian proteases from S1A family suggesting its role in vertebrate-host-parasite interactions. In this study, the structural and biochemical characterization of the recombinant T. cruzi ISP2 (rTcISP2), produced in E. coli was purified in soluble form and analyzed by circular dichroism, fluorescence spectroscopy, native electrophoresis, dynamic light scattering, low X-ray scattering and homology modeling. The obtained data revealed that rTcISP2 was biologically active and forms homodimers in solution. Furthermore, inhibitory activity of rTcISP2 against human neutrophil elastase (HNE) is the highest among ISP2 orthologous from bacteria and trypanosomatids. The role of NE to control T. cruzi parasites through modulation of cellular and humoral innate immune responses in vertebrate hosts, make TcISP2 a key molecular component for parasite infection efficiency, providing a useful basis for investigation of host-parasite interactions and the potential of TcISP2 for biotechnological applications.

Characterization of Staphylococcus intermedius Group Isolates Associated with Animals from Antarctica and Emended Description of Staphylococcus delphini.

Members of the genus Staphylococcus are widespread in nature and occupy a variety of niches, however, staphylococcal colonization of animals in the Antarctic environment has not been adequately studied. Here, we describe the first isolation and characterization of two Staphylococcus intermedius group (SIG) members, Staphylococcus delphini and Staphylococcus pseudintermedius, in Antarctic wildlife. Staphylococcus delphini were found exclusively in Adélie penguins. The report of S. pseudintermedius from Weddell seals confirmed its occurrence in all families of the suborder Caniformia. Partial RNA polymerase beta-subunit (rpoB) gene sequencing, repetitive PCR fingerprinting with the (GTG)5 primer, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry gave consistent identification results and proved to be suitable for identifying SIG members. Comparative genomics of S. delphini isolates revealed variable genomic elements, including new prophages, a novel phage-inducible chromosomal island, and numerous putative virulence factors. Surface and extracellular protein distribution were compared between genomes and showed strain-specific profiles. The pathogenic potential of S. delphini was enhanced by a novel type of exfoliative toxin, trypsin-like serine protease cluster, and enterotoxin C. Detailed analysis of phenotypic characteristics performed on six Antarctic isolates of S. delphini and eight reference strains from different animal sources enabled us to emend the species description of S. delphini.

Clip-Domain Serine Protease Gene (LsCLIP3) Is Essential for Larval-Pupal Molting and Immunity in Lasioderma serricorne.

Clip-domain serine proteases (CLIPs) play crucial roles in insect development and innate immunity. In this study, we identified a CLIP gene (designated LsCLIP3) from the cigarette beetle Lasioderma serricorne. LsCLIP3 contains a 1,773-bp open reading frame (ORF) encoding a 390-amino-acid protein and shows a conserved clip domain and a trypsin-like serine protease domain. Phylogenetic analysis indicated that LsCLIP3 was orthologous to the CLIP-B subfamily. LsCLIP3 was prominently expressed in larva, pupa, and early adult stages. In larval tissues, it was highly expressed in the integument and fat body. The expression of LsCLIP3 was induced by 20-hydroxyecdysone. A similar induction was also found by peptidoglycans from Escherichia coli and Staphylococcus aureus. RNA interference (RNAi)-mediated silencing of LsCLIP3 disrupted larval–pupal molting and specifically reduced the expression of genes in 20-hydroxyecdysone synthesis and signaling pathway. The chitin amounts of LsCLIP3 RNAi larvae were greatly decreased, and expressions of six chitin metabolic-related genes were significantly reduced. Knockdown of LsCLIP3 increased larval sensitivity to Gram-negative and Gram-positive bacteria. There was significantly decreased expression of four antimicrobial peptide (AMP) genes. The results suggest that LsCLIP3 is an important component of the larva to pupa molt and for the immunity of L. serricorne.

Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.).

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.

SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

Viruses possessing class I fusion proteins require proteolytic activation by host cell proteases to mediate fusion with the host cell membrane. The mammalian SPINT2 gene encodes a protease inhibitor that targets trypsin-like serine proteases. Here we show the protease inhibitor, SPINT2, restricts cleavage-activation efficiently for a range of influenza viruses and for human metapneumovirus (HMPV). SPINT2 treatment resulted in the cleavage and fusion inhibition of full-length influenza A/CA/04/09 (H1N1) HA, A/Aichi/68 (H3N2) HA, A/Shanghai/2/2013 (H7N9) HA and HMPV F when activated by trypsin, recombinant matriptase or KLK5. We also demonstrate that SPINT2 was able to reduce viral growth of influenza A/CA/04/09 H1N1 and A/X31 H3N2 in cell culture by inhibiting matriptase or TMPRSS2. Moreover, inhibition efficacy did not differ whether SPINT2 was added at the time of infection or 24 h post-infection. Our data suggest that the SPINT2 inhibitor has a strong potential to serve as a novel broad-spectrum antiviral.