Autoinhibition of suicidal capsid protease from O'nyong'nyong virus.

Alphaviruses pose a significant threat to public health. Capsid protein encoded in the alphaviral genomes constitutes an interesting therapy target, as it also serves as a protease (CP). Remarkably, it undergoes autoproteolysis, leading to the generation of the C-terminal tryptophan that localizes to the active pocket, deactivating the enzyme. Lack of activity hampers the viral replication cycle, as the virus is not capable of producing the infectious progeny. We investigated the structure and function of the CP encoded in the genome of O'nyong'nyong virus (ONNV), which has instigated outbreaks in Africa. Our research provides a high-resolution crystal structure of the ONNV CP in its active state and evaluates the enzyme's activity. Furthermore, we demonstrated a dose-dependent reduction in ONNV CP proteolytic activity when exposed to indole, suggesting that tryptophan analogs may be a promising basis for developing small molecule inhibitors. It's noteworthy that the capsid protease plays an essential role in virus assembly, binding viral glycoproteins through its glycoprotein-binding hydrophobic pocket. We showed that non-aromatic cyclic compounds like dioxane disrupt this vital interaction. Our findings provide deeper insights into ONNV's biology, and we believe they will prove instrumental in guiding the development of antiviral strategies against arthritogenic alphaviruses.

Significance of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) for the Monitoring of Treatment Response to Cyclosporine in Patients with Psoriasis.

Neutrophil gelatinase-associated lipocalin (NGAL) may promote development of inflammation in psoriasis, whereas proprotein convertase subtilisin/kexin type 9 (PCSK9) may account for dyslipidemia in some psoriatic patients. The aim of the study was to analyze the influence of cyclosporine therapy on serum levels of NGAL and PCSK9 in patients with psoriasis vulgaris. METHODS: Serum samples were obtained before and after three months cyclosporine therapy. Patients were grouped into responders and non-responders to cyclosporine depending on whether they achieved at least 50% reduction of Psoriatic Activity Score Index (PASI), or not. Serum levels of PCSK9 and NGAL were assayed using commercially available ELISA tests. Lipid levels were measured with an enzymatic method. RESULTS: There were 40 patients enrolled. A significant decrease in serum NGAL level was seen in cyclosporine responders. No similar dependance was found for PCSK9. Serum PCSK9 concentration correlated with total cholesterol (TChol) and LDL at baseline and after three month treatment. CONCLUSIONS: Cyclosporine therapy contributes to the reduction of the NGAL serum but not the PCSK9 concentration. Correlation between the PCSK9 serum level and TChol as well as LDL concentration may help to understand drug induced dyslipidemia after cyclosporine.

IgYs: on her majesty's secret service.

There has been an increasing interest in using Immunoglobulin Y (IgY) antibodies as an alternative to "classical" antimicrobials. Unlike traditional antibiotics, they can be utilized on a continual basis without leading to the development of resistance. The veterinary IgY antibody market is growing because of the demand for minimal antibiotic use in animal production. IgY antibodies are not as strong as antibiotics for treating infections, but they work well as preventative agents and are natural, nontoxic, and easy to produce. They can be administered orally and are well tolerated, even by young animals. Unlike antibiotics, oral IgY supplements support the microbiome that plays a vital role in maintaining overall health, including immune system function. IgY formulations can be delivered as egg yolk powder and do not require extensive purification. Lipids in IgY supplements improve antibody stability in the digestive tract. Given this, using IgY antibodies as an alternative to antimicrobials has garnered interest. In this review, we will examine their antibacterial potential.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

Camostat Does Not Inhibit the Proteolytic Activity of Neutrophil Serine Proteases.

Coronavirus disease 2019 (COVID-19) can lead to multi-organ failure influenced by comorbidities and age. Binding of the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-CoV-2 S protein) to angiotensin-converting enzyme 2 (ACE2), along with proteolytic digestion of the S protein by furin and transmembrane protease serine subtype 2 (TMPRSS2), provokes internalization of SARS-CoV-2 into the host cell. Productive infection occurs through viral replication in the cytosol and cell-to-cell transmission. The catalytic activity of TMPRSS2 can be blocked by the trypsin-like serine protease inhibitor camostat, which impairs infection by SARS-CoV-2. At the site of infection, immune cells, such as neutrophils, infiltrate and become activated, releasing neutrophil serine proteases (NSPs), including cathepsin G (CatG), neutrophil elastase (NE), and proteinase 3 (PR3), which promote the mounting of a robust immune response. However, NSPs might be involved in infection and the severe outcome of COVID-19 since the uncontrolled proteolytic activity is responsible for many complications, including autoimmunity, chronic inflammatory disorders, cardiovascular diseases, and thrombosis. Here, we demonstrate that camostat does not inhibit the catalytic activity of CatG, NE, and PR3, indicating the need for additional selective serine protease inhibitors to reduce the risk of developing severe COVID-19.

The Fellowship of Privileged Scaffolds-One Structure to Inhibit Them All.

Over the past few years, the application of privileged structure has emerged as a powerful approach to the discovery of new biologically active molecules. Privileged structures are molecular scaffolds with binding properties to the range of different biological targets. Moreover, privileged structures typically exhibit good drug-like properties, thus assuring more drug-like properties of modified compound. Our main objective is to discuss the privileged structures used for the development of antiviral agents.

The prevalence and role of functional autoantibodies to angiotensin-converting-enzyme-2 in patients with systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is an autoimmune disease caused by the imbalance between the activity of angiotensin II and angiotensin-(1-7). Their balance should be controlled by angiotensin-converting enzyme 2 (ACE2), which degrades angiotensin II into angiotensin-(1-7). Previously, autoantibodies to ACE2 (anti-ACE2) were identified in patients with vasculopathy due to different connective tissue diseases, including SSc, but their frequency in SSc was not further analyzed. The aim of the research was to investigate the prevalence and potential role of those anti-ACE2 antibodies in SSc patients. MATERIALS AND METHODS: There were enrolled 27 patients with SSc and 23 healthy donors. ELISA assay determined the presence of anti-ACE2 autoantibodies in serum samples. The results were compared to plasma measurements of angiotensin-(1-7) level via commercial ELISA. RESULTS: The presence of anti-ACE2 autoantibodies was confirmed in five patients with SSc and two healthy controls. Two of those SSc subjects were anti-Scl70+, another two were double anti-Scl70+ and anti-Ro/SSA+, and anti-PM/Scl antibodies were detected in one patient. Median plasma level of Ang-(1-7) in anti-ACE2 negative patients was 47.4 pg/ml and stayed below the detection level in anti-ACE2 positive subjects. The plasma level of Ang-(1-7) was undetectable in four SSc patients, and three of them were anti-ACE2 positive. CONCLUSIONS: Anti-ACE2 antibodies appear to be other functional autoantibodies with the potential to dysregulate the balance between Ang II and Ang-(1-7). They are non-specific for SSc and probably result from polyautoimmunity which affect some of SSc patients. Their occurrence in SSc settings may be associated with a severe depletion of plasma Ang-(1-7).

Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors.

Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50-90 % of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation, amyloid ß-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been proposed as an interesting target for the development of new potential anticancer agents. Here, we present the synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists.

One of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylalanine analogs, which displayed nanomolar Ki values. Their antiproliferative potential as well as their proapoptotic action, manifested by an increase in caspase-3 activity, was examined using various cell lines.

Impact of Prebiotics and Synbiotics Administered in ovo on the Immune Response against Experimental Antigens in Chicken Broilers.

The effect of the in ovo application of selected prebiotics and synbiotics on the humoral immune response against T-dependent (SRBC) and T-independent (dextran) antigens and delayed-type hypersensitivity (DTH) to phytohemagglutinin was studied. On the 12th day of incubation, 800 eggs (Ross 308) were divided into five groups and injected into the egg air chamber with prebiotic inulin (Pre1), Bi2tos (Pre2), a synbiotic composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1), a synbiotic composed of Bi2tos and L. lactis subsp. cremoris IBB SC1 (Syn2), and physiological saline (control group; C). The chickens were immunized twice at the 7th and 21st day of life with SRBC and dextran. A DTH test was performed on the 7th, 21st, and 35th day. The application of prebiotics and synbiotics had no significant effect on the humoral immune response. SRBC-immunized in ovo Pre1- and Pre2-treated chickens showed significantly higher serum IgG levels than the control. A significant effect on the DTH reaction was detected on the 7th (Pre1 < C) and 21st (Pre2 > Syn2) day. However; Bi2tos may transiently stimulate the cellular immune response on the 21st day. It may be concluded that the application of inulin in an egg air chamber on the 12th day of incubation may stimulate the secondary immune response. The inulin-treated group exhibited a lower mortality rate than the control group.

Human proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin.

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.

Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly.

Cathepsin G (CatG) is responsible for several distinct immune processes of adaptive and innate immunity depending on extra- or intracellular occurrence of CatG. Recently, we established a method to detect CatG activity at the cell surface of natural killer cells by using the activity-based probe MARS116-Bt in flow cytometry. MARS116-Bt consists of biotin, spacer, amino acid sequence, and a phosphonate warhead which binds covalently to the serine amino acid residue within the active center of CatG. Herein, MARS116 was conjugated to 5(6)-carboxyfluorescein (FAM) in order to limit non-specific signal-to-noise ratio generally resulting from binding of fluorescein-labelled avidin (avidin-FAM) to biotinylated, intracellular proteins; since MARS116-Bt is incubated with avidin-FAM in a second labelling step. MARS116-FAM was capable to detect intracellular CatG activity, in contrast to the control compound MARS116*-FAM which lacks the functional phosphonate warhead crucial for binding to the active-site of CatG and contains a carboxyl group instead. Furthermore, intracellular CatG activity was determined in CD4+ T cells, CD8+ T cells as well as in T regulatory cell (Treg) subsets. Thus, MARS116-FAM is a convenient activity-based probe to detect intracellular CatG activity in a flow cytometry approach.

Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy.

Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.

Selected prebiotics and synbiotics administered in ovo can modify innate immunity in chicken broilers.

BACKGROUND: A previous study showed that prebiotics and synbiotics administered in ovo into the egg air cell on the 12th day of incubation enhance the growth and development of chickens. However, the influence of this procedure on the development and efficiency of the innate immune system of broiler chickens is unclear. Therefore, the aim of this study was to evaluate whether the early (on the 12th day of embryo development) in ovo administration of selected prebiotics (inulin - Pre1 and Bi2tos - Pre2) and synbiotics (inulin + Lactococcus lactis subsp. lactis IBB SL1 - Syn1 and Bi2tos + L. lactis subsp. cremoris IBB SC1 - Syn2) influences the innate immune system. RESULTS: Chickens (broiler, Ross 308) that were treated with Pre1 exhibited a decreased H/L ratio on D7, but an increased H/L ratio was observed on D21 and D35. In the remaining experimental groups, an increase in the H/L ratio was observed on D21 and D35. The oxidative potential of leukocytes measured using the NBT test increased on D21 in Pre2 and Syn1 groups. The rate of the phagocytic ability of leukocytes increased in Pre1 and Syn1 groups on D21. The phagocytic index decreased in Pre1 and Syn2 groups on D21 and D35. Concurrently, the count of WBC in circulating blood decreased on D21 in Pre1, Pre2, and Syn1 groups. The hematocrit value was increased in Syn1 chickens on D21, in Pre1 chickens on D35, and in Syn2 chickens on both time points. CONCLUSIONS: Early in ovo treatment of chicken embryos with prebiotics and synbiotics may temporarily modulate not only the production/maturation of leukocytes but also their reactivity.

Proteinase 3 phosphonic inhibitors.

Neutrophils are one of the most important military services of the armed forces of the immune system, a crucial line of defense against bacterial or fungal onslaughts. One of their mechanisms of action relies on the production of serine proteases. One of these enzymes is proteinase 3 (PR3), which is engaged in the processing of pro-inflammatory cytokines, receptors, heat shock proteins and in the generation of antibacterial peptides. Despite its protective function, uncontrolled activity of PR3 has been associated with the progression of inflammation and tissue injury. Although PR3 was characterized at the beginning of 90's of the last century for the first time, the research on the development of its inhibitors is barely noticeable. Here we present the recent findings on the design, synthesis and the activity of phosphonic PR3 inhibitors together with the historical perspective.

Development and Evaluation of an Immunoglobulin Y-Based ELISA for Measuring Prostate Specific Antigen in Human Serum.

BACKGROUND: Measurement of serum prostate specific antigen (PSA) concentrations remains one of the leading methods for diagnosing prostate cancer. We developed and evaluated an immunoglobulin Y (IgY)-based ELISA to measure total PSA (tPSA) concentrations in human serum that could be used as an alternative to commercially available in vitro diagnostic assays that rely on mouse monoclonal IgG. METHODS: A sandwich ELISA based on an anti-PSA IgY antibody was developed. We evaluated the ability of the anti-PSA IgY antibody to detect free and complexed PSA at the same molar ratio. The assay was optimized, and its analytical performance was verified by calculating limit of background (LoB), limit of detection (LoD), and limit of quantification (LoQ). We performed correlation and regression analyses between tPSA concentrations measured by our ELISA and those from commercial assays: Cobas 6000 (Roche Diagnostics, Warszawa, Poland) and PSA total ELISA (IBL International, Hamburg, Germany). RESULTS: LoB, LoD, and LoQ, were 0.061, 0.083, and 0.100 ng/mL, respectively, and linearity range was 0.100-3.375 ng/mL. tPSA concentrations from our IgY-based ELISA strongly correlated with those from the commercial assays. CONCLUSIONS: Our IgY-based ELISA is an efficient equivalent to the above commercial assays. The use of IgY as the detecting agent could reduce the risk of false positive results, as well as decrease the overall cost of analysis.

Phosphonate inhibitors of West Nile virus NS2B/NS3 protease.

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M-1s-1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.

The Lord of the Bacteria: The Fellowship of the Leader and Other Serine Protease Inhibitors.

Since antibiotics use is currently limited due to undesired side effects and the increasing antibiotic resistance of various bacteria strains, there is a pressing need to develop new strategies and methods preventing epidemic outbreaks. The virulent potency of bacteria relies on a number of different extracellularly secreted factors among which proteases considered as promising, novel drug targets are of special interest. The first evidence that bacterial cysteine, serine and metalloproteinases contributed to the progression of infection was found in the early 70's. This extracellular proteolytic system allows bacteria to penetrate into tissues, escape detection by the host's immune mechanisms and grow despite limited access to nutrition. A molecule able to selectively inhibit the activity of bacterial proteases in the spread of infection may lead to designing novel therapeutics. Moreover, due to their mechanism of action, bacterial protease inhibitors can be used to fight antibiotic-resistant strains. Herein, we undertake a review of various bacterial proteases together with the design and development of their inhibitors (excluding ß-lactams) for the last ten years, and introduce the reader to a brief history of the subject.