Type II transmembrane serine proteases as potential target for anti-influenza drug discovery

Introduction: The outbreak of an influenza pandemic as well as the continued circulation of seasonal influenza highlights the need for effective antiviral therapies. The emergence of drug-resistant strains further necessitates the development of novel antivirals that target the host factors crucial for viral replication.

Area covered: This review summarizes the current understanding of the structural and functional properties of type II transmembrane serine proteases (TTSPs) as a proteolytic activator of influenza virus infection and discusses their potential as antiviral targets. It also explores the experimental evidence accumulated for inhibitors of TTSPs as novel, broad-spectrum antivirals against various influenza virus subtypes. The review also provides an overview of the properties of small molecules, proteins, and peptides that efficiently inhibit the proteolytic activation of the influenza virus.

Expert opinion: TTSPs activate a wide range of influenza virus subtypes including avian influenza viruses, both in vitro and in vivo, via proteolytic cleavage of influenza hemagglutinin (HA) into infection-competent fusogenic conformation. Other viruses such as SARS-, MERS-coronaviruses and human metapneumoviruses may use the same host cell proteases for activation, implying that TTSP inhibition might be a novel strategy for developing broad-spectrum antiviral agents for respiratory viral infections.     

Viral proteinases: targets of opportunity

During antiviral drug development, any essential stage of the viral life cycle can serve as a potential drug target. Since most viruses encode specific proteases whose cleavage activity is required for viral replication, and whose structure and activity are unique to the virus and not the host cell, these enzymes make excellent targets for drug development. Success using this approach has been demonstrated with the plethora of protease inhibitors approved for use against HIV. This discussion is designed to review the field of antiviral drug development, focusing on the search for protease inhibitors, while highlighting some of the challenges encountered along the way. Protease inhibitor drug discovery efforts highlighting progress made with HIV, HCV, HRV, and vaccinia virus as a model system are included. Drug Dev. Res. 67:501–510, 2006. © 2006 Wiley‐Liss, Inc.     

Is there still a role for aprotinin in cardiac surgery?

Cardiac surgery is associated with a systemic inflammatory response and systemic coagulopathy, which can result in significant organ dysfunction and bleeding. Aprotinin, a serine protease inhibitor, can limit systemic inflammation, and has been associated with myocardial, pulmonary and cerebral protection in addition to its proven haemostatic efficacy. Data are currently conflicting regarding the haemostatic efficacy of aprotinin relative to alternative agents including tranexamic acid. Recent studies have demonstrated aprotinin usage is associated with increased rates of thrombotic and renal complications, but these findings are at odds with the majority of studies relating to aprotinin safety to date. The lack of adequately powered, randomised studies evaluating aprotinin and alternative agents limits drawing conclusions about the complete use or disuse of aprotinin presently and requires individualised patient selection based on bleeding risk and co-morbidities for its usage.     

Host cellular proteases trigger the infectivity of the influenza virus in the airway and brain

The pathogenesis of the influenza and Sendai viruses is primarily determined by host cellular trypsin-type processing proteases that activate viral fusion activity and infectivity. We isolated three secretory trypsin-type proteases from rat lungs, such as tryptase Clara, mini-plasmin, and ectopic anionic trypsin, candidates for the processing proteases of viral envelope glycoproteins. These enzymes specifically cleave the precursor of fusion glycoprotein hemagglutinin (HA) of influenza virus at Arg(325) and the F(0) of Sendai virus at Arg(116) in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the induction of infectivity of these viruses. These proteases show different localization in the airway and susceptibility for the processing of various subtypes of influenza virus HA, suggesting that these processing proteases determine the viral pathogenicity. Influenza virus readily infects and replicates in the airway epithelial cells but occasionally replicates in the central nervous system, particularly in children below 5-6 years of age and Reye's syndrome patients. We found an invasion by a non-neurovirulent influenza virus in cerebral capillaries with progressive brain edema of mice having impaired mitochondrial fatty acid metabolism congenitally or posteriorly in the newborn period. In the brain of these mice, mini-plasmin, which potentiates viral-multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries but only a little in the control mice without impaired mitochondrial fatty acid metabolism.     

丙型肝炎病毒NS3/4A丝氨酸蛋白酶活性测定方法研究进展

丙型肝炎病毒（HCV）NS3／4A丝氨酸蛋白酶在病毒多聚蛋白体加工和RNA复制的过程中发挥着重要作用,是治疗HCV的理想靶点。因为NS3／4A丝氨酸蛋白酶在HCV多聚蛋白上负责四个位点的切割,所以可根据此特性设计其活性的测定方法。此文就目前应用于NS3／4A丝氨酸蛋白酶活性测定的方法进行综述,以期为筛选有效的抑制剂提供技术帮助。     

Anti-inflammatory actions of aprotinin provide dose-dependent cardioprotection from reperfusion injury

BACKGROUND AND PURPOSE: Myocardial injury following ischaemia and reperfusion has been attributed to activation and transmigration of polymorphonuclear leukocytes (PMNs) with release of mediators including oxygen-derived radicals and proteases causing damage. EXPERIMENTAL APPROACH: We studied the serine protease inhibitor aprotinin in an in vivo rabbit model of 1 h of myocardial ischaemia followed by 3 h of reperfusion (MI+R). Aprotinin (10,000 Ukg(-1)) or its vehicle were injected 5 min prior to the start of reperfusion. KEY RESULTS: Myocardial injury was significantly reduced with aprotinin treatment as indicated by a reduced necrotic area (11+/-2.7% necrosis as percentage of area at risk after aprotinin; 24+/-3.1% after vehicle; P<0.05) and plasma creatine kinase activity (12.2+/-1.5 and 17.3+/-2.3 IU g(-1) protein in aprotinin and vehicle groups, respectively, P<0.05). PMN infiltration (assessed by myeloperoxidase activity) was significantly decreased in aprotinin-treated animals compared to vehicle (P<0.01). Histological analysis also revealed a substantial increase in PMN infiltration following MI+R and this was significantly reduced by aprotinin therapy (44+/-15 vs 102+/-2 PMN mm2 in aprotinin vs vehicle-treated animals, P<0.05). In parallel in vitro experiments, aprotinin inhibited neutrophil-endothelium interaction by reducing PMN adhesion on isolated, activated aortic endothelium. Finally, immunohistochemical analysis illustrated aprotinin significantly reduced myocardial apoptosis following MI+R. CONCLUSIONS AND IMPLICATIONS: Inhibition of serine proteases by aprotinin inhibits an inflammatory cascade initiated by MI+R. The cardioprotective effect appears to be at least partly due to reduced PMN adhesion and infiltration with subsequently reduced myocardial necrosis and apoptosis.     

Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses

The 1918 pandemic H1N1 influenza virus and the recently emerged Southeast Asian H5N1 avian influenza virus are unique among influenza A virus isolates in their high virulence for humans and their lethality for a variety of animal species without prior adaptation. Reverse genetic studies have implicated several viral genes as virulence determinants. For both the 1918 and H5N1 viruses, the hemagglutinin and the polymerase complex contribute to high virulence. Non-structural proteins NS1 and PB1-F2, which block host antiviral responses, also influence pathogenesis. Additionally, recent studies correlate high levels of viral replication and induction of strong proinflammatory responses with the high virulence of these viruses. Defining how individual viral proteins promote enhanced replication, inflammation and severe disease will provide insight into the pathogenesis of severe influenza virus infections and suggest novel therapeutic approaches.     

Retro peptide-hybrids as selective inhibitors of the Dengue virus NS2B-NS3 protease

New chemotherapeutics against Dengue virus and related flaviviruses are of growing interest in antiviral drug discovery. The viral serine protease NS2B-NS3 is a promising target for the development of such agents. Drug-like inhibitors of this protease with high affinity to the target are not available at the moment. The present work describes the discovery of new retro di- and tripeptide hybrids that do not necessarily require an electrophilic "warhead" to achieve affinities in the low micromolar range. The most active sequence in this series is the tripeptide R-Arg-Lys-Nle-NH(2). By variation of the N-terminal groups (R) it could be shown that the previously described arylcyanoacrylamide moiety is a preferable group in this position. Retro tripeptide hybrids were found to be more active and more selective than retro dipeptide hybrids. A significant selectivity towards the Dengue virus protease could be shown in a counterscreen with thrombin and the West Nile virus protease. Alternative sequences to R-Arg-Lys-Nle-NH(2) did not have higher affinities towards the Dengue virus protease, similar to retro-inverse sequences with D-lysine and D-arginine residues. The results of a competition assay with the known inhibitor aprotinin indicate that the N-terminal arylcyanoacrylamide residue of this compound class binds near the catalytic center of the enzyme.     

West Nile Virus NS2B/NS3 protease as an antiviral target

West Nile Virus (WNV) has spread rapidly during the last decade across five continents causing disease and fatalities in humans and mammals. It highlights the serious threat to both our health and the economy posed by viruses crossing species, in this case from migratory birds via mosquitoes to mammals. There is no vaccine or antiviral drug for treating WNV infection. One attractive target for antiviral development is a viral trypsin-like serine protease, encoded by the N-terminal 184 amino acids of NS3, which is only active when tethered to its cofactor, NS2B. This protease, NS2B/NS3pro, cleaves the viral polyprotein to release structural and non-structural viral proteins that are essential in viral replication and assembly of new virus particles. Disruption of this protease activity is lethal for virus replication. The NS3 protein also has other enzymes within its sequence (helicase, nucleoside triphosphatase, RNA triphosphatase), all of which are tightly regulated through localisation within membranous compartments in the infected cell. This review describes the various roles of NS3, focussing on NS2B-NS3 protease and its function and regulation in WNV replication and infection. Current advances towards development of antiviral inhibitors of NS2B/NS3pro are examined along with obstacles to their development as an antiviral therapy.     

新型抗病毒RNA聚合酶PA亚基抑制剂临床研究进展

流行性感冒（简称流感）是流感病毒引起的对人类健康危害较重的呼吸道传染病。目前抗流感药物的应用主要面临病毒耐药性和对高致病性流感病毒的效价低的问题,如M2离子通道抑制剂和神经氨酸酶抑制剂存在药效低、时间窗口窄和耐药性等缺点。近来的抗流感药物研发的靶点聚焦于病毒RNA聚合酶。流感病毒RNA聚合酶是病毒在宿主细胞内完成复制和转录过程的关键酶,其中PA亚基通过内切酶活性为流感病毒的转录过程提供引物,成为潜在抗流感药物靶点。本文总结了以PA亚基内切酶为靶点的抗流感药物研究进展,旨在为开发安全有效的新型抗病毒RNA聚合酶抑制剂提供参考。     

Screening methods for influenza antiviral drug discovery

INTRODUCTION: Influenza antiviral high-throughput screens have been extensive, and yet no approved influenza antivirals have been identified through high-throughput screening. This underscores the idea that development of successful screens should focus on the exploitation of the underrepresented viral targets and novel, therapeutic host targets. AREAS COVERED: The authors review conventional screening applications and emerging technologies with the potential to enhance influenza antiviral discovery. Real-world examples from the authors' work in biocontained environments are also provided. Future innovations are discussed, including the use of targeted libraries, multiplexed assays, proximity-based endpoint methods, non-laboratory-adapted virus strains, and primary cells, for immediate physiological relevance and translational applications. EXPERT OPINION: The lack of successful anti-influenza drug discovery using high-throughput screening should not deter future efforts. Increased understanding of the functions of viral targets and host-pathogen interactions has broadened the target reservoir. Future screening efforts should focus on identifying new drugs against unexploited viral and host targets using currently developed assays, and on the development of novel, innovative assays to discover new drugs with novel mechanisms. Innovative screens must be designed to identify compounds that specifically inhibit protein-protein or protein-RNA interactions or other virus/host factor interactions that are crucial for viral replication. Finally, the use of recent viral isolates, increased biocontainment (for highly-pathogenic strains), primary cell lines, and targeted compound libraries must converge in efficient high-throughput primary screens to generate high-content, physiologically-relevant data on compounds with robust antiviral activity.     

抑肽酶基因的克隆和表达

抑肽酶作为天然非特异性丝氨酸蛋白酶抑制剂，用途广泛。目前抑肽酶制品主要从牛肺中提取。用基因重组技术，将抑肽酶结构基因导入表达载体pGrxA，并在抑肽酶基因上游引入FXa识别位点。将重组质粒转化至Origami^TM中，用异丙基硫代β-D半乳糖苷(IPTG)诱导表达目的蛋白，产物以可溶性形式存在于胞内。将菌体超声破壁和离心，融合蛋白经分子筛层析和离子交换层析纯化后，用FXa切割该融合蛋白可得到N端不含多余氨基酸残基且与天然构像一致的活性抑肽酶。     

Potent cationic inhibitors of West Nile virus NS2B/NS3 protease with serum stability, cell permeability and antiviral activity

West Nile virus (WNV) has spread rapidly around the globe, efficiently crossing species from migrating birds into humans and other mammals. The viral protease NS2B-NS3 is important for WNV replication and recognizes dibasic substrate sequences common to other flaviviral proteases but different from most mammalian proteases. Potent inhibitors of WNV protease with antiviral activity have been elusive to date. We report the smallest and most potent inhibitors known for this enzyme, cationic tripeptides with nonpeptidic caps at the N-terminus and aldehyde at the C-terminus. One of these, compound 3 ( Ki = 9 nM) is stable in serum (>90% intact after 3 h, 37 degrees C), cell permeable, and shows antiviral activity (IC 50 1.6 microM) without cytotoxicity (IC 50 >400 microM), thereby validating the approach of inhibiting WNV protease to suppress WNV replication.     

In vitro and in vivo assay systems for study of influenza virus inhibitors

Evaluation of potential influenza virus inhibitors may utilize multiple steps. First would be to determine if the viral target (e.g. influenza virus neuraminidase) being focused upon will be inhibited in the appropriate assay. Standard in vitro antiviral assays, used next in antiviral evaluations, may utilize inhibition of viral plaques, viral cytopathic effect (CPE), and viral hemagglutinin or other protein, with inhibition of viral yield used in follow-up evaluations. The CPE can be determined visually and by dye uptake. Animal models used for study of potential influenza virus inhibitors include the ferret, the laboratory mouse, and the chicken, with a variety of parameters used to indicate the severity of the infection and its inhibition by therapy. Multiple parameters are recommended in any in vivo antiviral evaluation. The ferret and the mouse infection models have been useful in studying the development of drug resistance and the relative virulence of drug-resistant viruses. The influenza mouse model has also been of value for the evaluation of immunomodulating effects of test compounds and for the study of the utility of antiviral drugs for use against influenza virus infections in the immunocompromised host. In considering the use of any animal model, species differences in drug pharmacology and metabolism must be taken into account. This review has described the systems which have been used most frequently by antiviral investigators, using, as examples, recent studies with the clinically approved influenza virus neuraminidase inhibitors oseltamivir and zanamivir.     

Antiviral effect of catechins in green tea on influenza virus

Polyphenolic compound catechins ((-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC)) from green tea were evaluated for their ability to inhibit influenza virus replication in cell culture and for potentially direct virucidal effect. Among the test compounds, the EGCG and ECG were found to be potent inhibitors of influenza virus replication in MDCK cell culture and this effect was observed in all influenza virus subtypes tested, including A/H1N1, A/H3N2 and B virus. The 50% effective inhibition concentration (EC50) of EGCG, ECG, and EGC for influenza A virus were 22-28, 22-40 and 309-318 microM, respectively. EGCG and ECG exhibited hemagglutination inhibition activity, EGCG being more effective. However, the sensitivity in hemagglutination inhibition was widely different among three different subtypes of influenza viruses tested. Quantitative RT-PCR analysis revealed that, at high concentration, EGCG and ECG also suppressed viral RNA synthesis in MDCK cells whereas EGC failed to show similar effect. Similarly, EGCG and ECG inhibited the neuraminidase activity more effectively than the EGC. The results show that the 3-galloyl group of catechin skeleton plays an important role on the observed antiviral activity, whereas the 5'-OH at the trihydroxy benzyl moiety at 2-position plays a minor role. The results, along with the HA type-specific effect, suggest that the antiviral effect of catechins on influenza virus is mediated not only by specific interaction with HA, but altering the physical properties of viral membrane.     

Aprotinin: a serine protease inhibitor with therapeutic actions: its interaction with ACE inhibitors

Aprotinin is an important member of a family of related protease inhibitors and has many clinically beneficial activities. These inhibitors have multiple functions, but not all of them are mediated by enzyme inhibition. Aprotinin has complex effects on many homeostatic functions including coagulation, platelet function and inflammation. It also has complex interactions with other drug therapies including angiotensin-converting enzyme inhibitors. Since patients with cardiovascular diseases are treated frequently with angiotensin-converting enzyme inhibitors and also often need cardiopulmonary bypass surgery and receive aprotinin, these interactions are potentially significant but often overlooked. Aprotinin is currently used to reduce the amount of transfused homologous blood (during cardiopulmonary bypass surgery) and thus, the risks associated with homologous blood transfusion. Aprotinin also has potential uses in acute pancreatitis, carcinoid tumors, sepsis, and other clinical situations. Future research will provide a definitive answer for the need to employ this inhibitor therapeutically in these situations. Aprotinin also has some potentially adverse effects in the kidney in special circumstances. For example, the use of aprotinin in diabetic patients may be related with an increased risk for renal dysfunction. It has also been associated with thrombosis, inadequate coagulation, and allergic reactions. In balance, the available information indicates that the advantages of its application outweigh its disadvantages in most patients.