A patent review of the antimicrobial applications of lectins: Perspectives on therapy of infectious diseases

Patents of lectins with antiviral, antibacterial and antifungal applications were searched and reviewed. Lectins are proteins that reversibly bind to specific carbohydrates and have the potential for therapy of infectious diseases as biopharmaceuticals, biomedical tools or in drug design. Given the rising concerns over drug resistance and epidemics, our patent review aims to add information, open horizons and indicate our view of the future perspectives about the antimicrobial applications of lectins. Patents with publications until December 2020 were retrieved from Espacenet using defined search terms and Boolean operators. The documents were used to identify the geographical and temporal distribution of the patents, characterize their lectins, and classify and summarize their antiviral, antibiotic and antifungal applications. Lectins are promising antiviral agents against viruses with epidemics and drug resistance concerns. Mannose-binding lectins were the most suggested antiviral agents since glycans with mannose residues are commonly involved in viral entry mechanisms. They were also immobilized onto surfaces to trap viral particles and inhibit their spread and replication. Many patents described the extraction, isolation, amino acid and nucleotide sequences, and expression vectors of lectins with antibiotic and/or antifungal activities in terms of MIC and IC₅₀ for in vitro assays. The inventions also included lectins as biological tools in nanosensors for antibiotics susceptibility tests, drug-delivery systems for the treatment of resistant bacteria, diagnostics of viral diseases and as a vaccine adjuvant. Although research and development of new medicines is highly expensive, antimicrobial lectins may be worth investments given the emergence of epidemics and drug resistance. For this purpose, less invasive routes should be developed as alternatives to the parenteral administration of biologics. While anti-glycan neutralizing antibodies are difficult to develop due to the low immunogenicity of carbohydrates, lectins can be produced more easily and have a broad-spectrum activity. Protein engineering technologies may make the antimicrobial applications of lectins more successful.     

TIM-1 acts a dual-attachment receptor for Ebolavirus by interacting directly with viral GP and the PS on the viral envelope

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%−90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.     

Sulfotyrosine dipeptide: Synthesis and evaluation as HIV-entry inhibitor

Human immunodeficiency virus type 1 (HIV-1) is responsible for the worldwide AIDS pandemic. Due to the lack of prophylactic HIV-1 vaccine, drug treatment of the infected patients becomes essential to reduce the viral load and to slow down progression of the disease. Because of drug resistance, finding new antiviral agents is necessary for AIDS drug therapies. The interaction of gp120 and co-receptor (CCR5/CXCR4) mediates the entry of HIV-1 into host cells, which has been increasingly exploited in recent years as the target for new antiviral agents. A conserved co-receptor binding site on gp120 that recognizes sulfotyrosine (sTyr) residues represents a structural target to design novel HIV entry inhibitors. In this work, we developed an efficient synthesis of sulfotyrosine dipeptide and evaluated it as an HIV-1 entry inhibitor.     

麻疹病毒受体与病毒侵入

麻疹病毒是一种具囊膜的负链RNA病毒,两种主要的囊膜蛋白血凝素蛋白(H)和膜融合蛋白(F)表达在膜表面负责病毒侵入过程中与宿主受体的结合和膜融合过程.病毒囊膜蛋白与受体的相互作用是病毒侵入宿主的关键步骤,决定了病毒感染能力、种属和组织嗜性.因此,囊膜病毒与受体的结合位点往往成为重要的抗病毒药物的靶点.目前已发现的3种麻疹病毒受体包括CD46、SLAM和Nectin-4.以下综述了麻疹病毒受体的特征及在病毒侵入中的作用、麻疹病毒H蛋白与受体的相互作用机制,为抗病毒药物设计及麻疹病毒作为肿瘤治疗性载体的应用提供理论依据.     

Characterization of Resistance to Rhabdovirus and Retrovirus Infection in a Human Myeloid Cell Line

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.     

A conserved molecular action of native and recombinant Epap-1 in inhibition of HIV-1 gp120 mediated viral entry

The early expression of Epap-1 (early pregnancy associated protein), a 90 kDa anti-HIV-1 active glycoprotein, in the first trimester placental tissue suggests that it is one of the innate immune factors/proteins protecting the fetus from HIV infections. In the present investigation, we have cloned and expressed Epap-1 in bacterial and baculovirus expression systems. The recombinant Epap-1 as well as native Epap-1 shows a conserved molecular mode of action. These proteins exhibit significant antiviral activity and inhibit the cell fusion reaction between gp120 expressing HeLa (HL2/3) cells and T cell line (SupT1). Further, the rhodamine labeled Epap-1 specifically bound to gp120 expressed on the surface of HL2/3 cells during fusion reaction thereby inhibiting viral entry. Analysis of the interacting gp120 epitopes revealed that Epap-1 binds specifically to epitopes of gp120, recognizing constant-5 (C5) region and the variable-3 (V3) epitope of gp120 expressed on HL2/3 cells; It exhibits specific interaction with C5 region of cell-free virus in four HIV-1 isolates suggesting that the molecular interaction of Epap-1 is specific and is highly conserved in binding to gp120 leading to inhibition of viral entry. Epap-1 can thus be a very efficient natural protection mechanism against cell-free and cell-associated viral infections during early pregnancy.     

红藻凝集素G研究进展

来源于海洋红藻的凝集素G已被证实对多种囊膜病毒都有抗病毒活性,可与囊膜病毒表面糖基结合而阻断病毒的入侵。以病毒入侵为作用靶点的抗病毒药物,不仅可以阻断病毒的自由传播途径,还可以阻断细胞间传播途径,红藻凝集素G还具有可溶性好、易表达、稳定性强、免疫原性低、安全性好等优点,所以红藻凝集素G作为一类新型抗病毒药物越来越受到科学家的青睐。     

病毒的细胞进入研究进展及其应用前景

病毒感染的初期事件包括病毒与细胞表面受体的相互作用和进入细胞的过程,而病毒的宿主细胞专一性很大程度上取决于这一阶段的专一识别特征和特殊要求。人乳头状瘤病毒、人免疫缺陷病毒和单纯疱疹病毒是感染人类的几种常见病原物,该文简要综述和讨论了与人体健康关系密切的这三种重要病毒表面的蛋白组分、宿主细胞表面受体及其相互作用和病毒的细胞进入的研究进展,以及在以病毒的细胞进入过程为靶点的抗病毒药物研发中的应用前景。     

New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides

Influenza A viral (IAV) fusion peptides are known for their important role in viral-cell fusion process and membrane destabilization potential which are compatible with those of antimicrobial peptides. Thus, by replacing the negatively or neutrally charged residues of FPs with positively charged lysines, we synthesized several potent antimicrobial peptides derived from the fusogenic peptides (FPs) of hemagglutinin glycoproteins (HAs) of IAV. The biological screening identified that in addition to the potent antibacterial activities, these positively charged fusion peptides (pFPs) effectively inhibited the replication of influenza A viruses including oseltamivir-resistant strain. By employing pseudovirus-based entry inhibition assays including H5N1 influenza A virus (IAV), and VSV-G, the mechanism study indicated that the antiviral activity may be associated with the interactions between the HA2 subunit and pFP, of which, the nascent pFP exerted a strong effect to interrupt the conformational changes of HA2, thereby blocking the entry of viruses into host cells. In addition to providing new peptide “entry blockers”, these data also demonstrate a useful strategy in designing potent antibacterial agents, as well as effective viral entry inhibitors. It would be meaningful in treatment of bacterial co-infection during influenza pandemic periods, as well as in our current war against those emerging pathogenic microorganisms such as IAV and HIV.     

Viral infection in hematopoietic stem cell transplantation: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee review on the role of cellular therapy in prevention and treatment

Despite recent advances in the field of HSCT, viral infections remain a frequent causeof morbidity and mortality among HSCT recipients. Adoptive transfer of viral specific T cells has been successfully used both as prophylaxis and treatment of viral infections in immunocompromised HSCT recipients. Increasingly, precise risk stratification of HSCT recipients with infectious complications should incorporate not only pretransplant clinical criteria, but milestones of immune reconstitution as well. These factors can better identify those at highest risk of morbidity and mortality and identify a population of HSCT recipients in whom adoptive therapy with viral specific T cells should be considered for either prophylaxis or second line treatment early after inadequate response to first line antiviral therapy. Broadening these approaches to improve outcomes for transplant recipients in countries with limited resources is a major challenge. While the principles of risk stratification can be applied, early detection of viral reactivation as well as treatment is challenging in regions where commercial PCR assays and antiviral agents are not readily available.     

Ebola virus entry requires the host-programmed recognition of an intracellular receptor

Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.     

Carnosine exhibits significant antiviral activity against Dengue and Zika virus

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito‐borne diseases whereas ZIKV infection occasionally re‐emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β‐alanyl‐l ‐histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell‐based assays were performed to validate the computational results. Mode‐of‐inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC₅₀ values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode‐of‐inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.     

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.     

Virus-host interactions during hepatitis C virus entry — implications for pathogenesis and novel treatment approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.      

Cholesterol conjugation potentiates the antiviral activity of an HIV immunoadhesin

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand‐binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand‐binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol‐conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41‐Fc‐chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV‐1 strains, it was considerably more potent than the unconjugated C41‐Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol‐conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat‐derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Viruses customize autophagy protein for efficient viral entry

While the cell imposes multiple barriers to virus entry, enveloped viruses are remarkably still able to gain entry to their cellular hosts by hitchhiking and remodeling the endomembrane system to traffic within, and eventually escape from, endosomal organelles for their genome release. Elucidating viral entry mechanisms and their interaction with the host trafficking network is necessary for antiviral therapy. Here, we focus on the use of host autophagy molecular factors during the entry of prototypic negative-stranded RNA viruses, and highlight recent progress in our understanding of the role of one such factor, UVRAG, in both viral and cellular endocytic membrane trafficking and fusion events.     

HIV‐1 ENV gp120 structural determinants for peptide triazole dual receptor site antagonism

Despite advances in HIV therapy, viral resistance and side‐effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV‐1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co‐receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide's affinity and its ability to inhibit binding of CD4 and the co‐receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single‐round cell infection assays. Two mutations, D474A and T257A, caused large‐scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co‐receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

An optimized MM/PBSA virtual screening approach applied to an HIV-1 gp41 fusion peptide inhibitor

VIRus Inhibitory Peptide (VIRIP), a 20 amino acid peptide, binds to the fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) gp41 and blocks viral entry. VIRIP derivatives with improved antiviral activity have been developed, and one of those derivatives has recently proven effective and safe in a phase 1/2 clinical trial. Here, molecular dynamics were executed in combination with molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) free energy calculations to explore the binding interaction between VIRIP derivatives and gp41 FP. A promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations, and the use of multiple internal dielectric constants for the MM/PBSA calculations depending on the amino acid sequence. Based on these results, a virtual screening experiment was carried out to design VIRIP analogs with further improved antiretroviral activity. A selection of peptides was tested for inhibitory activity and several VIRIP derivatives were identified with significantly enhanced activity compared to the reference peptides. The results demonstrate that computational modeling strategies using an adapted MM/PBSA methodology improve the accuracy of binding free energy calculations of peptide complexes compared to the classic MM/PBSA protocol. As such, this virtual screening approach generated HIV-1 gp41 FP inhibitors with improved antiviral activity that could be useful for future clinical applications.     

广谱抗病毒抑制剂研究进展

研制广谱抗病毒制剂是病毒学前瞻性方向,也是国际医药界倍受瞩目的热点之一.近年来,广谱抗病毒制剂相关研究获得长足进展,突破了抗病毒制剂单一宿主的瓶颈与限制,以及广谱类制剂活性相对较弱的原有思维,部分成果已面向临床应用.本文基于病毒侵染以及宿主细胞防御两个层面,全面综述新型广谱抗病毒抑制剂的研究进展与应用潜力,讨论现存的挑战,并展望了未来研究趋势.