Papers by Patrice Guillon
Glycobiology, 2008
It binds to the histo-blood group antigen (HBGA) H type 2 which requires an α1,2fucosyltransferas... more It binds to the histo-blood group antigen (HBGA) H type 2 which requires an α1,2fucosyltransferase for its synthesis. In rabbit, three α1,2fucosyltransferases genes are known, Fut1, Fut2, and Sec1. Nonfunctional alleles at any of these loci could potentially confer resistance to RHDV, similar to human FUT2 alleles that determine the nonsecretor phenotype and resistance to infection by various NoV strains. In this study, we looked for the presence of H type 2 on buccal epithelial cells of wild rabbits from two geographic areas under RHDV pressure and from one RHDV-free area. Some animals with diminished H type 2 expression were found in the three populations (nonsecretor-like phenotype). Their frequency markedly increased according to the RHDV impact, suggesting that outbreaks selected survivors with low expression of the virus ligand. Polymorphisms of the Fut1, Fut2, and Sec1 coding regions were determined among animals that either died or survived outbreaks. The Fut2 and Sec1 genes presented a high polymorphism and the frequency of one Sec1 allele was significantly elevated, over 6fold, among survivors. Sec1 enzyme variants showed either moderate, low, or undetectable catalytic activity, whereas all variant Fut2 enzymes showed strong catalytic activity. This functional analysis of the enzymes encoded by each Fut2 and Sec1 allele suggests that the association between one Sec1 allele and survival might be explained by a deficit of α1,2fucosyltransferase expression rather than by impaired catalytic activity.
The catalytic mechanism of human parainfluenza virus type 3 haemagglutinin-neuraminidase revealed
Angewandte Chemie (International ed. in English), Jan 2, 2015
Human parainfluenza virus type 3 (hPIV-3) is one of the leading causes for lower respiratory trac... more Human parainfluenza virus type 3 (hPIV-3) is one of the leading causes for lower respiratory tract disease in children, with neither an approved antiviral drug nor vaccine available to date. Understanding the catalytic mechanism of human parainfluenza virus haemagglutinin-neuraminidase (HN) protein is key to the design of specific inhibitors against this virus. Herein, we used (1) H NMR spectroscopy, X-ray crystallography, and virological assays to study the catalytic mechanism of the HN enzyme activity and have identified the conserved Tyr530 as a key amino acid involved in catalysis. A novel 2,3-difluorosialic acid derivative showed prolonged enzyme inhibition and was found to react and form a covalent bond with Tyr530. Furthermore, the novel derivative exhibited enhanced potency in virus blockade assays relative to its Neu2en analogue. These outcomes open the door for a new generation of potent inhibitors against hPIV-3 HN.
Cover Picture: The Catalytic Mechanism of Human Parainfluenza Virus Type 3 Haemagglutinin-Neuraminidase Revealed (Angew. Chem. Int. Ed. 10/2015)
Angewandte Chemie International Edition, 2015
Nature Communications, 2014
Human parainfluenza viruses (hPIVs) cause upper and lower respiratory tract disease in children t... more Human parainfluenza viruses (hPIVs) cause upper and lower respiratory tract disease in children that results in a significant number of hospitalizations and impacts health systems worldwide. To date, neither antiviral drugs nor vaccines are approved for clinical use against parainfluenza virus, which reinforces the urgent need for new therapeutic discovery strategies. Here we use a multidisciplinary approach to develop potent inhibitors that target a structural feature within the hPIV type 3 haemagglutinin-neuraminidase (hPIV-3 HN). These dual-acting designer inhibitors represent the most potent designer compounds and efficiently block both hPIV cell entry and virion progeny release. We also define the binding mode of these inhibitors in the presence of whole-inactivated hPIV and recombinantly expressed hPIV-3 HN by Saturation Transfer Difference NMR spectroscopy. Collectively, our study provides an antiviral preclinical candidate and a new direction towards the discovery of potential anti-parainfluenza drugs.
Emerging Microbes & Infections, 2014
Enterovirus 71 (EV71) causes severe central nervous system infections, leading to cardiopulmonary... more Enterovirus 71 (EV71) causes severe central nervous system infections, leading to cardiopulmonary complications and death in young children. There is an urgent unmet medical need for new pharmaceutical agents to control EV71 infections. Using a multidisciplinary approach, we found that the approved pediatric antiparasitic drug suramin blocked EV71 infectivity by a novel mechanism of action that involves binding of the naphtalentrisulonic acid group of suramin to the viral capsid. Moreover, we demonstrate that when suramin is used in vivo at doses equivalent to or lower than the highest dose already used in humans, it significantly decreased mortality in mice challenged with a lethal dose of EV71 and peak viral load in adult rhesus monkeys. Thus, suramin inhibits EV71 infection by neutralizing virus particles prior to cell attachment. Consequently, these findings identify suramin as a clinical candidate for further development as a therapeutic or prophylactic treatment for severe EV71 infection.
Exploring human parainfluenza virus type-1 hemagglutinin-neuraminidase as a target for inhibitor discovery
Journal of medicinal chemistry, Jan 25, 2014
Human parainfluenza virus type 1 is the major cause of croup in infants and young children. There... more Human parainfluenza virus type 1 is the major cause of croup in infants and young children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Neu5Ac2en)-based inhibitor development. In this study, we explore the effect of C-5 modifications on the potency of Neu5Ac2en derivatives that target the human parainfluenza type-1 hemagglutinin-neuraminidase protein. Our study demonstrates that the replacement of the Neu5Ac2en C-5 acetamido moiety with more hydrophobic alkane-based moieties improves the inhibitory potency for both hemagglutinin-neuraminidase functions. These findings shed light on the importance of C-5 substitution on Neu5Ac2en in the design of novel sialic acid-based inhibitors that target human parainfluenza type-1 hemagglutinin-neuraminidase.
In-vitro Antiviral Activity of a Novel Phthalic Acid Ester Derivative Isolated from the Bangladeshi Mangrove Fern Acrostichum aureum
Background: Over the past century dengue (DENV2), chikungunya (CHIKV) and human parainfluenza (hP... more Background: Over the past century dengue (DENV2), chikungunya (CHIKV) and human parainfluenza (hPiV3) viruses have profoundly impacted on human morbidity, mortality and the economy worldwide. Current therapy options to treat infections of these viruses have severe limitations leading to a continued search for novel drug candidates. Acrostichum aureum L. (Pteridaceae) is a mangrove fern, that has been used as a traditional medicine in Bangladesh and other various countries for a variety of diseases including infection.
Objectives: Isolation and structural elucidation of novel antiviral secondary metabolites from the methanol extract of the aerial parts of A. aureum.
Materials and methods: The novel phthalate acid ester was isolated (HPLC) and structurally elucidated using 1D and 2D NMR, MS and other spectroscopic methods. The compound was tested for antiviral activity against DENV2 andhPiV3in Vero cells using the fluorescent focus (FFA) assay and against CHIKV virus in LLC-MK2 cells using the plaque-forming unit assay (PFU). The activity of the isolated compound was further compared with its known derivative.
Results: In this study, we report on the isolation of a novel phthalic acid ester, 2’’-(methoxycarbonyl)-5’’- methylpentyl 2’-methylhexyl phthalate from the aerial parts of the Bangladeshi mangrove fern Acrostichumaureum and its in vitro antiviral activity. The novel phthalate showed antiviral activity against dengue virus, human parainfluenza virus and chikungunya. The most potent activity was recorded against hPiV3 (EC50 29.4 μM) and was slightly higher than the activity determined for the positive control BCX 2798 (EC50 44 μM). Cellulose acetate phthalate was also evaluated for antiviral activity against these viruses for the first time and was found to be inactive. Both compounds were found to be non-toxic against Vero and LLC-MK2 cells.
Conclusion: This study shows that some selected phthalates have potent antiviral activity and should be further investigated as potential novel antiviral agents.
Glycoconjugate Journal, 2010
The secretor (Se)/nonsecretor (se) histo-blood group variation depends on the action of the FUT2 ... more The secretor (Se)/nonsecretor (se) histo-blood group variation depends on the action of the FUT2 enzyme and has major implications for human susceptibility to infections. To characterize the functionality of FUT2 variants, we assessed the correlation between saliva phenotypes and sequence variation at the FUT2 gene in sixty seven individuals from northern Portugal. While most nonsecretor haplotypes were found to carry the 428G > A nonsense mutation in association with a 739G > A missense substitution, we have also identified a recombinant haplotype carrying the 739*A allele together with the efficient 428*G variant in individuals with the Se phenotype. This finding suggested, in contrast to previous results, that the 739*A allele encodes an efficient Se allele. To test this hypothesis we evaluated the in vivo enzyme activity of full coding expression constructs in transient transfection of CHO-K1 cells using FACS (fluorescence-activated cell sorting) analysis and expression of type 2 and type 3 chain H structures as read out. We detected FUT2 activity for the 739*A expression construct, demonstrating that the 739G > A substitution is indeed not inactivating. In accordance with the hypothesis that FUT2 is under long standing balancing selection, we estimated that the time depth of FUT2 global genetic variation is as old as 3 million years. Age estimates of specific variants suggest that the 428G > A mutation occurred at least 1.87 million years ago while the 739G > A substitution is about 816,000 years old. The 385A > T missense mutation underlying the non-secretor phenotype in East Asians appears to be more recent and is likely to have occurred about 256,000 years ago.
Journal of Molecular Evolution - J MOL EVOL, 2009
The alpha-2-fucosyltransferases (α2FTs) are enzymes involved in the biosynthesis of α2fucosylated... more The alpha-2-fucosyltransferases (α2FTs) are enzymes involved in the biosynthesis of α2fucosylated glycan structures. In mammalian genomes, there are three α2FT genes located in tandem—FUT1, FUT2, and Sec1—each contained within a single exon. It has been suggested that these genes originated from two successive duplications, with FUT1 being generated first and FUT2 and Sec1 second. Despite gene conversion being considered the main mechanism of concerted evolution in gene families, previous studies of primates α2FTs failed to detect it, although the occurrence of gene conversion between FUT2 and Sec1 was recently reported in a human allele. The primary aim of our work was to initiate a broader study on the molecular evolution of mammalian α2FTs. Sequence comparison leads us to confirm that the three genes appeared by two rounds of duplication. In addition, we were able to detect multiple gene-conversion events at the base of primates and within several nonprimate species involving FUT...
Glycobiology, 2009
RHDV (rabbit hemorrhagic disease virus) is a highly virulent calicivirus that has become a major ... more RHDV (rabbit hemorrhagic disease virus) is a highly virulent calicivirus that has become a major cause of mortality in wild rabbit populations (Oryctolagus cuniculus). It binds to the histo-blood group antigen (HBGA) H type 2 which requires an alpha1,2fucosyltransferase for its synthesis. In rabbit, three alpha1,2fucosyltransferases genes are known, Fut1, Fut2, and Sec1. Nonfunctional alleles at any of these loci could potentially confer resistance to RHDV, similar to human FUT2 alleles that determine the nonsecretor phenotype and resistance to infection by various NoV strains. In this study, we looked for the presence of H type 2 on buccal epithelial cells of wild rabbits from two geographic areas under RHDV pressure and from one RHDV-free area. Some animals with diminished H type 2 expression were found in the three populations (nonsecretor-like phenotype). Their frequency markedly increased according to the RHDV impact, suggesting that outbreaks selected survivors with low expres...
Glycobiology, 2008
Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly pathogenic emergent virus wh... more Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly pathogenic emergent virus which replicates in cells that can express ABH histo-blood group antigens. The heavily glycosylated SARS-CoV spike (S) protein binds to angiotensin-converting enzyme 2 which serves as a cellular receptor. Epidemiological analysis of a hospital outbreak in Hong Kong revealed that blood group O was associated with a low risk of infection. In this study, we used a cellular model of adhesion to investigate whether natural antibodies of the ABO system could block the S protein and angiotensin-converting enzyme 2 interaction. To this aim, a C-terminally EGFP-tagged S protein was expressed in chinese hamster ovary cells cotransfected with an alpha1,2-fucosyltransferase and an A-transferase in order to coexpress the S glycoprotein ectodomain and the A antigen at the cell surface. We observed that the S protein/angiotensin-converting enzyme 2-dependent adhesion of these cells to an angiotensin-conv...
Histo-blood group antigens ABH actors of the innate antiviral protection : examples of Caliciviruses (NV and RHDV) and of the SARS-CoV
Glycobiology, 2008
It binds to the histo-blood group antigen (HBGA) H type 2 which requires an α1,2fucosyltransferas... more It binds to the histo-blood group antigen (HBGA) H type 2 which requires an α1,2fucosyltransferase for its synthesis. In rabbit, three α1,2fucosyltransferases genes are known, Fut1, Fut2, and Sec1. Nonfunctional alleles at any of these loci could potentially confer resistance to RHDV, similar to human FUT2 alleles that determine the nonsecretor phenotype and resistance to infection by various NoV strains. In this study, we looked for the presence of H type 2 on buccal epithelial cells of wild rabbits from two geographic areas under RHDV pressure and from one RHDV-free area. Some animals with diminished H type 2 expression were found in the three populations (nonsecretor-like phenotype). Their frequency markedly increased according to the RHDV impact, suggesting that outbreaks selected survivors with low expression of the virus ligand. Polymorphisms of the Fut1, Fut2, and Sec1 coding regions were determined among animals that either died or survived outbreaks. The Fut2 and Sec1 genes presented a high polymorphism and the frequency of one Sec1 allele was significantly elevated, over 6fold, among survivors. Sec1 enzyme variants showed either moderate, low, or undetectable catalytic activity, whereas all variant Fut2 enzymes showed strong catalytic activity. This functional analysis of the enzymes encoded by each Fut2 and Sec1 allele suggests that the association between one Sec1 allele and survival might be explained by a deficit of α1,2fucosyltransferase expression rather than by impaired catalytic activity.
Biochemical Journal, 2006
Breast-feeding-associated protection against calicivirus diarrhoea is associated with the presenc... more Breast-feeding-associated protection against calicivirus diarrhoea is associated with the presence of high levels of 2-linked oligosaccharides in mother's milk, and human calicivirus strains including the NV (Norwalk virus) use gut 2-linked fucosylated glycans as receptors, suggesting the presence of decoy receptors in milk. Our aim was to analyse the ability of human milk to inhibit the attachment of rNV VLPs (recombinant NV-like particles) to their carbohydrate ligands and to characterize potential inhibitors found in milk. Milk from women with the secretor phenotype was strongly inhibitory, unlike milk from women that are non-secre-tors, which is devoid of 2-linked fucosylated structures. At least two fractions in human milk acted as inhibitors for the NV capsid attachment. The first fraction corresponded to BSSL (bile-saltstimulated lipase) and the second to associated mucins MUC1 and MUC4. These proteins present tandem repeat O-glycosylated sequences that should act as decoy receptors for the NV, depending on the combined mother/child secretor status.
Journal of Virology, 2006
Rotavirus (RV) is the main etiological agent of severe gastroenteritis in infants, and vaccinatio... more Rotavirus (RV) is the main etiological agent of severe gastroenteritis in infants, and vaccination seems the most effective way to control the disease. Recombinant rotavirus-like particles composed of the viral protein 6 (VP6) and VP2 (2/6-VLPs) have been reported to induce protective immunity in mice when administered by the intranasal (i.n.) route. In this study, we show that administration of 2/6-VLPs by the intrarectal (i.r.) route together with either cholera toxin (CT) or a CpG-containing oligodeoxynucleotide as the adjuvant protects adult mice against RV infection. Moreover, when CT is used, RV shedding in animals immunized by the i.r. route is even reduced in comparison with that in animals immunized by the i.n. route. Humoral and cellular immune responses induced by these immunization protocols were analyzed. We found that although i.r. immunization with 2/6-VLPs induces lower RV-specific immunoglobulin G (IgG) and IgA levels in serum, intestinal anti-RV IgA production is higher in mice immunized by the i.r. route. Cellular immune response has been evaluated by measuring cytokine production by spleen and Peyer's patch cells (PPs) after ex vivo restimulation with RV. Mice immunized by the i.n. and i.r. routes display higher gamma interferon production in spleen and PPs, respectively. In conclusion, we demonstrate that i.r. immunization with 2/6-VLPs protects against RV infection in mice and is more efficient than i.n. immunization in inducing an anti-RV immune response in intestinal mucosa.
Journal of Virological Methods, 2005
Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promisi... more Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.
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Papers by Patrice Guillon
Objectives: Isolation and structural elucidation of novel antiviral secondary metabolites from the methanol extract of the aerial parts of A. aureum.
Materials and methods: The novel phthalate acid ester was isolated (HPLC) and structurally elucidated using 1D and 2D NMR, MS and other spectroscopic methods. The compound was tested for antiviral activity against DENV2 andhPiV3in Vero cells using the fluorescent focus (FFA) assay and against CHIKV virus in LLC-MK2 cells using the plaque-forming unit assay (PFU). The activity of the isolated compound was further compared with its known derivative.
Results: In this study, we report on the isolation of a novel phthalic acid ester, 2’’-(methoxycarbonyl)-5’’- methylpentyl 2’-methylhexyl phthalate from the aerial parts of the Bangladeshi mangrove fern Acrostichumaureum and its in vitro antiviral activity. The novel phthalate showed antiviral activity against dengue virus, human parainfluenza virus and chikungunya. The most potent activity was recorded against hPiV3 (EC50 29.4 μM) and was slightly higher than the activity determined for the positive control BCX 2798 (EC50 44 μM). Cellulose acetate phthalate was also evaluated for antiviral activity against these viruses for the first time and was found to be inactive. Both compounds were found to be non-toxic against Vero and LLC-MK2 cells.
Conclusion: This study shows that some selected phthalates have potent antiviral activity and should be further investigated as potential novel antiviral agents.