Virus (2) Basic elements Structure of DNA and RNA: base = contains information phosphate = linker, charged gives solubility desoxyribose = 2 nd linker, gives helical structure nucleotide = basic element http://ici.cegep-ste-foy.qc.ca/profs/gbourbonnais/pascal/nya/genetique/notesadn/adn3.htm Double stranded b-dna helix : Virus (6) Basic elements minor groove major groove http://ici.cegep-ste-foy.qc.ca/profs/gbourbonnais/pascal/nya/genetique/notesadn/adn3.htm -1- -2- Prénom NomUniversité de Neuchâtel -1- Prénom Nom / 12/01/2010
Double stranded DNA helix : Virus (7) Basic elements http://ici.cegep-ste-foy.qc.ca/profs/gbourbonnais/pascal/nya/genetique/notesadn/adn3.htm Conformational Analysis (1) DNA is constructed of the following elements: 4 different heterocyclic bases A, G, T, C 1 sugar desoxyribose 1 phosphate DNA is a double stranded helix DNA is the molecule for information storage = permanent memory = exclusive function -3- -4- Prénom NomUniversité de Neuchâtel -2- Prénom Nom / 12/01/2010
Conformational Analysis (2) RNA is constructed of the following elements: 4 different heterocyclic bases A, G, U, C 1 sugar ribose 1 phosphate RNA is often single stranded with multiple structures and multiple functions RNA is the molecule for information transcription = active memory; structural unit = reading head; catalytic activity = functional unit Conformational Analysis (3) DNA is resistant to chemical hydrolysis = stable for years RNA is sensitive to chemical hydrolysis = degrades within minutes Reasons: internal nucleophile steric congestion DNA RNA -5- -6- Prénom NomUniversité de Neuchâtel -3- Prénom Nom / 12/01/2010
Conformational Analysis (4) RNA hydrolysis is helped by the presence of the additional H group Conformational Analysis (5) Homo-DNA contains 1 CH2 group more = homologous to DNA Therefore homo-dna contains a six-membered ring instead of a five-membered ring. This makes the conformational analysis much easier. DNA Homo-DNA -7- -8- Prénom NomUniversité de Neuchâtel -4- Prénom Nom / 12/01/2010
Conformational Analysis (5 bis) Synthesis of homo-dna and conformational analysis done by: Prof, Albert Eschenmoser (ETH-Z and Scripps) Homo-DNA Conformational Analysis (6) Three levels Diamond lattice -9- -10- Prénom NomUniversité de Neuchâtel -5- Prénom Nom / 12/01/2010
Conformational Analysis (7) Introduction of an idealized conformation of Homo-DNA-trinucleotide into a diamond lattice Conformational Analysis (8) Repetitive structure of DNA characterized by δ = 60 Gives an almost linear double strand -11- -12- Prénom NomUniversité de Neuchâtel -6- Prénom Nom / 12/01/2010
Conformational Analysis (9) Homo-DNA: δ = 60 DNA: δ > 60 ; δ(max) = 120 Conformational Analysis (10) DNA: δ ~ 80 The skeleton of the single strand is helical The helicity is fixed by the double strand formation (= base pair formation) -13- -14- Prénom NomUniversité de Neuchâtel -7- Prénom Nom / 12/01/2010
Virus (10) Structure of the Influenza Virus Figure 1: Transmission electron micrograph of avian influenza H5N1 virus Figure 2: Influenza Virus Anatomy STRUCTURE The structure of the influenza virus (see Figure 2) is somewhat variable, but the virion particles are usually spherical or ovoid in shape and 80 to 120 nanometers in diameter. http://www.sfcdcp.org/index.cfm?id=87 http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html Virus (11) Structure of the Influenza Virus Trimeric hemagglutinin Schematic representation STRUCTURE The influenza virion is an enveloped virus that derives its lipid bilayer from the plasma membrane of a host cell. Two different varieties of glycoprotein spike are embedded in the envelope. Approximately 80 percent of the spikes are hemagglutinin, a trimeric protein that functions in the attachment of the virus to a host cell. The remaining 20 percent of the glycoprotein spikes consist of neuraminidase, which is thought to be predominantly involved in facilitating the release of newly produced virus particles from the host cell. http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html -15- -16- Prénom NomUniversité de Neuchâtel -8- Prénom Nom / 12/01/2010
Virus (12) Structure of the Influenza Virus Electron microscopic pictures of the influence virus Electron microscopic picture showing the helical ribonucleo-protein STRUCTURE n the inner side of the envelope that surrounds an influenza virion is an antigenic matrix protein lining. Within the envelope is the influenza genome, which is organized into eight pieces of single-stranded RNA (A and B forms only; influenza C has 7 RNA segments). The RNA is packaged with nucleoprotein into a helical ribonucleoprotein form, with three polymerase peptides for each RNA segment. http://web.uct.ac.za/depts/mmi/stannard/fluvirus.html Virus (13) Structure of the Influenza Virus Life cycle of the influence virus STRUCTURE Specific varieties of the virus are generally named according to the particular antigenic determinants of hemagglutinin (13 major types) and neuraminidase (9 major types) surface proteins they possess, as in influenza A(H2N1) and A(H3N2). New strains of the influenza virus emerge due to a gradual process known as antigenic drift. Through this mechanism, the virus is able to largely circumvent the body's immune system. http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html -17- -18- Prénom NomUniversité de Neuchâtel -9- Prénom Nom / 12/01/2010
Virus (14) Classification and Nomenclature of Influenza Virus CLASSIFICATIN of virus STRAINS Is done on the basis of antigenicity of NP (nucleoprotein) and MP (matrix protein) into three main groups: Influenza A -HA undergoes minor and occasional major changes - very important. - NA some variation. Influenza B) Undergoes relatively slow change in HA with time. Known only in man. Influenza C) Uncommon strain, known only in man. NMENCLATURE Influenza strains are named in the following way: A TYPE of influenza SINGAPRE TWN where first isolated 6 NUMBER of isolates 86 YEAR of isolation (H1N1) MAJR TYPE of HA and NA http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html Virus (15) Epidemiology Influenza A virus is essentially an avian virus that has "recently" crossed into mammals. Birds have the greatest number and range of influenza strains. Avian haemagglutinins sometimes appear in pig human and horse influenza strains. Every now and then (10-15 years) a major new pandemic strain appears in man, with a totally new HA and sometimes a new NA as well (antigenic shift). This variant causes a major epidemic around the world (pandemic). ver the subsequent years this strain undergoes minor changes (antigenic drift) every two to three years, probably driven by selective antibody pressure in the populations of humans infected. http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html -19- -20- Prénom NomUniversité de Neuchâtel -10- Prénom Nom / 12/01/2010
Influenza A Evolution Virus (16) Epidemiology 1874 --- (H3N8) 1890 --- (H2N2)...Pandemic 1902 --- (H3N2) 1918 --- (H1N1)...Pandemic 1933 --- (H1N1)...First strains isolated 1947 --- (H1N1)...Variation detected 1957 --- (H2N2)..."Asian" Flu pandemic 1968 --- (H3N2)..."Hong Kong" Flu pandemic 1976 --- (H1N1)..."Swine" Flu, non-epidemic 1977 --- (H1N1) + (H3N2)..."Russian" Flu epidemic http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html Virus (17) Anti-Viral Chemotherapy Stages in virus replication which are possible targets for chemotherapeutic agents: Attachment to host cell Uncoating - (Amantadine) Synthesis of viral mrna - (Interferon) Translation of mrna - (Interferon) Replication of viral RNA or DNA - (Nucleoside anologues) Maturation of new virus proteins (Protease inhibitors) Budding, release Diseases for which effective therapy is available: Herpes Simplex virus (Acyclovir) Varicella-Zoster virus (Acyclovir) Cytomegalovirus (Gancyclovir, Foscarnet) AIDS (Zidovudine, Lamivudine[3TC], Protease inhibitors; in combination) Respiratory Syncitial virus (Ribavirin) Influenza (Amantadine) http://web.uct.ac.za/depts/mmi/jmoodie/welcome1.html -21- -22- Prénom NomUniversité de Neuchâtel -11- Prénom Nom / 12/01/2010
Virus (18) Anti-Viral Chemotherapy Nucleotide analogues: These are synthetic compounds which resemble nucleosides, but have an incomplete or abnormal deoxy-ribose /or ribose group.these compounds are phosphorylated to the tri-phosphate form within the infected cell. In this form, the drug competes with normal nucleotides for incorporation into viral DNA or RNA. http://web.uct.ac.za/depts/mmi/jmoodie/welcome1.html Virus (19) Treatment and Prophylaxis of Influenzavirus Zanamivir (Relenza) 1 st neuramidase inhibitor Sialic acid The release of sialic acid is inhibited by compounds like Zanamivir or seltamivir 1999 The drugs, Relenza and Tamiflu, are licensed in US and Europe. http://web.uct.ac.za/depts/mmi/stannard/fluvirus.html -23- -24- Prénom NomUniversité de Neuchâtel -12- Prénom Nom / 12/01/2010
http://www.biozentrum.unibas.ch/~schwede/teaching/bixii-ss05/boehm_i.pdf http://www.biozentrum.unibas.ch/~schwede/teaching/bixii-ss05/boehm_i.pdf -25- -26- Prénom NomUniversité de Neuchâtel -13- Prénom Nom / 12/01/2010
http://www.biozentrum.unibas.ch/~schwede/teaching/bixii-ss05/boehm_i.pdf Neuramidase X-ray structure from Swiss Protein Data bank Tetramer Asymetric Unit Swiss Protein Data Bank: Accession NumberQ8JT58 (http://www.expasy.org/uniprot/q8jt58) The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor Varghese, J.N., McKimm-Breschkin, J.L., Caldwell, J.B., Kortt, A.A., Colman, P.M. Proteins v14 pp. 327-32, 1992-27- -28- Prénom NomUniversité de Neuchâtel -14- Prénom Nom / 12/01/2010
Neuramidase X-ray structure from Swiss Protein Data bank Q8JT58 X-ray structure of Neuraminidase with a transition state analog inhibitor http://www.biozentrum.unibas.ch/~schwede/teaching/bixii-ss05/boehm_i.pdf -29- -30- Prénom NomUniversité de Neuchâtel -15- Prénom Nom / 12/01/2010
X-ray structure of Neuraminidase with Zanamivir http://www.biozentrum.unibas.ch/~schwede/teaching/bixii-ss05/boehm_i.pdf Comparison of the binding the transition state analog and seltamivir (Tamiflu) http://www.biozentrum.unibas.ch/~schwede/teaching/bixii-ss05/boehm_i.pdf -31- -32- Prénom NomUniversité de Neuchâtel -16- Prénom Nom / 12/01/2010
Tamiflu (1) Source: Coopération Tamiflu (2) Significant reduction in flu duration: Multicenter, double-blind, randomized and placebo-controlled studies of 253 otherwise healthy adults aged 18 to 65 years with naturally acquired influenza. Significant reduction in the duration of fever: Multicenter, double-blind, randomized and placebo-controlled studies (one in the U.S. and two outside of the U.S.) of 610 otherwise healthy adults aged 18 to 65 years with naturally acquired influenza. Tamiflu home page: http://www.tamiflu.com/hcp/treatment/treat_index.asp -33- -34- Prénom NomUniversité de Neuchâtel -17- Prénom Nom / 12/01/2010
Tamiflu (3) Reduction in viral shedding in challenge studies: Challenge studies support A antiviral activity in volunteers who received TAMIFLU or placebo shortly after influenza virus inoculation. Reduction in viral shedding in challenge studies : Challenge studies support B antiviral activity in volunteers who received TAMIFLU or placebo shortly after influenza virus inoculation. Tamiflu home page: http://www.tamiflu.com/hcp/treatment/treat_index.asp Synthesis of Tamiflu Phosphate d oseltamivir (R) N H (R) (S) NH 2. H 3 P 4-36- (3R,4R,5S)-4-Acetylamino-5-amino-3-(1-ethylpropoxy)-1- cyclohexene-1-carboxylic acid ethyl ester -35- Prénom NomUniversité de Neuchâtel -18- Prénom Nom / 12/01/2010
Synthesis : General Scheme (1) H CH 1) EtH, SCl 2 (0,5 eq) reflux, 3h 2) Evaporation H CEt 1) 3-pentanone (25 eq), TsH (0,05 eq) 2) Extraction 3) Evaporation CEt H 97% H 97% H H H 1 2 3 1) MsCl (1,3 eq) 2) Et 3N (2,0 eq), CH 2Cl 2, 0-5 C 3) Extraction 93% 1) KHC 3, EtH CEt H 2, 60 C 2) Evaporation 3) Extraction à l'hexane 1) BH CEt. 3 SMe 2, CH 2Cl 2 TMSTf, -20 C 2) Addition lente de NaHC 3 CEt 6 60 % de 4 H 5 Ms 4 Ms Synthesis : General Scheme (2) 1) NH 2 CEt MgBr 2.Et 2 (0,2 eq) t-bume / MeCN 9:1 55 C, 16h 2) (NH 4) 2S 4 / H 2 1) Pd/C, EtH CEt H 2NCH 2H reflux, 3h 2) H 2S 4 / H 2 CEt 97% 77% H H HN 6 7 8 NH 2 1) PhCH, t-bume, - H 2 2) MsCl, NEt 3, filtration 80% NH 3) 2, 112 C, 15h 4) HCl/H 2 CEt 1) 10% Pd/C, EtH H 2NCH 2CH 2H reflux, 3h 2) H 3P 4, EtH CEt Ac 2, AcH MsH t-bume, 15h, 20 C CEt HN 70 % HN 83% H 2N NH 2. H 3P 4 HN HN 11 10 9-37- -38- Prénom NomUniversité de Neuchâtel -19- Prénom Nom / 12/01/2010
Synthesis : General Scheme (1) Synthesis : General Scheme (2) -39- -40- Prénom NomUniversité de Neuchâtel -20- Prénom Nom / 12/01/2010
Natural Source of Shikimic acid: Star Anise The characteristically shaped fruits (pods), always used in dried state. The dried fruits may contain 5 to 8% of essential oil, which dominated by anethole (85 to 90%). The other components, phellandrene, safrole and terpineol, have only small effect on the aroma. Corey s Synthesis (2006) -41- -42- Prénom NomUniversité de Neuchâtel -21- Prénom Nom / 12/01/2010
Characteristics of the commercial product Sold as gelatinized capsule or as solution For anti-viral treatment, can beusedfor preventionof flu Inhibits neuraminidase. Reduces the capacity of the propagation of the virus «Commercial History» seltamivir sold by Gilead Sciences for 80 millions CHF and 10% in royalties, commercialised under the name of Tamiflu by Roche Estimated sells for 2005 : 1,12 billion CHF, 55 millions packages sold, corresponds to 3,3% of the total sells of Roche Estimated sells: 150 millions packages for 2006 and 300 millions for 2007-43- -44- Prénom NomUniversité de Neuchâtel -22- Prénom Nom / 12/01/2010
Sources J. rg. Chem. 2001, 66, 2044-2051 J. rg. Chem., 1998, 63,4545-4550 J. Am. Chem. Soc, 2006, 128, 6310-6311 rg. Process Res. Dev. 1999, 3, 266 Wikipedia -45- Prénom NomUniversité de Neuchâtel -23- Prénom Nom / 12/01/2010