Design of a Probe for Neuraminidase to Capture and Identify Influenza Viruses Shih-Hsiung Wu Institute of Biological Chemistry, Academia Sinica, Taipei, TAIWAN

Influenza A Virion Two glycoproteins : Hemagglutinin (HA, 80%) A trimeric protein that functions in the attachment of the virus to host cells. –14 major types Neuraminidase (NA, 20%) A tetrameric protein which is involved in facilitating the release of newly produced virus particles from host cell. –9 major types The Lancet 355 (2000) 827

Replicative cycle of Influenza virus The Lancet 355(2000) 827

Influenza Virus Neuraminidase Inhibitors NameManufacturerRoute 1 Zanamivir (Relenza) GlaxoInhalation 2 Oseltamivir (Tamiflu) RocheOral Two drugs that FDA approved : 12 Sialic Acid

Neuraminidase Inhibitor Normal assembly and budding of virus Lateral aggregation and formation of large bundles by virus Absence of NA inhibitorPresence of NA inhibitor The Lancet 355 (2000) 827

Zanamivir-Biotin Conjugates as Non-covalent Capture Ligand All three conjugates are good inhibitors of influenza NA. The compound successfully captured influenza A virus was demonstrated by ELISA method. Dr. J. L. McKimm-Breschkin et al. Angew. Chem. Int. Ed. 42 (2003)

Nature 437 (2005) 1108 Relenza probably lost its binding ability for drug-resistant mutants Is it possible to design a probe for capturing not only wild-type influenza viruses but also its mutant viruses? Generation of Drug-resistant Mutants

Mechanism-Based Inhibitor or probe for Glycosidase Suicide substrate inhibitors –  -Glucosidase Danzin et al –Neuraminidase Quash et al Ki=1.7 mM Ki=3.7 mM Ki=0.15 mM

Activity Probes –N-Acetylglucosaminidase Ichikawa et. al. (2001) –  -Glucosidase L.-C. Lo et al. (2002) –  -Galactosidase S. Nishimura, Y.-C. Lee et al. (2004) Neuraminidase (Vibrio cholerae) S. Nishimura et al., (2005) Mechanism-Based Inhibitor or probe for Glycosidase

RecognitionReporterLinker group Latent reactive Hydrolytic Activity Probes vs Suicide Substrates Activity Probes Recognition group Latent reactive Suicide Substrates

Design of a Mechanism-Based activity probe for Neuraminidase Sialic acid biotin The biotin group serves as a reporter (or tracer) : 1. Western blotting : it is used to visualize the labeled NA with streptavidin - conjugated peroxidase chemiluminescence. 2. Virus-capturing study by ELISA method

Mechanism of neuraminidase activity probe

Synthesis of activity probe for Neuraminidase Conditions: (i) MeOH, IR-120 (H + ) resin, 92 %; (ii) AcCl, AcOH; (iii) 2-hydroxy-5-nitrobenzaldehyde, Cs 2 CO 3, Bu 4 NBr, H 2 O-CHCl 3, 67 % for two steps; (iv) DAST, CH 2 Cl 2, 47 %; (v) H 2, 5% Pd/C, MeOH, 95 %; (vi) succinic ahydride, TEA, CH 2 Cl 2, 94 %

Conditions: (vii) EDCI, HOBt, 8, DIEA, DMF, 75 %; (viii) Na 2 CO 3, MeOH; aqueous Na 2 CO 3, 52 %.

Neuraminidase labeling assay NA : Arthrobacter ureafaciens neuraminidase Three biotinylated bands corresponding to isoenzymes at 88, 66, and 52 KDa were observed. The probe had no effect on BSA. In the absence of the probe, neither NA nor BSA was biotinylated. Western blotting

Mechanism-Based inhibition AU : Arthrobacter ureafaciens CP : Clostridium perfringens VC : Vibrio cholerae Inf A : influenza A virus probe and zanamivir : 3.3 mM  Zanamivir : transition state analog  NA Probe : mechanism-based inhibitor ＊ ＊ ＊

The Capture and Identification of Influenza Virus ELISART-PCR Anti-HA Ab IgG-HRP

The Detection of influenza A virus by ELISA NA probe could successfully capture influenza A virus and the intensity of the responding signal was proportional to the number of virus particles. Only influenza A virus was selectively captured and detected on the plate as compared with non-NA containing Japanese encephalitis virus (JEV).

RT-PCR Template Vol. RNA isolation RT-PCR (nucleoprotein) SYBR Safe TM Gel Stain A/WSN/33 NP 307 bp Virus particles (PFU) 500 NP 307 bp

Application of NA activity probe Activity probe for detection of microorganism –Virus Orthomyxoviridae (influenza virus) Paramyxoviridae (Newcastle disease virus, Sendai virus, parainfluenza virus, Mumps virus, porcine rubulavirus virus) –Pathogenic bacteria or parasite Clostridium perfringens Vibrio cholerae Shigella dysenteriae Salmonella typhimurium Streptococcus pneumoniae Trypanosoma cruzi

Application of activity-based probe Tumor Target Discovery Novel Enzyme Discovery Detection of Microorganism Inhibitor Discovery Characterization of Enzyme Mechanism Activity-based probe Proteomics