Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation and they consist of the discovery of norovirus protease inhibitors, modification of graphene oxides (GO) for the detection of norovirus, and design and fabrication of nanoelectronic device based on nanocarbon fibers for the detection of breast cancer proteases, legumain and cathepsin B. A novel class of tripeptidyl anti-noroviral compounds which strongly inhibit NV3CL[superscript]pro in enzyme and cell based assays was discovered. An example of one of the most active compounds is (1-{3-methyl-1-[2-oxo-1-(2-oxo-pyrrolidin-3-ylmethyl)-ethylcarbamoyl]-butylcarbamoyl}-2-naphthalen-1-yl-ethyl)-carbamic acid benzyl ester, which showed an IC₅₀ value of 0.14 ± 0.2 μM (enzyme assay) and EC₅₀ value of 0.04 ± 0.02 μM (cell based assay). This compound has an aldehyde warhead, a P1 glutamine surrogate, a P2 leucine, a P3 L-1-napthylalanine and an N-terminal carboxybenzyl cap. The corresponding bisulfite adduct, 2-[2-(2-benzyloxycarbonylamino-3-naphthalen-1-yl-propionylamino)-4-methyl-pentanoylamino]-1-hydroxy-3-(2-oxo-pyrrolidin-3-yl)-propane-1-sulfonic acid monosodium salt, has a comparable activity in enzyme and cell based assays (IC₅₀ 0.24 ± 0.1 μM; EC₅₀ 0.04 ± 0.03 μM). (1-{3-methyl-1-[2-oxo-1-(2-oxo-pyrrolidin-3-ylmethyl)-ethylcarbamoyl]-butylcarbamoyl}-2-naphthalen-1-yl-ethyl)-carbamic acid benzyl ester and its ketoamide derivative, (1-{1-[2-isopropylcarbamoyl-2-oxo-1-(2-oxo-pyrrolidin-3-ylmethyl)-ethylcarbamoyl]-3-methyl-butylcarbamoyl}-2-naphthalen-1-yl-ethyl)-carbamic acid benzyl ester, exhibited very good broad spectrum anti-viral activity, especially in human rhino virus and severe acute respiratory syndrome bioassays. We demonstrated that the surface of graphene oxide can be chemically modified with t-butylester and carboxylic acid functionalities. Fourier transform infrared spectroscopy, Raman spectroscopy and solid state nuclear magnetic resonance spectroscopy confirmed the presence of t-butylester and carboxylic acid functional groups. One sided oligonucleotide functionalized graphene oxide was synthesized using a solid state technique. A carboxylic acid functionalized graphene oxide was deposited onto the surface of electronic chips to bridge two gold electrodes, using a direct deposition technique. The carboxylic acid functionalized graphene oxide displayed semi-conductive properties and its use in an electronic biosensor device to detect noroviral RNA was investigated. Novel redox-active protease substrate peptides H₂N-(CH₂)₄CO-Ala-Ala-Asn-Leu-NHCH₂-ferrocene and H₂N-(CH₂)₄CO-Leu-Arg-Phe-Gly-NHCH₂-ferrocene were synthesized successfully and used in an alternating current voltammetry technique to facilitate the detection of the cancer related protease enzymes legumain and cathepsin B. After attachment of these peptides to the tips of carbon nanofiber nanoelectrode arrays, the presence of active protease enzymes could be detected as manifest by an exponential decay in current signal detect when monitored by alternating current voltammetry, at initial enzyme concentrations of 80.1 nM (legumain) and 30.7 nM (cathepsin B). The peptide cleavage sites were confirmed by analyses of the cleaved fragments using high performance liquid chromatography and mass spectrometry. Results showed that the cleavage of H₂N-(CH₂)₄CO-Ala-Ala-Asn-Leu-NHCH₂-ferrocene at the C-terminal side of asparagine residues by legumain and cleavage of H₂N-(CH₂)₄CO-Leu-Arg-Phe-Gly-NHCH₂-ferrocene at the C-terminal side of arginine residues by cathepsin B. Legumain exhibited a specificity constant (k[subscript]cat/K[subscript]m) of 11.3 x 10ᶟ M⁻¹S⁻¹ while cathepsin B exhibited a higher value of specificity constant (4.3 x 10⁴ M⁻¹S⁻¹) which agreed with the values obtained from fluorescence enzyme assay.

PML et les corps nucléaires (CN) sont impliqués dans la défense antivirale. En effet, notre équipe a montré que la surexpression de PMLIII confère la résistance au virus de la stomatite vésiculaire, au virus de l'influenza, au virus foamy mais pas au virus de l’encéphalomyocardite (EMCV). J’ai montré dans mon travail de thèse que l’EMCV contrecarre le pouvoir antiviral de PMLIII en induisant sa dégradation par un processus dépendant du protéasome et de SUMO. Cependant, les cellules de souris invalidées pour PML sont plus sensibles à l’infection par l’EMCV que les cellules issues de souris parentales. Pour déterminer l’isoforme de PML responsable de cet effet antiviral, j’ai analysé l’effet des sept isoformes de PML (PMLI-VII) et j’ai montré que seule l’expression en stable de PMLIV confère la résistance à l’EMCV en séquestrant la polymérase virale 3Dpol au sein des CN PML. De plus la déplétion de PMLIV augmente la production de l’EMCV dans les cellules traitées par l’interféron. Ces données indiquent le mécanisme par lequel PML confère la résistance à l’EMCV et révèlent que PML est l’une des protéines médiatrices des effets anti-EMCV de l’interféron. / PML and nuclear bodies (NBs) are implicated in antiviral defense. Indeed, our team showed that overexpression of PMLIII confers resistance to vesicular stomatitis virus, influenza virus, foamy virus but not to encephalomyocarditis virus (EMCV). I have shown during my thesis that EMCV counteracts the antiviral effect of PMLIII by inducing its degradation in SUMO and proteasome-dependent way. However, cells derived from PML knockout mice are more susceptible to EMCV infection than wild-type cells. To determine the isoforme of PML implicated in this antiviral effect, I analysed the effect of the seven PML isoforms (PMLI-PMLVII) and I showed that only stable expression of PMLIV confers resistance to EMCV by sequestring the viral polymérase 3Dpol in PML Nbs. In addition, depletion of PMLIV boosted EMCV production in interferon-treated cells. These finding sindicate the mechanism by which PML confers resistance to EMCV and reveal a new pathway mediating the antiviral activity of interferon against EMCV.

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