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dc.contributor.advisorMesecar, Andrewen_US
dc.contributor.authorBaez, Yahira M.en_US
dc.date.accessioned2012-12-13T21:45:58Z
dc.date.available2012-12-13T21:45:58Z
dc.date.created2012-08en_US
dc.date.issued2012-12-13
dc.date.submitted2012-08en_US
dc.identifier.urihttp://hdl.handle.net/10027/9527
dc.description.abstractCoronaviruses virus-encoded proteases are essential for viral replication. The research studies presented in this dissertation focused on the virus-encoded papain-like proteases, PLpro and PLP2, from the human coronaviruses SARS-CoV and HCoV-NL63, respectively. Inhibition of the enzymatic activity of PLpro has been shown to halt SARS-CoV viral replication in cell culture, validating coronaviral PLPs as antiviral drug targets. The structure-based design of inhibitors yielded the most potent PLpro inhibitor, compound 5b (IC50 = 0.2 µM, 2-fold potency improvement relative to the starting lead compound), displaying selectivity over related coronaviral and human deubiquitinating homolog enzymes. A new mechanism-based enzymatic assay for the rapid identification of PLpro and PLP2 inhibitors (also amenable to high-throughput screening (HTS)) was developed. From a screen of 100,000 compounds against PLP2, five lead compounds were discovered. Structure-activity relationships indicated that the dialkylaminobezene group of lead 1 and 2 (IC50 = 19.1 and 13.8 µM) is optimally substituted with a chlorine at position 3 (3-chlorophenyl). Both compounds share a 1-4,dimethylpiperazine chemical core linking the 3-chlorocyclohexanamine to the dimethoxyphenol (lead 1), or to an indole (lead 2). Lead 3 also contains the 4-ethyl-2,6-dimethoxyphenol but linked to conjugated aromatic groups containing a pyridine. All compounds display similar electronic properties at physiological conditions with a pronated piperazine and pyridine core. PLpro HTS resulted in new lead compounds (IC50 = 18 and 75 µM) with a peptide-like linker scaffold. PLpro substrate specificity towards viral peptides and ubiquitin substrates was investigated in detail using site-directed mutagenesis (SDM). The residues within the oxyanion hole were found to be most important for stability of the enzyme and for hydrolysis of both peptide and ubiquitin substrates. Palm domain mutants (R167E/A/M) showed a correlation in which increasing the domain hydrophobicity, increases the catalytic activity towards ubiquitin substrates. Zinc finger domain mutant M209A, decreased the deubiquitinating activity to less than 50%. The combination of M209A with F70S/H74R (located within the thumb domain) decreased deubiquitinating activity to 10% while maintaining the protease activity. These engineered PLpro enzymes are a useful tool for elucidating the in vivo effect of PLpro deubiquitinating activity and PLpro-mediated inactivation of the host innate immune response.en_US
dc.language.isoenen_US
dc.rightsCopyright 2102 Yahira M. Baezen
dc.subjectProteaseen_US
dc.subjectPLproen_US
dc.subjectCoronavirusen_US
dc.subjectSARSen_US
dc.subjectpapain-likeen_US
dc.subjectinhibitoren_US
dc.subjectstructureen_US
dc.subjectsubstrateen_US
dc.subjectSevere acute respiratory syndromeen
dc.titleInsight into the Substrate Specificity and Inhibition of Human Coronavirus Papain-Like Proteasesen_US
thesis.degree.departmentMedicinal Chemistry and Pharmacognosyen_US
thesis.degree.disciplinePharmacognosyen_US
thesis.degree.grantorUniversity of Illinois at Chicagoen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePhD, Doctor of Philosophyen_US
dc.type.genrethesisen_US
dc.contributor.committeeMemberOrjala, Jimmyen_US
dc.contributor.committeeMemberFederle, Michaelen_US
dc.contributor.committeeMemberVolz, Karlen_US
dc.contributor.committeeMemberBaker, Susanen_US
dc.type.materialtexten_US


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