GSBS Dissertations and Theses
Approval Date
9-1-2016
Document Type
Doctoral Dissertation
Academic Program
Biochemistry and Molecular Pharmacology
Department
Biochemistry and Molecular Pharmacology
First Thesis Advisor
Celia Schiffer, PhD
Keywords
Dengue Virus, Viral Drug Resistance, Hepacivirus, HIV Infections, HIV Protease
Subjects
Dissertations, UMMS; Dengue Virus; Drug Resistance, Viral; Hepacivirus; HIV Infections; HIV Protease
Abstract
Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.
First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.
Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.
Repository Citation
Lin, K. Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation. (2016). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 841. DOI: 10.13028/M2GW26. https://escholarship.umassmed.edu/gsbs_diss/841
DOI
10.13028/M2GW26
DOI Link
Rights and Permissions
Copyright is held by the author, with all rights reserved.
Included in
Biochemistry Commons, Cellular and Molecular Physiology Commons, Enzymes and Coenzymes Commons, Immunoprophylaxis and Therapy Commons, Pharmacology Commons, Structural Biology Commons, Virology Commons, Virus Diseases Commons