ABSTRACT OF THE DISSERTATION
Genetically Engineered Protein Modules: Development and Applications in Anti-Viral Agent Screening and Cancer Marker Detection
Doctor of Philosophy
Cell Molecular and Developmental Biology Graduate Program
University of California, Riverside, August 2010
Dr. Wilfred Chen, Chairperson
One of the most critical aspects in drug discovery is the bioactivity screening assay, by which compounds that most effectively inhibit the target are identified. During the past decade, the antiviral marketplace had experienced tremendous growth and is expected to grow further as new viral targets are identified. The persistent problem of viral resistance requires developing new-generation drugs to replace the existing ones. The pharmaceutical industry faces huge cost due to the failure of promising drug candidates to pass the test of potency and safety in humans. The quest for successful antiviral drugs is further hampered by the fact that successful drugs must often enter an infected cell and neutralize the replicating virus without causing harm to the host cell. To meet these challenges, the screening assays should be representative of the in vivo setting so that the results can be interpreted, with sufficient confidence, from the viewpoint of clinical efficacy. To this end, cell-based high-throughput screening (HTS) assays are preferred since they can provide additional information on the adverse effects of the drug and transport efficiency to the target cells.
The overall objective of this thesis is to develop a genetically programmable module that is easily adaptable for screening inhibitors for a wide range of proteases. Proteases are proteolytic enzymes that catalyze the cleavage of peptide bonds. They play important role in many essential intracellular and extracellular processes such as viral infection, blood coagulation, caner development, fibrinolysis, hormone maturation, and apoptosis. These properties make proteases a prime target for detailed investigation in order for better understanding the disease development process and to identify targets for drug treatment.
The approach was to generate a quantum dot (QD)-modified, protease-specific protein module that can be used as a Fluorescence Resonance Energy Transfer (FRET) based nanoprobe for probing protease activity. The site-specific incorporation of an acceptor fluorescent dye was accomplished using the thiol group of cysteine. While conjugation to QDs was facilitated by the presence of a hexa-histidine tag, due to its affinity for the Zn shell of QDs. Presence of an elastin domain within the module enabled the simple purification of the QD-modified FRET substrate. The modular nature of the design allowed easy alteration of the recognition sequence without significantly modifying other domains. Moreover, the FRET efficiency within the nanoassemblies was easily controlled by changing the QD- peptide dye ratios. The flexibility of the approach was demonstrated by generating nanoprobes for rapid and sensitive detection of the cancer-specific matrix metalloprotease (MMP-7) and the West Nile virus protease (NS3). Intracellular delivery of the substrates was facilitated by the use of a flanking TAT peptide. The effectiveness of the FRET substrate was investigated by monitoring the whole-cell fluorescence ratio between the QD and the acceptor fluorescence dye when introduced into a HeLa cell line. The utility of the assay system was validated for the HTS of HIV protease inhibitors.
This modular QD based FRET assay provides a future platform useful for general HTS of a wide range of protease activities relating to viral infection, blood coagulation, fibrinolysis, hormone maturation, and apoptosis. The ease of using this genetic approach also significantly reduced the cost associated with peptide synthesis. This could ultimately result in a low-cost screening drug platform for a wide range of protease targets important for human health.
The integration of the development of tunable biomolecules with FRET-based HTS screening represents a unique effort that expands the fundamental development of protein engineering with the implementation of drug discovery. Through this research an integrated perspective of the important interfaces and synergies connecting biochemistry, modern genetics, and HTS was gained.