High-Throughput Screening (HTS) of Potential Lead Compounds for Target Proteins with No False Identifications Using LC/MS
Issue Date
2017-08-31Author
Imaduwage, Kasun Prabodha
Publisher
University of Kansas
Format
125 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Chemistry
Rights
Copyright held by the author.
Metadata
Show full item recordAbstract
Developing effective high throughput screening (HTS) methods is of paramount importance in the early stage of drug discovery. When a protein binding event can be detected via spectroscopy, then absorbance, fluorescence or chemiluminescence based assays can be used to screen for tens of thousands of compounds per day. However, this is not feasible for many protein targets. To screen for drug candidates for target proteins where standard light based assays are not viable, we developed a high throughput screening method using LC-MS. Our assay is label free, which allows for rapid assay development and eliminates the risk of label interfering with the drug-protein complex. The main challenge of MS based screening methods is the high rate of false positives; our MS based screening method identifies binding partners for a target protein with no detection of false positives. In this method, ligands are mixed with the immobilized target protein and the non-binding analytes are detected, binding lignads are identified by comparing with a control where no protein was added. The tightest binders completely disappear from the ligand sea after the protein is added, therefore, these high affinity ligands are readily detected as peaks that are absent in the MS data. We then extended our study to eliminate not only the false positives but also the false negatives under high throughput conditions. The assay, which is similar to a ligand fishing experiment, mitigates false positives by selectively identifying positive hits when a ligand at the binding site of interest is displaced. The reporter molecule ionizes well, eliminating false negatives caused by non-ionizing compounds. Finally, we further extended our study to develop MS-based HTS assay using minute amount of target proteins to identify weak affinity ligands while still mitigating false positives and negatives. In summary, this dissertation addresses the major challenges in the field of MS-based HTS to identify binders for target proteins with no false positives or negatives. These improvements meet the current requirements of the drug discovery field better than any existing MS-based method.
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- Chemistry Dissertations and Theses [335]
- Dissertations [4701]
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