| High throughput screening (HTS) is an integral part of the drug discovery process, enabling researchers to screen thousands of compounds in a fast and facile manner. After the advent of combinatorial chemistry the need to develop HTS is greater than ever. It has been hypothesized there are between 3000-10000 possible targets for therapeutic intervention with only 300-500 of these currently utilized. To explore these therapeutic possibilities, it is necessary to quickly develop primary assays for these targets.; The primary goal of this dissertation was to develop methods of screening that are inexpensive, facile, and broadly applicable. A library of ∼10000 benzodiazepine tethered small molecules was screened in a multiplexed direct-binding assay against seven potential protein targets. The information gathered was sufficient to develop a structure-activity relationship (SAR) used to design a second generation focused library and more thoroughly describe the SAR of the compounds. A second type of binding assay was developed utilizing a microarray printing technology that allows the screening of ∼1000 member library in a single assay plate with 5 replicates for each library member. A library of 840 SH3 domain binding peptides was designed and synthesized to show discriminatory binding to Src and Abl SH3 proteins. The library was synthesized on resin containing peptide epitopes that allow the resulting library to self-sort to the array locations based on interactions between those epitopes and monoclonal antibodies.; The secondary goal of this dissertation is to apply high throughput methods to the development of a sandwich ELISA for the peptide hormone ghrelin to increase the throughput and improve the data quality over the available commercial assays. To develop the sandwich assay the epitopes of several antibodies were mapped using synthetic peptides in maleimide capture ELISA. Additionally the sandwich assay parameters were optimized for buffer contents, incubation times, and capture direction. Samples from clinical studies were assayed to measure ghrelin stability, enable a comparison to commercial assays, and monitor ghrelin under fed and fasting conditions. The result was a specific assay that led to the observation of a previously unseen relationship between endogenous ghrelin and growth hormone. |
