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Rutgers University

1. Inoyama, Daigo, 1981-. The discovery of small-molecule inhibitors of keap1-nrf2 interaction with homogeneous fluorescence-based high throughput screening assays.

Degree: PhD, Medicinal Chemistry, 2014, Rutgers University

Keap1-Nrf2 interaction is a key protein-protein interaction involved in the activation of antioxidant response element which regulates the expression of cytoprotective enzymes in response to oxidative stress conditions. To identify direct inhibitors of Keap1-Nrf2 interaction, a series of fluorescently-labeled Nrf2 peptides were synthesized and evaluated as tracers in the development of a fluorescence polarization (FP) assay. The optimized tracer, FITC-9mer Nrf2 amide, was determined to have the highest binding affinity and dynamic range of all tracers evaluated. The FP assay has considerable tolerance towards DMSO and was capable of distinguishing the inhibitory Nrf2 peptides with a Z'-factor of 0.70 as determined under the FP assay conditions. Taken together, these results demonstrate that the FP assay is suitable for application in high-throughput screening (HTS). In addition, a series of novel lanthanide chelates were designed, synthesized, and evaluated for the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay using an in situ labeling approach. The cyclam and EDTA-based tris-NTA were found to have the strongest affinity to the His6-tag of Keap1 Kelch domain with a Kd of 1.6 nM and 2.2 nM, respectively. Among the tris-NTA conjugates evaluated as donors of TR-FRET, the EDTA-tris-NTA Acp6-linker conjugate was found to have the highest TR-FRET signal. The in situ labeling technique was also evaluated for Eu(III) chelate and through biotin-streptavidin interaction, increasing the choice of fluorophores and extending the application of the assay to biotinylated biomolecules. Thus, the novel labeling reagent provides an excellent solution for facile development of TR-FRET assays in drug discovery. The FP assay was adapted for HTS of the NIH MLPCN library. Eight confirmed hits were obtained and one of the promising hits (LH602) was targeted for preliminary structure-activity relationship (SAR) studies. Among the synthesized compounds, 2,4,6-trimethylbenzenesulfonamide substituted analog 81 showed the highest affinity to Keap1 Kelch domain. However, LH602 was found to be unstable due to its naphthoquinone structure with a half-life of 6 hours in the assay buffer. Further SAR studies are needed to improve the binding affinity as well as stability of the compound in our effort to identify potent inhibitors of Keap1-Nrf2 interaction.

Advisors/Committee Members: Hu, Longqin (chair), LaVoie, Edmond J. (internal member), Rice, Joseph E. (internal member), Freundlich, Joel S. (outside member).

Subjects/Keywords: Protein-protein interactions; High throughput screening (Drug development)

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APA (6th Edition):

Inoyama, Daigo, 1. (2014). The discovery of small-molecule inhibitors of keap1-nrf2 interaction with homogeneous fluorescence-based high throughput screening assays. (Doctoral Dissertation). Rutgers University. Retrieved from https://rucore.libraries.rutgers.edu/rutgers-lib/45302/

Chicago Manual of Style (16th Edition):

Inoyama, Daigo, 1981-. “The discovery of small-molecule inhibitors of keap1-nrf2 interaction with homogeneous fluorescence-based high throughput screening assays.” 2014. Doctoral Dissertation, Rutgers University. Accessed November 20, 2019. https://rucore.libraries.rutgers.edu/rutgers-lib/45302/.

MLA Handbook (7th Edition):

Inoyama, Daigo, 1981-. “The discovery of small-molecule inhibitors of keap1-nrf2 interaction with homogeneous fluorescence-based high throughput screening assays.” 2014. Web. 20 Nov 2019.

Vancouver:

Inoyama, Daigo 1. The discovery of small-molecule inhibitors of keap1-nrf2 interaction with homogeneous fluorescence-based high throughput screening assays. [Internet] [Doctoral dissertation]. Rutgers University; 2014. [cited 2019 Nov 20]. Available from: https://rucore.libraries.rutgers.edu/rutgers-lib/45302/.

Council of Science Editors:

Inoyama, Daigo 1. The discovery of small-molecule inhibitors of keap1-nrf2 interaction with homogeneous fluorescence-based high throughput screening assays. [Doctoral Dissertation]. Rutgers University; 2014. Available from: https://rucore.libraries.rutgers.edu/rutgers-lib/45302/

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