Publication Date

12-2020

Date of Final Oral Examination (Defense)

8-18-2020

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Chemistry

Department

Chemistry

Major Advisor

Don Warner, Ph.D.

Advisor

Matthew King, Ph.D.

Advisor

Lisa Warner, Ph.D.

Advisor

Cheryl Jorcyk, Ph.D.

Abstract

Inflammation is one of the body's most important natural defense mechanisms involved in wound healing. It is usually triggered by a harmful event, such as physical trauma or exposure to external stimuli including bacteria, fungi, viruses, harmful chemicals, or environmental particulates. The inflammatory process brings blood containing inflammatory mediators consisting of leukocytes, hormones, and cytokines to the site of trauma to begin healing. However, the lack of a proper inflammatory response or an overactive response can lead to further progressive tissue damage resulting in chronic inflammatory conditions or death. The cytokine oncostatin M (OSM) is of particular interest due to the pivotal role it plays in chronic inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, and various forms of cancer. These diseases have a detrimental impact on a person’s quality of life and life expectancy, as well as the economy and health care system.

There is currently no clinically approved treatment targeting OSM. Thus, we propose the development of a small molecule inhibitor (SMI) targeting OSM. Using the known crystal structure of OSM combined with computational methods, a sample of 10,000 randomly selected molecules from online databases were docked in the OSM binding site 3, the site presumably responsible for binding to its receptor. The most energetically favorable binding poses were used to create a weighted density map (WDM) that shows the probability of aromatic carbons, hydrogen bond acceptors, and hydrogen bond donors to bind to OSM at particular locations in site 3. A 2,4-disubstituted quinazoline SMI was rationally designed that constructively overlaid with the WDM and was predicted to bind with high affinity based on computational docking studies.

The SMI and analogs thereof, termed the SMI-27 series, were synthesized using a 4-step reaction sequence to create a small library to be tested against OSM. In order to evaluate the ability of the SMIs to inhibit OSM activity and to determine cytokine binding specificity, enzyme-linked immunosorbent assays (ELISAs) and western blot assays were performed. Fluorescence quenching experiments were used to determine the binding affinity of SMI analogs toward OSM. Finally, chemical shift perturbation NMR experiments were used to identify the important amino acids required for binding of the SMI to OSM.

All of the SMI-27 analogs tested by ELISA inhibited OSM induced pSTAT3 expression below the level of the control. Additionally, SMIs 27B3 and 27B5 showed specific binding to OSM, and not to leukemia inhibitory factor (LIF) or interleukin-6 (IL-6), structurally related cytokines. The fluorescence quenching assays indicate that all SMIs exhibited direct binding to OSM, with 27B12 having a Kd of 5.1 ± 2.7 uM. Finally, the chemical shift perturbation assay identified several amino acids that appear to be involved in SMI binding. Importantly, three of these, tentatively assigned as Arg91, Leu92, and Gly166, are all located in OSM site 3. These experiments support our hypothesis that an SMI can be used to inhibit OSM activity and lay a solid foundation for the development of an SMI drug candidate that would provide a significant advancement in clinical treatments of OSM-related diseases.

DOI

10.18122/td/1746/boisestate

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