Design and Synthesis of Thiazole-Based Small Molecules to Inhibit the Inflammatory Cytokine Oncostatin M

The body’s innate ability to produce an inflammatory response due to tissue irritation or damage is essential for growth and survival. The inflammatory response can be triggered by numerous stimuli, including physical trauma, inhalation of dangerous debris (such as silica and asbestos), tobacco/alcohol consumption, microbiota, diet, and other lifestyle stressors. In a well-orchestrated inflammatory response, the circulatory system increases blood flow and capillary permeability in the inflamed area to deliver nutrients, white blood cells, and inflammatory mediating molecules. However, a dysregulated inflammatory response can lead to chronic inflammation. Emerging evidence continues to frame oncostatin M (OSM) as a pro-inflammatory cytokine that drives chronic inflammation. The overexpression of OSM has been implicated in the maintenance of several chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, metabolic syndrome, systemic sclerosis, lupus, and many different forms of cancer. Therefore, OSM presents itself as a potential therapeutic target. To the best of our research group’s knowledge, no clinically approved drugs inhibit OSM signaling. Therefore, 26 lead small molecule inhibitors (SMIs) were generated by high-throughput virtual screening of ~1.65 million compounds targeting the putative receptor binding site of OSM. Out of the 26 compounds subjected to an enzyme-linked immunosorbent assay, SMI-8 was identified as one of the most potent inhibitors of OSM signaling. However, the predicted toxicity and stereoselective synthesis of SMI-8 makes the compound a poor drug candidate. So, mono- and bisthiazole-based analogs of SMI-8 were designed and synthesized using five distinct synthetic pathways to produce a total of 35 compounds. Enzyme-linked immunosorbent assay (ELISA) experiments and Western blot assays were performed to determine the inhibitory activity of several of the analogs. In addition, fluorescence quenching assays, differential scanning fluorimetry experiments, and chemical shift perturbation experiments identified direct binding properties of the SMIs. ELISA experiments revealed that almost all the SMI-8 analogs tested effectively decreased the amount of pSTAT3 expressed in T47D human breast cancer cells. Tryptophan fluorescence quenching assays determined the dissociation constant (K_D) values of SMI-8 and a structurally similar analog, SMI-8S, which were 6 ± 1 μM, and 14 ± 3 μM, respectively. Unfortunately, the fluorescence quenching assay could not be used to study the binding affinity of other analogs, as they were found to fluoresce at the same wavelength as the tryptophan residue of the OSM protein. In an attempt to solve this problem, differential scanning fluorimetry (DSF) experiments were used to determine the relative binding characteristics of the compounds. The data obtained from the DSF experiments suggest that most of the analogs tested bind to the unfolded state of OSM, as the ligands decrease the melting temperature of the protein. In addition, a chemical shift perturbation (CSP) experiment suggests that SMI-8S interacts with the amino acids Gln90, Arg91, and Leu 92 at the putative receptor binding site of OSM. The data, findings, and research within this thesis provide enough evidence to support the idea that SMIs can target OSM as a practical approach to treating various chronic inflammatory diseases.