Abstract
Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Recently, elevated OSM levels have been found in the serum of COVID-19 patients in intensive care units. Multiple anti-OSM therapeutics have been investigated, but to date no OSM small molecule inhibitors are clinically available. To pursue a high-throughput screening and structure-based drug discovery strategy to design a small molecule inhibitor of OSM, milligram quantities of highly pure, bioactive OSM are required. Here, we developed a reliable protocol to produce highly pure unlabeled and isotope enriched OSM from E. coli for biochemical and NMR studies. High yields (ca. 10 mg/L culture) were obtained in rich and minimal defined media cultures. Purified OSM was characterized by mass spectrometry and circular dichroism. The bioactivity was confirmed by induction of OSM/OSM receptor signaling through STAT3 phosphorylation in human breast cancer cells. Optimized buffer conditions yielded 1H, 15N HSQC NMR spectra with intense, well-dispersed peaks. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 μM. These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening.
Highlights
Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis
We have further shown that OSM promotes the expression of proteases that can contribute to metastasis[14] as well as other metastasis-related proangiogenic proteins such as vascular endothelial growth factor (VEGF)[16] and hypoxia-inducible factor-1α (HIF1α)[16,17]
This fragment was chosen for three reasons: (1) it has the same sequence used in the crystal structure and supports a structure-based drug design approach[28], (2) the binding site we are interrogating with small molecules is distal to the C-terminus, and (3) the C-terminus of the mature protein, comprising residues 188–196, does not contribute to OSMRβ b inding[30]
Summary
Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 μM These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening. OSM was identified as one of the proteins that are found at high levels in the serum of COVID-19 intensive care unit (ICU) patients that correlated with disease severity, potentially pointing to a role for OSM in cytokine storm reactions during SARS-CoV-2 infections[9]. Our work in defining the role of OSM in breast cancer metastasis has shown that OSM signaling increases tumor cell detachment in a COX2-dependent fashion[13], epithelial-mesenchymal transition[14], and the invasive potential of human and mouse mammary carcinoma cells[13,14,15]. ERK1/2, PI3K, and STAT1/3 can be activated by OSM binding the LIFR or OSMR, but only signaling through the OSMR can activate STAT5, p38, JNK, and possibly PKC-δ and S TAT622
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