Bioactive recombinant human oncostatin M for NMR-based screening in drug discovery

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.

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Fig: OSM structure overview. (A) Schematic representation of the OSM gene product. Signal peptide and propeptide are removed post-translationally. (B) Domain architecture of OSM showing the four helices A, B, C and D and disulfide bonds. The Loop CD and the C-terminus were not observed in the crystal structure and are colored gray. (C) Cartoon representation of OSM from PDBID 1EVS, Site II, Site III, and disulfides are shown as sticks, colored according to domain architecture. (D) Schematic representation of the expression constructs used in this study. A Tobacco Etch Virus protease site (scissors) was engineered into pD441-NH (6-His) and pD441-MBP (maltose binding protein) fusion constructs that included OSM 1–187.

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.

Mass, O.A., Tuccinardi, J., Woodbury, L. et al. Bioactive recombinant human oncostatin M for NMR-based screening in drug discovery. Sci Rep 11, 16174 (2021). https://doi.org/10.1038/s41598-021-95424-6

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