A Small Molecule Screen to Dissect the Secretory Biology of CHO-S Cells

Constitutive secretion is a fundamental process in cells that delivers extracellular proteins such as antibodies and matrix proteins to the extracellular space. Aberrations in constitutive secretion are implicated in numerous diseases. These include, but are not limited to, cancer, the bone disorder osteogenesis imperfecta, and neuronal tube defects. Constitutive secretion is a rate limiting step in the industrial production of recombinant therapeutic antibodies in Chinese Hamster Ovary (CHO) cells. Although the core machinery involved in constitutive secretion has been elucidated, a detailed understanding of how the process is regulated is lacking.

We performed a small molecule screen using a GFP-tagged CHO-S secretory reporter cell line. The primary screen identified multiple inhibitors of constitutive secretion. As expected, disruptors of microtubules and calcium channel blockers reduced secretion. Inhibitors of Akt/PKB and retinoic acid receptors also reduced secretion, suggesting a potential role of these proteins in regulating constitutive secretion. Several compounds were identified that inhibited secretion of multiple cargos across a diverse range of cell types. We provide evidence that our most potent secretion inhibitor, IMD 0354, may inhibit PI4K activity at the Golgi. IMD 0354 appears to block secreted cargo In the Golgi or in post-Golgi vesicles. Rescreening of compounds identified several enhancers of the secretion of the monoclonal antibody Herceptin in CHO-S cells. However, this enhancement was not sustained over several days of treatment. One of these compounds, the PPAR-δ/β agonist GW 0742 ,was subsequently identified as a novel enhancer of exodosis, the process through which normally ER-resident proteins are secreted.

The novel secretion modulators identified by this project may provide new tools for research into secretory biology. Additionally, characterisation of the mechanisms of these modulators may elucidate novel proteins involved in the regulation of constitutive secretion, which may be of relevance to improving recombinant protein production in CHO cells.