Scopton, A. P. | bioRxiv Science

bioRxiv2022

A small molecule screen reveals the essentiality of p38 kinase for choanoflagellate cell proliferation

Animal kinases regulate cellular responses to environmental stimuli, including cell differentiation, migration, survival, and response to stress, but the ancestry of these functions is poorly understood. Choanoflagellates, the closest living relatives of animals, encode homologs of diverse animal kinases and have emerged as model organisms for reconstructing animal origins. However, efforts to identify key kinase regulators in choanoflagellates have been constrained by the limitations of currently available genetic tools. Here, we report on a framework that combines small molecule-driven kinase discovery with targeted genetics to reveal kinase function in choanoflagellates. To study the physiological roles of choanoflagellate kinases, we established two high-throughput platforms to screen the model choanoflagellate Salpingoeca rosetta with a curated library of human kinase inhibitors. We identified 95 diverse kinase inhibitors that disrupt S. rosetta cell proliferation. By focusing on one inhibitor, sorafenib, we identified a p38 kinase as a regulator of the heat shock response in S. rosetta. This finding reveals a conserved p38 function between choanoflagellates, animals, and fungi. Moreover, this study demonstrates that existing kinase inhibitors can serve as powerful tools to examine the ancestral roles of kinases that regulate modern animal development.

biochemistry↗

bioRxiv2018

Integrated in silico and Drosophila cancer model platform captures previously unappreciated chemicals perturbing a kinase network

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) with disease-promoting kinase network activated by mutant dRetM955T. Structural models for kinases relevant to MTC were generated for virtually screening to identify initial hits that were dissimilar to known kinase inhibitors yet suppressed dRetM955T-induced oncogenicity. We then combined features from the hits and known inhibitors to create a hybrid molecule with improved dRetM955T phenotypic outcome. Our platform provides a framework to efficiently explore novel chemical spaces, develop compounds outside of the current inhibitor chemical space, and correct cancer-causing signaling networks to improve disease prognosis while minimizing whole body toxicity.\n\nAUTHOR SUMMARYEffective and safe treatment of multigenic diseases often involves drugs that modulate whole systems by interacting with multiple nodes in pathways and networks, i.e., polypharmacology. Polypharmacology is increasingly appreciated as a potential desirable property of kinase drugs; however, most known drugs that interact with multiple targets have been identified as such by chance, and most polypharmacological compounds are not chemically unique resembling to structures of known kinase inhibitors. The fruit fly Drosophila has been established as a robust screening platform that provides an inexpensive, rapid, and quantitative measure of whole animal drug response, complementing computational approaches. We present a chemical genetics approach that efficiently combines Drosophila with structural prediction and virtual screening, creating a unique discovery platform. We demonstrate the utility of our approach by developing useful small molecules targeting a kinase network in a Drosophila model of Medullary Thyroid Cancer (MTC) driven by the active mutant dRetM955T.

cancer biology↗