Gabriel Castro-Falcón1, Grant S Seiler1,2, Özlem Demir2, Manoj K Rathinaswamy3, David Hamelin3, Reece M Hoffmann3, Stefanie L Makowski4, Anne-Catrin Letzel1, Seth J Field4, John E Burke3, Rommie E Amaro2, Chambers C Hughes1. 1. Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography , University of California, San Diego , La Jolla , California 92093 , United States. 2. Department of Chemistry and Biochemistry , University of California, San Diego , La Jolla , California 92093 , United States. 3. Department of Biochemistry and Microbiology , University of Victoria , Victoria , British Columbia V8W 2Y2 , Canada. 4. School of Medicine , University of California, San Diego , La Jolla , California , 92093 , United States.
Abstract
Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/pan class="Gene">mTOR dual inhibitor pan class="Chemical">neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
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