Abstract 646: Proteome wide target deconvolution of pharmacologically active natural products by CETSA® coupled to mass spectrometry

Abstract Nature has provided a large plethora of different molecules that in the hands of man has been ascribed to a vast range of pharmacological actions. In some cases, pharmacology is clearly connected to a phenotype e. g. , Golgi apparatus dissolution by Brefeldin A, whereas in other cases the proposed phenotypes are many and disparate e. g. , Quercetin. Many claims are made around natural compounds as having various positive therapeutic effects on cancer e. g. , curcumin and quercetin. Intriguingly, claims about therapeutic effects of various natural compounds keep on surfacing, and we wanted to address this issue by performing target deconvolution of a selection of natural compounds in Hepatic cancer cells: HepG2. We took advantage of the CETSA® (CEllular Thermal Shift Assay) method coupled to Mass spectrometry detection in order to deconvolute the mode of action of seven highly cited natural compounds that comprised both well annotated compounds with an established mode of action: Brefeldin A and Wortmannin; and compounds with a less well established mode of action: Carnosol, Curcumin, Resveratrol, Rhoifolin, and Quercetin. CETSA is a method that allows detection of compound-induced thermal shifts of proteins in intact living cells as well as in cell lysates. Subsequently, a thermal shift of a protein in the presence of a compound then implicates a direct or in the case of intact cells, also an indirect interaction between the protein in question and compound. As CETSA can be performed in intact living cells we could detect proteins that were potential direct binders as well as proteins that were part of the downstream signaling events or part of the phenotypic response. The Natural compounds were assessed in a screening format in which three bio-replicates were analyzed at a single concentration, 30 µM. Good proteome coverage was obtained in the HepG2 cells and all compounds tested induced thermal shifts of proteins at 30 µM. Most of the compounds displayed many protein shifts of both suggested direct targets and proteins presumed to be part of a phenotypic response, as well as novel targets/off-targets. Curcumin and Rhoifolin were the exceptions, which only had a few shifted proteins. Brefeldin A, Quercetin, and Wortmannin engaged proteins associated to their annotated mode of action pathways, whereas Carnosol, Curcumin, and Resveratrol was less specific in their responses with regards to shifted proteins i. e. , no clear signaling pathway was determined. In this study we use CETSA coupled to MS detection to delineate mode of action of natural compounds, which shed light on why they could be implicated to have a role in tumor biology. We also found that compounds with a well-established mode of action give a clearer target engagement profile compared to less annotated compounds. Citation Format: Tomas Friman, Tuomas Tolvanen, Erin Gilson, Alexey Chernobrovkin, Stina Lundgren, Victoria Brehmer, Daniel Martinez Molina. Proteome wide target deconvolution of pharmacologically active natural products by CETSA® coupled to mass spectrometry abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 646.