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CRISPRi meets metabolomics: a platform for rapid functional annotation of compound libraries

ABSTRACT: Molecular profiling of small-molecules offers invaluable insights on compound functionality and allows for hypothesis generation of targets. However, current profiling methods are either limited in the number of measurable parameters or throughput. Here, we developed a multiplexed, unbiased framework that by linking genetic to drug-induced changes in nearly a thousand metabolites allows for high-throughput functional annotation of compound libraries in Escherichia Coli. First, we generated a reference map of metabolic changes from (CRISPR) interference with 352 genes in all major essential biological processes. Next, based on the comparison of essential gene knockdown metabolic profiles with 1342 drug-induced metabolic changes we demonstrated the ability to make de novo predictions of compound functionality and revealed drugs interfering with unconventional antibacterial targets. The same framework that combines dynamic gene silencing with metabolomics we implemented in E. coli can be adapted and applied as a general strategy for comprehensive high-throughput analysis of compound functionality, from bacteria to human cells.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli

SUBMITTER: Ludovic Gillet

LAB HEAD: Mattia Zampieri

PROVIDER: PXD024133 | Pride | 2021-12-16

REPOSITORIES: pride

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			Action	DRS
	200922_EX1_lgillet_LG005_085.raw	Raw
	200922_EX1_lgillet_LG005_086.raw	Raw
	200922_EX1_lgillet_LG005_087.raw	Raw
	200922_EX1_lgillet_LG005_088.raw	Raw
	200922_EX1_lgillet_LG005_089.raw	Raw

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Project description:Cervical cancer is still the leading cause of cancer mortality worldwide even after introduction of vaccine against Human papillomavirus (HPV), due to low vaccine coverage, especially in the developing world. Cervical cancer is primarily treated by Chemo/Radiotherapy, depending on the disease stage, with Carboplatin/Cisplatin-based drug regime. These drugs being non-specific, target rapidly dividing cells, including normal cells, so safer options are needed for lower off-target toxicity. Natural products offer an attractive option compared to synthetic drugs due to their well-established safety profile and capacity to target multiple oncogenic hallmarks of cancer like inflammation, angiogenesis, etc. In the current study, we investigated the effect of Bergenin (C-glycoside of 4-O-methylgallic acid), a natural polyphenol compound that is isolated from medicinal plants such as Bergenia crassifolia, Caesalpinia digyna, and Flueggea leucopyrus . Bergenin has been shown to have anti-inflammatory, anti-ulcerogenic, and wound healing properties but its anticancer potential has been realized only recently. We performed a proteomic analysis of cervical carcinoma cells treated with bergenin and found it to influence multiple hallmarks of cancers, including apoptosis, angiogenesis, and tumor suppressor proteins. It was also involved in many different cellular processes unrelated to cancer, as shown by our proteomic analysis. Further analysis showed bergenin to be a potent -angiogenic agent by reducing key angiogenic proteins like Galectin 3 and MMP-9 (Matrix Metalloprotease 9) in cervical carcinoma cells. Further understanding of this interaction was carried out using molecular docking analysis, which indicated MMP-9 has more affinity for bergenin as compared to Galectin-3. Cumulatively, our data provide novel insight into the anti-angiogenic mechanism of bergenin in cervical carcinoma cells by modulation of multiple angiogenic proteins like Galectin-3 and MMP-9 which warrant its further development as an anticancer agent in cervical cancer.

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CRISPRi meets metabolomics: a platform for rapid functional annotation of compound libraries

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