Natural products represent an inexhaustible source of novel therapeutic agents. Their complex and constrained three-dimensional structures endow these molecules with exceptional biological properties, thereby giving them a major role in drug discovery programs. However, the search for new bioactive metabolites is hampered by the chemical complexity of the biological matrices in which they are found. The purification of single constituents from such matrices requires such a significant amount of work that it should be ideally performed only on molecules of high potential value (i.e., chemical novelty and biological activity). Recent bioinformatics approaches based on mass spectrometry metabolite profiling methods are beginning to address the complex task of compound identification within complex mixtures. However, in parallel to these developments, methods providing information on the bioactivity potential of natural products prior to their isolation are still lacking and are of key interest to target the isolation of valuable natural products only. In the present investigation, we propose an integrated analysis strategy for bioactive natural products prioritization. Our approach uses massive molecular networks embedding various informational layers (bioactivity and taxonomical data) to highlight potentially bioactive scaffolds within the chemical diversity of crude extracts collections. We exemplify this workflow by targeting the isolation of predicted active and nonactive metabolites from two botanical sources (Bocquillonia nervosa and Neoguillauminia cleopatra) against two biological targets (Wnt signaling pathway and chikungunya virus replication). Eventually, the detection and isolation processes of a daphnane diterpene orthoester and four 12-deoxyphorbols inhibiting the Wnt signaling pathway and exhibiting potent antiviral activities against the CHIKV virus are detailed. Combined with efficient metabolite annotation tools, this bioactive natural products prioritization pipeline proves to be efficient. Implementation of this approach in drug discovery programs based on natural extract screening should speed up and rationalize the isolation of bioactive natural products.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Receptors of the Fz (Frizzled) family initiate Wnt ligand-dependent signalling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains, and their signalling depends on heterotrimeric G-proteins in various organisms; however, Fz proteins constitute a distinct group within the GPCR (G-protein-coupled receptor) superfamily, and Fz signalling can be G-protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. In the present study, we demonstrate that mammalian Fz proteins act as GPCRs on heterotrimeric G(o/i) proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine-nucleotide exchange on G(o/i) proteins. These responses were sensitive to a Wnt antagonist and to pertussis toxin, which decouples the G(o/i) proteins from their receptors through covalent modification. The results of the present study provide the long-awaited biochemical proof of the GPCR nature of Fz receptors.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis on G protein α subunits, restricting their activity downstream from G protein-coupled receptors. Here we identify Drosophila Double hit (Dhit) as a dual RGS regulator of Gαo. In addition to the conventional GTPase-activating action, Dhit possesses the guanine nucleotide dissociation inhibitor (GDI) activity, slowing the rate of GTP uptake by Gαo; both activities are mediated by the same RGS domain. These findings are recapitulated using homologous mammalian Gαo/i proteins and RGS19. Crystal structure and mutagenesis studies provide clues into the molecular mechanism for this unprecedented GDI activity. Physiologically, we confirm this activity in Drosophila asymmetric cell divisions and HEK293T cells. We show that the oncogenic Gαo mutant found in breast cancer escapes this GDI regulation. Our studies identify Dhit and its homologs as double-action regulators, inhibiting Gαo/i proteins both through suppression of their activation and acceleration of their inactivation through the single RGS domain.

Rab5 is a small guanosine triphosphatase (GTPase) that regulates the early stages of endocytosis and is conserved in eukaryotes. Rab5 regulates the internalization of receptors and other membrane-associated signaling proteins. The function of Rab5 in these processes is considered relatively passive, so that the endocytic capacity of Rab5 is used during, for example, beta-arrestin-dependent internalization of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs). Direct recruitment or activation of Rab5 by the components of these signaling pathways has not been reported. Here, we demonstrate an interaction of Drosophila Rab5 and an immediate transducer of GPCR signaling, the G protein G(o), in vitro and in vivo. Rab5 and G(o) bound to each other as purified proteins, as well as in fly extracts. In cellular assays, G(o) led to Rab5 activation and endosome fusion. We further showed that the G(o)-Rab5 interaction functioned in Drosophila planar cell polarity and Wingless signal transduction, pathways initiated by GPCRs of the Frizzled (Fz) family. Additionally, the recycling Rab GTPases Rab4 and Rab11 functioned in Fz- and G(o)-mediated signaling to favor planar cell polarity over canonical Wingless signaling. The interplay between heterotrimeric G proteins and Rab GTPases controlled receptor internalization, revealing a previously uncharacterized regulatory mechanism in GPCR signaling.

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Wnt signaling is important for breast development and remodeling during pregnancy and lactation. Epigenetic modifications change expression levels of components of the Wnt pathway, underlying oncogenic transformation. However, no clear Wnt component increasing expression universally across breast cancer (BC) or its most Wnt-dependent triple-negative BC (TNBC) subgroup has been identified, delaying development of targeted therapies. Here we perform network correlation analysis of expression of >100 Wnt pathway components in hundreds of healthy and cancerous breast tissues. Varying in expression levels among people, Wnt components remarkably coordinate their production; this coordination is dramatically decreased in BC. Clusters with coordinated gene expression exist within the healthy cohort, highlighting Wnt signaling subtypes. Different BC subgroups are identified, characterized by different remaining Wnt signaling signatures, providing the rational for patient stratification for personalizing the therapeutic applications. Key pairwise interactions within the Wnt pathway (some inherited and some established de novo) emerge as targets for future drug discovery against BC.

Cardamonin (CD), an active chalconoid, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of CD for the treatment of triple negative breast cancer (TNBC) is unclear. This study aims to examine the cytotoxic effects of CD and investigate the underlying mechanism in human TNBC cells. The results show that CD exhibits cytotoxicity by inducing apoptosis and cell cycle arrest in TNBC cells via modulation of Bcl-2, Bax, cyt-C, cleaved caspase-3, and PARP. We find that CD significantly increases expression of the epithelial marker E-cadherin, while reciprocally decreasing expression of mesenchymal markers such as snail, slug, and vimentin in BT-549 cells. In parallel with epithelial-mesenchymal transition (EMT) reversal, CD down regulates invasion and migration of BT-549 cells. CD markedly reduces stability and nuclear translocation of β-catenin, accompanied with downregulation of β-catenin target genes. Using the TopFlash luciferase reporter assay, we reveal CD as a specific inhibitor of the Wnt3a-induced signaling. These results suggest the involvement of the Wnt/β-catenin signaling in the CD-induced EMT reversion of BT-549 cells. Notably, CD restores the glycogen synthase kinase-3β (GSK3β) activity, required for β-catenin destruction via the proteasome-mediated system, by inhibiting the phosphorylation of GSK3β by Akt. These occurrences ultimately lead to the blockage of EMT and the invasion of TNBC cells. Further antitumor activity of CD was tested in 4T1 (TNBC cells) induced tumor and it was found that CD significantly inhibited the tumor volume at dose of 5 mg/kg-treated mice. © 2016 BioFactors, 43(2):152-169, 2017.