Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization ...and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.
Colorectal cancer (CRC) often involves wild-type p53 inactivation by MDM2 and MDM4 overexpression, promoting tumor progression and resistance to 5-fluoruracil (5-FU). Disrupting the MDM2/4 ...heterodimer can proficiently reactivate p53, sensitizing cancer cells to 5-FU. Herein, we developed 16 peptides based on Pep3 (1), the only known peptide acting through this mechanism. The new peptides, notably 3 and 9, showed lower IC50 values than 1. When incorporated into tumor-targeted biodegradable nanoparticles, these exhibited cytotoxicity against three different CRC cell lines. Notably, NPs/9 caused a significant increase in p53 levels associated with a strong increment of its main downstream target p21 inducing apoptosis. Also, the combined treatment of 9 with 5-FU caused the activation of nucleolar stress and a synergic apoptotic effect. Hence, the co-delivery of MDM2/4 heterodimer disruptors with 5-FU through nanoparticles might be a promising strategy to overcome drug resistance in CRC.
The poor prognosis of Glioblastoma Multiforme (GBM) is due to a high resistance to conventional treatments and to the presence of a subpopulation of glioma stem cells (GSCs). Combination therapies ...targeting survival/self-renewal signals of GBM and GSCs are emerging as useful tools to improve GBM treatment. In this context, the hyperactivated AKT/mammalian target of the rapamycin (AKT/mTOR) and the inhibited wild-type p53 appear to be good candidates. Herein, the interaction between these pathways was investigated, using the novel AKT/mTOR inhibitor FC85 and ISA27, which re-activates p53 functionality by blocking its endogenous inhibitor murine double minute 2 homologue (MDM2). In GBM cells, FC85 efficiently inhibited AKT/mTOR signalling and reactivated p53 functionality, triggering cellular apoptosis. The combined therapy with ISA27 produced a synergic effect on the inhibition of cell viability and on the reactivation of p53 pathway. Most importantly, FC85 and ISA27 blocked proliferation and promoted the differentiation of GSCs. The simultaneous use of these compounds significantly enhanced GSC differentiation/apoptosis. These findings suggest that FC85 actively enhances the downstream p53 signalling and that a combination strategy aimed at inhibiting the AKT/mTOR pathway and re-activating p53 signalling is potentially effective in GBM and in GSCs.
The discovery of selective agonists of cannabinoid receptor 2 (CB2) is strongly pursued to successfully tuning endocannabinoid signaling for therapeutic purposes. However, the design of selective CB2 ...agonists is still challenging because of the high homology with the cannabinoid receptor 1 (CB1) and for the yet unclear molecular basis of the agonist/antagonist switch. Here, the 1,3-benzoxazine scaffold is presented as a versatile chemotype for the design of CB2 agonists from which 25 derivatives were synthesized. Among these, compound 7b5 (CB2 EC50 = 110 nM, CB1 EC50 > 10 μM) demonstrated to impair proliferation of triple negative breast cancer BT549 cells and to attenuate the release of pro-inflammatory cytokines in a CB2-dependent manner. Furthermore, 7b5 abrogated the activation of extracellular signal-regulated kinase (ERK) 1/2, a key pro-inflammatory and oncogenic enzyme. Finally, molecular dynamics studies suggested a new rationale for the in vitro measured selectivity and for the observed agonist behavior.
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•Targeting cannabinoid receptor 2 for therapeutic purposes.•1,3-benzoxazin-4-one scaffold as chemotype for the design of selective CB2 agonists.•Promising anti-cancer therapeutic.
High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ...ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvβ3, α5β1, or αvβ6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvβ3 (L2b) and an αvβ6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.
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The αvβ6 integrin binds the RGD‐containing peptide of the foot and mouth disease virus with high selectivity. In this study, the long binding helix of this ligand was downsized to an enzymatically ...stable cyclic peptide endowed with sub‐nanomolar binding affinity toward the αvβ6 receptor and remarkable selectivity against other integrins. Computational studies were performed to disclose the molecular bases underlying the high binding affinity and receptor subtype selectivity of this peptide. Finally, the utility of the ligand for use in biomedical studies was also demonstrated here.
The search for binding: The αvβ6 integrin binds the RGD‐containing peptide of the foot and mouth disease virus with high selectivity. The long binding helix of this ligand was downsized to an enzymatically stable cyclic peptide endowed with sub‐nanomolar binding affinity toward the αvβ6 receptor and remarkable selectivity against other integrins.
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates ...the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
αvβ6 Integrin is an epithelial transmembrane protein that recognizes latency‐associated peptide (LAP) and primarily activates transforming growth factor beta (TGF‐β). It is overexpressed in ...carcinomas (most notably, pancreatic) and other conditions associated with αvβ6 integrin‐dependent TGF‐β dysregulation, such as fibrosis. We have designed a trimeric Ga‐68‐labeled TRAP conjugate of the αvβ6‐specific cyclic pentapeptide SDM17 (cycloRGD‐Chg‐E‐CONH2) to enhance αvβ6 integrin affinity as well as target‐specific in‐vivo uptake. Ga‐68‐TRAP(SDM17)3 showed a 28‐fold higher αvβ6 affinity than the corresponding monomer Ga‐68‐NOTA‐SDM17 (IC50 of 0.26 vs. 7.4 nM, respectively), a 13‐fold higher IC50‐based selectivity over the related integrin αvβ8 (factors of 662 vs. 49), and a threefold higher tumor uptake (2.1 vs. 0.66 %ID/g) in biodistribution experiments with H2009 tumor‐bearing SCID mice. The remarkably high tumor/organ ratios (tumor‐to‐blood 11.2; ‐to‐liver 8.7; ‐to‐pancreas 29.7) enabled high‐contrast tumor delineation in PET images. We conclude that Ga‐68‐TRAP(SDM17)3 holds promise for improved clinical PET diagnostics of carcinomas and fibrosis.
See three with glee: Tethering three rather than a single peptidic αvβ6 integrin ligand to a radionuclide lets tumor tissue light up brightly in tomographic images, potentially enabling a more sensitive diagnosis of various cancers.
A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and ...Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.
The inhibition of the PD-1/PD-L1 axis by monoclonal antibodies has achieved remarkable success in treating a growing number of cancers. However, a novel class of small organic molecules, with BMS-202 ...(1) as the lead, is emerging as direct PD-L1 inhibitors. Herein, we report a series of 2,4,6-tri- and 2,4-disubstituted 1,3,5-triazines, which were synthesized and assayed for their PD-L1 binding by NMR and homogeneous time-resolved fluorescence. Among them, compound 10 demonstrated to strongly bind with the PD-L1 protein and challenged it in a co-culture of PD-L1 expressing cancer cells (PC9 and HCC827 cells) and peripheral blood mononuclear cells enhanced antitumor immune activity of the latter. Compound 10 significantly increased interferon γ release and apoptotic induction of cancer cells, with low cytotoxicity in healthy cells when compared to 1, thus paving the way for subsequent preclinical optimization and medical applications.
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