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Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. [ABSTRACT FROM AUTHOR]

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms. particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity. [ABSTRACT FROM AUTHOR]

Sugar amino acids (SAAs) are useful building blocks for the design of peptidomimetics and peptide scaffolds. The three-dimensional structures of cyclic hybrid molecules containing the furanoid ∈-SAA III and several amino acids were elucidated to study the preferred conformation of such an ∈-SAA and its conformational influence on the backbone of cyclic peptides. NMR-based molecular dynamics simulations and empirical calculations of the cyclic tetramer 1, consisting of two copies of the SAA residue and two amino acids, revealed that it is conformationally restrained. The two SAA residues adopt different conformations. One of them forms an unusual turn, stabilized by an intraresidue nine-member hydrogen bond. The methylene functionalities of the other SAA residue are positioned in such a way that an intraresidue H bond is not possible. The X-ray crystal structure of I strongly resembles the solution conformation. Molecular dynamics calculations in combination with NMR analysis were also performed for compounds 2 and 3, which contain the RGD (Arg-Gly-Asp) consensus sequence and were previously shown to inhibit α[sub IIb]β[sub 3]-receptor-mediated platelet aggregation. The biologically most active compound 2 adopts a preferred conformation with the single SAA residue folded into the nine-member H bond-containing turn. Compound 3, containing an additional valine residue, as compared with compound 2, is conformational flexible. Our studies demonstrate that the furanoid ∈-SAA III is able to introduce an unusual intraresidue hydrogen bond-stabilized β-turn-like conformation in two of the three cyclic structures. [ABSTRACT FROM AUTHOR]

Despite progress in the prevention and diagnosis of cancer, current technologies for tumor detection present several limitations including invasiveness, toxicity, inaccuracy, lengthy testing duration and high cost. Therefore, innovative diagnostic techniques that integrate knowledge from biology, oncology, medicinal and analytical chemistry are now quickly emerging in the attempt to address these issues. Following this approach, here we developed a paper-based electrochemical device for detecting cancer-derived Small Extracellular Vesicles (S-EVs) in fluids. S-EVs were obtained from cancer cell lines known to express, at a different level, the αvβ6 integrin receptor, a well-established hallmark of numerous epithelial cancer types. The resulting biosensor turned out to recognize αvβ6-containing S-EVs down to a limit of 0.7*103 S-EVs/mL with a linear range up to 105 S-EVs /mL, and a relative standard deviation of 11%, thus it may represent a novel opportunity for αvβ6 expressing cancers detection. The detection of cancer in its early stages can greatly prevent disease development, however, current technologies for tumor detection present several limitations. Here, the authors develop a paper-based electrochemical device for detecting cancer-derived small extracellular vesicles (S-EVs) in fluids, recognizing αvβ6-containing S-EVs down to a limit of 0.7*103 S-EVs/mL with a linear range up to 105 S-EVs/mL. [ABSTRACT FROM AUTHOR]

The article presents the research that evaluates the efficacy of Virtual Screening (VS) and nuclear magnetic resonance experiments in identifying molecular chemotypes to bind the grooves of DNA quadruplex structures. Researchers used the two schemes because it furnished valuable alternative series in lead optimization to identify new pharmacological tools. Moreover, these tools are also useful in clarifying mechanism and targeting and as therapeutic potential of G-quadruplexes.

The recent disclosure of the ability of aromatic isocyanides to harvest visible light and act as single electron acceptors when reacting with tertiary aromatic amines has triggered a renewed interest in their application to the development of green photoredox catalytic methodologies. Accordingly, the present work explores their ability to promote the generation of both alkyl and acyl radicals starting from radical precursors such as Hantzsch esters, potassium alkyltrifluoroborates, and α‐oxoacids. Mechanistic studies involving UV‐visible absorption and fluorescence experiments, electrochemical measurements of the ground‐state redox potentials along with computational calculations of both the ground‐ and the excited‐state redox potentials of a set of nine different aromatic isocyanides provide key insights to promote a rationale design of a new generation of isocyanide‐based organic photoredox catalysts. Importantly, the green potential of the investigated chemistry is demonstrated by a direct and easy access to deuterium labeled compounds. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53–MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2–p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2–p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection. [ABSTRACT FROM AUTHOR]

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C‐X‐C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4‐ and 1,5‐ disubstituted 1,2,3‐triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12‐mediated cell migration, the 1,4‐triazole variant conserved the antagonistic effect in the low‐mid nanomolar range, while the 1,5‐triazole one displayed the ability to activate the migration, becoming the first in class low‐molecular‐weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR‐interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors. [ABSTRACT FROM AUTHOR]

Abstract The osteoblast generation from Mesenchymal stem cells (MSCs) is tightly coordinated by transcriptional networks and signalling pathways that control gene expression and protein stability of osteogenic "master transcription factors". Among these pathways, a great attention has been focused on p53 and its physiological negative regulator, the E3 ligase Murine double minute 2 (Mdm2). Nevertheless, the signalling that regulates Mdm2-p53 axis in osteoblasts remain to be elucidated, also considering that Mdm2 possesses numerous p53-independent activities and interacts with additional proteins. Herein, the effects of Mdm2 modulation on MSC differentiation were examined by the use of short- and long-lasting inhibitors of the Mdm2-p53 complex. The long-lasting Mdm2-p53 dissociation was demonstrated to enhance the MSC differentiation into osteoblasts. The increase of Mdm2 levels promoted its association to G protein-coupled receptors kinase (GRK) 2, one of the most relevant kinases involved in the desensitization of G protein-coupled receptors (GPCRs). In turn, the long-lasting Mdm2-p53 dissociation decreased GRK2 levels and favoured the functionality of A 2B Adenosine Receptors (A 2B ARs), a GPCR dictating MSC fate. EB148 facilitated cAMP accumulation, and mediated a sustained activation of extracellular signal–regulated kinases (ERKs) and cAMP response element-binding protein (CREB). Such pro-osteogenic effects were not detectable by using the reversible Mdm2-p53 complex inhibitor, suggesting the time course of Mdm2-p53 dissociation may impact on intracellular proteins involved in cell differentiation fate. These results suggest that the long-lasting Mdm2 binding plays a key role in the mobilization of intracellular proteins that regulate the final biological outcome of MSCs. Graphical abstract Unlabelled Image Highlights • Long-lasting dissociation of Mdm2-p53 induces mesenchymal stem cell differentiation. • Long-lasting dissociation of Mdm2-p53 decreases GRK2 levels. • Long-lasting dissociation of Mdm2-p53 induced sustained ERK and CREB activation. [ABSTRACT FROM AUTHOR]

Abstract New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different combinations of substituents (H, Cl, OCH 3) at R2-R4 positions and protonatable R1-dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell lines, showing significant antiproliferative activity (GI 50 values spanning from 0.31 to 6.93 μM) and pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores to form a molecular complex with DNA, following an intercalative mode of binding. All compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with topoisomerase I revealed a significant poisoning effect for compounds 5g , 5h , 5s , and 5t. A theoretical model provided by hydrated docking calculations clarified the role of the R1-R4 substituents on the topoisomerase I poison activity, revealing a crucial role of the R2-OCH 3 group. Graphical abstract Image 1 Highlights • New benzo- and pyrido-thiopyranoindoles were investigated as antiproliferative agents. • Most compounds showed antiproliferative activity and pro-apoptotic effect. • All compounds displayed intercalative properties. • A number of compounds revealed a significant poisoning effect on topoisomerase I. [ABSTRACT FROM AUTHOR]

The discovery of selective agonists of cannabinoid receptor 2 (CB 2) is strongly pursued to successfully tuning endocannabinoid signaling for therapeutic purposes. However, the design of selective CB 2 agonists is still challenging because of the high homology with the cannabinoid receptor 1 (CB 1) 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 CB 2 agonists from which 25 derivatives were synthesized. Among these, compound 7b5 (CB 2 EC 50 = 110 nM, CB 1 EC 50 > 10 μM) demonstrated to impair proliferation of triple negative breast cancer BT549 cells and to attenuate the release of pro-inflammatory cytokines in a CB 2 -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. [Display omitted] • Targeting cannabinoid receptor 2 for therapeutic purposes. • 1,3-benzoxazin-4-one scaffold as chemotype for the design of selective CB 2 agonists. • Promising anti-cancer therapeutic. [ABSTRACT FROM AUTHOR]

Das RGD‐erkennende Integrin αvβ6 trat in jüngster Zeit als vielbeachtete Zielstruktur für die Diagnostik und Therapie von Krebserkrankungen in Erscheinung. Infolgedessen besteht derzeit ein dringender Bedarf an niedermolekularen Liganden für diesen Rezeptor. Ein auf Metadynamik beruhender Ansatz ermöglichte die erfolgreiche Konvertierung eines helikalen Nonapeptids in ein zyklisches Pentapeptid (6) mit bemerkenswerter Affinität und Spezifität für αvβ6‐Integrin. Die Gründe hierfür konnten mittels NMR‐ und Docking‐Untersuchungen aufgeklärt werden und bilden einen Ausgangspunkt für die zielgerichtete Konzeption neuer αvβ6‐spezifischer Peptide oder sogar Peptidomimetika. Des Weiteren wurde die Möglichkeit einer medizinischen Anwendung von 6 mittels PET‐Bildgebungsexperimenten im lebenden Organismus veranschaulicht. Eine Metadynamik‐basierte Wirkstoff‐Design‐Strategie wurde eingesetzt, um ein helikales Nonapeptid in ein cyclisches Pentapeptid als potenten und selektiven Liganden für das RGD‐erkennende αvβ6‐Integrin zu transformieren. Diese Verbindung wurde erfolgreich zu einem Tracer für die spezifische αvβ6‐PET‐Bildgebung in vivo weiterentwickelt, was ihre Verwendbarkeit für die Krebsdiagnose nahelegt. [ABSTRACT FROM AUTHOR]