Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

Specific targeting of the NRF2/ß-TrCP axis promotes beneficial effects in NASH.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease that compromises liver function, for which there is not a specifically approved medicine. Recent research has identified transcription factor NRF2 as a potential therapeutic target. However, current NRF2 activators, designed to inhibit its repressor KEAP1, exhibit unwanted side effects. Alternatively, we previously introduced PHAR, a protein-protein interaction inhibitor of NRF2/ß-TrCP, which induces a mild NRF2 activation and selectively activates NRF2 in the liver, close to normal physiological levels. Herein, we assessed the effect of PHAR in protection against NASH and its progression to fibrosis. We conducted experiments to demonstrate that PHAR effectively activated NRF2 in hepatocytes, Kupffer cells, and stellate cells. Then, we used the STAM mouse model of NASH, based on partial damage of endocrine pancreas and insulin secretion impairment, followed by a high fat diet. Non-invasive analysis using MRI revealed that PHAR protects against liver fat accumulation. Moreover, PHAR attenuated key markers of NASH progression, including liver steatosis, hepatocellular ballooning, inflammation, and fibrosis. Notably, transcriptomic data indicate that PHAR led to upregulation of 3 anti-fibrotic genes (Plg, Serpina1a, and Bmp7) and downregulation of 6 pro-fibrotic (including Acta2 and Col3a1), 11 extracellular matrix remodeling, and 8 inflammatory genes. Overall, our study suggests that the mild activation of NRF2 via the protein-protein interaction inhibitor PHAR holds promise as a strategy for addressing NASH and its progression to liver fibrosis.

Fatty acid synthase (FASN) signalome: A molecular guide for precision oncology.

The initial excitement generated more than two decades ago by the discovery of drugs targeting fatty acid synthase (FASN)-catalyzed de novo lipogenesis for cancer therapy was short-lived. However, the advent of the first clinical-grade FASN inhibitor (TVB-2640; denifanstat), which is currently being studied in various phase II trials, and the exciting advances in understanding the FASN signalome are fueling a renewed interest in FASN-targeted strategies for the treatment and prevention of cancer. Here, we provide a detailed overview of how FASN can drive phenotypic plasticity and cell fate decisions, mitochondrial regulation of cell death, immune escape and organ-specific metastatic potential. We then present a variety of FASN-targeted therapeutic approaches that address the major challenges facing FASN therapy. These include limitations of current FASN inhibitors and the lack of precision tools to maximize the therapeutic potential of FASN inhibitors in the clinic. Rethinking the role of FASN as a signal transducer in cancer pathogenesis may provide molecularly driven strategies to optimize FASN as a long-awaited target for cancer therapeutics.

Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors.

Epithelial-to-mesenchymal transition (EMT) may drive the escape of ALK-rearranged non-small-cell lung cancer (NSCLC) tumors from ALK-tyrosine kinase inhibitors (TKIs). We investigated whether first-generation ALK-TKI therapy-induced EMT promotes cross-resistance to new-generation ALK-TKIs and whether this could be circumvented by the flavonolignan silibinin, an EMT inhibitor. ALK-rearranged NSCLC cells acquiring a bona fide EMT phenotype upon chronic exposure to the first-generation ALK-TKI crizotinib exhibited increased resistance to second-generation brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK-TKIs, which was partially recapitulated upon chronic TGFß stimulation, was less pronounced in ALK-rearranged NSCLC cells solely acquiring a partial/hybrid E/M transition state. Silibinin overcame EMT-induced resistance to brigatinib and lorlatinib and restored their efficacy involving the transforming growth factor-beta (TGFß)/SMAD signaling pathway. Silibinin deactivated TGFß-regulated SMAD2/3 phosphorylation and suppressed the transcriptional activation of genes under the control of SMAD binding elements. Computational modeling studies and kinase binding assays predicted a targeted inhibitory binding of silibinin to the ATP-binding pocket of TGFß type-1 receptor 1 (TGFBR1) and TGFBR2 but solely at the two-digit micromolar range. A secretome profiling confirmed the ability of silibinin to normalize the augmented release of TGFß into the extracellular fluid of ALK-TKIs-resistant NSCLC cells and reduce constitutive and inducible SMAD2/3 phosphorylation occurring in the presence of ALK-TKIs. In summary, the ab initio plasticity along the EMT spectrum may explain the propensity of ALK-rearranged NSCLC cells to acquire resistance to new-generation ALK-TKIs, a phenomenon that could be abrogated by the silibinin-driven attenuation of the TGFß/SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells.

The current status and future prospects for therapeutic targeting of KEAP1-NRF2 and ß-TrCP-NRF2 interactions in cancer chemoresistance.

Drug resistance is one of the biggest challenges in cancer treatment and limits the potential to cure patients. In many tumors, sustained activation of the protein NRF2 makes tumor cells resistant to chemo- and radiotherapy. Thus, blocking inappropriate NRF2 activity in cancers has been shown to reduce resistance in models of the disease. There is a growing scientific interest in NRF2 inhibitors. However, the compounds developed so far are not target-specific and are associated with a high degree of toxicity, hampering clinical applications. Compounds that can enhance the binding of NRF2 to its ubiquitination-facilitating regulator proteins, either KEAP1 or ß-TrCP, have the potential to increase NRF2 degradation and may be of value as potential chemosensitising agents in cancer treatment. Approaches based on molecular glue-type mechanisms, in which ligands stabilise a ternary complex between a protein and its binding partner have shown to enhance ß-catenin degradation by stabilising its interaction with ß-TrCP. This strategy could be applied to rationally discover degradative ß-TrCP-NRF2 and KEAP1-NRF2 protein-protein interaction enhancers. We are proposing a novel approach to selectively suppress NRF2 activity in tumors. It is based on recent methodology and has the potential to be a promising new addition to the arsenal of anticancer agents.

Silibinin Suppresses the Hyperlipidemic Effects of the ALK-Tyrosine Kinase Inhibitor Lorlatinib in Hepatic Cells.

The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug−drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.

An inhibitor of interaction between the transcription factor NRF2 and the E3 ubiquitin ligase adapter ß-TrCP delivers anti-inflammatory responses in mouse liver.

It is widely accepted that activating the transcription factor NRF2 will blast the physiological anti-inflammatory mechanisms, which will help combat pathologic inflammation. Much effort is being put in inhibiting the main NRF2 repressor, KEAP1, with either electrophilic small molecules or disrupters of the KEAP1/NRF2 interaction. However, targeting ß-TrCP, the non-canonical repressor of NRF2, has not been considered yet. After in silico screening of ∼1 million compounds, we now describe a novel small molecule, PHAR, that selectively inhibits the interaction between ß-TrCP and the phosphodegron in transcription factor NRF2. PHAR upregulates NRF2-target genes such as Hmox1, Nqo1, Gclc, Gclm and Aox1, in a KEAP1-independent, but ß-TrCP dependent manner, breaks the ß-TrCP/NRF2 interaction in the cell nucleus, and inhibits the ß-TrCP-mediated in vitro ubiquitination of NRF2. PHAR attenuates hydrogen peroxide induced oxidative stress and, in lipopolysaccharide-treated macrophages, it downregulates the expression of inflammatory genes Il1b, Il6, Cox2, Nos2. In mice, PHAR selectively targets the liver and greatly attenuates LPS-induced liver inflammation as indicated by a reduction in the gene expression of the inflammatory cytokines Il1b, TNf, and Il6, and in F4/80-stained liver resident macrophages. Thus, PHAR offers a still unexplored alternative to current NRF2 activators by acting as a ß-TrCP/NRF2 interaction inhibitor that may have a therapeutic value against undesirable inflammation.

The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases.

The high proliferation of microorganisms in aquatic environments has allowed their coevolution for billions of years with other living beings that also inhabit these niches. Among the different existing types of interaction, the eternal competition for supremacy between the susceptible species and their pathogens has selected, as part of the effector division of the immune system of the former ones, a vast and varied arsenal of efficient antimicrobial molecules, which is highly amplified by the broad biodiversity radiated, above any others, at the marine habitats. At present, the great recent scientific and technological advances already allow the massive discovery and exploitation of these defense compounds for therapeutic purposes against infectious diseases of our interest. Among them, antimicrobial peptides and antimicrobial metabolites stand out because of the wide dimensions of their structural diversities, mechanisms of action, and target pathogen ranges. This revision work contextualizes the research in this field and serves as a presentation and scope identification of the Special Issue from Marine Drugs journal "The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases".

G protein-coupled estrogen receptor activation by bisphenol-A disrupts the protection from apoptosis conferred by the estrogen receptors ERα and ERß in pancreatic beta cells.

17ß-estradiol protects pancreatic ß-cells from apoptosis via the estrogen receptors ERα, ERß and GPER. Conversely, the endocrine disruptor bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular-initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERß, or in dispersed islet cells from ERß knockout (BERKO) mice. However, the ERα and ERß agonists PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERß form homodimers and heterodimers. Molecular dynamics simulations together with proximity ligand assays and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERß as well as GPER activation by G1 decreased ERαß heterodimers. We propose that ERαß heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decreases in ERαß heterodimers lead to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors.

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

Metformin Is a Pyridoxal-5'-phosphate (PLP)-Competitive Inhibitor of SHMT2.

The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5'-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

Quercetin metabolites from Hibiscus sabdariffa contribute to alleviate glucolipotoxicity-induced metabolic stress in vitro.

Polyphenols from Hibiscus sabdariffa (HS) alleviate obesity-related metabolic complications but the metabolites responsible for such effects are unknown. We aimed to elucidate which of the potential plasma metabolites from a polyphenol-enriched HS (PEHS) extract contributed for the reversion of glucolipotoxicity-induced metabolic stress using 3T3-L1 adipocyte and INS 832/13 pancreatic ß-cell models under glucolipotoxic conditions. PEHS extract, quercetin (Q) and quercetin-3-O-glucuronide (Q3GA) showed stronger capacity to decrease glucolipotoxicity-induced ROS generation than ascorbic acid or chlorogenic acid. PEHS extract, Q and Q3GA decreased secretion of cytokines (leptin, TNF-α, IGF-1, IL-6, VEGF, IL-1α, IL-1ß and CCL2) and reduced CCL2 expression at transcriptional level. In addition, PEHS extract, Q and Q3GA reduced triglyceride accumulation, which occurred through fatty acid synthase (FASN) downregulation, AMPK activation and mitochondrial mass and biogenesis restoration via PPARα upregulation. Electron microscopy confirmed that PEHS extract and Q3GA decreased mitochondrial remodeling and mitophagy. Virtual screening leads us to postulate that Q and Q3GA might act as agonists of these protein targets at specific sites. These data suggest that Q and Q3GA may be the main responsible compounds for the capacity of PEHS extract to revert glucolipotoxicity-induced metabolic stress through AMPK-mediated decrease in fat storage and increase in fatty acid oxidation, though other compounds of the extract may contribute to this capacity.

Silibinin and SARS-CoV-2: Dual Targeting of Host Cytokine Storm and Virus Replication Machinery for Clinical Management of COVID-19 Patients.

COVID-19, the illness caused by infection with the novel coronavirus SARS-CoV-2, is a rapidly spreading global pandemic in urgent need of effective treatments. Here we present a comprehensive examination of the host- and virus-targeted functions of the flavonolignan silibinin, a potential drug candidate against COVID-19/SARS-CoV-2. As a direct inhibitor of STAT3-a master checkpoint regulator of inflammatory cytokine signaling and immune response-silibinin might be expected to phenotypically integrate the mechanisms of action of IL-6-targeted monoclonal antibodies and pan-JAK1/2 inhibitors to limit the cytokine storm and T-cell lymphopenia in the clinical setting of severe COVID-19. As a computationally predicted, remdesivir-like inhibitor of RNA-dependent RNA polymerase (RdRp)-the central component of the replication/transcription machinery of SARS-CoV-2-silibinin is expected to reduce viral load and impede delayed interferon responses. The dual ability of silibinin to target both the host cytokine storm and the virus replication machinery provides a strong rationale for the clinical testing of silibinin against the COVID-19 global public health emergency. A randomized, open-label, phase II multicentric clinical trial (SIL-COVID19) will evaluate the therapeutic efficacy of silibinin in the prevention of acute respiratory distress syndrome in moderate-to-severe COVID-19-positive onco-hematological patients at the Catalan Institute of Oncology in Catalonia, Spain.

Potential Drugs Targeting Early Innate Immune Evasion of SARS-Coronavirus 2 via 2'-O-Methylation of Viral RNA.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing the COVID-19 respiratory disease pandemic utilizes unique 2'-O-methyltransferase (2'-O-MTase) capping machinery to camouflage its RNA from innate immune recognition. The nsp16 catalytic subunit of the 2'-O-MTase is unusual in its requirement for a stimulatory subunit (nsp10) to catalyze the ribose 2'-O-methylation of the viral RNA cap. Here we provide a computational basis for drug repositioning or de novo drug development based on three differential traits of the intermolecular interactions of the SARS-CoV-2-specific nsp16/nsp10 heterodimer, namely: (1) the S-adenosyl-l-methionine-binding pocket of nsp16, (2) the unique "activating surface" between nsp16 and nsp10, and (3) the RNA-binding groove of nsp16. We employed ≈9000 U.S. Food and Drug Administration (FDA)-approved investigational and experimental drugs from the DrugBank repository for docking virtual screening. After molecular dynamics calculations of the stability of the binding modes of high-scoring nsp16/nsp10-drug complexes, we considered their pharmacological overlapping with functional modules of the virus-host interactome that is relevant to the viral lifecycle, and to the clinical features of COVID-19. Some of the predicted drugs (e.g., tegobuvir, sonidegib, siramesine, antrafenine, bemcentinib, itacitinib, or phthalocyanine) might be suitable for repurposing to pharmacologically reactivate innate immune restriction and antagonism of SARS-CoV-2 RNAs lacking 2'-O-methylation.

The LSD1 inhibitor iadademstat (ORY-1001) targets SOX2-driven breast cancer stem cells: a potential epigenetic therapy in luminal-B and HER2-positive breast cancer subtypes.

SOX2 is a core pluripotency-associated transcription factor causally related to cancer initiation, aggressiveness, and drug resistance by driving the self-renewal and seeding capacity of cancer stem cells (CSC). Here, we tested the ability of the clinically proven inhibitor of the lysine-specific demethylase 1 (LSD1/KDM1A) iadademstat (ORY-100) to target SOX2-driven CSC in breast cancer. Iadademstat blocked CSC-driven mammosphere formation in breast cancer cell lines that are dependent on SOX2 expression to maintain their CSC phenotype. Iadademstat prevented the activation of an LSD1-targeted stemness-specific SOX2 enhancer in CSC-enriched 3-dimensional spheroids. Using high-throughput transcriptional data available from the METABRIC dataset, high expression of SOX2 was significantly more common in luminal-B and HER2-enriched subtypes according to PAM50 classifier and in IntClust1 (high proliferating luminal-B) and IntClust 5 (luminal-B and HER2-amplified) according to integrative clustering. Iadademstat significantly reduced mammospheres formation by CSC-like cells from a multidrug-resistant luminal-B breast cancer patient-derived xenograft but not of those from a treatment-naïve luminal-A patient. Iadademstat reduced the expression of SOX2 in luminal-B but not in luminal-A mammospheres, likely indicating a selective targeting of SOX2-driven CSC. The therapeutic relevance of targeting SOX2-driven breast CSC suggests the potential clinical use of iadademstat as an epigenetic therapy in luminal-B and HER2-positive subtypes.

Resveratrol targets PD-L1 glycosylation and dimerization to enhance antitumor T-cell immunity.

New strategies to block the immune evasion activity of programmed death ligand-1 (PD-L1) are urgently needed. When exploring the PD-L1-targeted effects of mechanistically diverse metabolism-targeting drugs, exposure to the dietary polyphenol resveratrol (RSV) revealed its differential capacity to generate a distinct PD-L1 electrophoretic migration pattern. Using biochemical assays, computer-aided docking/molecular dynamics simulations, and fluorescence microscopy, we found that RSV can operate as a direct inhibitor of glyco-PD-L1-processing enzymes (α-glucosidase/α-mannosidase) that modulate N-linked glycan decoration of PD-L1, thereby promoting the endoplasmic reticulum retention of a mannose-rich, abnormally glycosylated form of PD-L1. RSV was also predicted to interact with the inner surface of PD-L1 involved in the interaction with PD-1, almost perfectly occupying the target space of the small compound BMS-202 that binds to and induces dimerization of PD-L1. The ability of RSV to directly target PD-L1 interferes with its stability and trafficking, ultimately impeding its targeting to the cancer cell plasma membrane. Impedance-based real-time cell analysis (xCELLigence) showed that cytotoxic T-lymphocyte activity was notably exacerbated when cancer cells were previously exposed to RSV. This unforeseen immunomodulating mechanism of RSV might illuminate new approaches to restore T-cell function by targeting the PD-1/PD-L1 immunologic checkpoint with natural polyphenols.

Antimicrobial Capacity of Plant Polyphenols against Gram-positive Bacteria: A Comprehensive Review.

BACKGROUND: Multi-drug-resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA) disseminate rapidly amongst patients in healthcare facilities and suppose an increasingly important cause of community-associated infections and associated mortality. The development of effective therapeutic options against resistant bacteria is a public health priority. Plant polyphenols are structurally diverse compounds that have been used for centuries for medicinal purposes, including infections treatment and possess, not only antimicrobial activity, but also antioxidant, anti-inflammatory and anticancer activities among others. Based on the existing evidence on the polyphenols' antibacterial capacity, polyphenols may be postulated as an alternative or complementary therapy for infectious diseases. OBJECTIVE: To review the antimicrobial activity of plant polyphenols against Gram-positive bacteria, especially against S. aureus and its resistant strains. Determine the main bacterial molecular targets of polyphenols and their potential mechanism of action. METHODOLOGY: The most relevant reports on plant polyphenols' antibacterial activity and their putative molecular targets were studied. We also performed virtual screening of thousand different polyphenols against proteins involved in the peptidoglycan biosynthesis to find potential valuable bioactive compounds. The bibliographic information used in this review was obtained from MEDLINE via PubMed. RESULTS: Several polyphenols: phenolic acids, flavonoids (especially flavonols), tannins, lignans, stilbenes and combinations of these in botanical mixtures, have exhibited significant antibacterial activity against resistant and non-resistant Gram-positive bacteria at low µg/mL range MIC values. Their mechanism of action is quite diverse, targeting cell wall, lipid membrane, membrane receptors and ion channels, bacteria metabolites and biofilm formation. Synergic effects were also demonstrated for some combinations of polyphenols and antibiotics. CONCLUSION: Plant polyphenols mean a promising source of antibacterial agents, either alone or in combination with existing antibiotics, for the development of new antibiotic therapies.

The Potential Synergistic Modulation of AMPK by Lippia citriodora Compounds as a Target in Metabolic Disorders.

Lippia citriodora (LC) represents a complex plant-derived source of polyphenols and iridoids that has shown beneficial properties against obesity-related metabolic disorders. The complete extract and its major compound, verbascoside, have shown AMPK-activating capacity in cell and animal models. In this work, we aimed to elucidate the contribution of the different compounds present in the LC extract on the AMPK activation capacity of the whole extract. Semipreparative reversed-phase high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RP-HPLC-ESI-TOF-MS) was used to identify the major compounds with bioassay-guided fractionation in an adipocyte cell model for the measurement of AMPK activity. Twenty-two compounds were identified and purified almost to homogeneity in 16 fractions, and three compounds, namely verbascoside, luteolin-7-diglucuronide and loganic acid, showed the highest AMPK-activating capacity. The synergy study using the checkerboard and fractional inhibitory concentration index (FICI) methods exhibited synergistic behavior between loganic acid and luteolin-7-diglucuronide. Molecular docking experiments revealed that these three compounds might act as direct agonists of AMPK, binding to the AMP binding sites of the gamma subunit and/or the different sites of the interaction zones between the gamma and beta subunits. Although our findings conclude that the bioactivity of the extract is mainly due to verbascoside, the synergy found between loganic acid and luteolin-7-diglucuronide deserves further research aimed to develop optimized combinations of polyphenols as a new nutritional strategy against obesity-related metabolic disorders.

Extra Virgin Olive Oil Contains a Phenolic Inhibitor of the Histone Demethylase LSD1/KDM1A.

The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC50 as low as 2.5 µmol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors.