Computation-Assisted Identification of Bioactive Compounds in Botanical Extracts: A Case Study of Anti-Inflammatory Natural Products from Hops.

The slow pace of discovery of bioactive natural products can be attributed to the difficulty in rapidly identifying them in complex mixtures such as plant extracts. To overcome these hurdles, we explored the utility of two machine learning techniques, i.e., Elastic Net and Random Forests, for identifying the individual anti-inflammatory principle(s) of an extract of the inflorescences of the hops (Humulus lupulus) containing hundreds of natural products. We fractionated a hop extract by column chromatography to obtain 40 impure fractions, determined their anti-inflammatory activity using a macrophage-based bioassay that measures inhibition of iNOS-mediated formation of nitric oxide, and characterized the chemical composition of the fractions by flow-injection HRAM mass spectrometry and LC-MS/MS. Among the top 10 predictors of bioactivity were prenylated flavonoids and humulones. The top Random Forests predictor of bioactivity, xanthohumol, was tested in pure form in the same bioassay to validate the predicted result (IC50 7 µM). Other predictors of bioactivity were identified by spectral similarity with known hop natural products using the Global Natural Products Social Networking (GNPS) algorithm. Our machine learning approach demonstrated that individual bioactive natural products can be identified without the need for extensive and repetitive bioassay-guided fractionation of a plant extract.

Xanthohumol Pyrazole Derivative Improves Diet-Induced Obesity and Induces Energy Expenditure in High-Fat Diet-Fed Mice.

The energy intake exceeding energy expenditure (EE) results in a positive energy balance, leading to storage of excess energy and weight gain. Here, we investigate the potential of a newly synthesized compound as an inducer of EE for the management of diet-induced obesity and insulin resistance. Xanthohumol (XN), a prenylated flavonoid from hops, was used as a precursor for the synthesis of a pyrazole derivative tested for its properties on high-fat diet (HFD)-induced metabolic impairments. In a comparative study with XN, we report that 4-(5-(4-hydroxyphenyl)-1-methyl-1H-pyrazol-3-yl)-5-methoxy-2-(3-methylbut-2-en-1-yl)benzene-1,3-diol (XP) uncouples oxidative phosphorylation in C2C12 cells. In HFD-fed mice, XP improved glucose tolerance and decreased weight gain by increasing EE and locomotor activity. Using an untargeted metabolomics approach, we assessed the effects of treatment on metabolites and their corresponding biochemical pathways. We found that XP and XN reduced purine metabolites and other energy metabolites in the plasma of HFD-fed mice. The induction of locomotor activity was associated with an increase in inosine monophosphate in the cortex of XP-treated mice. Together, these results suggest that XP, better than XN, affects mitochondrial respiration and cellular energy metabolism to prevent obesity in HFD-fed mice.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

Exploring the Interaction of Curaxin CBL0137 with G-Quadruplex DNA Oligomers.

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.

Plant-Derived Stilbenoids as DNA-Binding Agents: From Monomers to Dimers.

Stilbenoids are natural compounds endowed with several biological activities, including cardioprotection and cancer prevention. Among them, (±)-trans-δ-viniferin, deriving from trans-resveratrol dimerization, was investigated in its ability to target DNA duplex and G-quadruplex structures by exploiting NMR spectroscopy, circular dichroism, fluorescence spectroscopy and molecular docking. (±)-trans-δ-Viniferin proved to bind both the minor and major grooves of duplexes, whereas it bound the 3'- and 5'-ends of a G-quadruplex by stacking on the outer quartets, accompanied by rearrangement of flanking residues. Specifically, (±)-trans-δ-viniferin demonstrated higher affinity for the investigated DNA targets than its monomeric counterpart. Additionally, the methoxylated derivatives of (±)-trans-δ-viniferin and trans-resveratrol, i. e. (±)-pterostilbene-trans-dihydrodimer and trans-pterostilbene, respectively, were evaluated, revealing similar binding modes, affinities and stoichiometries with the DNA targets as their parent analogues. All tested compounds were cytotoxic at µM concentration on several cancer cell lines, showing DNA damaging activity consistent with their ability to tightly interact with duplex and G-quadruplex structures.

Natural and nature-inspired stilbenoids as antiviral agents.

Viruses continue to be a major threat to human health. In the last century, pandemics occurred and resulted in significant mortality and morbidity. Natural products have been largely screened as source of inspiration for new antiviral agents. Within the huge class of plant secondary metabolites, resveratrol-derived stilbenoids present a wide structural diversity and mediate a great number of biological responses relevant for human health. However, whilst the antiviral activity of resveratrol has been extensively studied, little is known about the efficacy of its monomeric and oligomeric derivatives. The purpose of this review is to provide an overview of the achievements in this field, with particular emphasis on the source, chemical structures and the mechanism of action of resveratrol-derived stilbenoids against the most challenging viruses. The collected results highlight the therapeutic versatility of stilbene-containing compounds and provide a prospective insight into their potential development as antiviral agents.

Structural Requirements of Benzofuran Derivatives Dehydro-δ- and Dehydro-ε-Viniferin for Antimicrobial Activity Against the Foodborne Pathogen Listeria monocytogenes.

In a recent study, we investigated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers against a series of foodborne pathogens. Out of the tested molecules, dehydro-δ-viniferin and dehydro-ε-viniferin emerged as the most promising derivatives. To define the structural elements essential to the antimicrobial activity against the foodborne pathogen L. monocytogenes Scott A as a model Gram-positive microorganism, the synthesis of a series of simplified benzofuran-containing derivatives was carried out. The systematic removal of the aromatic moieties of the parent molecules allowed a deeper insight into the most relevant structural features affecting the activity. While the overall structure of compound 1 could not be altered without a substantial loss of antimicrobial activity, the structural simplification of compound 2 (minimal inhibitory concentration (MIC) 16 µg/mL, minimal bactericidal concentration (MBC) >512 µg/mL) led to the analogue 7 with increased activity (MIC 8 µg/mL, MBC 64 µg/mL).

Antimicrobial activity of resveratrol-derived monomers and dimers against foodborne pathogens.

Plant polyphenolic compounds are considered a promising source for new antibacterial agents. In this study, we evaluated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers screened as single molecules against a panel of nine foodborne pathogens. The results demonstrated that two monomers (i.e., pterostilbene 2 and (E)-3-hydroxy-4',5-dimethoxystilbene 9) and three dimers (i.e., δ-viniferin 10, viniferifuran 14 and dehydro-δ-viniferin 15) were endowed with significant antibacterial activity against gram-positive bacteria. The exposure of gram-positive foodborne pathogens to 100 µg/mL of 2, 9 and 15 induced severe cell membrane damage, resulting in the disruption of the phospholipid bilayer. The most promising dimeric compound, dehydro-δ-viniferin 15, was tested against Listeria monocytogenes, resulting in a loss of cultivability, viability and cell membrane potential. TEM analysis revealed grave morphological modifications on the cell membrane and leakage of intracellular content, confirming that the cell membrane was the principal biological target of the tested derivative.

Inhibition of Pancreatic α-amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity between Viniferin Enantiomers.

To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids-in particular, viniferin isomers- were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.