Determining the Effect of Pterostilbene on Insulin Secretion Using Chemoproteomics.

Pterostilbene, the 3,5-dimethoxy derivative of resveratrol, is a well-known polyphenolic compound, mainly found in blueberries, grapevines, and heartwood, which has recently attracted a great deal of attention due to its wide bio-pharmacological profile. Moreover, pterostilbene is more lipophilic than resveratrol, with a consequently better bioavailability and a more interesting therapeutic potential. In this work, a chemoproteomic approach, based on affinity chromatography, was applied on pterostilbene in the attempt to identify the biological targets responsible for its bioactivity.

Targeting mPGES-1 by a Combinatorial Approach: Identification of the Aminobenzothiazole Scaffold to Suppress PGE Levels.

Microsomal prostaglandin E synthase-1 (mPGES-1), the terminal enzyme responsible for the production of inducible prostaglandin E, has become an attractive target for the treatment of inflammation and cancer pathologies. Starting from an aminobenzothiazole scaffold, used as an unprecedented chemical core for mPGES-1 inhibition, a Combinatorial Virtual Screening campaign was conducted, using the X-ray crystal structure of human mPGES-1. Two combinatorial libraries (6 × 10) were obtained by decorating the aminobenzothiazole scaffold with all acyl chlorides and boronates available at the Merck database.

DFT/NMR Approach for the Configuration Assignment of Groups of Stereoisomers by the Combination and Comparison of Experimental and Predicted Sets of Data.

Quantum mechanical/nuclear magnetic resonance (NMR) approaches are widely used for the configuration assignment of organic compounds generally comparing one cluster of experimentally determined data (e.g., C NMR chemical shifts) with those predicted for all possible theoretical stereoisomers.

Elucidating heteroatom influence on homonuclear J coupling constants by DFT/NMR approach.

We report the structural dependency of long range scalar J-coupling constant across four bonds as function of the dihedral angles Φ1 and Φ3. The calculated homonuclear coupling constants J( ), obtained at a density functional theory level, were measured between C(1)─X(2) and X(2)─C(3) bonds in three-term models, where C, N, O, and S were systematically used as the second atom of the alkyl structures (1-4). The J calculated values, tabulated for variation of 30° for both Φ1 and Φ3, have disclosed an unexpected detectable coupling constant ( J( ) ≥ 1 Hz) across heteroatoms, useful to provide valuable structural information.

Quantitative proteomics discloses monacolin K-induced alterations in triple-negative breast cancer cell proteomes and phosphoproteomes.

A positive prognosis of triple-negative breast cancer can be considered as one of the major challenges in clinical studies; accordingly, scientific research has the mission to find out novel chemotherapeutics to make it curable. In recent times, a good potential of dietary bioactive natural substances, called nutraceuticals, in suppressing cancer cell proliferation via gene expression regulation has been discovered: this effect and the lack of toxicity make nutraceuticals potentially effective agents against cancers. Monacolin K from red rice, a FDA-approved and well-tolerated compound generally employed to treat hypercholesterolemia, has been proved to have anti-proliferative and apoptotic effects in a wide panel of triple-negative breast cancers.

Garcinol and Related Polyisoprenylated Benzophenones as Topoisomerase II Inhibitors: Biochemical and Molecular Modeling Studies.

Garcinol, a polyisoprenylated benzophenone isolated from genus, has been reported to inhibit eukaryotic topoisomerase I and topoisomerase II at concentrations comparable to that of etoposide (∼25-100 μM). With the aim to clarify the underlying molecular mechanisms by which garcinol inhibits human topoisomerase IIα and topoisomerase IIβ, biochemical assays along with molecular docking and molecular dynamics studies were carried out on garcinol and six congeners. The biochemical results revealed that garcinol derivatives appear to act as catalytic inhibitors of topoisomerase II and to inhibit ATP hydrolysis by topoisomerase II via some form of mixed inhibition.

Protein Preparation Automatic Protocol for High-Throughput Inverse Virtual Screening: Accelerating the Target Identification by Computational Methods.

Structure-based virtual screening is highly used in the early stages of drug discovery to identify new putative lead compounds for a given target. However, when a small molecule elicits a biological effect, but its target is unknown, or the side effects it causes arise from its undesired interaction with unknown counterparts, the identification of its interacting targets represents an indispensable task. The computational procedure named inverse virtual screening, which relies on docking a molecule (or a small set of compounds) against panels of target proteins to select the most promising complexes, could be useful to overcome these issues.

Identification of the 2-Benzoxazol-2-yl-phenol Scaffold as New Hit for JMJD3 Inhibition.

JMJD3 is a member of the KDM6 subfamily and catalyzes the demethylation of lysine 27 on histone H3 (H3K27). This protein was identified as a useful tool in understanding the role of epigenetics in inflammatory conditions and in cancer as well. Guided by a virtual fragment screening approach, we identified the benzoxazole scaffold as a new hit suitable for the development of tighter JMJD3 inhibitors.

Immunomodulatory Biscembranoids and Assignment of Their Relative and Absolute Configurations: Data Set Modulation in the Density Functional Theory/Nuclear Magnetic Resonance Approach.

Five new biscembranoids, bistrochelides A-E (3-7), were isolated together with glaucumolides A (1) and B (2) from the soft coral Sarcophyton trocheliophorum. Their structures and absolute configurations were determined by spectroscopic methods, X-ray crystal diffraction, and DFT/NMR (density functional theory/nuclear magnetic resonance) and TDDFT/ECD (time-dependent density functional theory/electronic circular dichroism) calculations. A new approach is introduced to determine the relative configuration of a stereocenter through the dynamic evaluation of the mean absolute errors (MAEs) between the investigated diastereoisomers, moving from an "extended" to a more diagnostic "restricted" set of atoms.

Chemical Proteomics-Guided Identification of a Novel Biological Target of the Bioactive Neolignan Magnolol.

Understanding the recognition process between bioactive natural products and their specific cellular receptors is of key importance in the drug discovery process. In this outline, some potential targets of Magnolol, a natural bioactive compound, have been identified by proteomic approaches. Among them, Importin-β1 has been considered as the most relevant one.

Discovery of 3-hydroxy-3-pyrrolin-2-one-based mPGES-1 inhibitors using a multi-step virtual screening protocol.

Targeting microsomal prostaglandin E synthase-1 (mPGES-1) represents an efficient strategy for the development of novel drugs against inflammation and cancer with potentially reduced side effects. With this aim, a virtual screening was performed on a large library of commercially available molecules using the X-ray structure of mPGES-1 co-complexed with a potent inhibitor. Combining fast ligand-based shape alignment, molecular docking experiments, and qualitative analysis of the binding poses, a small set of molecules was selected for the subsequent steps of validation of the biological activity.

Chemoproteomic fishing identifies arzanol as a positive modulator of brain glycogen phosphorylase.

The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors.

The natural product magnolol (1) and a selection of its bioinspired derivatives 2-5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3-5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.

Discovery and synthesis of the first selective BAG domain modulator of BAG3 as an attractive candidate for the development of a new class of chemotherapeutics.

BAG3 protein has emerged as a key regulator of important cellular processes and its expression is increased in some tumor types; however, despite its potential value for future chemotherapeutics, no selective BAG3 modulators have been yet reported. Here we report the 2,4-thiazolidinedione derivative 28 as the first BAG3 protein modulator.

Discovery of new erbB4 inhibitors: Repositioning an orphan chemical library by inverse virtual screening.

Inverse Virtual Screening (IVS) is a docking based approach aimed to the evaluation of the virtual ability of a single compound to interact with a library of proteins. For the first time, we applied this methodology to a library of synthetic compounds, which proved to be inactive towards the target they were initially designed for. Trifluoromethyl-benzenesulfonamides 3-21 were repositioned by means of IVS identifying new lead compounds (14-16, 19 and 20) for the inhibition of erbB4 in the low micromolar range.

Virtual Fragment Screening Identification of a Quinoline-5,8-dicarboxylic Acid Derivative as a Selective JMJD3 Inhibitor.

The quinoline-5,8 dicarboxylic acid scaffold has been identified by a fragment-based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3-(2,4-dimethoxypyrimidin-5-yl)quinoline-5,8-dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain-containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.

Discovery of new molecular entities able to strongly interfere with Hsp90 C-terminal domain.

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone deeply involved in the complex network of cellular signaling governing some key functions, such as cell proliferation and survival, invasion and angiogenesis. Over the past years the N-terminal protein domain has been fully investigated as attractive strategy against cancer, but despite the many efforts lavished in the field, none of the N-terminal binders (termed "classical inhibitors"), currently in clinical trials, have yet successfully reached the market, because of the detrimental heat shock response (HSR) that showed to induce; thus, recently, the selective inhibition of Hsp90 C-terminal domain has powerfully emerged as a more promising alternative strategy for anti-cancer therapy, not eliciting this cell rescue cascade. However, the structural complexity of the target protein and, mostly, the lack of a co-crystal structure of C-terminal domain-ligand, essential to drive the identification of new hits, represent the largest hurdles in the development of new selective C-terminal inhibitors.

Discovery of new potent molecular entities able to inhibit mPGES-1.

mPGES-1, a glutathione-dependent membrane protein is involved in the last step of PGE production and has been well recognized as a strategic target for the development of anti-inflammatory and anti-cancer agents. It has been proven to selectively control the PGE levels induced by inflammatory stimuli, with neither affecting PGE constitutively produced, nor homeostatic prostanoids, so that its modulation can represent a better strategy to control PGE related disorders, compared to the use of the classical anti-inflammatory drugs, endowed with severe side effects. Despite the intensive research on the identification of potent mPGES-1 inhibitors as attractive candidates for drug development, none of the disclosed molecules, except for LY3023705, which recently entered clinical trials, are available for clinical use, therefore the discovery of new effective mPGES-1 inhibitors with increased drug-like properties are urgently needed.

Identification of Trombospondin-1 as a Novel Amelogenin Interactor by Functional Proteomics.

Amelogenins are a set of low molecular-weight enamel proteins belonging to a group of extracellular matrix (ECM) proteins with a key role in tooth enamel development and in other regeneration processes, such as wound healing and angiogenesis. Since only few data are actually available to unravel amelogenin mechanism of action in chronic skin healing restoration, we moved to the full characterization of the human amelogenin isoform 2 interactome in the secretome and lysate of Human Umbilical Vein Endothelial cells (HUVEC), using a functional proteomic approach. Trombospondin-1 has been identified as a novel and interesting partner of human amelogenin isoform 2 and their direct binding has been validated thought biophysical orthogonal approaches.

Discovering the Biological Target of 5-epi-Sinuleptolide Using a Combination of Proteomic Approaches.

Sinuleptolide and its congeners are diterpenes with a norcembranoid skeleton isolated from the soft coral genus . These marine metabolites are endowed with relevant biological activities, mainly associated with cancer development. 5--sinuleptolide has been selected as a candidate for target discovery studies through the application of complementary proteomic approaches.

Chemistry and Selective Tumor Cell Growth Inhibitory Activity of Polyketides from the South China Sea Sponge Plakortis sp.

Simplextone E (), a new metabolite of polyketide origin, was isolated with eight known analogues (-) from the South China Sea sponge sp. The relative configuration of the new compound was elucidated by a detailed analysis of the spectroscopic data and quantum mechanical calculation of NMR chemical shifts, aided by the newly reported DP4+ approach. Its absolute configuration was determined by the TDDFT/ECD calculation.

Determination of Gymnemic Acid I as a Protein Biosynthesis Inhibitor Using Chemical Proteomics.

The plant Gymnema sylvestre has been used widely in traditional medicine as a remedy for several diseases, and its leaf extract is known to contain a group of bioactive triterpene saponins belonging to the gymnemic acid class. Gymnemic acid I (1) is one of the main components among this group of secondary metabolites and is endowed with an interesting bioactivity profile. Since there is a lack of information about its specific biological targets, the full interactome of 1 was investigated through a quantitative chemical proteomic approach, based on stable-isotope dimethyl labeling.

Bissubvilides A and B, Cembrane-Capnosane Heterodimers from the Soft Coral Sarcophyton subviride.

Two new biscembranoid-like compounds, bissubvilides A (1) and B (2), were isolated together with sarsolilide B (3), the proposed biogenetic precursor to 1, from the soft coral Sarcophyton subviride. The structures and absolute configurations were solved by spectroscopic analysis and TDDFT/ECD and DFT/NMR calculations. The bissubvilides represent a novel biscembranoid-like skeleton presumed to derive from a cembrane-type diene and a capnosane-type dienophile via a Diels-Alder reaction.

Identification of novel microsomal prostaglandin E synthase-1 (mPGES-1) lead inhibitors from Fragment Virtual Screening.

Identification of new microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors is currently sought for the treatment of cancer and inflammation. Here we show the results of a Fragment Virtual Screening campaign using the X-ray crystal structure of human mPGES-1 (PDB code: 4AL0). Among the fragments selected and biologically tested, 6 (9H-indeno [1,2-b] [1,2,5]oxadiazolo [3,4-e]pyrazin-9-one) showed the most promising mPGES-1 inhibitory activity (∼30% inhibition at 10 μM).

Biomolecular proteomics discloses ATP synthase as the main target of the natural glycoside deglucoruscin.

Extracts of Ruscus aculeatus are a rich source of bioactive steroidal glycosides, such as ruscogenins which are reported to act against chronic venous disorders. Nowadays, several preparations of its roots, commonly used in traditional medicine, are on the market as food supplements for health care and maintenance. Although spirostanol deglucoruscin is one of the main metabolites in these extracts, literature reports about its pharmacological profile are scarce.