Phenolic Compounds and Capsaicinoids in Three Capsicum annuum Varieties: From Analytical Characterization to In Silico Hypotheses on Biological Activity.

The affinity of specific phenolic compounds (PCs) and capsaicinoids (CAPs) present in three Capsicum annuum varieties (Friariello, Cayenne and Dzuljunska Sipka) to the transient receptor potential vanilloid member 1 (TRPV1) was investigated by integrating an analytic approach for the simultaneous extraction and analysis through high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC/ITMS) and UV detection (HPLC-UV) of PCs and CAPs and structural bioinformatics based on the protein modelling and molecular simulations of protein-ligand docking. Overall, a total of 35 compounds were identified in the different samples and CAPs were quantified. The highest content of total polyphenols was recorded in the pungent Dzuljunska Sipka variety (8.91 ± 0.05 gGAE/Kg DW) while the lowest was found in the non-pungent variety Friariello (3.58 ± 0.02 gGAE/Kg DW). Protein modelling generated for the first time a complete model of the homotetrameric human TRPV1, and it was used for docking simulations with the compounds detected via the analytic approach, as well as with other compounds, as an inhibitor reference. The simulations indicate that different capsaicinoids can interact with the receptor, providing details on the molecular interaction, with similar predicted binding energy values. These results offer new insights into the interaction of capsaicinoids with TRPV1 and their possible actions.

Sirtuin Inhibitor Cambinol Induces Cell Differentiation and Differently Interferes with SIRT1 and 2 at the Substrate Binding Site.

Epigenetic mechanisms finely regulate gene expression and represent potential therapeutic targets. Cambinol is a synthetic heterocyclic compound that inhibits class III histone deacetylases known as sirtuins (SIRTs). The acetylating action that results could be crucial in modulating cellular functions via epigenetic regulations. The main aim of this research was to investigate the effects of cambinol, and its underlying mechanisms, on cell differentiation by combining wet experiments with bioinformatics analyses and molecular docking simulations. Our in vitro study evidenced the ability of cambinol to induce the differentiation in MCF-7, NB4, and 3T3-L1 cell lines. Interestingly, focusing on the latter that accumulated cytoplasmic lipid droplets, the first promising results related to the action mechanisms of cambinol have shown the induction of cell cycle-related proteins (such as p16 and p27) and modulation of the expression of Rb protein and nuclear receptors related to cell differentiation. Moreover, we explored the inhibitory mechanism of cambinol on human SIRT1 and 2 performing in silico molecular simulations by protein-ligand docking. Cambinol, unlike from other sirtuin inhibitors, is able to better interact with the substrate binding site of SIRT1 than with the inhibition site. Additionally, for SIRT2, cambinol partially interacts with the substrate binding site, although the inhibition site is preferred. Overall, our findings suggest that cambinol might contribute to the development of an alternative to the existing epigenetic therapies that modulate SIRTs.

Molecular Docking of Natural Compounds for Potential Inhibition of AhR.

The aryl hydrocarbon receptor (AhR) is a highly conserved environmental sensor, historically known for mediating the toxicity of xenobiotics. It is involved in numerous cellular processes such as differentiation, proliferation, immunity, inflammation, homeostasis, and metabolism. It exerts a central role in several conditions such as cancer, inflammation, and aging, acting as a transcription factor belonging to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) protein family. A key step in the canonical AhR activation is AhR-ARNT heterodimerization followed by the binding to the xenobiotic-responsive elements (XREs). The present work aims to investigate the potential AhR inhibitory activity of selected natural compounds. Due to the absence of a complete structure of human AhRs, a model consisting of the bHLH, the PAS A, and the PAS B domains was constructed. Blind and focused docking simulations revealed the presence of further binding pockets, different from the canonical one presented in the PAS B domain, which could be important for AhR inhibition due to the possibility to impede AhR:ARNT heterodimerization, either preventing conformational changes or masking crucial sites necessary for protein-protein interaction. Two of the compounds retrieved from the docking simulations, i.e., ß-carotene and ellagic acid, confirmed their capacity of inhibiting benzo[a]pyrene (BaP)-induced AhR activation in in vitro tests on the human hepatoma cell line HepG2, validating the efficacy of the computational approach.

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells.

BACKGROUND: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. METHODS: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. RESULTS: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD's catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. CONCLUSIONS: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.

Investigating the Effects of Amino Acid Variations in Human Menin.

Human menin is a nuclear protein that participates in many cellular processes, as transcriptional regulation, DNA damage repair, cell signaling, cell division, proliferation, and migration, by interacting with many other proteins. Mutations of the gene encoding menin cause multiple endocrine neoplasia type 1 (MEN1), a rare autosomal dominant disorder associated with tumors of the endocrine glands. In order to characterize the structural and functional effects at protein level of the hundreds of missense variations, we investigated by computational methods the wild-type menin and more than 200 variants, predicting the amino acid variations that change secondary structure, solvent accessibility, salt-bridge and H-bond interactions, protein thermostability, and altering the capability to bind known protein interactors. The structural analyses are freely accessible online by means of a web interface that integrates also a 3D visualization of the structure of the wild-type and variant proteins. The results of the study offer insight into the effects of the amino acid variations in view of a more complete understanding of their pathological role.

A hypothesis on the capacity of plant odorant-binding proteins to bind volatile isoprenoids based on in silico evidences.

Volatile organic compounds (VOCs) from 'emitting' plants inform the 'receiving' (listening) plants of impending stresses or simply of their presence. However, the receptors that allow receivers to detect the volatile cue are elusive. Most likely, plants (as animals) have odorant-binding proteins (OBPs), and in fact, a few OBPs are known to bind 'stress-induced' plant VOCs. We investigated whether these and other putative OBPs may bind volatile constitutive and stress-induced isoprenoids, the most emitted plant VOCs, with well-established roles in plant communication and defense. Molecular docking simulation experiments suggest that structural features of a few plant proteins screened in databases could allow VOC binding. In particular, our results show that monoterpenes may bind the same plant proteins that were described to bind other stress-induced VOCs, while the constitutive hemiterpene isoprene is unlikely to bind any investigated putative OBP and may not have an info-chemical role. We conclude that, as for animal, there may be plant OBPs that bind multiple VOCs. Plant OBPs may play an important role in allowing plants to eavesdrop messages by neighboring plants, triggering defensive responses and communication with other organisms.

Cytotoxic, Antioxidant, and Enzyme Inhibitory Properties of the Traditional Medicinal Plant Matthiola incana (L.) R. Br.

Matthiola incana (L.) R. Br. (Brassicaceae) is widely cultivated for ornamental purposes and utilized as a medicinal plant. In the present work, the hydroalcoholic extract from the aerial parts of this species has been evaluated in different bioassays in order to detect potential pharmacological applications. The cytotoxic capacity against the human colorectal adenocarcinoma (CaCo-2) and breast cancer (MCF-7) cell lines was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The extract was investigated as a neuroprotective inhibitor of central nervous system (CNS) enzymes such as monoamine oxidase A, tyrosinase, acetylcholinesterase, and as a natural enzyme inhibitor of α-glucosidase and lipase involved in some metabolic disorders such as obesity or type 2 diabetes. The antioxidant ability was also evaluated in an enzymatic system (xanthine/xanthine oxidase assay). Results showed that the M. incana extract displayed moderate to low cytotoxicity vs. CaCo-2 cells. The extract acted as a superoxide radical scavenger and enzymatic inhibitor of monoamine oxidase A, tyrosinase, α-glucosidase, and lipase. The best results were found in the α-glucosidase assay, as M. incana hydroalcoholic extract was able to inhibit the enzyme α-glucosidase up to 100% without significant differences, compared to the antidiabetic drug acarbose. Matthiola incana has been demonstrated to exert different biological properties. These are important in order to consider this species as a source of bioactive compounds.

Synthesis and biological evaluation of the progenitor of a new class of cephalosporin analogues, with a particular focus on structure-based computational analysis.

We present the synthesis and biological evaluation of the prototype of a new class of cephalosporins, containing an additional isolated beta lactam ring with two phenyl substituents. This new compound is effective against Gram positive microorganisms, with a potency similar to that of ceftriaxone, a cephalosporin widely used in clinics and taken as a reference, and with no cytotoxicity against two different human cell lines, even at a concentration much higher than the minimal inhibitory concentration tested. Additionally, a deep computational analysis has been conducted with the aim of understanding the contribution of its moieties to the binding energy towards several penicillin-binding proteins from both Gram positive and Gram negative bacteria. All these results will help us developing derivatives of this compound with improved chemical and biological properties, such as a broader spectrum of action and/or an increased affinity towards their molecular targets.

Local-regional staging of endometrial carcinoma: role of MR imaging in surgical planning.

PURPOSE: To assess magnetic resonance (MR) imaging in depicting the depth of myometrial infiltration, cervical invasion, and presence of enlarged lymph nodes in patients with endometrial adenocarcinoma compared with surgicopathologic findings. MATERIALS AND METHODS: Thirty-seven consecutive patients with endometrial carcinoma were included in this prospective study. All patients underwent MR imaging and surgery. Qualitative image analysis included the depth of myometrial infiltration, infiltration of the uterine cervix, and presence of enlarged lymph nodes. Quantitative image analysis included tumor and myometrium contrast-to-noise ratios during different phases of dynamic imaging. MR imaging findings were compared with surgicopathologic findings. Sensitivity, specificity, diagnostic accuracy, and positive and negative predictive values of MR imaging in depicting myometrial and cervical infiltration and in lymph node assessment were calculated. RESULTS: Respective sensitivity, specificity, diagnostic accuracy, and positive and negative predictive values in assessing myometrial infiltration were 87%, 91%, 89%, 87%, and 91%; those for cervical infiltration, 80%, 96%, 92%, 89%, and 93%; and those for lymph node assessment, 50%, 95%, 90%, 50%, and 95%. There was significant agreement between MR imaging and surgicopathologic findings in assessment of myometrial invasion (P <.001). Myometrial and cervical invasion and lymph node enlargement were correctly assessed with MR imaging in 28 (76%) of 37 patients. Quantitative analysis showed a significant improvement in tumor and myometrium contrast-to-noise ratios during the equilibrium phase compared with the arterial and precontrast phases (P <.001). CONCLUSION: MR imaging coupled with contrast material-enhanced dynamic MR imaging is highly accurate in local-regional staging of endometrial carcinoma; more challenging is the assessment of pelvic and lumboaortic lymph nodes.