Chemoproteomics Reveals USP5 (Ubiquitin Carboxyl-Terminal Hydrolase 5) as Promising Target of the Marine Polyketide Gracilioether A.

Mass spectrometry-based chemical proteomic approaches using limited proteolysis have become a powerful tool for the identification and analysis of the interactions between a small molecule (SM) and its protein target(s). Gracilioether A (GeA) is a polyketide isolated from a marine sponge, for which we aimed to trace the interactome using this strategy. DARTS (Drug Affinity Responsive Target Stability) and t-LiP-MS (targeted-Limited Proteolysis-Mass Spectrometry) represented the main techniques used in this study. DARTS was applied on HeLa cell lysate for the identification of the GeA target proteins, and t-LiP-MS was employed to investigate the protein's regions involved in the binding with GeA. The results were complemented through the use of binding studies using Surface Plasmon Resonance (SPR) and in silico molecular docking experiments. Ubiquitin carboxyl-terminal hydrolase 5 (USP5) was identified as a promising target of GeA, and the interaction profile of the USP5-GeA complex was explained. USP5 is an enzyme involved in the pathway of protein metabolism through the disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. This activity is connected to different cellular functions concerning the maintenance of chromatin structure and receptors and the degradation of abnormal proteins and cancerogenic progression. On this basis, this structural information opens the way to following studies focused on the definition of the biological potential of Gracilioether A and the rational development of novel USP5 inhibitors based on a new structural skeleton.

Identification of Mortalin as the Main Interactor of Mycalin A, a Poly-Brominated C-15 Acetogenin Sponge Metabolite, by MS-Based Proteomics.

Mycalin A (MA) is a polybrominated C-15 acetogenin isolated from the marine sponge Mycale rotalis. Since this substance displays a strong antiproliferative bioactivity towards some tumour cells, we have now directed our studies towards the elucidation of the MA interactome through functional proteomic approaches, (DARTS and t-LIP-MS). DARTS experiments were performed on Hela cell lysates with the purpose of identifying MA main target protein(s); t-LiP-MS was then applied for an in-depth investigation of the MA-target protein interaction. Both these techniques exploit limited proteolysis coupled with MS analysis. To corroborate LiP data, molecular docking studies were performed on the complexes. Finally, biological and SPR analysis were conducted to explore the effect of the binding. Mortalin (GRP75) was identified as the MA's main interactor. This protein belongs to the Hsp70 family and has garnered significant attention due to its involvement in certain forms of cancer. Specifically, its overexpression in cancer cells appears to hinder the pro-apoptotic function of p53, one of its client proteins, because it becomes sequestered in the cytoplasm. Our research, therefore, has been focused on the possibility that MA might prevent this sequestration, promoting the re-localization of p53 to the nucleus and facilitating the apoptosis of tumor cells.

Fatty Acid Synthase as Interacting Anticancer Target of the Terpenoid Myrianthic Acid Disclosed by MS-Based Proteomics Approaches.

This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of Myrianthus arboreus and from Oenothera maritima Nutt. (Onagraceae), using MS-based chemical proteomic techniques. Application of drug affinity responsive target stability (DARTS) and targeted-limited proteolysis coupled to mass spectrometry (t-LiP-MS) led to the identification of the enzyme fatty acid synthase (FAS) as an interesting macromolecular counterpart of myrianthic acid. This result, confirmed by comparison with the natural ursolic acid, was thoroughly investigated and validated in silico by molecular docking, which gave a precise picture of the interactions in the MA/FAS complex. Moreover, biological assays showcased the inhibitory activity of myrianthic acid against the FAS enzyme, most likely related to its antiproliferative activity towards tumor cells. Given the significance of FAS in specific pathologies, especially cancer, the myrianthic acid structural moieties could serve as a promising reference point to start the potential development of innovative approaches in therapy.

Analysis of Hsp90 allosteric modulators interactome reveals a potential dual action mode involving mitochondrial MDH2.

Hsp90 (i.e., Heat shock protein 90) is a well-established therapeutic target for several diseases, ranging from misfolding-related disfunctions to cancer. In this framework, we have developed in recent years a family of benzofuran compounds that act as Hsp90 allosteric modulators. Such molecules can interfere with the stability of some relevant Hsp90 client oncoproteins, showing a low µM cytotoxic activity in vitro in cancer cell lines. Here we identify the target profile of these chemical probes by means of chemical proteomics, which established MDH2 (mitochondrial malate dehydrogenase) as an additional relevant cellular target that might help elucidate the molecular mechanism of their citotoxicity. Western blotting, DARTS (i.e., Drug Affinity Responsive Target Stability) and enzymatic assays data confirmed a dose-dependent interaction of MDH2 with several members of the benzofuran Hsp90 modulators family and a computational model allowed to interpret the observed interactions.

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 Pterocarpus marsupium 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. On this basis, syntaxins, a group of proteins involved in the formation of SNARE complexes mediating vesicles exocytosis, were selected among the most interesting pterostilbene interactors. In vitro and in cell assays gave evidence of the pterostilbene ability to reduce insulin secretion on glucose-stimulated pancreatic beta cells, opening the way to potential applications of pterostilbene as a supplement in the care of insulin-dependent metabolic disorders.

Proteasome as a New Target for Bio-Inspired Benzo[k,l]xanthene Lignans.

Mass spectrometry-based chemical proteomics is a powerful tool for the target discovery of small molecules. Here, the application of this approach is presented to define the target profile of bio-inspired synthetic benzo[k,l]xanthene lignans endowed with interesting biological properties. Proteasome has been identified as a new main interactor for this class of compounds. A combination of molecular docking with in vitro and in cell fluorescence assays gave insights on the molecular mechanism of the interaction, highlighting the tendency of these lignans to inhibit the proteasome.

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. The ribosome complex was found to be the main partner of compound 1, and a full validation of the proteomics results was achieved by orthogonal approaches. Further biochemical and biological investigations revealed an inhibitory effect of 1 on the ribosome machinery.

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 Sinularia. These marine metabolites are endowed with relevant biological activities, mainly associated with cancer development. 5-epi-sinuleptolide has been selected as a candidate for target discovery studies through the application of complementary proteomic approaches. Specifically, a combination of conventional chemical proteomics based on affinity chromatography, coupled with high-resolution mass spectrometry and bioinformatics, as well as drug affinity responsive target stability (DARTS), led to a clear identification of actins as main targets for 5-epi-sinuleptolide. Subsequent in-cell assays, performed with cytochalasin D as reference compound, gave information on the ability of 5-epi-sinuleptolide to disrupt the actin cytoskeleton by loss of actin fibers and formation of F-actin amorphous aggregates. These results suggest the potential application of 5-epi-sinuleptolide as a useful tool in the study of the molecular processes impaired in several disorders in which actin is thought to play an essential role.

Mechanistic insights on petrosaspongiolide M inhibitory effects on immunoproteasome and autophagy.

The proteasome, a complex multimeric structure strictly implicated in cell protein degradation, has gained the status of privileged drug target since its functional involvement in relevant pathways ruling the cell life, such as cell cycle, transcription and protein quality control, and the recent marketing of bortezomib as proteasome inhibitor for anti-cancer therapy. The marine γ-hydroxybutenolide terpenoid petrosaspongiolide M has been recently discovered as new proteasome inhibitor through a chemical proteomic approach and in cell biological assays. In this study a deep investigation has been carried out on the molecular mechanism of interaction of petrosaspongiolide M with the immunoproteasome, a proteasomal variant mainly involved in the immune responses. The results define a picture in which petrosaspongiolide M exerts its inhibitory activity by binding the active sites in the inner core of the immunoproteasome and/or covalently linking a Lys residue at the proteasome core/11S activator particle interface. Moreover, petrosaspongiolide M is also able to impair autophagy, a complementary pathway involved in protein degradation and cross-talking with the proteasome system. On this basis, petrosaspongiolide M could represent an interesting molecule for its propensity to modulate intracellular proteolysis through a dual inhibition of the immunoproteasome and autophagy.

Antiangiogenic effects of N6-isopentenyladenosine, an endogenous isoprenoid end product, mediated by AMPK activation.

N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway with an isopentenyl chain, is already known to exert a suppressor effect against various tumors. In this work, we investigated whether iPA also directly interferes with the angiogenic process, which is fundamental to tumor growth and progression. To this end, using human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, we evaluated their viability, proliferation, migration, invasion, tube formation in response to iPA, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 µM. We demonstrated for the first time, by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), that iPA was monophosphorylated into 5'-iPA-monophosphate (iPAMP) by the adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pretreatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. In conclusion, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.

Biomolecular Fishing for Calixarene Partners by a Chemoproteomic Approach.

MS-based chemical-proteomics technology is introduced herein as a third general strategy to study the biomolecular recognition properties of given calixarene derivatives. In particular, we demonstrate that a simply designed calix[4]arene derivative 1 a bearing acetamido groups at the exo rim (pAC), when linked to a solid support, is able to fish out a specific protein (PDI protein) from a crude extract of HeLa cells. Western blot and surface plasmon resonance studies confirmed the direct interaction between PDI and the linker-free pAC derivative 1 b with considerable affinity, and in vitro tests showed its inhibition of PDI chaperone activity. In accordance with the role of PDI in a variety of human cancers, biological tests showed that pAC 1 b was cytotoxic and cytostatic toward CAL-27 and PC-3 cancer cell lines in vitro. Docking studies showed that H bonds and hydrophobic interactions contribute to the stabilization of the PDI/pAC complex.

Anion-induced dimerization in p-squaramidocalix[4]arene derivatives.

Spherical anions induce the dimerization of calix[4]arene derivatives 3 and 4 bearing squaramide moieties at the exo rim (p-squaramidocalixarenes). (1)H NMR titration experiments showed that unlike the distal isomer 3, proximal p-squaramidocalixarene 4 is also able to form dimeric complexes with trigonal-planar anions.

Analysis of the Fecal Metabolomic Profile in Breast vs. Different Formula Milk Feeding in Late Preterm Infants.

Human milk is the gold standard for infant nutrition, but when it is not available or insufficient to satisfy the needs of the infant, formula milk is proposed as an effective substitute. A prospective observational cohort study was conducted on late preterm infants fed with breast and two different formula milks. On this basis, they were divided into three groups: group FMPB (fed with formula + postbiotic), group FM (fed with standard formula), and group BM (breastfed). Stool samples for a metabolomic study were collected at T0 (5-7 days after birth), T1 (30 days of life), and T2 (90 days of life), giving rise to 74 samples analyzed via liquid chromatography coupled with high-resolution mass spectrometry. The T0, T1, and T2 LC-MS raw data were processed for Partial Least Square Discriminant Analysis (PLS-DA), followed by a statistical analysis. This preliminary study highlighted a good overlapping between the fecal metabolome of breast and substitute feeding systems, confirming the efficacy of the formula preparations as breast milk substitutes. Moreover, several similarities were also detected between the FMPB and BM metabolome, highlighting that the addition of a postbiotic to standard formula milk could be more effective and considered a better alternative to breast milk.

NMR Metabolomics and Chemometrics of Commercial Varieties of Phaseolus vulgaris L. Seeds from Italy and In Vitro Antioxidant and Antifungal Activity.

The metabolite fingerprinting of four Italian commercial bean seed cultivars, i.e., Phaseolus Cannellino (PCANN), Controne (PCON), Vellutina (PVEL), and Occhio Nero (PON), were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate data analysis. The hydroalcoholic and organic extract analysis disclosed more than 32 metabolites from various classes, i.e., carbohydrates, amino acids, organic acids, nucleosides, alkaloids, and fatty acids. PVEL, PCON, and PCANN varieties displayed similar chemical profiles, albeit with somewhat different quantitative results. The PON metabolite composition was slightly different from the others; it lacked GABA and pipecolic acid, featured a higher percentage of malic acid than the other samples, and showed quantitative variations of several metabolites. The lipophilic extracts from all four cultivars demonstrated the presence of omega-3 and omega-6 unsaturated fatty acids. After the determination of the total phenolic, flavonoids, and condensed tannins content, in vitro antioxidant activity was then assessed using the DPPH scavenging activity, the ABTS scavenging assay, and ferric-reducing antioxidant power (FRAP). Compared to non-dark seeds (PCON, PCANN), brown seeds (PVEL, PON) featured a higher antioxidant capacity. Lastly, only PON extract showed in vitro antifungal activity against the sclerotia growth of S. rolfsii, by inhibiting halo growth by 75%.

Through-the-annulus threading of the larger calix[8]arene macrocycle.

A complete study of the through-the-annulus threading of the larger calix[8]arene macrocycle with di-n-alkylammonium cations has been performed in the presence of the "superweak" TFPB counterion. Thus, it was found that such threading occurs only upon partial preorganization of the calix[8]arene macroring by intramolecular bridging. In particular, 1,5-bridged calix[8]arenes with a meta- or para-xylylene bridge (2 and 3) gave pseudo[2]rotaxanes in which one dialkylammonium axle (4a-4e(+)) was threaded into one of the two subcavities of the calix[8]-wheel. Conformational studies by using chemical shift surface maps and DFT calculations evidenced a 3/4-cone geometry for these subcavities. Higher pseudorotaxane K(ass) values were obtained for calix[8]-wheels 2 and 3, with respect to calix[6]-host 1a, due to the cooperative effect of their two subcavities. Dynamic NMR studies on calix[8]-pseudorotaxanes evidenced a direct correlation between K(ass) (and ΔG(ass)) values and energy barriers for calix inversion due to the effectiveness of thread templation. In accordance with DFT calculations, an endo-alkyl preference, over the endo-benzyl one, was observed by threading calix[8]-wheel 3 with the directional n-butylbenzylammonium axle 4d(+).

Differential in gel electrophoresis (DIGE) comparative proteomic analysis of macrophages cell cultures in response to perthamide C treatment.

Secondary metabolites contained in marine organisms disclose diverse pharmacological activities, due to their intrinsic ability to recognize bio-macromolecules, which alter their expression and modulate their function. Thus, the identification of the cellular pathways affected by marine natural products is crucial to provide important functional information concerning their mechanism of action at the molecular level. Perthamide C, a marine sponge metabolite isolated from the polar extracts of Theonella swinhoei and endowed with a broad and interesting anti-inflammatory profile, was found in a previous study to specifically interact with heat shock protein-90 and glucose regulated protein-94, also disclosing the ability to reduce cisplatin-mediated apoptosis. In this paper, we evaluated the effect of this compound on the whole proteome of murine macrophages cells by two-dimensional DIGE proteomics. Thirty-three spots were found to be altered in expression by at least 1.6-fold and 29 proteins were identified by LC ESI-Q/TOF-MS. These proteins are involved in different processes, such as metabolism, structural stability, protein folding assistance and gene expression. Among them, perthamide C modulates the expression of several chaperones implicated in the folding of proteins correlated to apoptosis, such as Hsp90 and T-complexes, and in this context our data shed more light on the cellular effects and pathways altered by this marine cyclo-peptide.

Modulation of proteasome machinery by natural and synthetic analogues of the marine bioactive compound petrosaspongiolide M.

Natural or synthetic? Several petrosaspongiolide M natural and synthetic analogues have been tested as proteasome inhibitors and apoptosis modulators. The natural petrosaspongiolide M congeners gave a consistent decrease in activity. Among the synthetic analogues, the introduction of the benzothiophene ring resulted in a bioequivalent alternative of the petrosaspongiolide M terpenoid system.

Chemical proteomics reveals heat shock protein 60 to be the main cellular target of the marine bioactive sesterterpene suvanine.

Marine bioactive compounds are potential drug leads because of their diverse pharmacological effects against human diseases. The identification of their cellular targets is crucial for a rational approach to their application in medicinal chemistry. Thus, we have analyzed the cell interactome of suvanine, a sulfated tricyclic terpenoid of marine origin endowed with an interesting anti-inflammatory activity, by application of a chemical proteomic approach. Heat Shock Protein 60, a chaperone involved in the inflammatory response, is the main cellular target of suvanine, which is also able to interfere with protein chaperone activity, giving evidence for its anti-inflammatory properties.

The inactivation mechanism of human group IIA phospholipase A(2) by Scalaradial.

Secretory phospholipases A(2) (sPLA(2)s) are implicated in the pathogenesis of several inflammation diseases, such as rheumatoid arthritis, septic shock, psoriasis, and asthma. Thus, an understanding of their inactivation mechanisms could be useful for the development of new classes of chemical selective inhibitors. In the marine environment, several bioactive terpenoids possess interesting anti-inflammatory activity, often through covalent and/or noncovalent inactivation of sPLA(2). Herein, we report the molecular mechanism of human group IIA phospholipase A(2) (sPLA(2)-IIA) inactivation by Scalaradial (SLD), a marine 1,4-dialdehyde terpenoid isolated from the sponge Cacospongia mollior and endowed with a significant anti-inflammatory profile. Our results have been collected by a combination of biochemical approaches, advanced mass spectrometry, surface plasmon resonance, and molecular modeling. These suggest that SLD acts as a competitive inhibitor. Indeed, the sPLA(2)-IIA inactivation process seems to be driven by the noncovalent recognition process of SLD in the enzyme active site and by chelation of the catalytic calcium ion. In contrast, covalent modification of the enzyme by the SLD dialdehyde moiety emerges as only a minor side event in the ligand-enzyme interaction. These results could be helpful for the rational design of new PLA(2) inhibitors that would be able to selectively target the enzyme active site.

Modulation of tau protein fibrillization by oleocanthal.

Among the phenolic compounds extracted from extra virgin olive oil, oleocanthal (1) has attracted considerable attention in the modulation of many human diseases, such as inflammation and Alzheimer's disease (AD). Indeed, 1 is capable of altering the fibrillization of tau protein, which is one of the key factors at the basis of neurodegenerative diseases, and of covalently reacting with lysine ε-amino groups of the tau fragment K18 in an unspecific fashion. In the present study, an investigation of the recognition process and the reaction profile between 1 and the wild-type tau protein has been conducted by a circular dichroism, surface plasmon resonance, fluorescence, and mass spectrometry combined approach. As a result, 1 has been found to interact with tau-441, inducing stable conformational modifications of the protein secondary structure and also interfering with tau aggregation. These findings provide experimental support for the potential reduced risk of AD and related neurodegenerative diseases associated with olive oil consumption and may offer a new chemical scaffold for the development of AD-modulating agents.