The agro-industrial sector produces large amounts of by-products that have a high environmental impact, so it has become essential to recover food waste at all levels. This is because it often contains bioactive molecules that can be a valuable source of new products such as animal feed, biopolymers, or products for human use, (e.g., cosmetics and nutraceuticals) due to its antioxidant, antimicrobial, and anti-inflammatory properties. Advanced analytical methodologies such as liquid chromatography coupled to mass spectrometry (LC-MS) are crucial for the characterisation of bioactive chemicals in these waste materials. LC-MS enables both targeted and untargeted metabolomic approaches, facilitating the identification and quantification of a wide range of secondary metabolites, including polyphenols, flavonoids, alkaloids, and terpenoids. The choice of extraction methodology is essential for the precise identification and quantification of these metabolites. This study provides an overview of LC-MS as an effective tool for analysing complex extracts derived from plant waste, discussing both methodological aspects and typical bioactive metabolites identified, and offering examples of their potential applications in cosmeceutics.

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.

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.

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.

The ultra-high-performance liquid chromatography high-resolution mass spectrometry (LC-ESI-HR-MS/MS) technique was used to characterize the polyphenolic fraction of the hot water infusion (WI) of inflorescences of Cannabis sativa L. Kompolti variety, commercially used for food preparations or cosmetic purposes. On water infusion extract, we applied a multidisciplinary approach, where NMR, MS, in vitro cell-free and cell-based assays coupled with in silico studies, were used to rationalize at the molecular level the effects of the major component Cannabidiol (CBD), in a model of Parkinson’s disease (PD). The phytochemical analysis by LC-MS/MS led to the tentative identification of many components belonging to different classes of polyphenols, such as phenolic acids, flavonoids, and their glycosides. CBD and cannabidiolic acid (CBDA) were also detected in good amounts in the infusion, together with several minor cannabinoids. In addition, the water infusion WI was evaluated for mineral content, total phenolic content, flavonoid content, and antioxidant capacity by DPPH and FRAP methods. Notably, our results in a cellular model of PD highlight that CBD protects against rotenone-induced cell death without recovering neuronal morphology. These biological outcomes were rationalized by an in silico approach, where we hypothesize that CBD could influence the cellular response to oxidative stress via its interaction with the Keap1/Nrf2 pathway. In summary, these results enriched the nutraceutical profile of the water infusion of the inflorescences of the Kompolti cultivar, which demonstrated a high CBD content. This study could lead to the development of dietary supplements that could help in the management of clinical symptoms related to the antioxidant activity of CBD in the pathophysiology of PD, which remains poorly characterized.

In the complex network of cellular physiology, the maintenance of cellular proteostasis emerges as a critical factor for cell survival, particularly under stress conditions. This homeostasis is largely governed by a sophisticated network of molecular chaperones and co-chaperones, among which Bcl-2-associated athanogene 3 (BAG3), able to interact with the ATPase domain of Heat Shock Protein 70 (HSP70), plays a pivotal role. The BAG3-HSP70 functional module is not only essential for cellular homeostasis but is also involved in the pathogenesis of various diseases, including cancer, neurodegenerative disorders, and cardiac dysfunction, making it an attractive target for therapeutic intervention. Inspired by our continuous interest in the development of new chemical platforms able to interfere with BAG3 protein, herein we report the discovery of compound 16, the first-in-class BAG3/HSP70 dual modulator, obtained by combining the multicomponent Ugi reaction with the alkyne-azide Huisgen procedure in a sequential tandem reaction approach. Through a combination of biophysical analysis, biochemical assays, and cell-based studies, we elucidated the mechanism of action of this inhibitor and assessed its potential as a therapeutic agent. Hence, this study can open new avenues for the development of novel anticancer strategies that leverage the simultaneous disruption of multiple chaperone pathways.