A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Chiral natural compounds are often biosynthesized in an enantiomerically pure fashion, and stereochemistry plays a pivotal role in biological activity. Herein, we investigated the significance of chirality for nature-inspired 3-Br-acivicin (3-BA) and its derivatives. The three unnatural isomers of 3-BA and its ester and amide derivatives were prepared and characterized for their antimalarial activity. Only the (5S, αS) isomers displayed significant antiplasmodial activity, revealing that their uptake might be mediated by the L-amino acid transport system, which is known to mediate the acivicin membrane’s permeability. In addition, we investigated the inhibitory activity towards Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) since it is involved in the multitarget mechanism of action of 3-BA. Molecular modeling has shed light on the structural and stereochemical requirements for an efficient interaction with PfGAPDH, leading to covalent irreversible binding and enzyme inactivation. While stereochemistry affects the target binding only for two subclasses (1a–d and 4a–d), it leads to significant differences in the antimalarial activity for all subclasses, suggesting that a stereoselective uptake might be responsible for the enhanced biological activity of the (5S, αS) isomers.

Protein-based biosensors require controlled and site-selective functionalization strategies to enable stable and oriented immobilization without compromising protein structure and signal transduction efficiency. We evaluated a chemoselective linchpin-directed modification (LDM) approach targeting Lys–His pairs as a tool for site-specific labeling of the model fluorescent biosensor green fluorescent protein (GFP). LDM molecules with variable spacer lengths were prepared, and a structure-guided computational workflow was implemented to map Lys–His distances on the protein and identify candidate modification sites. Experimental validation by UV-Vis spectroscopy and mass spectrometry demonstrated efficient conjugation and a final degree of labeling close to unity, consistent with single-site modification, with all LDM molecules selectively targeting the same histidine residue (His181), independently of spacer length. Structural analysis revealed that this residue is located within an accessible internal cavity that favors productive interactions with the reactive group. Importantly, the modification preserves GFP fluorescence and pH response, confirming retention of sensing functionality. These results demonstrate that LDM enables selective modification not only of surface residues, but also of structurally guided, non-surface residues. This approach provides the proof of concept of a new, promising strategy for the controlled functionalization and immobilization of protein-based biosensors.

In the pharmaceutical field, lipid-based nanoparticles are extensively used for drug or vaccine delivery, particularly for treating respiratory disorders. However, their physico-chemical instability, particularly associated with lipid degradation through hydrolysis or oxidation, can affect their encapsulation properties. To monitor the stability of lipid-based formulations over time, we prepared acrylodan-labeled bovine serum albumin (here called albuminodan), and showed it is a fluorescent biosensor capable of concomitantly detect phospholipids as well as their degradation products, i.e., fatty acids and lysophospholipids. We demonstrated that this tool can be used to follow the distribution of lipids in an aqueous phase and hence could be suitable to characterize the hydrolysis of phospholipids in a lipid-based formulation to monitor the stability of nanoparticles.

Protein-based biosensors require controlled and site-selective functionalization strategies to enable stable immobilization and signal transduction without compromising protein structure and activity. Here, we evaluate a chemoselective linchpin-directed modification (LDM) approach targeting Lys–His pairs as a tool for site-specific labeling of the model fluorescent biosensor green fluorescent protein (GFPmut2). A panel of LDM molecules with variable spacer lengths was prepared, and a structure-guided computational workflow was implemented to map Lys–His distances on the protein and predict potential modification sites. Experimental validation by UV–Vis spectroscopy and mass spectrometry demonstrated efficient conjugation and a final degree of labeling close to unity, consistent with single-site modification, with all LDM molecules selectively targeted the same histidine residue (His181), independently of spacer length. Structural analysis revealed that this residue is located within an accessible internal cavity, enabling a funneling effect that enhances local reactivity. Importantly, the modification preserves the fluorescence and pH response of GFP, confirming retention of sensing functionality. These results demonstrate that LDM enables selective modification not only of surface residues, but also of structurally guided, non-surface residues. This approach provides a novel strategy for the controlled functionalization and immobilization of protein-based biosensors, improving their stability and performance.