Stefano Bruno

Staphylococcus aureus is a widespread human pathogenic bacterium that poses an increasingly serious threat to public health worldwide, due to the emergence and spread of antibiotic‐resistant strains. A key virulence mechanism is the acquisition of iron from the heme group of human host's hemoglobin, initiated by hemoglobin binding to receptors exposed on the bacterial surface. Although this interaction is a promising target for new antimicrobials, quantitative data on hemoglobin capture by single bacteria are lacking. In this work, single‐molecule localization microscopy is used to directly quantify the number of hemoglobin molecules captured by individual S. aureus cells and resolve their nanoscale spatial distribution at the sub‐cellular level. This quantitative approach provides a methodological framework to monitor, with single‐molecule sensitivity, changes in surface density of functional receptors in response to iron limitations. This piece of information, in turn, is critical to define the overall efficacy of antimicrobials targeting bacterial receptors. As proof of this concept, we develop a photo‐activated molecular system based on hemoglobin for targeted photodynamic inactivation of S. aureus .

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.

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.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, driven by metabolic reprogramming. Since direct glycolytic enzyme inhibition is limited by toxicity, indirect glycolysis modulation through inhibition of the kinase activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) may offer a safer therapeutic strategy. Herein, we report the first-in-class covalent PFKFB3 inhibitor (6), targeting a previously unexplored cysteine. Enzyme assays, site-directed mutagenesis, and mass spectrometry confirmed covalent binding and kinetic selectivity for PFKFB3. Compound 6 reduced viability across multiple PDAC cell lines and suppressed PDAC growth in zebrafish xenografts. Its combination with standard chemotherapeutics revealed synergistic effects. Although the limited cellular activity of 6 restricts its use as a chemical probe in biological studies, we proved for the first time the druggability of a previously unexplored cysteine in PFKFB3. Our work represents a significant achievement in the selective targeting of this kinase, paving the way for an innovative mechanism of action for PFKFB3 inhibitors.

Marine organisms have proven to be excellent sources of bioactive natural products with potential therapeutic applications. To date, seventeen marine-derived molecules are on the market for the treatment of human diseases, mainly cancer. While multiple bioactivities of marine compounds have been consecutively reported, peptides represent promising candidates for these applications. This review focuses on peptides from marine organisms living in extreme marine environments, such as the deep ocean, polar regions, and tropical ecosystems. These are particularly promising for further bioprospecting, since their distinctive conditions have driven the evolution of unique biomolecules, as well as unique stability profile that can improve efficacy, shelf life, and performance under a wide range of industrial conditions. Ziconotide (Prialt), a neurotoxic peptide derived from the venom of a marine snail (Conus sp.) found at depths greater than 1000 m, is already commercially available for the treatment of severe pain. Recent technologies and computational tools are speeding up the discovery of new peptides and enzymes (very few from extreme environments). Overall, the review reports about eight peptides with anticancer properties from deep environments, nine, two and seven from polar habitats with antioxidants, anti-inflammatory and anticancer properties, respectively, and approximately ninety peptides from tropical waters (five antioxidant, thirty-five anti-inflammatory and fifty-four anticancer peptides). However, future studies in extreme environments will need to develop and apply sampling technologies, cultivation systems, as well as methods to assess efficacy, side effects and mechanisms of action, in vitro and in vivo.

Prostate cancer (PCa) remains a major global health burden. Although androgen deprivation and receptor-targeted therapies initially benefit patients, resistance often leads to metastatic castration-resistant prostate cancer, with limited treatment options. Aldehyde dehydrogenase 7A1 (ALDH7A1) is an emerging oncogenic driver of PCa, but selective inhibitors are lacking. Here, we report irreversible ALDH7A1 inhibitors targeting the catalytic Cys330, identified from a library of 3-bromo-4,5-dihydroisoxazole derivatives. These compounds show minimal inhibition of ALDH4A1 and GAPDH, supporting selectivity for ALDH7A1. Compounds 3b and 4b reduced DU145 cell viability (low μM IC₅₀), impaired migration, and altered the cell cycle. No significant effects were observed in LNCaP cells (low ALDH7A1 expression) or Hs27 fibroblasts. Treatment of DU145 cells resulted in the inhibition of intracellular ALDH activity and accumulation of malondialdehyde, consistent with increased oxidative stress. These findings validate ALDH7A1 as a druggable target and introduce a new chemical space for selective covalent ALDH inhibitors in oncology.

Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of 3‐phosphoserine, which is the final step in the de novo biosynthesis of l ‐serine ( l ‐Ser) via the phosphorylated pathway in human astrocytes. Individuals who are homozygous or compound heterozygous for functionally defective PSP variants exhibit reduced cerebrospinal fluid l ‐Ser levels and severe neurological symptoms. In the present study, single nucleotide polymorphisms in PSP were identified in hippocampal samples from Alzheimer's disease (AD) patients. Two single nucleotide polymorphisms, likely forming a haplotype (chr7:56088825 T>A and chr7:56088811 T>C, encoding R27S and D32G PSP variants, respectively), were detected exclusively in AD patients (three females and one male). Biochemical characterization of the recombinant R27S/D32G PSP enzyme revealed a slight decrease in thermostability, a 38‐fold reduction in catalytic efficiency and a two‐fold increase in IC ₅₀ for l ‐Ser, with the D32G substitution being the primary contributor to these effects. Despite its reduced enzyme activity, the R27S/D32G variant did not impair l ‐Ser biosynthesis either in an in vitro reconstructed pathway or in U251 human glioblastoma cells ectopically expressing the variant under heterozygous conditions. In these cells, PSP colocalized extensively with the other two phosphorylated pathway enzymes, namely phosphoglycerate dehydrogenase and phosphoserine aminotransferase, suggesting that they assemble into a functional complex, known as the serinosome. Notably, the R27S/D32G PSP variant exhibited increased nuclear localization compared to the wild‐type enzyme. This mislocalization raises the intriguing possibility that PSP's moonlighting functions, including its putative role as a protein phosphatase, may be affected, potentially implicating it in pathways beyond l ‐Ser biosynthesis.

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.

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold‐adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph‐2/2HbO‐2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics.

Photo-immunotherapy uses antibodies conjugated to photosensitizers to produce nanostructured constructs endowed with targeting properties and photo-inactivation capabilities towards tumor cells. The superficial receptor density on cancer cells is considered a determining factor for the efficacy of the photodynamic treatment. In this work, we propose the use of a photoactive conjugate that consists of the clinical grade PD-L1-binding monoclonal antibody Atezolizumab, covalently linked to either the well-known photosensitizer eosin or the fluorescent probe Alexa647. Using single-molecule localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM), and an anti-PD-L1 monoclonal antibody labelled with Alexa647, we quantified the density of PD-L1 receptors exposed on the cell surface in two human non-small-cell lung cancer lines (H322 and A549) expressing PD-L1 to a different level. We then investigated if this value correlates with the effectiveness of the photodynamic treatment. The photodynamic treatment of H322 and A549 with the photo-immunoconjugate demonstrated its potential for PDT treatments, but the efficacy did not correlate with the PD-L1 expression levels. Our results provide additional evidence that receptor density does not determine a priori the level of photo-induced cell death.