Giulio Fracasso

The mitochondrial chaperone TRAP1 is a key regulator of cellular homeostasis and its activity has important implications in neurodegeneration, ischemia and cancer. Recent evidence has indicated that TRAP1 mutations are involved in several disorders, even though the structural basis for the impact of point mutations on TRAP1 functions has never been studied. By exploiting a modular structure-based framework and molecular dynamics simulations, we investigated the effect of five TRAP1 mutations on its structure and stability. Each mutation differentially impacts long-range interactions, intra and inter-protomer dynamics and ATPase activity. Changes in these parameters influence TRAP1 functions, as revealed by their effects on the activity of the TRAP1 interactor succinate dehydrogenase (SDH). In keeping with this, TRAP1 point mutations affect the growth and migration of aggressive sarcoma cells, and alter sensitivity to a selective TRAP1 inhibitor. Our work provides new insights on the structure-activity relationship of TRAP1, identifying crucial amino acid residues that regulate TRAP1 proteostatic functions and pro-neoplastic activity.

Previously, we demonstrated that the ¹⁷⁷Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to ¹⁷⁷Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a ^99mTc-/¹⁷⁷Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel ^99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH₂ (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(−) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and ^99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of ^99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of ^99mTc-HscFv1 and ^99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of ^99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of ^99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging.

The human genome counts hundreds of GPCRs specialized to sense thousands of different extracellular cues, including light, odorants and nutrients in addition to hormones. Primordial GPCRs were likely glucose transporters that became sensors to monitor the abundance of nutrients and direct the cell to switch from aerobic metabolism to fermentation. Human β cells express multiple GPCRs that contribute to regulate glucose homeostasis, cooperating with many others expressed by a variety of cell types and tissues. These GPCRs are intensely studied as pharmacological targets to treat type 2 diabetes in adults. The dramatic rise of type 2 diabetes incidence in pediatric age is likely correlated to the rapidly evolving lifestyle of children and adolescents of the new century. Current pharmacological treatments are based on therapies designed for adults, while youth and puberty are characterized by a different hormonal balance related to glucose metabolism. This review focuses on GPCRs functional traits that are relevant for β cells function, with an emphasis on aspects that could help to differentiate new treatments specifically addressed to young type 2 diabetes patients.

Green nanotechnology produces biocompatible gold nanoparticles (AuNPs) with intrinsic anti-microbial, anti-viral, and anti-tumour properties. Green AuNPs (g-AuNPs) are gaining a solid ground in prostate cancer (PC) nanotherapy. Hence, in this review, we summarise and compare data from studies published between 2015 and 2022 to highlight major biological compounds and outputs from in vitro and in vivo applications of green or biosynthesised AuNPs in PC theranostics. In fact, g-AuNPs can be easily generated with effective antiproliferative PC characteristics. Taken together, g-AuNPs exert various apoptosis-related mechanisms, such as reactive oxygen species (ROS) generation, cell cycle arrest, cytotoxicity, mitochondrial disruption, and anti-tumour immune cell activation, with additional successful delivery of the biological compounds in green materials. g-AuNPs could offer another modality for targeted cancer therapy using antibodies and targeting ligands to specifically recognise and destroy PC tumours. In conclusion, g-AuNPs applications drive forth sustainable nanotechnology while minimising ethical concerns and toxicity risks in clinical trials.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l -arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8 ⁺ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8 ⁺ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

Despite the multitude of therapeutic agents available to treat prostate cancer (PC), there are still no effective and safe measures to treat the tumor. It remains a challenge to develop a simple approach to target PC with specific antibodies. In our study, D2B monoclonal antibodies against a prostate-specific membrane antigen (PSMA) were used. We investigated the functionalization of gold nanoparticles (AuNPs) with D2B to generate favorable physicochemical and biological properties that mediate specific binding to PC. For this purpose, AuNPs with a size of about 25 nm were synthesized in water using sodium citrate as a reducing and stabilizing agent and then coated with D2B. Major physicochemical properties of naked and D2B-coated AuNPs were investigated by ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. The successful binding of D2B to AuNPs-citrate caused a 15 nm red shift in the UV-vis. This was assessed by DLS as an increase in zeta potential from ∼-45 to ∼-23 mV and in the size of AuNPs from ∼25 to ∼63 nm. Scanning electron microscopy confirmed the size shift of AuNPs, which was detected as an exterior organic layer of D2Bs surrounding each AuNP. Even at high exposure levels of the bioconjugates, PSMA-PC-3 cells exhibited minimal cytotoxicity. The specific and dose-dependent binding of AuNPs-D2B to PC-3-PSMA cells was validated by flow cytometry analysis. Our data provide effective drug delivery systems in PC theranostics.

Single chain variable D2B antibody fragments (scFvD2Bs) exhibit high affinity binding to prostate specific membrane antigens overexpressed in metastatic prostate cancer (PC). Conjugation of scFvD2B to gold nanoparticles (AuNPs) would enhance its stability and plasma half-life circulation to shuttle theranostic agents in PC. In this study, we synthesized PEGylated scFvD2B-AuNPs (AuNPs-scFvD2B-PEG) and tested their integrity, biocompatibility, and immunogenicity in freshly withdrawn human blood. Prior to blood incubation, Zeta potential measurements, UV–Vis spectroscopy, and dynamic light scattering (DLS) were used to assess the physicochemical properties of our nano-complexes in the presence or absence of PEGylation. A surface plasmon resonance band shift of 2 and 4 nm confirmed the successful coating for AuNPs-scFvD2B and AuNPs-scFvD2B-PEG, respectively. Likewise, DLS revealed a size increase of ∼3 nm for AuNPs-scFvD2B and ∼19 nm for AuNPs-scFvD2B-PEG. Zeta potential increased from −34 to −19 mV for AuNPs-scFvD2B and reached −3 mV upon PEGylation. Similar assessment measures were applied post-incubation in human blood with additional immunogenicity tests, such as hemolysis assay, neutrophil function test, and pyridine formazan extraction. Interestingly, grafting PEG chains on AuNPs-scFvD2B precluded the binding of blood plasma proteins and reduced neutrophil activation level compared with naked AuNPs-citrate counterparts. Most likely, a hydrated negative PEG cloud shielded the NPs rendering blood compatiblility with less than 10% hemolysis. In conclusion, the biocompatible AuNPs-scFvD2B-PEG presents promising characteristics for PC targeted therapy, with minimal protein adsorption affinity, low immunorecognition, and reduced hemolytic activity.

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) encapsulated in human H-chain Ferritin (HFt), designated as HFt-MP-PASE-MIT, has shown excellent tumor distribution and extended serum half-life meriting further investigation for PC treatment. Thus, in this study, we used the same nano-formulation to test its cytotoxicity using both in vitro and in vivo assays. Interestingly, both encapsulated and free-MIT drugs demonstrated similar killing capabilities on PaCa44 cell line. Conversely, in vivo assessment in a subcutaneous PaCa44 tumor model of PC demonstrated a remarkable capability for encapsulated MIT to control tumor growth and improve mouse survival with a median survival rate of 65 vs. 33 days for loaded and free-MIT, respectively. Interestingly, throughout the course of mice treatment, MIT encapsulation did not present any adverse side effects as confirmed by histological analysis of various murine tissue organs and body mass weights. Our results are promising and pave the way to effective PC targeted chemotherapy using our HFt nanodelivery platforms.