Human Neutrophil Elastase and the Protein-Storm Axis: Reversible Synthetic Inhibitors in Inflammatory Disease Simona Viglio, Maria Antonietta Grignano, Marilena Gregorini, Teresa Rampino, Giampiero Pietrocola, et al. Molecules, 2026 Human neutrophil elastase (HNE) is a central mediator of neutrophil-driven inflammation. Yet, despite decades of research and drug development, therapies targeting HNE have not consistently translated into clear clinical benefits. We suggest that this translational gap partly arises from how HNE has traditionally been conceptualized, as a single enzyme to inhibit. In biological systems, however, HNE operates within a complex and tightly regulated network of proteases and inflammatory mediators. This network is spatially compartmentalized and strongly influenced by local redox conditions, making HNE activity highly context-dependent. From a systems perspective, HNE acts as an amplifier of inflammation. Its extracellular activity connects several pathological processes, including activation of innate immunity, extracellular matrix degradation, disruption of epithelial and endothelial barriers, and the transition toward chronic inflammation. In this review, we integrate insights from enzymology, systems biology, and clinical research to reassess the development of HNE inhibitors, ranging from endogenous antiproteases to more recent reversible synthetic compounds. Despite their chemical and pharmacological diversity, many of these strategies have encountered similar limitations. We therefore argue that future therapeutic approaches should move beyond the inhibition of HNE as an isolated target and instead aim to modulate the broader protease network, with particular attention to drug–target kinetics and precise delivery to disease-relevant microenvironments.
The Differential Redox Resilience of Alvelestat and Sivelestat: A Mechanistic Hypothesis for Inhibitor Performance Under Oxidative Stress Maura D’Amato, Pasquale Linciano, Laurent R. Chiarelli, Giampiero Pietrocola, Paolo Iadarola, et al. Molecules, 2026 Human neutrophil elastase (HNE) is a key driver of inflammatory lung disorders, promoting extracellular matrix degradation and tissue damage. Although inhibitors such as Sivelestat and Alvelestat are clinically relevant, their performance within the oxidative microenvironment of diseased lungs remains poorly understood. Here, we employed an integrated in vitro and in silico approach to investigate their behavior under physiological and oxidative conditions and to provide a molecular-level interpretation. Under physiological conditions, enzymatic assays and steady-state kinetics confirmed that both compounds act as competitive inhibitors, with Sivelestat displaying higher baseline potency. Under oxidative stress, however, Sivelestat exhibited a marked reduction in inhibitory potency, whereas Alvelestat retained its efficacy. Molecular modeling and molecular dynamics simulations of native and oxidized HNE variants provided a structural rationale for this divergence. Alvelestat, as a non-covalent inhibitor, maintains stable binding despite increased flexibility of the active site, whereas Sivelestat, acting via a reversible covalent mechanism, requires a precise pre-acylation geometry. Oxidation-induced remodeling of the S1 pocket disrupts the near-attack configuration required for covalent bond formation, thereby impairing inhibition. Overall, these findings indicate that oxidative stress may selectively compromise covalent inhibition while preserving enzymatic activity, and suggest that context-dependent redox-related structural effects may represent a consideration for the design of next-generation HNE inhibitors.
Immune cell changes following chemotherapy in advanced pancreatic cancer with variations based on gender Roberto Aquilani, Salvatore Corallo, Roberto Maestri, Silvia Brugnatelli, Paolo Iadarola, et al. Scientific Reports, 2025 To explore the changes in blood inflammatory cells (neutrophils, monocytes, platelets) and adaptive immune cells (lymphocytes) during chemotherapy, we retrospectively analysed medical records from 66 patients with unresectable Pancreatic Ductal Adenocarcinoma (PDAC) treated with the Gemcitabine-nab-Paclitaxel (GnP) regimen. Evaluations were conducted at baseline (pre-GnP, TA), after the first cycle (TB), and after the third cycle (TC) of treatment. In metastatic PDAC (mPDAC), the monocyte-to-lymphocyte ratio significantly increased at both TB and TC compared to TA, whereas no such change was observed in locally advanced PDAC (laPDAC) (interaction: p = 0.006). Platelet levels rose over time in both phenotypes, with a more pronounced increase in mPDAC (intergroup: p = 0.008). When stratified by gender, males with mPDAC showed an increase in monocyte percentages among total white blood cells (intergroup: p = 0.018), while both phenotypes exhibited rising platelet-to-lymphocyte ratios over time. In females, the platelet-to-lymphocyte ratio increased more significantly in laPDAC than in mPDAC (interaction: p = 0.046). GnP treatment notably increased circulating inflammatory cells and their relationships with lymphocytes in a manner dependent on both disease phenotype and gender. Pretreatment factors such as monocyte counts < 0.6 × 10 3 /µl, lymphocyte counts > 1 × 10 3 /µl, and a monocyte-to-lymphocyte ratio < 0.43 were identified as independent predictors of survival.
A Pre-Clinical Study on the Use of the Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitor PEP 2-8 to Mitigate Ischemic Injury in a Rat Marginal Donor Model Maria Antonietta Grignano, Marilena Gregorini, Chiara Barisione, Caterina Ivaldo, Daniela Verzola, et al. International Journal of Molecular Sciences, 2025 Proprotein Convertase Subtilisin/Kexin type 9 PCSK9 inhibitors (PCSK9i) are a novel class of cholesterol-lowering agents that also offer protection against tissue ischemia by reducing apoptosis, pyroptosis, and myocardial infarct size. This study evaluated the effects of the PCSK9 inhibitor PEP 2-8 during hypothermic perfusion (HP) in a rat model of donation after circulatory death (DCD) kidney transplantation. DCD kidneys were perfused at 4 °C for six hours with either Perf-Gen solution alone (control) or Perf-Gen supplemented with PEP 2-8. Glucose and lactate dehydrogenase (LDH) levels were measured at baseline and after six hours (T6h). At T6h, kidneys were evaluated for ischemic injury, tubular cell proliferation, apoptosis, nitrotyrosine (N-Tyr) staining, tissue ATP and LDH levels, and gene expression of PCSK9 and NOX4. Metabolomic profiling was also performed. PEP 2-8 treatment significantly reduced PCSK9 expression, decreased tubular ischemic injury and necrosis, and lowered LDH release. Treated kidneys showed enhanced tubular cell proliferation, reduced apoptosis, and diminished oxidative stress, indicated by decreased N-Tyr staining and NOX4 expression. Energy metabolism was improved, with higher tissue ATP and glucose levels observed in the PEP 2-8 group. Metabolomic analysis further supported the antioxidant effects of PEP 2-8. This is the first study to demonstrate that PEP 2-8 administered during pre-transplant hypothermic perfusion provides renal protection by improving energy metabolism and reducing oxidative stress in the context of ischemic injury.
Mass Spectrometric Proteomics 3.0 Paolo Iadarola, Simona Viglio International Journal of Molecular Sciences, 2025 Mass spectrometry (MS) has undergone a profound transformation over the past two decades, expanding in both technical capability and scientific impact [...]
Can Proteomics Play a Significant Role in the Identification of Biomarkers for Alpha1-Antitrypsin Deficiency? Maria Antonietta Grignano, Maura D’Amato, Marilena Gregorini, Teresa Rampino, Paolo Iadarola, et al. International Journal of Molecular Sciences, 2025 Alpha-1 antitrypsin deficiency (AATD) is a common genetic disorder that can manifest in a broad spectrum of clinical symptoms, ranging from asymptomatic cases to severe, progressive systemic diseases, primarily affecting the lungs and liver. Despite its prevalence, AATD is often perceived as a rare condition, which can lead to a lack of awareness among primary care physicians and even some respiratory specialists. This misconception may result in missed opportunities for diagnosis, particularly in mild or asymptomatic patients. Consequently, it is vital for healthcare providers to familiarize themselves with the various presentations, diagnostic techniques, and management strategies for AATD. This review explores the current understanding of AATD, emphasizing the valuable role of liquid chromatography-mass spectrometry in identifying biomarkers that could enhance early diagnosis and help predict disease outcomes. As knowledge about the complexities of AATD continues to grow, physicians may begin to view the disorder not as a fatal pathology, but as a treatable inherited condition with the potential for improved management.
Identification and Structural Characterization of a Novel COL3A1 Gene Duplication in a Family With Vascular Ehlers–Danlos Syndrome Gianmaria Miolo, Piernicola Machin, Marco De Conto, Sara Fortuna, Simona Viglio, et al. Molecular Genetics and Genomic Medicine, 2025 BackgroundVascular Ehlers–Danlos syndrome (vEDS) is caused by alterations in the COL3A1 gene, typically involving missense variants that replace glycine residues. In contrast, short in‐frame insertions, deletions, and duplications are rare and pose significant challenges for investigation.MethodsThe histological examination of vascular tissue from a 26‐year‐old man, who died from a common iliac artery aneurysm and whose mother died at age 60 from an abdominal aortic dissection, strongly suggested a diagnosis of Ehler–Danlos type IV. Ex vivo collagen phenotype assessment, molecular analysis, and in silico structural studies of type III collagen were subsequently performed.ResultsEx vivo analysis of the patient's fibroblasts revealed altered collagen synthesis, whereas the molecular testing identified a novel 18‐nucleotide in‐frame duplication (c.2868_2885dup‐GGGTCTTGCAGGACCACC) in the COL3A1 gene, resulting in a six‐amino acid insertion, p.(Leu958_Gly963dup). Structural investigation indicated that this duplication led to a local perturbation of the collagen triple helix near a metalloproteinase cleavage site.ConclusionThis study highlights the pathogenic role of a novel in‐frame duplication in the COL3A1 gene, demonstrating how this seemingly benign alteration significantly compromises collagen turnover and contributes to the development of vEDS.
Engineered ATP-Loaded Extracellular Vesicles Derived from Mesenchymal Stromal Cells: A Novel Strategy to Counteract Cell ATP Depletion in an In Vitro Model Maria Antonietta Grignano, Silvia Pisani, Marilena Gregorini, Giorgia Rainaudo, Maria Antonietta Avanzini, et al. International Journal of Molecular Sciences, 2025 The use of adenosine triphosphate (ATP) has shown promising effects in alleviating ischemic damage across various tissues. However, the penetration of ATP into kidney tubular cells presents a challenge due to their unique anatomical and physiological properties. In this study, we introduce a novel bioinspired drug delivery system utilizing extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) and engineered to carry ATP. ATP-loaded liposomes (ATP-LPs) and ATP-loaded EVs (ATP-EVs) were prepared using microfluidic technology, followed by characterization of their morphology (DLS, NTA, SEM, TEM), ATP content, and release rate at 37 °C (pH 7.4). Additionally, the delivery efficacy of ATP-LPs and ATP-EVs was evaluated in vitro on renal cells (HK2 cells) under chemically induced ischemia. The results indicated successful ATP enrichment in EVs, with ATP-EVs showing no significant changes in morphology or size compared to naïve EVs. Notably, ATP-EVs demonstrated superior ATP retention compared to ATP-LPs, protecting the ATP from degradation in the extracellular environment. In an ATP-depleted HK2 cell model, only ATP-EVs effectively restored ATP levels, preserving cell viability and reducing apoptotic gene expression (BCL2-BAX). This study is the first to successfully demonstrate the direct delivery of ATP into renal tubular cells in vitro using EVs as carriers.
Antarctic Soil Metabolomics: A Pilot Study Carlotta Ciaramelli, Alessandro Palmioli, Maura Brioschi, Simona Viglio, Maura D’Amato, et al. International Journal of Molecular Sciences, 2023
Alveolar inflammation in cystic fibrosis Martina Ulrich, Dieter Worlitzsch, Simona Viglio, Nanna Siegmann, Paolo Iadarola, et al. Journal of Cystic Fibrosis, 2010
