Ischemic stroke is the leading cause of death and permanent disability. Despite recent substantial progress in prevention and management, its successful treatment remains a large unmet medical need. Studies carried out in the Laboratory of Stroke and Vascular Dysfunctions, in collaboration with our Lab, suggested the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. These data support the hypothesis that MBL inhibition may be a relevant therapeutic target in humans. Our group has developed new methods based on Surface Plasmon Resonance which have been and are currently applied to identify molecules or functionalized nanoparticles able to counteract the detrimental effects of lectins. These compounds have neuroprotective activity in animal models undergoing ischemia, suggesting a promising treatment strategy of ischemic brain stroke.

Many biological drugs, including monoclonal antibodies, are being approved in the clinical practice for their therapeutic benefit. One of the issues with these drugs is the immunogenicity, i.e. the production of anti-drug antibodies which can affect the activity (if neutralizing antibodies) and/or the pharmacokinetic properties, resulting in a loss of response. We have developed a new test that uses Surface Plasmon Resonance technology, to simultaneously measure the drug and anti-drug antibodies in the blood of patients, unveiling important pitfalls of the classical ELISA tests. This test has been successfully applied to study antibodies developed by patients against Infliximab, an anti-TNF biological used for different autoimmune diseases. We are currently applying this method to measure the antibodies developed by patients treated with PEGylated drugs, either anti-drug or anti-PEG antibodies, to determine their relevance for the response to therapy. The knowledge of these data for each patient might allow doctors to customize and optimize the therapy. With this in mind, we also plan to develop, in collaboration with engineering departments, new miniaturized and portable devices to allow rapid, easy and cheap point-of-care analysis.

We are currently developing a comprehensive platform for the rapid and reliable design, production, and validation of miniproteins—small, engineered proteins with high specificity and stability. The platform integrates AI-assisted de novo protein design with classical biophysical and biochemical approaches. The design phase relies on machine learning models and generative AI tools like RFdiffusion and BindCraft to create optimised miniproteins tailored for specific biological targets. Different methods, including solid-phase peptide synthesis (SPPS), cell-free expression systems, and recombinant production in cells, are then tested to produce the computationally designed molecules. These methods enable the efficient synthesis of miniproteins, which are then characterised using biophysical techniques such as Surface Plasmon Resonance (SPR) and Circular Dichroism (CD) to assess their binding affinity, stability, and structural properties. The versatility of our miniproteins platform enables its application across multiple disease-related targets, focusing on proteins with key pathological roles. Here are the main current applications: - miniproteins targeting gelsolin, a protein implicated in amyloidosis and cancer, to investigate its pathological interactions and aggregation mechanisms. - binders for protein disulphide isomerase (PDI) and ERO1a, focusing on their interaction and its implications in cancer, aiming to modulate oxidative stress and protein folding pathways involved in tumor progression. - miniproteins to direct extracellular Clusterin to lysosomal degradation pathways, allowing its selective degradation through LYTAC (Lysosome-Targeting Chimera) technology. This approach provides a novel strategy for those diseases in which excessive Clusterin accumulation plays a pathological role, such as neurodegenerative disorders and certain cancers. - development of miniproteins as diagnostic tools, including projects focused on detecting zoonotic diseases.

In addition to miniproteins engineering, our laboratory is actively engaged in small-molecule drug discovery, including research on the modulation of thyroid hormone receptor alpha (TRα1). This research area combines AI-assisted molecular docking, classical pharmacological screening, and medicinal chemistry approaches to identify, refine, and de novo create compounds with potential therapeutic applications.

The laboratory has an extensive experience in solid phase peptide synthesis (SPPS), carried out with two automated peptide synthesizers that allow the synthesis of up to 96 peptides simultaneously, with lengths of up 70-100 amino acids, or specific modifications in the amino acid (e.g., with D-amino acids, cyclic peptides or peptides functionalized with probes such as a fluorophore or biotin). The sequence and the purity of the peptides is confirmed using HPLC and MALDI-TOF mass spectrometer. Some examples of the application of this expertise are reported below: - synthesis of the beta-amyloid peptides (Aβ 1-40, Aβ 1-42) and derivatives designed to reduce Aβ aggregation; - synthesis of cyclic peptides capable of preventing the SARS-COV 2 entry in cells, designed to impair the interaction between the virus Spike protein and its target angiotensin-converting enzyme 2 (ACE2); - synthesis of musclin-derived peptides potentially useful against cachexia (muscle loss) and bone wasting in cancer; - synthesis of oncolytic peptides as therapeutic agents for breast and gastric cancer

Data obtained in the Department of Cardiovascular Medicine (now Department of Acute Brain and Cardiovascular Injury), in collaboration with our lab, revealed that a metabolic pathway, called kynurenine pathway (KP), is activated immediately after cardiac arrest (CA), and that it is associated with severity of post–cardiac arrest shock, early death, and poor long-term outcome. Additionally, our data in a rodent model of CA showed alteration of KP metabolites in the brain, suggesting a role in the cascade of molecular events underlying the CA-induced brain injury. We are currently measuring the KP metabolites in patients with CA treated with Argon (CPAr trial, NCT05482945), to unveil possible association between the predicted beneficial effect of Argon and the KP response. Moreover, considering the role of the kynurenine pathway in several neurological disorders as consequence of neuro-inflammation and brain insult, we plan to exploit the developed UHPLC-MS/MS method to investigate the kynurenine pathway dysregulation in other clinical conditions affecting brain health.

Antibiotic treatment in critically ill patients is challenging due to the pathophysiological changes occurring in these patients that can alter the pharmacokinetic properties of antibiotics, resulting in ineffective or incorrect treatments. The Laboratory of Clinical Data Sciences and the GiViTI group are conducting a multicentric clinical study, AbioKin (Antibiotics Pharmacokinetics, NCT02609646) with the aim to develop an advanced mathematical model integrating patient characteristics and clinical parameters, to optimize antibiotics therapy. In this context, we have developed and validated a rapid and sensitive UHPLC-MS/MS method able to simultaneously detect and quantify five antibiotics commonly used in Intensive Care Units (i.e., Vancomycin, Linezolid, Piperacillin/Tazobactam and Meropenem). The method is currently applied to measure the antibiotics concentrations in thousands of plasma samples from the patients enrolled in the AbioKin study, to investigate the pharmacokinetic of these antibiotics in relationship to patients’ characteristics, clinical parameters and extracorporeal therapies.

The laboratory contributes to different studies in animal models by developing and applying new bioanalytical methods for measuring drug levels in plasma and relevant tissues (e.g., brain or liver), in order to better understand their pharmacological effects and the PD-PK relationships. As examples of these studies: - PK of two tyrosine kinase inhibitors (Imatinib and Nintedanib) and of a Janus kinase inhibitor (Ruxolitinib) carried by inhalator targeted liposomal formulations in mouse models of pulmonary hypertension and post-inflammatory fibrosis - PK and dose optimization of two heat shock protein inhibitors (K5 and K4) as potential senolytic drugs in a transgenic mouse model of senescence - PK of antibodies, evaluated with novel methods developed in the Lab and based on Surface Plasmon Resonance

The laboratory has been involved in different studies regarding the possible effects of Doxycycline for the treatment of central and peripheral amyloidosis (Alzheimer diseases, Prion disease, β2-microglobuline or transthyretin amyloidosis). In particular, we have evaluated both the pharmacodynamic effects of the drug (e.g., interaction with - and effects on – amyloidogenic proteins and their aggregates), as well its pharmacokinetic properties in animal models and in patients. The integration of pharmacodynamics and pharmacokinetics data allowed to propose a new potential mechanism of action of the drug.