Utilizzo pratico e significato clinicopatologico delle frazioni escrete degli elettroliti nel riconoscimento del danno renale acuto (AKI) in diversi setting clinici

Ricerche nel campo del danno renale acuto e frazioni urinarie escrete. La prima parte verte sull’analisi dei dati provenienti da una casistica di cani con leptospirosi, sono stati confrontati due gruppi di cani, il primo con AKI da leptospirosi e l’altro con AKI per eziologie differenti. In queste due popolazioni di pazienti abbiamo valutato alcuni analiti sierici ed urinari come ad esempio l’escrezione elettrolitica frazionata e biomarker di AKI come NGAL. I cani con leptospirosi, hanno mostrato maggiore kaliuresi e più grave glicosuria rispetto a quelli non affetti da leptospirosi, così come erano più frequentemente glicosurici rispetto agli altri. Questi dati sono in analogia con quanto è riportato nell’uomo e dimostrano un pattern di danno tubulare tipico in corso di questa malattia se paragonato appunto ad altre cause di danno tubulare acuto e AKI La seconda parte riguarda la valutazione della funzionalità renale e del danno renale acuto in cani affetti da insufficienza valvolare mitralica. E’ stata incentrata sul danno renale in corso di cardiopatie e per questa ragione abbiamo pensato di valutare esclusivamente pazienti con MVD per varie ragioni: poiché sono pazienti che si presentano frequentemente nella pratica clinica; la malattia è tipicamente cronica e il paziente rimane stabile a lungo con un andamento progressivo della malattia; questi pazienti possono presentare frequenti episodi di AKI legati allo scompenso cardiaco e/o alla terapia con diuretico 3/o ace-i che questi animali ricevono. Abbiamo valutato prospetticamente l'impatto della terapia orale con furosemide sulla chimica urinaria, nei cani con malattia della valvola mitrale mixomatosa. Tali differenze sono state attribuite all'effetto della terapia con furosemide sugli elettroliti renali. La chimica urinaria è utile per stimare la risposta diuretica nei cani con malattie cardiache. I dati suggeriscono differenze significative tra i diversi stadi ACVIM con particolare riferimento all’escrezione elettrolitica di Sodio, Potassio e Cloro.

Applications of network-based approaches in drug research

Recent research trends in computer-aided drug design have shown an increasing interest towards the implementation of advanced approaches able to deal with large amount of data. This demand arose from the awareness of the complexity of biological systems and from the availability of data provided by high-throughput technologies. As a consequence, drug research has embraced this paradigm shift exploiting approaches such as that based on networks. Indeed, the process of drug discovery can benefit from the implementation of network-based methods at different steps from target identification to drug repurposing. From this broad range of opportunities, this thesis is focused on three main topics: (i) chemical space networks (CSNs), which are designed to represent and characterize bioactive compound data sets; (ii) drug-target interactions (DTIs) prediction through a network-based algorithm that predicts missing links; (iii) COVID-19 drug research which was explored implementing COVIDrugNet, a network-based tool for COVID-19 related drugs. The main highlight emerged from this thesis is that network-based approaches can be considered useful methodologies to tackle different issues in drug research. In detail, CSNs are valuable coordinate-free, graphically accessible representations of structure-activity relationships of bioactive compounds data sets especially for medium-large libraries of molecules. DTIs prediction through the random walk with restart algorithm on heterogeneous networks can be a helpful method for target identification. COVIDrugNet is an example of the usefulness of network-based approaches for studying drugs related to a specific condition, i.e., COVID-19, and the same ‘systems-based’ approaches can be used for other diseases. To conclude, network-based tools are proving to be suitable in many applications in drug research and provide the opportunity to model and analyze diverse drug-related data sets, even large ones, also integrating different multi-domain information.

Ai confini del senso. La schizofrenia tra semiotica, psicopatologia e scienze cognitive

La schizofrenia rappresenta uno dei più grandi enigmi per l’impresa conoscitiva umana: non si conosce la sua eziologia, né le sue basi biologiche e cerebrali. Non è neanche chiaro cosa accada nell’esperienza di chi ne soffre, che sembra vivere in un mondo altro. La scarsa conoscenza dell’esperienza schizofrenica e la distanza tra questa e il senso comune hanno portato molti studiosi a inquadrare questo disturbo come illogico, irrazionale, insensato. Il presente lavoro tenta di confutare tale impostazione, mostrando come il mondo di senso dello schizofrenico si altera, non si disgrega; si trasforma, non si annulla. Il campo di studi all’interno del quale si colloca la ricerca è la semiotica, disciplina che studia i sistemi e i processi di significazione e i modi attraverso cui l’essere umano dà senso al mondo. L’intera indagine è inserita in un quadro interdisciplinare in costante dialogo con la psicopatologia fenomenologica e le scienze cognitive contemporanee, e si sviluppa a partire da numerosi testi autobiografici di pazienti schizofrenici, report psichiatrici, articoli di giornale, film e romanzi sul tema. L’ipotesi su cui si muove il lavoro è che sia possibile comprendere la schizofrenia come un problema costitutivamente semiotico, il cui nucleo è da rintracciarsi in una radicale metamorfosi delle modalità di produrre e interpretare il significato. La scommessa sottesa è che la semiotica possa contribuire in modo sostanziale alla comprensione delle modalità attraverso cui la nostra cultura concettualizza la schizofrenia e dei modi in cui gli schizofrenici danno senso al mondo. Il lavoro indaga, quindi, i legami tra schizofrenia e cultura, la storia del concetto nosografico, e le alterazioni dei processi di significazione nei casi di eloquio disorganizzato, nei racconti autobiografici e nei deliri, cercando anche di fornire strumenti utili alla pratica clinica.

Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis

In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia. Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2. Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.

Structural and biophysical characterization of RAD52 as novel pharmacological target for synthetic lethality

RAD52 is a protein involved in various DNA reparation mechanisms. In the last few years, RAD52 has been proposed as a novel pharmacological target for cancer synthetic lethality strategies. Hence, this work has the purpose to investigate RAD52 protein, with biophysical and structural tools to shed light on proteins features and mechanistic details that are, up to now poorly described, and to design novel strategies for its inhibition. My PhD work had two goals: the structural and functional characterization of RAD52 and the identification of novel RAD52 inhibitors. For the first part, RAD52 was characterized both for its DNA interaction and oligomerization state together with its propensity to form high molecular weight superstructures. Moreover, using EM and Cryo-EM techniques, additional RAD52 structural hallmarks were obtained, valuable both for understanding protein mechanism of action and for drug discovery purpose. The second part of my PhD project focused on the design and characterization of novel RAD52 inhibitors to be potentially used in combination therapies with PARPi to achieve cancer cells synthetic lethality, avoiding resistance occurrence and side effects. With this aim we selected and characterized promising RAD52 inhibitors through three different approaches: 19F NMR fragment-based screening; virtual screening campaign; aptamers computational design. Selected hits (fragments, molecules and aptamers) were investigated for their binding to RAD52 and for their mechanism of inhibition. Collected data highlighted the identification of hits worthy to be developed into more potent and selective RAD52 inhibitors. Finally, a side project carried out during my PhD is reported. GSK-3β protein, an already validated pharmacological target was investigated using biophysical and structural biology tools. Here, an innovative and adaptable drug discovery screening pipeline able to directly identify selective compounds with binding affinities not higher than a reference binder was developed.

Dalla continuita' alle transizioni: una ricerca-formazione nei servizi 0-6

Il progetto di ricerca relativo a questa tesi prende le mosse dalla più recente normativa italiana (l. 107/15; d.l. 65/2017) ed europea (EU Council Recommendation on high quality ECEC systems, 2019) e dalla riflessione teorica ad essa collegata in relazione all’istituzione e al riconoscimento del Sistema Integrato 0-6. La ricerca, metodologicamente impostata come ricerca-formazione, si è posta come obiettivo la definizione di categorie di analisi connesse a temi educativi e didattici emergenti nelle sperimentazioni di percorsi 0-6, e alla loro declinazione per fini formativi. La cornice teorica di riferimento è stata identificata nella riflessione sul concetto di continuità, con particolare riferimento alle declinazioni di curricolo verticale (Venturelli & Cigala, 2017; Cerini et al., 2019) e curricolo implicito (Gariboldi, 2007; Prott & Pressing, 2007). Si è giunti, grazie ai risultati di ricerca, a proporre una riflessione rispetto all’operalizzazione del costrutto di continuità nell’ambito del sistema integrato 0-6, fino ad arrivare a definire il suo progressivo superamento all’interno di percorsi di progettazione che caratterizzano le sperimentazioni in corso di attuazione all’interno di Poli 0-6, attraverso la definizione del tema delle transizioni. Tale tema, emerso durante la ricerca, come cornice di riferimento tra i temi caratteristici lo 0-6 potrebbe, infatti, andare ad affiancare la riflessione sul tema della continuità, portando a risignificare la declinazione pratica dello stesso. Dentro un approccio qualitativo più ampio, la ricerca-formazione è stata eletta come cornice metodologica di riferimento per questa ricerca, coniugando alcuni strumenti propri della stessa (identificazione degli obiettivi specifici, co-costruzione dei dati e analisi congiunta insieme ai soggetti coinvolti, focus group sul materiale raccolto) con alcuni strumenti di raccolta dati della ricerca etnografica (osservazioni, interviste semi-strutturate). La scelta di queste strategie metodologiche ha avuto sempre come obiettivo quello di sostenere la riflessività del gruppo di lavoro, rendendo continuamente espliciti i nessi tra teorie e prassi.

3D bioprinted organ models for drug screening

In recent years, 3D bioprinting has emerged as an innovative and versatile technology able to produce in vitro models that resemble the native spatial organization of organ tissues, by employing or more bioinks composed of various types of cells suspended in hydrogels. Natural and semi-synthetic hydrogels are extensively used for 3D bioprinting models since they can mimic the natural composition of the tissues, they are biocompatible and bioactive with customizable mechanical properties, allowing to support cell growth. The possibility to tailor hydrogels mechanical properties by modifying the chemical structures to obtain photo-crosslinkable materials, while maintaining their biocompatibility and biomimicry, make their use versatile and suitable to simulate a broad spectrum of physiological features. In this PhD Thesis, 3D bioprinted in vitro models with tailored mechanical properties and physiologically-like features were fabricated. AlgMa-based bioinks were employed to produce a living platform with gradient stiffness, with the aim to create an easy to handle and accessible biological tool to evaluate mechanobiology. In addition, GelMa, collagen and IPN of GelMa and collagen were used as bioinks to fabricate a proof-of-concept of 3D intestinal barrier, which include multiple cell components and multi-layered structure. A useful rheological guide to drive users to the selection of the suitable bioinks for 3D bioprinting and to correlate the model’s mechanical stability after crosslinking is proposed. In conclusion, a platform capable to reproduce models with physiological gradient stiffness was developed and the fabrication of 3D bioprinted intestinal models displaying a good hierarchical structure and cells composition was fully reported and successfully achieved. The good biological results obtained demonstrated that 3D bioprinting can be used for the fabrications of 3D models and that the mechanical properties of the external environment plays a key role on the cell pathways, viability and morphology.

Nanostructured smart materials as functional components of medical devices

The field of medical devices has experienced, more than others, technological advances, developments and innovations, thanks to the rapidly expanding scientific knowledge and collaboration between different disciplines such as biology, engineering and materials science. The design of functional components can be achieved by exploiting composite materials based on nanostructured smart materials, that due to the inherent characteristics of single constituents develop unique properties that make them suitable for different applications preserving excellent mechanical proprieties. For instance, recent developments have focused on the fabrication of piezoelectric devices with multiple biomedical functions, as actuation and sensing functions in one component for monitoring pressure signals. The present Ph.D. Thesis aims at investigating nanostructured smart materials embedded into a polymeric matrix to obtain a composite material that can be used as a functional component for medical devices. (i) Nanostructured piezoelectric material with self-sensing capability was successfully manufactured by using ceramic (i.e. lead zirconate titanate (PZT)) and (ii) polymeric (i.e. poly(vinylidene fluoride-trifluoro ethylene (PVDF-TRFE)) piezoelectric materials. PZT nanofibers were obtained by sol-gel electrospinning starting from synthetized PZT precursor solution. Synthesis, sol-gel electrospinning process, and thermal treatment were accurately controlled to obtain PZT nanofibers dimensionally stable with densely packed grains in the perovskite phase. To guarantee the impact resistance of the laminate, the morphology and size of the hosting filler were accurately designed by increasing the surface area to volume ratio. Moreover, to solve the issue relative to the mechanical discrepancy between rigid electronic materials/soft human tissues/different material of the device (iii) a nanostructured flexible composite material based on a network of Poly-L-lactic acid (PLLA) made of curled nanofibers that present a tuneable mechanical response as a function of the applied stress was successful fabricated.

Impacts of trace element pollution on sewage sludge and soil-plant systems: developing remediation techniques and assessing plant uptake prevention measures

Trace Elements (TEs) pollution is a significant environmental concern due to its toxic effects on human and ecosystem health and its potential to bioaccumulate in the food chain and to threaten species survival, leading to a decline in biodiversity. Urban areas, industrial and mining activities, agricultural practices, all contribute to the release of TEs into the environment posing a significant risk to human health and ecosystems. Several techniques have been developed to control TEs into the environment. This work presents the findings of three-year PhD program that focused on research on TEs pollution. The study discusses three fundamental aspects related to this topic from the perspective of sustainable development, environmental and human health. (1) High levels of TEs contamination prevent the use of sewage sludge (SS) as a fertilizer in agriculture, despite its potential as a soil amendment. Developing effective techniques to manage TEs contamination in SS is critical to ensure its safe use in agriculture and promote resource efficiency through sludge reuse. Another purpose of the study was to evaluate different strategies to limit the TEs uptake by horticultural crops (specifically, Cucumis Melo L.). This study addressed the effect of seasonality, Trichoderma inoculation and clinoptilolite application on chromium (Cr), copper (Cu) and lead (Pb) content of early- and late-ripening cultivars of Cucumis Melo L.. Finally, the accumulation of copper and the effect of its bioavailable fraction on bacterial and fungal communities in the rhizosphere soil of two vineyards, featuring two different varieties of Vitis vinifera grown for varying lengths of time, were evaluated.

Sewage sludge as organic fertilizer in agriculture. A study of physico-chemical properties, agronomic efficiency and environmental safety, with a specific focus on tannery sludge

In recent decades, the use of organic fertilizers has gained increasing interest mainly for two reasons: their ability to improve soil fertility and the need to find a sustainable alternative to mineral and synthetic fertilizers. In this context, sewage sludge is a useful organic matrix that can be successfully used in agriculture, due to its chemical composition rich in organic matter, nitrogen, phosphorus and other micronutrients necessary for plant growth. This work investigated three indispensable aspects (i.e., physico-chemical properties, agronomic efficiency and environmental safety) of sewage sludge application as organic fertilizer, emphasizing the role of tannery sludge. In a comparison study with municipal sewage sludge, results showed that the targeted analyses applied (total carbon and nitrogen content, isotope ratio of carbon and nitrogen, infrared spectroscopy and thermal analysis) were able to discriminate tannery sludge from municipal ones, highlighting differences in composition due to the origin of the wastewater and the treatment processes used in the plants. Regarding agronomic efficiency, N bioavailability was tested in a selection of organic fertilizers, including tannery sludge and tannery sludge-based fertilizers. Specifically, the hot-water extractable N has proven to be a good chemical indicator, providing a rapid and reliable indication of N bioavailability in soil. Finally, the behavior of oxybenzone (an emerging organic contaminant detected in sewage sludge) in soils with different physico-chemical properties was studied. Through adsorption and desorption experiments, it was found that the mobility of oxybenzone is reduced in soils rich in organic matter. Furthermore, through spectroscopic methods (e.g., infrared spectroscopy and surface-enhanced Raman spectroscopy) the mechanisms of oxybenzone-humic acids interaction were studied, finding that H-bonds and π-π stacking were predominantly present.

Strategies to hijack MTs dysfunction in neurodegenerative tauopathies: Design and synthesis of novel CNS-disease-modifying tools

Neuronal microtubules assembly and dynamics are regulated by several proteins including (MT)-associated protein tau, whose aberrant hyperphosphorylation promotes its dissociation from MTs and its abnormal deposition into neurofibrillary tangles, a common neurotoxic hallmarks of neurodegenerative tauopathies. To date, no disease-modifying drugs have been approved to combat CNS tau-related diseases. The multifactorial etiology of these conditions represents one of the major limits in the discovery of effective therapeutic options. In addition, tau protein functions are orchestrated by diverse post-translational modifications among which phosphorylation mediated by PKs plays a leading role. In this context, conventional single-target therapies are often inadequate in restoring perturbed networks and fraught with adverse side-effects. This thesis reports two distinct approaches to hijack MT defects in neurons. The first is focused on the rational design and synthesis of first-in-class triple inhibitors of GSK-3β, FYN, and DYRK1A, three close-related PKs, which act as master regulators of aberrant tau hyperphosphorylation. A merged multi-target pharmacophore strategy was applied to simultaneously modulate all three targets and achieve a disease-modifying effect. Optimization of ARN25068 by a computationally and crystallographic driven SAR exploration, allowed to rationalize the key structural modifications to maintain a balanced potency against all three targets and develop a new generation of quite well-balanced analogs exhibiting improved physicochemical properties, a good in vitro ADME profile, and promising cell-based anti-tau phosphorylation activity. In Part II, MT-stabilizing compounds have been developed to compensate MT defects in tau-related pathologies. Intensive chemical effort has been devoted to scaling up BL-0884, identified as a promising MT-normalizing TPD, which exhibited favorable ADME-PK, including brain penetration, oral bioavailability, and brain pharmacodynamic activity. A suitable functionalization of the exposed hydroxyl moiety of BL-0884 was carried out to generate corresponding esters and amides possessing a wide range of applications as prodrugs and active targeting for cancer chemotherapy.

Unveiling the molecular mechanism of BRCA2-RAD51 interaction to tackle cancer onset

Different kinds of lesions can occur to DNA, and among them, one of the most dangerous is the double strand breaks (DSBs). Actually, DSBs can result in mutations, chromosome translocation or deletion. For this kind of lesions, depending on cell cycle phase as well as DNA-end resection, cells have developed specific repair pathways. Among these the error-free homologous recombination (HR) plays a crucial role. HR takes place during S/G2 phases, since the sister chromatids can be used as homologous templates. In this process, hRAD51 and BRCA2 are key players. hRAD51 is a recombinase of 339 amino-acids highly conserved through evolution which displays an intrinsic tendency to form oligomeric structures. BRCA2 is a very large protein of 3418 amino-acids, essential for the recruitment and accumulation of hRAD51 in the nucleus repairing-foci. BRCA2 interacts with hRAD51 through eight, so-called, BRC repeats, composed of 35-40 amino-acids. Mutations within this region have been linked to an increased risk of ovarian cancer development. In particular, several reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with hRAD51. To date the only interaction that has been structurally elucidated, is the one taking place amid the fourth BRC repeat and hRAD51. Only very little biophysical information is available on the interaction of the other BRC repeats with hRAD51. This thesis aims at elucidating the mechanism of hRAD51-BRCA2 interaction, by means of biophysical and structural approaches.

Inhibition and characterization of two-metal ion enzymes to treat cancer: dna polymerase Ƞ and topoisomerase II α

Chemotherapeutic drugs can in many ways disrupt the replication machinery triggering apoptosis in cancer cells: some act directly on DNA and others block the enzymes involved in preparing DNA for replication. Cisplatin-based drugs are common as first-line cancer chemotherapics. Another example is etoposide, a molecule that blocks topoisomerase II α leading to the inhibition of dsDNA replication. Despite their efficacy, cancer cells can respond to these treatments over time by overtaking their effects, leading to drug resistance. Chemoresistance events can be triggered by the action of enzymes like DNA polymerase ƞ (Pol η). This polymerase helps also to bypass drug-induced damage in cancer cells, allowing DNA replication and cancer cells proliferation even when cisplatin-based chemotherapeutic drugs are in use. Pol ƞ is a promising drug discovery target, whose inhibition would help in overcoming of drug resistance. This study aims to identify a potent and selective Pol ƞ inhibitor able to improve the efficacy of platinum-based chemotherapeutic drugs. We report the discovery of compound 64 (ARN24964), after an extensive SAR reporting 35 analogs. We evaluated compound 64 on four different cell lines. Interestingly, the molecule is a Pol η inhibitor able to act synergistically with cisplatin. Moreover, we also synthesized a prodrug form that allowed us to improve its stability and the bioavailability. This compound represents an advanced scaffold featuring good potency and DMPK properties. In addition to this central theme, this thesis also describes our efforts in developing and characterize a novel hybrid inhibitor/poison for the human topoisomerase II α enzyme. In particular, we performed specific assays to study the inhibiton of Topoisomesare II α and we evaluated compounds effect on three cancer cell lines. These studies allowed us to identify a compound that is able to inhibit the enzyme with a good pK and a good potency.

New chemical modalities for leishmaniasis drug discovery

Leishmaniasis is one of the major parasitic diseases among neglected tropical diseases with a high rate of morbidity and mortality. Human migration and climate change have spread the disease from limited endemic areas all over the world, also reaching regions in Southern Europe, and causing significant health and economic burden. The currently available treatments are far from ideal due to host toxicity, elevated cost, and increasing rates of drug resistance. Safer and more effective drugs are thus urgently required. Nevertheless, the identification of new chemical entities for leishmaniasis has proven to be incredibly hard and exacerbated by the scarcity of well-validated targets. Trypanothione reductase (TR) represents one robustly validated target in Leishmania that fulfils most of the requirements for a good drug target. However, due to the large and featureless active site, TR is considered extremely challenging and almost undruggable by small molecules. This scenario advocates the development of new chemical entities by unlocking new modalities for leishmaniasis drug discovery. The classical toolbox for drug discovery has enormously expanded in the last decade, and medicinal chemists can now strategize across a variety of new chemical modalities and a vast chemical space, to efficiently modulate challenging targets and provide effective treatments. Beyond others, Targeted p Protein Degradation (TPD) is an emerging strategy that uses small molecules to hijack endogenous proteolysis systems to degrade disease-relevant proteins and thus reduce their abundance in the cell. Based on these considerations, this thesis aimed to develop new strategies for leishmaniasis drug discovery while embracing novel chemical modalities and navigating the chemical space by chasing unprecedented chemotypes. This has been achieved by four complementary projects. We believe that these next-generation chemical modalities for leishmaniasis will play an important role in what was previously thought to be a drug discovery landscape dominated by small molecules.

Design, synthesis and biological evaluation of dual inhibitors of GSK-3B and Fyn kinases for the study of inflammatory pathways in neurodegenerative diseases

Neuroinflammation represents a key hallmark of neurodegenerative diseases and is the result of a complex network of signaling cascades within microglial cells. A positive feedback loop exists between inflammation, microglia activation and protein misfolding processes, that, together with oxidative stress and excitotoxicity, lead to neuronal degeneration. Therefore, targeting this vicious cycle can be beneficial for mitigating neurodegeneration and cognitive decline in central nervous system disorders. At molecular level, GSK-3B and Fyn kinases play a crucial role in microglia activation and their deregulation has been associated to many neurodegenerative diseases. Thus, we envisioned their combined targeting as an effective approach to disrupt this toxic loop. Specifically in this project, a hit compound, based on a 7-azaindole-3-aminothiazole structure, was first identified in a virtual screening campaign, and displayed a weak dual inhibitory activity on GSK-3B and Fyn, unbalanced towards the former. Then, in a commitment to uncover the structural features required for modulating the activity on the two targets, we systematically manipulated this compound by inserting various substitution patterns in different positions. The most potent compounds obtained were advanced to deeper investigations to test their ability of tackling the inflammatory burden also in cellular systems and to unveil their binding modes within the catalytic pocket. The new class of molecules synthesized emerged as a valuable tool to deepen our understanding of the complex network governing the inflammatory events in neurodegenerative disorders.