Applicazione di un protocollo di screening differenziale nel sistema Vitis vinifera per l’identificazione di peptidi con attività antimicrobica

Le piante e gli animali presentano elementi comuni nel loro sistema di difesa contro gli agenti patogeni, come la sintesi diretta di enzimi idrolitici (chitinasi, glucanasi, proteinasi e ossidasi) e di peptidi antimicrobici (AMPs). Gli AMPs sono peptidi ampiamente espressi negli organismi animali (vertebrati e invertebrati) e nelle piante. Possono essere espressi costitutivamente o rapidamente indotti inseguito ad uno stimolo biotico, a differenti livelli cellulari, per interagire direttamente con l’agente infettante e/o per modulare la risposta immunitaria contro i patogeni. Tali peptidi sono oggi classificati in relazione alle loro caratteristiche biochimiche (carica netta) e/ o alle loro caratteristiche strutturali (composizione amminoacidica, struttura lineare o circolare). In base a queste caratteristiche le molecole possono essere distinte nei seguenti gruppi: 1) peptidi lineari ad alfa elica; 2) peptidi ciclici con β-sheets e due o più ponti disolfuro; 3) peptidi con alfa elica e β-sheets stabilizzati da ponti disolfuro; 4) peptidi con hairpin o loop stabilizzati da ponti disolfuro; 5) peptidi lineari con residui aminoacidici ripetuti, come prolina, glicina, triptofano o istidina; 6) piccoli peptidi con struttura avvolta o con una struttura secondaria non definita. Nonostante la loro diversità strutturale, i peptidi antimicrobici presentano la caratteristica comune di inibire la crescita di un largo spettro di microbi, quali Gram-positivi, Gram-negativi, funghi e in alcuni casi anche virus, tanto da far coniare il termine di “antibiotici naturali”. Negli ultimi anni è notevolmente incrementato l’interesse verso tali peptidi dal momento che dati scientifici hanno mostrato che questi non inducono lo sviluppo di meccanismi di resistenza nei microrganismi patogeni. Gli AMPs quindi potrebbero costituire una valida alternativa non solo in ambito sanitario, per la sostituzione di antibiotici di sintesi chimica e di origine microbiologica, ma potrebbero avere un importante utilizzo in campo industriale e nello sviluppo di nuovi sistemi di conservazione degli alimenti al fine di incrementare la loro “shelf-life”.

Ottimizzazione di materiali e trattamenti per componenti critici destinati a motociclette da competizione

L’attività svolta durante il dottorato è stata incentrata su due tematiche riguardanti: (i) la modifica della composizione chimica delle classiche leghe di alluminio da fonderia per incrementarne la resistenza e stabilità termica; (ii) lo studio del comportamento a fatica di acciai innovativi alto-resistenziali, allo scopo di valutarne il loro utilizzo per la produzione di alberi motore e distribuzione in sostituzione dei tradizionali acciai utilizzati dopo bonifica e trattamento superficiale di nitrurazione. La messa a punto di una lega di alluminio da fonderia con elevata resistenza in temperatura ha richiesto, oltre all’individuazione della composizione chimica, l’ottimizzazione del trattamento termico e una completa caratterizzazione meccanica statica a fatica sia a temperatura ambiente sia a 200°C. L’attività ha permesso di sviluppare una lega, ottenuta aggiungendo 1,3% in peso di rame alla classica lega A357 (Al-Si-Mg), cha ha mostrato avere proprietà meccaniche superiori a quelle delle tradizionali leghe Al-Si-Mg-Cu quali la A354 e C355 sia a temperatura ambiente che a 200 °C dopo lunga esposizione in temperatura. Per quanto riguarda gli acciai innovativi, dopo una preliminare analisi di mercato per individuare quali acciai potessero essere oggetto di studio, è stato valutato come migliorarne le prestazioni a fatica, anche in presenza d’intaglio, attraverso la scelta del trattamento termico più opportuno e del processo di pallinatura. I risultati delle caratterizzazioni microstrutturale e meccanica svolte hanno permesso di individuare due acciai (nomi commerciali K890 e ASP2017) ottenuti per metallurgia delle polveri, ad oggi utilizzati solo per la produzione di stampi e/o utensili, in grado di sostituire gli acciai con cui vengono oggi realizzati i componenti, senza la necessità di eseguire il trattamento di nitrurazione

Soil enzyme activities and biochemical indexes to assess soil quality in agronomic and forested ecosystems

The soil carries out a wide range of functions and it is important study the effects of land use on soil quality in order to provide most sustainable practices. Three fields trial have been considered to assess soil quality and functionality after human alteration, and to determine the power of soil enzymatic activities, biochemical indexes and mathematical model in the evaluation of soil status. The first field was characterized by conventional and organic management in which were tested also tillage effects. The second was characterized by conventional, organic and agro-ecological management. Finally, the third was a beech forest where was tested the effects of N deposition on soil organic carbon sequestration. Results highlight that both enzyme activities and biochemical indexes could be valid parameters for soil quality evaluation. Conventional management and plowing negatively affected soil quality and functionality with intensive tillage that lead to the downturn of microbial biomass and activity. Both organic and agro-ecological management revealed to be good practices for the maintenance of soil functionality with better microbial activity and metabolic efficiency. This positively affected also soil organic carbon content. At the eutrophic forest, enzyme activities and biochemical indexes positively respond to the treatments but one year of experimentation resulted to be not enough to observe variation in soil organic carbon content. Mathematical models and biochemical indicators resulted to be valid tools for assess soil quality, nonetheless it would be better including the microbial component in the mathematical model and consider more than one index if the aim of the work is to evaluate the overall soil quality and functionality. Concluding, the forest site is the richest one in terms of organic carbon, microbial biomass and activity while, the organic and the agro-ecological management seem to be the more sustainable but without taking in consideration the yield.

Novel dual D3R/GSK-3β modulators: an innovative multitarget strategy for bipolar disorder

Among the psychiatric diseases, bipolar disorder (BD) is the sixth leading cause of disability with a prevalence up to 4 % worldwide. BD is a complex neuropsychiatric condition which alternates episodes of mania with symptoms of depression. Although the neurobiological pathways are not completely clarified, the dopamine (DA) hypothesis, recognized as the leading theory explaining the pathophysiology of the malady, states that the dramatically compromised homeostatic regulation of dopaminergic circuits leads to alternated changes in DA neurotransmission. Modulation of D2 and D3 receptors (D2/3R) through partial agonists represents the first-line therapeutic strategy for psychiatric diseases. Moreover, a deregulation of the enzyme glycogen synthase kinase-3β (GSK-3β) has been reported as peculiar feature of BD. In this scenario, the concomitant modulation of D3R and GSK-3β, by employing multitarget compounds, could offer promises to achieve an effective cure of this illness. In the light of these findings, we rationally envisaged the pharmacophoric model at the basis of the design of several D3R partial agonists, suitable to be exploited for the dual D3R/GSK-3β ligand design. Thus, synthetic efforts were addressed to develop a first set of hybrid molecules able to concurrently modulate the selected targets. For a chemical structure point of view, we employed different spacers to combine a substituted aryl-piperazine moiety, reported in previously discovered D3R modulators, with a pyrazole-based fragment, already identified in GSK-3β inhibitors. A fluorescent and a cellular functional assays were carried out to assess the activity of all synthetized compounds against GSK-3β and on D3R, respectively. Most of the derivatives proved to effectively modulate both GSK-3β and D3R with potencies in the low-µM and low-nM range, respectively. The consistent biological data allowed us to identify some lead candidates worth to be further modified with the aim to optimize their biological profile and to perform a structure-activity relationship (SAR) study.

Biological analysis of the in-vitro effects of homologous recombination inhibition and its use in cancer treatment through synthetic lethality

Synthetic lethality represents an anticancer strategy that targets tumor specific gene defects. One of the most studied application is the use of PARP inhibitors (e.g. olaparib) in BRCA1/2-less cancer cells. In BRCA2-defective tumors, olaparib (OLA) inhibits DNA single-strand break repair, while BRCA2 mutations hamper homologous recombination (HR) repair. The simultaneous impairment of those pathways leads BRCA-less cells to death by synthetic lethality. The projects described in this thesis were aimed at extending the use of OLA in cancer cells that do not carry a mutation in BRCA2 by combining this drug with compounds that could mimic a BRCA-less environment via HR inhibition. We demonstrated the effectiveness of our “fully small-molecule induced synthetic lethality” by using two different approaches. In the direct approach (Project A), we identified a series of neo-synthesized compounds (named RAD51-BRCA2 disruptors) that mimic BRCA2 mutations by disrupting the RAD51-BRCA2 interaction and thus the HR pathway. Compound ARN 24089 inhibited HR in human pancreatic adenocarcinoma cell line and triggered synthetic lethality by synergizing with OLA. Interestingly, the observed synthetic lethality was triggered by tackling two biochemically different mechanisms: enzyme inhibition (PARP) and protein-protein disruption (RAD51-BRCA2). In the indirect approach (Project B), we inhibited HR by interfering with the cellular metabolism through inhibition of LDH activity. The obtained data suggest an LDH-mediated control on HR that can be exerted by regulating either the energy supply needed to this repair mechanism or the expression level of genes involved in DNA repair. LDH inhibition also succeeded in increasing the efficiency of OLA in BRCA-proficient cell lines. Although preliminary, these results highlight a complex relationship between metabolic reactions and the control of DNA integrity. Both the described projects proved that our “fully small-molecule-induced synthetic lethality” approach could be an innovative approach to unmet oncological needs.

Design and Synthesis of Novel Kinase Inhibitors for the Treatment of Chronic Respiratory Diseases

In 2017, Chronic Respiratory Diseases accounted for almost four million deaths worldwide. Unfortunately, current treatments are not definitive for such diseases. This unmet medical need forces the scientific community to increase efforts in the identification of new therapeutic solutions. PI3K delta plays a key role in mechanisms that promote airway chronic inflammation underlying Asthma and COPD. The first part of this project was dedicated to the identification of novel PI3K delta inhibitors. A first SAR expansion of a Hit, previously identified by a HTS campaign, was carried out. A library of 43 analogues was synthesised taking advantage of an efficient synthetic approach. This allowed the identification of an improved Hit of nanomolar enzymatic potency and moderate selectivity for PI3K delta over other PI3K isoforms. However, this compound exhibited low potency in cell-based assays. Low cellular potency was related to sub optimal phys-chem and ADME properties. The analysis of the X-ray crystal structure of this compound in human PI3K delta guided a second tailored SAR expansion that led to improved cellular potency and solubility. The second part of the thesis was focused on the rational design and synthesis of new macrocyclic Rho-associated protein kinases (ROCKs) inhibitors. Inhibition of these kinases has been associated with vasodilating effects. Therefore, ROCKs could represent attractive targets for the treatment of pulmonary arterial hypertension (PAH). Known ROCK inhibitors suffer from low selectivity across the kinome. The design of macrocyclic inhibitors was considered a promising strategy to obtain improved selectivity. Known inhibitors from literature were evaluated for opportunities of macrocyclization using a knowledge-based approach supported by Computer Aided Drug Design (CADD). The identification of a macrocyclic ROCK inhibitor with enzymatic activity in the low micro molar range against ROCK II represented a promising result that validated this innovative approach in the design of new ROCKs inhibitors.

Design and synthesis of (pro)electrophilic compounds for investigating the multifactorial nature of neurodegenerative diseases: focus on inflammation-driven events

Neuroinflammation constitutes a major player in the etiopathology of neurodegenerative diseases (NDDs), by orchestrating several neurotoxic pathways which in concert lead to neurodegeneration. A positive feedback loop occurs between inflammation, microglia activation and misfolding processes that, alongside excitotoxicity and oxidative events, represent crucial features of this intricate scenario. The multi-layered nature of NDDs requires a deepen investigation on how these vicious cycles work. This could further help in the search for effective treatments. Electrophiles are critically involved in the modulation of a variety of neuroprotective responses. Thus, we envisioned their peculiar ability to switch on/off biological activities as a powerful tool for investigating the neurotoxic scenario driven by inflammation in NDDs. In particular, in this thesis project, we wanted to dissect at a molecular level the functional role of (pro)electrophilic moieties of previously synthesized thioesters of variously substituted trans-cinnamic acids, to identify crucial features which could interfere with amyloid aggregation as well as modulate Nrf2 and/or NF-κB activation. To this aim, we first synthesized new compounds to identify bioactive cores which could specifically modulate the intended target. Then, we systematically modified their structure to reach additional pathogenic pathways which could in tandem contribute to the inflammatory process. In particular, following the investigation of the mechanistic underpinnings involving the catechol feature in amyloid binding through the synthesis of new dihydroxyl derivatives, we incorporated the identified antiaggregating nucleus into constrained frames which could contrast neuroinflammation also through the modulation of CB2Rs. In parallel, Nrf2 and/or NF-κB antinflammatory structural requirements were combined with the neuroprotective cores of pioglitazone, an antidiabetic drug endowed with MAO-B inhibitory properties, and memantine, which notably contrasts excitotoxicity. By acting as Swiss army knives, the new set of molecules emerge as promising tools to deepen our insights into the complex scenario regulating NDDs.

The human gut microbiome in disease: Role in chemotherapy treatments and relationship with clinical outcomes

The gut microbiome (GM) is a plastic entity, capable of adapting in response to intrinsic and extrinsic factors. However, several circumstances can disrupt this homeostatic balance, forcing the GM to shift from a health-associated mutualistic configuration to a disease-associated profile. Nowadays, a new frontier of microbiome research is understanding the GM role in chemo-immunotherapies and clinical outcomes. Here, the role of the genotoxin‐producing pathogen Salmonella in colorectal carcinogenesis was characterized by in-vitro models. A synergistic effect of Salmonella and the CRC-associated mutation (APC gene) promoted a tumorigenic microenvironment by increasing cellular genomic instability. Subsequently, the GM involvement in anti-cancer therapies was investigated via next-generation sequencing in different patient cohorts. The GM trajectory during treatments was characterized for women with epithelial ovarian cancer and pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). The results highlighted the loss of GM homeostasis, with diversity reduction, decrease in health-associated microorganisms and pathobiont bloom. Interestingly, a distinctive GM profile was identified in ovarian cancer patients with a poor response to chemotherapy compared to patients in remission. Moreover, maintenance of GM homeostasis through enteral feeding in pediatric HSCT patients highlighted a better prognosis, with reduced risk of clinical complications. In this context, the gut resistome – the pattern of GM antibiotic-resistance genes (ARGs) – was evaluated longitudinally in HSCT patients. The results showed new acquisitions and consolidation of ARGs already present in patients developing clinical complications. Antibiotic exposure was also evaluated in infants under low-dose antibiotic prophylaxis for vesico-ureteral reflux showing an impairment of the GM configuration with possible long-term health implications. Dramatic GM dysbiosis was finally observed in critically ill patients with COVID-19 (undergoing multiple drug therapies) and correlated with increased risk of bloodstream infection. All these findings pointed out the importance of maintaining GM homeostasis during chemotherapy treatments for improving patients’ clinical outcomes.

Small molecules exploiting emerging therapeutic opportunities for breast cancer treatment

Among the different types of breast cancer (BC), the estrogen receptor positive (ER+) subtype, which requires estrogens for its growth and proliferation, is the most common, while triple negative BC, characterized by the absence of ER, progesterone receptor and human epidermal growth factor receptor 2, often leads to poor prognosis. First-line therapies for the treatment of ER+ BC act either by suppressing estrogen production, through the inhibition of aromatase (AR) enzyme, or by blocking estrogen prooncogenic activity, via the modulation/degradation of ERs. The serious side effects and the intrinsic or acquired resistance phenomena that arise with prolonged use of these drugs limit their therapeutic application, stimulating the search for new strategies to face this disease. In this context, the development of dual acting aromatase inhibitors, able to target both the orthosteric and the recently identified allosteric pockets of AR could be an opportunity to fight ER+ BC. Another promising strategy could be the development of multitarget compounds, targeting both AR and ERs. In this scenario, here we designed and synthesized two series of new xanthones or more flexible benzophenones as potential dual acting aromatase inhibitors. Moreover, inspired from tamoxifen metabolites and a literature compound endowed with activity on both AR and ER, different structurally related series of potential multitarget compounds were developed. The biological results showed that some of the new molecules were promising candidates for further development. It was recently observed that the lately discovered histamine H4 receptor is expressed in human breast tissue, displaying a key role in biological processes mediated by histamine such as cell proliferation, senescence, and apoptosis in malignant cells, representing a potential target in triple negative BC. Thus, a broad series of methyl quinazoline sulfonamides, carrying different functional groups on the sulfonamide moiety, were designed and synthesized as potential H4 receptor ligands.

Design, development and characterization of DHA-based emulsion for Nutrilipidomics strategies

The industrial PhD project presented here is part of the R&D strategies of the Lipinutragen company. The innovation brought by the company concerns nutrilipidomics, i.e. the correlation between the lipid composition (in fatty acids) of the cell membrane and lipid-based nutraceuticals, especially starting from the well-known dependence of the lipid composition on the intake of essential fats, omega- 6 and omega-3 polyunsaturated fatty acids. Among the results obtained from the membrane lipidomic profiles, the case of autistic subjects is here highlighted, showing the significant deficiency of docosahexaenoic acid (DHA). The activity during the PhD was devoted to the nutrilipidomic approach. Part of the activities were devoted to scientific research in lipidomics: a) the study of lipidomic profiles in the frame of two collaboration projects: one with the group of Dr. I. Tueros at AZTI, Bilbao, regading obese population, and the other one regarding seed germination with the changes of the fatty acid profiles with the group of prof. A. Balestrazzi of the University of Parma; b) the liposome preparation for protection and lifetime prolongation of the peptide somatostatin, which was an important premise to the formulation of the DHA-containing microemulsion. The activities was also focused on the development of DHA-containing nutraceutical formulations in the form of emulsion, overcoming the difficulty of the capsule ingestion, to be administered orally. The work pointed to study the combination of active ingredients, based on the previous know-how regarding the bioavailability for the cell membrane incorporation. The ingredients of the formulation were studied and tested in vitro for the bioavailability of DHA to be incorporated in the cell membranes of different types of cultured cells. Part of this study is covered by non-disclosure agreement since it belongs to the know-how of Lipinutragen.

Foglio 027 Bolzano. Studio petrografico delle vulcaniti permiane del settore Val di Tires – Passo Nigra, Alta Val d’Ega, Bolzano

Il presente lavoro di tesi si inquadra nel programma di rilevamento geologico del Foglio 027 Bolzano, nell’area della Val di Tires – Passo Nigra, Alta Val d’Ega (Bolzano), la cui realizzazione è in corso a cura della Provincia Autonoma di Bolzano, nell’ambito del progetto CARG. Durante la campagna di rilevamento sono stati prelevati 33 campioni presso Tires, in Alta Val d’Ega, appartenenti alle vulcaniti permiane del Gruppo Vulcanico Atesino (GVA), 18 dei quali sono stati sottoposti ad analisi ottico-petrografica e chimica in XRF. I campioni sono stati prelevati dalle tre principali formazioni geologiche affioranti nell’area: la Formazione di Gargazzone (IGG), la Formazione di Cornedo (COR) e la Formazione di Ora (ORA). Dalle analisi è emerso che i campioni prelevati dalla Formazione di Gargazzone risultano di composizione riodacitica, quelli della Formazione di Cornedo hanno composizione andesitica e andesitico-dacitica, quelli della Formazione di Ora hanno composizione riolitica e riodacitica. Sono state distinte tre litofacies all’interno della Formazione di Ora: una litofacies basale, caratterizzata da un’elevata concentrazione di componenti litiche; una litofacies intermedia, decisamente più massiccia ed omogenea, che presenta al suo interno sporadici corpi lenticolari di ignimbriti ricche in pomici; una litofacies di tetto, ricca in pomici. Generalmente le ignimbriti della Formazione di Ora si caratterizzano per la composizione riolitica ricca in sanidino, ma la presenza di ignimbriti di composizione riodacitica all’interno della formazione e precisamente nell’area di studio, si può imputare alla modificazione della composizione mineralogica e chimica originaria a causa dei fenomeni metasomatici, particolarmente intensi lungo il bordo calderico. Sono stati inoltre prelevati cinque campioni che tagliano le formazioni affioranti, due dei quali hanno composizione riolitica, due hanno composizione basaltica e uno ha composizione alcali-basaltica.

Design di nuovi poliesteri a base di polibutilene succinato per la preparazione di scaffold per l’ingegneria tissutale del miocardio: struttura chimica e architettura molecolare come tool efficienti per modulare le proprietà chimico-fisiche

L’ingegneria dei tessuti molli, quali il miocardio, sta sempre più emergendo come approccio alternativo alle terapie tradizionali. In questo ambito, i poliesteri costituiscono una classe di polimeri promettente, poiché le variegate strutture chimiche che li caratterizzano permettono di soddisfare un’ampia gamma di esigenze. Negli ultimi anni, l’attenzione della ricerca si è incentrata sul poli(butilene succinato)(PBS). Il PBS, tuttavia, possiede proprietà meccaniche non ottimali per l’ingegneria dei tessuti molli; inoltre i tempi di degradazione sono lunghi; ciò è dovuto al grado di cristallinità e all’idrofobicità, entrambi elevati. Nell’ottica di migliorare le proprietà non soddisfacenti di tale omopolimero, sono stati sintetizzati e caratterizzati nuovi copoliesteri alifatici a base di PBS biocompatibili e biodegradabili. In particolare, sono stati realizzati un copolimero a blocchi e uno statistico a base di Pripol 1009, un diacido commerciale (Croda), e un copolimero a blocchi a base di neopentil glicole, valutando sia l’effetto del tipo di comonomero introdotto nel PBS (Pripol 1009 vs. neopentil glicole) che quello dell’architettura molecolare (copolimero statistico vs. copolimero multiblocco). I materiali sintetizzati sono stati processati in forma di film attraverso pressofusione e di scaffold tramite elettrofilatura. Oltre alla caratterizzazione molecolare, film e scaffold sono stati sottoposti anche ad analisi termica, diffrattometrica, meccanica e a studi di degradazione idrolitica in condizioni fisiologiche. I risultati ottenuti hanno evidenziato la possibilità di modulare sia le proprietà meccaniche che la velocità di degradazione in condizioni fisiologiche. Tutti i copolimeri, infatti, presentano caratteristiche di elastomeri termoplastici e dei profili di degradazione variabili rispetto all’omopolimero, che li rendono adatti per applicazioni nel campo dell’ingegneria dei tessuti molli.

Studio dell’effetto di stabilizzanti polimerici su catalizzatori di oro nanostrutturati

Negli ultimi anni ha riscosso particolare interesse l’utilizzo di nanoparticelle metalliche nella catalisi per via delle loro eccellenti proprietà. Le limitazioni al loro utilizzo sono connesse alla complessità della loro sintesi a causa dei molteplici parametri che possono influenzare le proprietà e la morfologia del sistema finale. Recenti studi hanno permesso di individuare la possibilità che polimeri con opportune caratteristiche possano influenzare sia la morfologia che l’attività catalitica dei catalizzatori nanostruturati. Queste ricerche hanno aperto alla possibilità di controllare le proprietà delle nanoparticelle attraverso la sintesi di macromolecole con specifiche caratteristiche. Nel corso dell’attività di tirocinio si è studiato come il peso molecolare e la natura dello stabilizzante polimerico influenzino le dimensioni e l’attività delle nanoparticelle d’oro in termini di conversione ed accessibilità dei siti attivi del catalizzatore attraverso la reazione di riduzione del 4-nitrofenolo. Si sono confrontati risultati ottenuti da nanoparticelle sintetizzate utilizzando come stabilizzante campioni a differente peso molecolare di poli-etilenglilcole, poli-vinilammina e poli-vinilalcol.

Development of new chemical entities to target neuroinflammatory pathways.

Neuroinflammatory pathways are main culprits of neurodegenerative diseases' onset and progression, including Alzheimer’s disease (AD). On this basis, several anti-inflammatory drugs were repurposed in clinical trials. However, they have failed, probably because neuroinflammation is a complex network, still not fully understood. From these evidences, this thesis focused on the design and synthesis of new chemical entities as potential neuroinflammatory drugs or chemical probes. Projects 1 and 2 aimed to multi-target-directed ligand (MTDL) development to target neuroinflammation in AD. Polypharmacology by MTDLs is considered one of the most promising strategies to face the multifactorial nature of neurodegenerative diseases. Particularly, Project 1 took inspiration from a cromolyn-ibuprofen drug combination polypharmacological approach, which was recently investigated in AD clinical trials. Based on that, two cromolyn-(S)-ibuprofen codrug series were designed and synthesized. Parent drugs were combined via linking or fusing strategies in 1:2 or 1:1 ratio, by means of hydrolyzable bonds. Project 2 started from a still ongoing AD clinical trial on investigational drug neflamapimod. It is a selective inhibitor of p38α-MAPK, a kinase strictly involved in neuroinflammatory pathways. On the other side, rasagiline, an anti-Parkinson drug, was also repurposed as AD treatment. Indeed, rasagiline’s propargylamine fragment demonstrated to be responsible not only for the MAO-B selective inhibition, but also for the neuroprotective activity. Thus, to synergistically combine these two effects into single-molecules, a small set of neflamapimod-rasagiline hybrids was developed. In the end BMX, a poorly investigated kinase, which seems to be involved in pro-inflammatory mediator production, was explored for the development of new chemical probes. High-quality chemical probes are a powerful tool in target validation and starting points for the development of new drug candidates. Thus, Project 3 focused on the design and synthesis of two series of optimized BMX covalent inhibitors as selective chemical probes.