Considering the crucial involvement of matrix metalloproteinases’ (MMPs) misregulated activity in the pathogenesis of several degenerative diseases, this class of enzymes has been considered a highly active set of targets for the design of new therapeutic agents. However, the scant success of synthetic MMP inhibitors, largely due to the disappointing results obtained in both clinical and preclinical studies, makes medicinal plants a valuable source of new active compounds able to modulate MMPs activity. In this work, a consistent number of plants, selected on the base of an ethnobotanical research, were tested as inhibitors of collagenase, the founding member of the MMPs family. 1H-NMR-based metabolomic analysis combined with multivariate data treatment (PLS and OPLS) was used to correlate the biological activity to the phytochemical profiles, suggesting tannins as an important class of collagenase inhibitors. Thus, a tannin-removal procedure was developed, which allowed to prove this hypothesis and to identify another class of active metabolites, the glucuronide-conjugated flavonoids (especially quercetin-3-O-β-glucoronide), whose the plant Alchemilla vulgaris was found to be a good source. In another stage of the project, different varieties of tea were investigated as collagenase inhibitors, finding black tea samples particularly potent. Then, an OPLS model was developed with the aim of correlating the biological activity to the UV-Vis spectra of teas, showing that a high activity was related to absorption values in the range 350-440 nm. A subsequent fractionation of the most active tea sample was carried out, and this approach allowed to corroborate the results obtained by the metabolomic analysis. Considering that the absorbance measurement of an extract represents a cheap and simple procedure, the proposed method can be suitable, for instance, to select the best tea variety to be developed as an anti-wrinkles cosmetic or food supplement.

The experiments described in the thesis for my PhD were addressed to the study of the anticancer activity of a conjugate of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) on hepatocellular carcinomas (HCCs) induced in rats by diethylnitrosamine. L-HSA is a neoglycoprotein exposing galactosyl residues. The conjugate was prepared to improve the chemo therapeutic index of DOXO in the treatment of the well differentiated (WD) HCCs whose cells mantain the receptor for galactosyl terminating glycoproteins and consequently can actively internalize L-HSA. In my first experiments I found that L-HSA coupled DOXO produced concentrations of DOXO higher than those raised by an equal dose of free drug, not only in WD HCCs, but also in the poorly differentiated forms (PD) of these tumors which do no express the receptor for galactosyl terminating glycoproteins. Subsequently I provided evidence that penetration of L-HSA-DOXO in PD HCCs was due to a non-specific adsorption mediated by the DOXO residues of the conjugate which interact with the cell surface mainly because at physiological pH they are positively charged and bind to anionic phospholipids of the cell membrane. In subsequent experiments, by ultrasound technique, I studied the action of free and L-HSA coupled DOXO on the growth of rat HCCs. I found that L-HSA coupled DOXO hindered the development of new neoplastic nodules and inhibited the growth of the established tumors. In contrast, the free drug neither inhibited the development of HCCs nor prevented the growth of the established tumors. Moreover, the free drug produced a severe loss of weight of rats, a sign of severe toxicity, which was not caused by the conjugate. In conclusion assuming that the results obtained in rats can be applied to patients, the results of my thesis suggest that the conjugate by increasing the efficacy and tolerability of DOXO could improve the value of this drug in the treatment of human HCCs.

No description available.

Background. Neuroblastoma is the most deadly solid tumor of childhood. In the 25% of cases it is associated with MYCN amplification (MA), resulting in the disregulation of several genes involved in cancer progression, chemotherapy resistance and poor prognosis causing the disregulation of several genes involved in cancer progression and chemotherapy resistance and resulting in a poor prognosis. Moreover, in this contest, therapy-related p53 mutations are frequently found in relapsed cases conferring an even stronger aggressiveness. For this reason, the actual therapy requires new antitumor molecules. Therefore, rapid, accurate, and reproducible preclinical models are needed to evaluate the evolution of the different subtypes and the efficacy of new pharmacological strategies. Procedures. We report the real-time tumorigenesis of MA Neuroblastoma mouse models: transgenic TH-MYCN mice and orthotopic xenograft models with either p53wt or p53mut, by non-invasive micro PET and bioluminescent imaging, respectively. Characterization of MYCN amplification and expression was performed on every collected sample. We tested the efficacy of a new MYCN inhibitor in vitro and in vivo. Results. MicroPET in TH-MYCN mice permitted the identification of Neuroblastoma at an early stage and offered a sensitive method to follow metabolic progression of tumors. The MA orthotopic model harboring multitherapy-related p53 mutations showed a shorter latency and progression and a stronger aggressiveness respect to the p53wt model. The presence of MA and overexpression was confirmed in each model and we saw a better survival in the TH-MYCN homozigous mice treated with the inhibitor. Conclusions. The mouse models obtained show characteristics of non-invasiveness, rapidity and sensitivity that make them suitable for the in vivo preclinical study of MA-NB. In particular, our firstly reported p53mut BLI xenograft orthotopic mouse model offers the possibility to evaluate the role of multitherapy-related p53 mutations and to validate new p53 independent therapies for this highly aggressive Neuroblastoma subtype. Moreover, we have shown potential clinical suitability of an antigene strategy through its cellular and molecular activity, ability to specifically inhibit transcription and in vivo efficacy with no evidence of toxicity.

In this work particular attention was given to the study of secondary metabolites produced by some plants belonging to the Amaryllidaceae family, in the specific case isoquinoline alkaloids. At the first instance were characterized both qualitatively and quantitatively three different plants belonging to Amaryllidaceae family, such as: Crinum angustum Steud., Pancratium illyricum L., and Leucojum nicaeense Ard. The alkaloids extracts obtained were separately tested against enzymes involved in specific diseases or liable in multifactorial pathologies, like: MMPs, AChE,and PPO. From leaves extract of P.illyricum was isolated a new compound, 11α-hydroxy-O-methylleucotamine, with important role in AChE inbition. Considering the protection role against external bodies carried out by these metabolites in plant, extracts were also assayed against ATCC microorganisms and clinical isolates. Plants with promising pharmacological activities have been the basis for development of in vitro plant models.

Neurodegenerative diseases are linked to altered splicing mechanisms (Mills et al., 2012). Fronto temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is one such disease that stems from the differential splicing caused due to mutations in Microtubule associated protein tau (MAPT) gene (Esther et al., 2002). This PhD thesis focuses on developing RNA-based therapeutic approaches to address FTDP-17. CHAPTER 1 introduces a broad range of topics such as splicing mechanism, neurodegenerative diseases associated with splice defects, therapeutic tools to modulate such splice defects in the context of neurogenetic diseases and possible applications of available tools for FTDP-17. CHAPTER 2 explores an exon skipping strategy to modulate splice defects in the context of FTD-17 using small nuclear RNAs (snRNAs). CHAPTER 3 is based on a short interfering RNA (siRNA) approach to modulate post-transcriptional gene silencing of specific isoform associated to FTDP-17. CHAPTER 4 employs long non coding RNA (lncRNA) to mediate post transcriptional repression of tau protein associated to FTDP-17 and deciphers its auxiliary role in splicing of exon 10 CHAPTER 5 elaborates on the future perspectives of all the above mentioned approaches to find a cure for FTDP-17.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. The search for new effective chemotherapeutic agents to treat GBM has proven challenging throughout the last few decades. As a result, very limited pharmacological treatment is currently available. GBM aggressiveness is associated with its glioblastoma stem cells (GSCs) component, which is responsible for resistance to therapy. Therefore, new specific pharmacological approaches directed to eradicate GSCs are endowed with a great therapeutic potential. GSCs have been shown to rely on mitochondrial respiration for their high energy demand. In order to have a functional mitochondrial respiration process, the five complexes forming the oxidative phosphorylation (OXPHOS) chain have to be built by the coordinate assembly of proteins translated by either the cytosolic or the mitochondrial ribosomes. Given their endosymbiotic origin and despite the evolutionary changes occurred the mitochondrial ribosomes (mitoribosomes) still share structural and functional similarities with the bacterial ones, particularly considering the functional ribosomal core. In the light of these similarities, we hypothesized that antibiotics targeting bacterial ribosomes could be exploited to inhibit mitoribosomes, affecting mitochondrial translation and OXPHOS assembly, and hence leading to detrimental effect on GSCs viability. We performed a high-content imaging driven screening of several bacterial ribosome targeting antibiotics and identified Drug A as the most promising compound due to its cytotoxic and mitotoxic effects on GSCs. We demonstrated that Drug A effectively prevents GSCs expansion, resulting to be over an order of magnitude more effective in GSCs growth inhibition than temozolomide, the only drug used in first line GBM therapy. We then investigated the mechanism of action of Drug A, proving that it inhibits mitochondrial translation and, as a consequence, it decreases the functionality of the OXPHOS complexes reducing mitochondrial respiration capacity. Moreover, we obtained the structure of this compound bound to the human mitoribosome using cryo-electron microscopy, which provides the basis for further development of more potent analogs. Finally we proved the efficacy of Drug A in vivo using a xenograft mouse model of GBM. Our results suggest that mitochondrial translation represents a therapeutic target for GBM and show that Drug A, acting via inhibition of mitochondrial translation, is extremely effective against GSCs. Given the urgent medical need for novel therapeutic approaches in GBM treatment, Drug A represents a promising therapeutic solution that is worth further preclinical and clinical investigations.

In recent years, advancements in computational methodologies have shed light on the complex process that makes proteins fold into their three-dimensional shapes. These new tools have helped us understand the steps proteins take to achieve these structures, revealing the presence of metastable intermediates along the folding pathways. This newfound understanding has led to the development of a novel drug discovery strategy known as Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT). This approach specifically targets folding intermediates to modulate protein expression levels, thus opening new opportunities for pharmacological intervention. This approach could be particularly relevant for diseases linked to targets that were previously considered "undruggable." A promising outcome of the PPI-FIT strategy is the identification of SM875, a compound that has been shown to lower prion protein (PrP) levels, positioning it as a potential therapeutic candidate for prion diseases. This study describes the initial phase of optimization of the SM875 scaffold. It encompasses the chemical diversification of SM875, followed by systematic evaluations of its biological activity and toxicity, with the aim of establishing structure-activity relationships (SAR). This knowledge is instrumental in guiding the synthesis of analogs with enhanced properties, advancing them through the development pipeline toward clinical application. Furthermore, this work investigates the potential regulatory function of folding intermediates in physiological processes, hypothesizing that they may serve as substrates for post translational modifications (PTMs). This hypothesis proposes an expansion of the current paradigm, suggesting that folding intermediates could constitute an additional layer of regulation within the complex network of proteostasis.