Design and Synthesis of Naphthalene Diimides Based Small Molecules as Anticancer Agents: Targeting the Polyamine Transporter, G-quadruplex Structures and HDAC

Cancer is a multifactorial disease characterized by a very complex etiology. Basing on its complex nature, a promising therapeutic strategy could be based by the “Multi-Target-Directed Ligand” (MTDL) approach, based on the assumption that a single molecule could hit several targets responsible for the pathology. Several agents acting on DNA are clinically used, but the severe deriving side effects limit their therapeutic application. G-quadruplex structures are DNA secondary structures located in key zones of human genome; targeting quadruplex structures could allow obtaining an anticancer therapy more free from side effects. In the last years it has been proved that epigenetic modulation can control the expression of human genes, playing a crucial role in carcinogenesis and, in particular, an abnormal expression of histone deacetylase enzymes are related to tumor onset and progression. This thesis deals with the design and synthesis of new naphthalene diimide (NDI) derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development, such as HDACs. NDI-polyamine and NDI-polyamine-hydroxamic acid conjugates have been designed with the aim to provide potential MTDLs, in order to create molecules able simultaneously to interact with different targets involved in this pathology, specifically the G-quadruplex structures and HDAC, and to exploit the polyamine transport system to get selectively into cancer cells. Macrocyclic NDIs have been designed with the aim to improve the quadruplex targeting profile of the disubstituted NDIs. These compounds proved the ability to induce a high and selective stabilization of the quadruplex structures, together with cytotoxic activities in the micromolar range. Finally, trisubstituted NDIs have been developed as G-quadruplex-binders, potentially effective against pancreatic adenocarcinoma. In conclusion, all these studies may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Development and optimization of techniques and design parameters for the engineering of atmospheric pressure plasma devices for biomedical applications and plasma medicine

In the last decades, the possibility to generate plasma at atmospheric pressure gave rise to a new emerging field called plasma medicine; it deals with the application of cold atmospheric pressure plasmas (CAPs) or plasma-activated solutions on or in the human body for therapeutic effects. Thanks to a blend of synergic biologically active agents and biocompatible temperatures, different CAP sources were successfully employed in many different biomedical applications such as dentistry, dermatology, wound healing, cancer treatment, blood coagulation, etc.… Despite their effectiveness has been verified in the above-mentioned biomedical applications, over the years, researchers throughout the world described numerous CAP sources which are still laboratory devices not optimized for the specific application. In this perspective, the aim of this dissertation was the development and the optimization of techniques and design parameters for the engineering of CAP sources for different biomedical applications and plasma medicine among which cancer treatment, dentistry and bioaerosol decontamination. In the first section, the discharge electrical parameters, the behavior of the plasma streamers and the liquid and the gas phase chemistry of a multiwire device for the treatment of liquids were performed. Moreover, two different plasma-activated liquids were used for the treatment of Epithelial Ovarian Cancer cells and fibroblasts to assess their selectivity. In the second section, in accordance with the most important standard regulations for medical devices, were reported the realization steps of a Plasma Gun device easy to handle and expected to be mounted on a tabletop device that could be used for dental clinical applications. In the third section, in relation to the current COVID-19 pandemic, were reported the first steps for the design, realization, and optimization of a dielectric barrier discharge source suitable for the treatment of different types of bioaerosol.

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 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.

Applied deep learning and data science with a human-centric and data-centric approach

The term Artificial intelligence acquired a lot of baggage since its introduction and in its current incarnation is synonymous with Deep Learning. The sudden availability of data and computing resources has opened the gates to myriads of applications. Not all are created equal though, and problems might arise especially for fields not closely related to the tasks that pertain tech companies that spearheaded DL. The perspective of practitioners seems to be changing, however. Human-Centric AI emerged in the last few years as a new way of thinking DL and AI applications from the ground up, with a special attention at their relationship with humans. The goal is designing a system that can gracefully integrate in already established workflows, as in many real-world scenarios AI may not be good enough to completely replace its humans. Often this replacement may even be unneeded or undesirable. Another important perspective comes from, Andrew Ng, a DL pioneer, who recently started shifting the focus of development from “better models” towards better, and smaller, data. He defined his approach Data-Centric AI. Without downplaying the importance of pushing the state of the art in DL, we must recognize that if the goal is creating a tool for humans to use, more raw performance may not align with more utility for the final user. A Human-Centric approach is compatible with a Data-Centric one, and we find that the two overlap nicely when human expertise is used as the driving force behind data quality. This thesis documents a series of case-studies where these approaches were employed, to different extents, to guide the design and implementation of intelligent systems. We found human expertise proved crucial in improving datasets and models. The last chapter includes a slight deviation, with studies on the pandemic, still preserving the human and data centric perspective.

Innovation in conceptual design of chemical processes

Nowadays, the chemical industry has reached significant goals to produce essential components for human being. The growing competitiveness of the market caused an important acceleration in R&D activities, introducing new opportunities and procedures for the definition of process improvement and optimization. In this dynamicity, sustainability is becoming one of the key aspects for the technological progress encompassing economic, environmental protection and safety aspects. With respect to the conceptual definition of sustainability, literature reports an extensive discussion of the strategies, as well as sets of specific principles and guidelines. However, literature procedures are not completely suitable and applicable to process design activities. Therefore, the development and introduction of sustainability-oriented methodologies is a necessary step to enhance process and plant design. The definition of key drivers as support system is a focal point for early process design decisions or implementation of process modifications. In this context, three different methodologies are developed to support design activities providing criteria and guidelines in a sustainable perspective. In this framework, a set of key Performance Indicators is selected and adopted to characterize the environmental, safety, economic and energetic aspects of a reference process. The methodologies are based on heat and material balances and the level of detailed for input data are compatible with available information of the specific application. Multiple case-studies are defined to prove the effectiveness of the methodologies. The principal application is the polyolefin productive lifecycle chain with particular focus on polymerization technologies. In this context, different design phases are investigated spanning from early process feasibility study to operative and improvements assessment. This flexibility allows to apply the methodologies at any level of design, providing supporting guidelines for design activities, compare alternative solutions, monitor operating process and identify potential for improvements.