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The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5–5.3 μM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 μM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets., Graphical abstract Image 1

In the last few decades, the constant development of novel microscopy techniques have created the basis for a new paradigm in the field of biophysics. Single-molecule techniques enabled to carry out experiments providing new information: the nanomanipulation of individual biomolecules revealed unknown insights into the elasticity and mechanics of molecules, improving the understanding of the fundamental relation between structural properties and biological functions. In particular, an AFM and mostly a MT setup were used during this thesis work, both located in biophysics laboratory of Prof. Francesco Mantegazza, at the University of Milano-Bicocca. Similar issues were encountered at the cellular level, because bulk experiments of conventional microscopy techniques provide information on average only, without taking into account the intrinsic biological heterogeneity. Recent developments in microfluidics enabled to follow individual cells over a long time and under controlled conditions. During the last part of this thesis project I used one of these microfluidic devices to perform time-lapse microscopy experiments at the single-cell level. These experiments were carried out during a visiting period of seven months in Prof. Pietro Cicuta’s laboratory, in Cavendish laboratory at University of Cambridge. In this thesis I dealt with three main research topics: • DNA structural polymorphism • nanomechanics of DNA-ligand interactions • the dual role of H-NS protein: DNA condensation and gene regulation The study of the conformational changes of DNA, namely the property of structural polymorphism, is addressed during two projects: one about the nanomechanics of a DNA analogue and another concerning the behavior of DNA at high supercoiling. The study of a DNA analogue enables to observe how a chemical modification of nucleotides can induce structural re- arrangements of the double-helix, biasing towards an A-like-form of DNA. The regimes of high supercoiling, both positive and negative supercoiling, show instead how an applied torsion at a certain forces can promote the formation of plectonemes or denaturation bubbles, which are conditions that favor particular structural transitions. The second major theme concerns the analysis of the nanomechanics of DNA-ligand complexes, particularly the interactions of DNA with anticancer drugs or with the H-NS protein and the crowding agent PEG. The project about the interactions between DNA and drugs clearly shows how the mechanical properties and the stability of DNA change due to the binding with compounds commonly used in clinics to treat tumors. On the other hand, the H-NS protein forms relatively stable DNA loops and influences the stability of the double helix, as well as the crowding agent. The protein binding mechanism has a preference for some DNA sequences and an unexpected concentration-dependent behavior. The analysis of the the DNA-H-NS interactions also enables, particularly in crowding conditions, to better understand the mechanism of DNA condensation inside the cell, one of the biological roles of H-NS. The second important function of this NAP is the gene regulation. To investigate the dual role of H-NS in great detail two complementary techniques have been combined. The nanoma- nipulation technique is employed to observe the structural role of H-NS and its combined activity with a crowding agent leading to a clear and abrupt compaction of DNA. Time-lapse fluorescence microscopy is instead used to study the regulatory role of the protein, more precisely the gene silencing mechanism, at the single-cell level. This activity has also a strong influence in the cell physiology, by significantly changing the growth rate of bacteria. In the last few decades, the constant development of novel microscopy techniques have created the basis for a new paradigm in the field of biophysics. Single-molecule techniques enabled to carry out experiments providing new information: the nanomanipulation of individual biomolecules revealed unknown insights into the elasticity and mechanics of molecules, improving the understanding of the fundamental relation between structural properties and biological functions. In particular, an AFM and mostly a MT setup were used during this thesis work, both located in biophysics laboratory of Prof. Francesco Mantegazza, at the University of Milano-Bicocca. Similar issues were encountered at the cellular level, because bulk experiments of conventional microscopy techniques provide information on average only, without taking into account the intrinsic biological heterogeneity. Recent developments in microfluidics enabled to follow individual cells over a long time and under controlled conditions. During the last part of this thesis project I used one of these microfluidic devices to perform time-lapse microscopy experiments at the single-cell level. These experiments were carried out during a visiting period of seven months in Prof. Pietro Cicuta’s laboratory, in Cavendish laboratory at University of Cambridge. In this thesis I dealt with three main research topics: • DNA structural polymorphism • nanomechanics of DNA-ligand interactions • the dual role of H-NS protein: DNA condensation and gene regulation The study of the conformational changes of DNA, namely the property of structural polymorphism, is addressed during two projects: one about the nanomechanics of a DNA analogue and another concerning the behavior of DNA at high supercoiling. The study of a DNA analogue enables to observe how a chemical modification of nucleotides can induce structural re- arrangements of the double-helix, biasing towards an A-like-form of DNA. The regimes of high supercoiling, both positive and negative supercoiling, show instead how an applied torsion at a certain forces can promote the formation of plectonemes or denaturation bubbles, which are conditions that favor particular structural transitions. The second major theme concerns the analysis of the nanomechanics of DNA-ligand complexes, particularly the interactions of DNA with anticancer drugs or with the H-NS protein and the crowding agent PEG. The project about the interactions between DNA and drugs clearly shows how the mechanical properties and the stability of DNA change due to the binding with compounds commonly used in clinics to treat tumors. On the other hand, the H-NS protein forms relatively stable DNA loops and influences the stability of the double helix, as well as the crowding agent. The protein binding mechanism has a preference for some DNA sequences and an unexpected concentration-dependent behavior. The analysis of the the DNA-H-NS interactions also enables, particularly in crowding conditions, to better understand the mechanism of DNA condensation inside the cell, one of the biological roles of H-NS. The second important function of this NAP is the gene regulation. To investigate the dual role of H-NS in great detail two complementary techniques have been combined. The nanoma- nipulation technique is employed to observe the structural role of H-NS and its combined activity with a crowding agent leading to a clear and abrupt compaction of DNA. Time-lapse fluorescence microscopy is instead used to study the regulatory role of the protein, more precisely the gene silencing mechanism, at the single-cell level. This activity has also a strong influence in the cell physiology, by significantly changing the growth rate of bacteria.

Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.