In Silico Methodologies for Selection and Prioritization of Compounds in Drug Discovery

Ph.DDOCTOR OF PHILOSOPH

Development and application of modelling techniques in drug design

Structure-based drug design is a creative process that displays several features that make it closer to human reasoning than to machine automation. However, very often the user intervention is limited to the preparation of the input and analysis of the output of a computer simulation. In some cases, allowing human intervention directly in the process could improve the quality of the results by applying the researcher intuition directly into the simulation. Haptic technology has proven to be a useful method to interact in realtime with a virtual environment, enriching the user's experience and allowing for a more natural and direct interaction with three-dimensional systems. Reported in this thesis is the design and implementation of a user-driven computer program for structure-based drug design based on haptic technology and char acterised by a trimodal feedback system and its application alongside more traditional approaches to drug design projects in the anticancer and antiviral area. The software proved to be very useful in several projects, validating the applicability of haptic technology to drug design. The results were in good agreement with those obtained by traditional techniques. Moreover the approach resulted in the identification of novel HCV inhibitors and a putative inhibitor of the dimerisation of EGFR which resulted active in vitro tests

Computer aided drug design

Hepatitis C virus (HCV) chronic infection represents one of the major and still unresolved health problems. HCV infecting 3% of the world population, leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in addition to the extrahepatic manifestations. No efficient therapy exists; the standard dual treatment with peg IFN-alpha and ribavirin is effective only in 55% of the selected cases with substantial side effects in addition to the high cost. To date, there is no vaccine against HCV due to the high variability of the RNA genome. NS3 helicase is one of the non-structural proteins whose activity is indispensable for viral RNA replication and its inhibition is estimated to arrest viral proliferation and indirectly stimulate a cellular antiviral response against ds RNA. In our project we proposed to use structure based knowledge of the x-ray crystal structure of helicase enzyme to design and synthesise different scaffolds of novel potential HCV NS3 helicase inhibitors. Using different computer software packages, we manage to design a number of small focused libraries of compounds, which were used for docking simulations. The results obtained in silico guided the selection of two series of promising compounds for synthesis. In the first series; several quinazoline derivatives were prepared and evaluated for antiviral activity in subgenomic replicon assay showing EC50 in the low muM range with relatively high selectivity index. In the second series of pyrrole or phenyl based compounds, irreversible inhibition of helicase is assumed through addition to the electrophilic warheads of the alpha,beta-unsaturated ketones, thiols or 1,2,4 thiadiazoles based inhibitors. Among the synthesised compounds a number showed a sub muM activity in the helicase enzyme assay. These promising findings are considered to be a starting point for further optimisation of structure, activity and toxicity relationships

A computational approach for identification and development of novel inhibitors targeting viral polymerases

Positive strand RNA viruses, which include hepatitis C virus (HCV), human immunodeficiency virus (HIV, and Bovine Viral Diarrhea Virus (BVDV), are known to create havoc for humans and animal health alike. Although vaccines have helped to control several of the most important viral pathogens, there is currently little prospect of an effective vaccine for either HCV or HIV. These pathogens infect ~170 million and ~40 million people worldwide, respectively, hastening the need for effective antiviral drugs. Likewise BVDV infects domesticated livestock causing significant economic losses worldwide. The development of new, effective antiviral compounds for combating these debilitating human (HIV and HCV) and animal pathogen (BVDV) is therefore of paramount importance, and is the focus of this thesis. Herein, polymerases of three positive strand RNA viruses, viz HCV, BVDV and HIV have been targeted with the goal of improving the efficacy of antivirals against wide range of resistant mutations. Lack of effective therapies for these viral infections as most of the established treatments are not always effective or well tolerated, highlights an urgent need for further refinement and development of antiviral drugs. It is not only the specific need that has inspired this work but also the idea to test and develop protocols that might enable a more rational structurebased drug design to be performed by keeping a tradeoff among rapidity, accuracy, and efficacy. Traditional methods for general drug discovery typically include evaluating random compound libraries for activity in relevant cellfree or cellbased assays. Success in antiviral development has emerged from the discovery of more focused libraries that provide clues about structureactivity relationships. Combining these with more recent approaches including structural biology and computational modeling can work efficiently to hasten discovery of active molecules. The ability to design drugs interfering with the progression of infection of virus comes with i)the knowledge of pathological, cellular and molecular mechanism involved in the disease; and ii)the identification of macromolecule (i.e possible drug target) involved in pathological pathways, their 3D structures and their functions. The biological activity of drug molecules is dependent on the threedimensional arrangement of its functional groups, which specifically bind to their target. Consequently, the structural information of the target protein is essential in drug development. Proteins are dynamic molecules and often undergo conformational change upon ligand binding. The flexible loop regions and in general the flexibility of the structure have a critical functional role in enzymes, but those features and their connection with the functionality of protein are hard to retrieve from xray, NMR techniques and cryoEM techniques. Being aware of the importance of the relationship structurefunction and structureactivity at large, i.e., including dynamics and interactions with solvent, in our work we are trying to address some of the relevant problems of drug development; basic key determinants in proteinligand stability, mechanism of inhibition, why and how, flexibility and collective motion of the protein is essential part in improvement of rational drug design, how mutation renders the protein resistant again potent drugs; the effect of resistance mutation on the flexibility and stability of protein, what is the mechanism of drug resistance, change in energetics consequences, affecting the conformation in wild and mutant systems. Various biophysical techniques of the computational arsenal we have applied have provided huge wealth of information related to protein dynamics and proteinligand recognition. These methods have grown in their effectiveness not only by offering a deeper understanding of the basic science, the biological events and molecular interactions that define a target for therapeutic intervention, but also because of advances in algorithms, representations, and mathematical procedures for studying such processes. This work represents the application of several computational techniques, such as docking, molecular dynamics, algorithms to calculate free energy of binding of ligands into the binding pocket (ex MMPBSA) and algorithms to study rare events (for ex. binding and unbinding of ligand from the binding site, Metadynamics) to explore, at microscopic level, the key pattern of interaction between protein and ligand, to understand the effect of mutations, to get an insight of the full docking and undocking path and to calculate binding energetics.

Computer-aided design, synthesis and evaluation of potential anti-HCV agents

Hepatitis C virus (HCV) is a major cause of chronic liver disease, leading to hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. A vaccine is currently not available, while the standard of care is effective in only 50% of treated patients. The first specific anti-HCV drugs have been recently approved, and new classes of targeted agents are under clinical trials/investigation. Nevertheless, improved treatment strategies are needed, in order to bypass the rapid emergence of resistance. All the viral non-structural proteins are a possible target for the identification of novel and selective antivirals. Among them, the NS3 helicase is still underexploited, with no known inhibitor under pre-clinical or clinical development. This enzyme plays a crucial role in the virus life cycle: it catalyses the separation of double-stranded RNA strands, which is necessary for genome amplification and translation. Due to its essential function, the NS3 helicase was chosen as a target for the identification of new, specific anti-HCV compounds. Different computer-aided techniques were employed to identify potential smallmolecule inhibitors of the enzyme. Two structure-based virtual screenings of commercially available compounds were performed on the main nucleic acid binding site. A series of candidate inhibitors was evaluated in the HCV replicon assay, yielding two primary hits with low μM activity. Secondly, the model of the one known inhibitor co-crystallised with the enzyme was used as a starting point for a shape-comparison screening of small molecule libraries. A new series of compounds was selected and evaluated for anti-HCV activity, and one of them was found to inhibit the viral replication at a low μM concentration. Several new derivatives of the initial hits were synthesised, belonging to four main structural families: bis-aromatic piperazine derivatives, symmetrical phenylendiamine compounds, differently substituted thieno-pyrimidines, and triphenyl-pyrrolone analogues. Inhibition of HCV replication in the replicon assay was evaluated for the new compounds prepared and several structures showed a range of activity from low-μM to n

Computer-aided design and synthesis of novel anti-DENV nucleoside analogues

Dengue virus (DENV) is one of the most important human pathogens among the genus flavivirus, with 3.9 billion people at risk of infection through mosquitoes, such as the widely spread ‘Asian tiger’ mosquitoes, and the four serotypes of DENV are endemic in over 100 countries in tropical and subtropical regions. Clinical manifestations of infection with DENV range from flu-like symptoms to the life-threatening dengue haemorrhagic fever. The dramatic increase in the incidence of the DENV infection, the rapid spread of DENV to new areas and the recent re-emergence of another member of the genus flavivirus, Zika virus (ZIKV), have highlighted the urgent need for specific antiviral therapies against infections with DENV and related viruses, which are not currently available. DENV RNA-dependent RNA polymerase (RdRp), the enzyme responsible for the synthesis of the viral genome, is one of the most attractive targets for the development of direct acting antiviral agents but its molecular mechanisms are poorly understood. Thefore, the aims of this PhD project were i) to build a model of the de novo initiation complex of DENV RdRp, of which there is currently no crystal structure available, ii) in silico design and synthesis of novel nucleoside and nucleotide analogues as potential inhibitors of DENV replication, iii) and finally to investigate the mechanism of the RNA synthesis by DENV RdRp. Molecular modelling techniques allowed for the creation of a model of the de novo initiation complex. The application of in silico drug design approaches resulted in the identification of three families of promising adenosine analogues: ribose-modified, nucleobase-modified and acyclic adenosine analogues. Strategies for the preparation of these nucleosides were investigated and ten adenosine analogues and eight nucleotide prodrugs, which are phosphoramidate ProTides, of specific nucleosides were synthesised and sent for biological evaluation in vitro. Innovative microwave irradiation conditions for the preparation of phosphoramidate ProTides were developed and successfully applied to synthesised nucleoside analogues. Finally, the application of molecular dynamics simulation methods on different complexes of DENV RdRp provided insights on the conformational changes of DENV RdRp during the synthesis of the viral genome. These results contributed to the understanding of DENV RdRp activity and will aid the design of inhibitors of the viral replication

Synthesis and biological evaluation of nitrogen heterocycle systems as potential antiviral agents

Viruses are obligate intracellular parasites that consist of either double- or single-stranded DNA or RNA enclosed in a protein coat called capsid. Some viruses also possess a lipid envelope that, like the capsid, may contain antigenic glycoproteins. Most of them contain or encode enzymes essential for their replicative cycle inside host cells, sometimes usurping their metabolic machinery. Traditional therapeutic approaches have mostly focused on targeting specific viral components or enzymes. This pathogen-directed strategy, while successful in numerous cases, in many others results ineffective due to the emergence of drug-resistance. A different approach, addressed to target host-factors essential for viral replication, has recently draw an increasing attention. My PhD project aimed at synthesizing new nitrogen heterocycle systems, designed especially against RNA viruses, such as those belonging to Flaviviridae, Orthomyxoviridae and Paramyxoviridae families. Among them there are, respectively, pathogens responsible for diseases with a high epidemiological impact, as BVDV in cattle and HCV in humans, influenza A and B viruses and respiratory syncytial virus (RSV). The project has been organized into the following phases: 1. Chemical synthesis of the novel compound series. During my PhD I designed and synthesized diverse chemical series of different chemotypes, in order to obtain new antiviral agents: the acridine nucleus, the dihydrotriazine scaffold, the benzimidazole ring as well as anilino and benzenesulfonamide derivatives. Previous studies performed by the research group where I develop my Ph.D. thesis identified some prototypes for the different classes endowed with intrinsic antiviral activity; thus, during my Ph.D. research work I explored various possibilities of functionalisation with the aim of increasing their potency and selectivity profiles towards the respective antiviral target. 2. Characterization of the new compounds. Each newly synthesized compound have been characterised by spectroscopic methods (such as UV, IR, 1H-NMR and 13C-NMR) and elemental analysis. 3. Evaluation of cytotoxicity, antiviral activity in vitro, enzymatic assays and computational studies have been performed in collaboration with several national and international research groups, to assess the biological activity and to identify/confirm the respective molecular targets

Structure-based approaches applied to the study of pharmaceutical relevant targets

Computer Aided Drug Design/Discovery methods became complementary to traditional and modern drug discovery approaches. Indeed CADD is useful to improve and speed up the detection and the optimization of bioactive molecules. The present study is focused on the application of structure-based approaches to the study of pharmaceutical relevant targets. The introduction provides a quick overview on the fundamentals of computational chemistry and structure-based methods, while in the successive chapters the main targets investigated through these methods are treated. In particular we focused our attention on Reverse Transcriptase of HIV-1, Monoamine oxidase B and VP35 of Ebola virus. The last chapter is dedicated to the validation of covalent docking performed with Autodock