Tesi Definitiva DMST Elisa Rita Ceresola

Tesi Definitiva DMST Elisa Rita Ceresola

CHARACTERIZATION OF NEW SYNTHETIC OR NATURAL COMPOUNDS WITH BROAD SPECTRUM OR DUAL ANTIVIRAL ACTIVITY IN HIV-1 TREATMENT PhD Degree Program in Experimental and Translational Medicine UNIVERSITY OF INSUBRIA Ph.D. Candidate: Elisa Rita Ceresola Supervisor: Prof. Filippo Canducci Academic year 2017-2018 Alla Luce che guida i miei passi, ai sorrisi innocenti che danno gioia alla mia vita 2 SUMMARY 1 INTRODUCTION ................................................................................................................6 1.1 CURRENTLY AVAILABLE DRUGS FOR HIV-1 TREATMENT ...........................6 1.2 HIV INTEGRASE ...................................................................................................... 10 1.2.1 MECHANISM OF VIRAL INTEGRATION ........................................................ 10 1.2.2 STRUCTURE OF HIV INTEGRASE ................................................................... 12 1.2.3 HIV INTEGRASE INIBITHORS (INI) ................................................................. 13 1.2.3.1 Integrase Strand Transfer Inhibitors (INSTIs) .................................................... 14 1.2.3.2 Allosteric Integrase Inhibitors (ALLINIs) .......................................................... 17 1.2.3.3 Dual inhibitors .................................................................................................. 20 1.3 HIV-1 MICROBICIDES ............................................................................................ 23 1.3.1 THE ENTRY PROCESS....................................................................................... 23 1.3.1.1 HIV-1 Entry Process ......................................................................................... 23 1.3.1.2 The relationship between HIV-1 and HSV-2...................................................... 25 1.3.1.3 HSV-2 entry process ......................................................................................... 28 1.3.2 TOPICAL MICROBICIDES ................................................................................. 29 1.3.2.1 Past, present and future of anti-HIV-1 and HSV-2 microbicides ........................ 31 1.3.2.2 The delivery system .......................................................................................... 35 1.4 AIM OF THE WORK ................................................................................................ 38 2 MATERIAL AND METHODS .......................................................................................... 40 2.1 MATERIALS AND METHODS (PART I) ................................................................ 40 2.1.1 BIOCHEMICAL STUDIES .................................................................................. 40 2.1.1.1 Preparation of recombinant proteins .................................................................. 40 2.1.1.2 HTRF LEDGF-dependent and -independent assay ............................................. 40 2.1.1.3 HTRF-based integrase-LEDGF interaction assay ............................................... 41 2.1.1.4 HTRF-based IN subunit exchange assay ............................................................ 41 2.1.1.5 RT Protein expression and purification .............................................................. 42 2.1.1.6 RNase H polymerase-independent cleavage assay ............................................. 42 2.1.1.7 DNA polymerase assay ..................................................................................... 42 2.1.2 CELLULAR BASED EXPERIMENT................................................................... 43 2.1.2.1 Cell-based assays .............................................................................................. 43 2.1.2.2 Time-of-addition assay (TOA) .......................................................................... 43 2.2 MATERIALS AND METHODS (PART II) .............................................................. 45 3 2.2.1 CHEMISTRY ....................................................................................................... 45 2.2.2 ANTIVIRAL ACTIVITY AND CELL TOXICITY ............................................... 48 2.2.2.1 Molecular cloning ............................................................................................. 48 2.2.2.2 Phenotypic analyses with fully replicating recombinant HIV-1 strains and clonal viral variants selected in patients failing integrase inhibitors on TZM-bl cells and human CD4+ lymphocyte ............................................................................................................ 49 2.2.2.3 HSV-1 and HSV-2 antiviral activity in Vero Cells ............................................. 50 2.2.2.4 Vero Cell viability ............................................................................................. 51 2.2.2.5 Human papilloma pseudovirus (HPV16 PsV) production ................................... 51 2.2.2.6 HPV-16 inhibition assay in HeLa cells .............................................................. 52 2.2.2.7 Inhibition of antiviral activity by fetal bovine serum .......................................... 52 2.2.2.8 Inhibition of antiviral activity by purified bovine serum albumin ....................... 53 2.2.2.9 Antiviral activity of compound 2 in a gel formulation ........................................ 53 2.2.2.10 Time of addition assay on compound 2 .......................................................... 54 2.2.3 ADME ASSAY .................................................................................................... 54 2.2.3.1 Chemicals and Excipients .................................................................................. 54 2.2.3.2 UV/HPLC-MS method ...................................................................................... 54 2.2.3.3 Aqueous Solubility ............................................................................................ 55 2.2.3.4 Parallel Artificial Membrane Permeability Assay (PAMPA) .............................. 55 2.2.3.5 Metabolic Stability in HLM (Human Liver Microsomes) ................................... 56 2.2.3.6 Solubility assessment ........................................................................................ 56 2.2.3.7 Gel formulation ................................................................................................. 57 2.2.3.8 Storage Stability and rheological characterization .............................................. 57 2.2.3.9 Binding Fluorimetric Assay ............................................................................... 57 3 RESULTS ........................................................................................................................... 59 3.1 RESULTS (PART I) ................................................................................................... 59 3.1.1 HOMOGENEOUS TIME-RESOLVED FLUORESCENCE (HTRF) ASSAYS ..... 59 3.1.2 CELL-BASED HIV REPLICATION ASSAY....................................................... 61 3.1.2.1 Cell based assay on fully replicating HIV-1 laboratory strain ............................. 61 3.1.2.2 Cell based assay on INSTI-resistant mutants...................................................... 61 3.1.2.3 TOA experiment ............................................................................................... 62 3.1.3 BIOCHEMICAL ASSAYS ON RT FUNCTIONS ................................................ 63 3.2 RESULTS (PART II).................................................................................................. 65 3.2.1 ANTIVIRAL ACTIVITIES OF RHODANINE DERIVATIVES ........................... 65 3.2.1.1 Antiviral activity and cytotoxicity on TZM-bl cell line infected with HIV-1 laboratory strains .............................................................................................................. 65 3.2.1.2 Antiviral activity and cytotoxicity on human CD4+ lymphocytes ....................... 67 4 3.2.1.3 Antiviral activity and cytotoxicity on Vero cell line infected with HSV-2 laboratory strain and HeLa infected with HPV-16 PsVs .................................................... 68 3.2.1.4 Binding to Fetal Bovine Serum.......................................................................... 70 3.2.1.5 Binding to Bovine purified serum Albumin ....................................................... 71 3.2.1.6 Antiviral activity of compound 2 in a microbicide gel formulation ..................... 72 3.2.1.7 Time of addition assay on compound 2 .............................................................. 72 3.2.2 IN VITRO ADME STUDIES ................................................................................. 74 3.2.2.1 Solubility, permeability and metabolic stability ................................................. 74 3.2.2.2 Albumin binding fluorimetric assay ................................................................... 74 3.2.2.3 Storage Stability of compound 2 Gel Formulation ............................................. 75 4 DISCUSSION ..................................................................................................................... 76 5 CONCLUSIONS ................................................................................................................ 85 6 REFERENCES ................................................................................................................... 87 7 ACKNOLEDGMENTS ...................................................................................................... 98 5 1 INTRODUCTION 1.1 CURRENTLY

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