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I Junaid M, Lapins M, Eklund M, Spjuth O, Wikberg JE (2010) Proteochemometric modeling of the susceptibility of mutated variants of the HIV-1 virus to reverse transcriptase inhibitors. PLoS ONE 5: 1-10. II Salaemae W, Junaid M, Angsuthanasombat C, Katzenmeier G (2010) Struc- ture-guided mutagenesis of active site residues in the dengue virus two- component protease NS2B-NS3. J Biomed Sci 17: 68. III Junaid M, Chalayut C, Torrejon AS, Angsuthasombat C, Shutava I, Lapins M, Wikberg JE, Katzenmeier G (2012) Enzymatic analysis of recombinant Japanese encephalitis virus NS2B(H)-NS3pro with model peptide substrates. PloS ONE (Accepted). IV Prusis P, Junaid M, Petrovska R, Yahorava S, Yahorau A, Katzenmeier G, Lapins M, Wikberg JE (2012) Design and evaluation of substrate-based octapeptide and nonsubstrate-based tetrapeptide inhibitors of dengue virus NS2B/NS3 proteases. (Manuscript). V Junaid M, Lapins M, Katzenmeier G, Cui T, Wikberg JE (2012) Design and Evaluation of Dengue NS3 Protease Peptide Inhibitors. (Manuscript). Reprints were made with permission from the respective publishers. I have also contributed to the following article, which is not included in this thesis. This publication reports further development of the research de- scribed in paper I. VI Spjuth O, Eklund M, Lapins M, Junaid M, Wikberg JES (2010) Services for prediction of drug susceptibility for HIV proteases and reverse transcriptases at the HIV Drug Research Centre. Bioinformatics 27: 1719- 1720. Contents 1 Introduction ......................................................................................... 11 1.1 Modern Drug Discovery and Development (MDDD) Process ....... 11 1.2 Computational Methods in Drug Discovery and Design ................ 14 1.2.1 Virtual High-throughput Screening (V-HTS) ........................ 14 1.2.2 Structure-Based Drug Design (SBD) ..................................... 15 1.2.3 Quantitative Structure-Activity Relationship (QSAR) .......... 15 1.2.4 Statistical Molecular Design (SMD) ...................................... 15 1.2.5 Proteochemometrics (PCM) .................................................. 16 1.3 Enzyme as Drug Target in Modern Medicine ................................. 20 1.4 The Virus in Modern Medicine ....................................................... 20 1.5 Retroviruses .................................................................................... 22 1.5.1 HIV/AIDS .............................................................................. 22 1.5.2 Life Cycle of HIV .................................................................. 25 1.5.3 HIV/AIDS Drug Targets and Drugs ...................................... 26 1.5.4 Highly Active Antiretroviral Therapy (HAART) .................. 29 1.5.5 HIV Drug Resistance: The Bottle Neck ................................. 29 1.6 Flaviviruses ..................................................................................... 31 1.6.1 Dengue ................................................................................... 32 1.6.2 Japanese encephalitis (JE) ..................................................... 35 1.6.3 Life Cycle of Flavivirus ......................................................... 38 1.6.4 Flaviviral Drug Targets: NS2B-NS3 Protease ....................... 40 1.6.5 Drug Development for Flaviviral NS2B-NS3 Proteases ....... 42 2 Aims ..................................................................................................... 44 3 Results Discussion ............................................................................... 45 3.1 PCM analysis of HIV mutants susceptibility to NRTIs (paper I) ... 45 3.2 Kinetics of DEN NS2B(H)-NS3pro mutants (paper II) ................. 46 3.3 JEV NS2B(H)-NS3pro substrate specificity (paper III) ................ 48 3.4 DEN NS2B(H)-NS3pro inhibitors design & evaluation (papers IV & V) .............................................................................. 50 4 Conclusions and Perspectives .............................................................. 53 5 Acknowledgements.............................................................................. 55 6 References ........................................................................................... 57 Abbreviations Abz o-aminobenzoic acid Ac Acetyl ADE Antibody-dependent enhancement ADMET Absorption, distribution, metabolism, excretion, toxicity AIDS Acquired immunodeficiency syndrome amc 4-Methylcoumaryl-7-amide ARV Antiretroviral Boc tert-butoxycarbonyl CV Cross validation DEN Dengue virus DF Dengue fever DHF Dengue hemorrhagic fever DoE Design of experiments DSS Dengue shock syndrome ECV External cross validation FBD Fragment-based design FDA Food and drug administration FDS Fold decrease in susceptibility FeLV Feline leukemia virus GRT Genotypic resistance testing HAART Highly active anti-retroviral therapy HIV-1 Human immunodeficiency virus type 1 HIV-1RT HIV-1 Reverse transcriptase HTLV Human T-cell leukemia virus ICTV International Committee on Taxonomy of Viruses IND Investigational new drug JEV Japanese encephalitis virus kcat Turnover number, Rate of catalysis Ki Inhibition constant Km Michaelis constant LOOCV Leave-one-out cross validation NDA New drug application NNRTIs Non-nucleoside reverse transcriptase inhibitors NRTIs Nucleos(t)ide reverse transcriptase inhibitors NS2B(H) Non-structural protein 2B hydrophilic sequence NS3 Non-structural protein 3 NS3pro NS3 protease PAGE Polyacrylamide gel electrophoresis PCA Principal component analysis PCM Proteochemometrics PLS Partial least squares projections to latent structures PRT Phenotypic resistance testing QSAR Quantitative structure-activity relationship QSPR Quantitative structure-property relationship RdRp RNA-dependent RNA polymerase RS Rough sets SBD Structure-based drug design SED Statistical experimental design SIV Simian immunodeficiency virus SMD Statistical molecular design SOE-PCR Splicing over extension polymerase chain reaction SVM Support vector machine TBEV Tick-borne encephalitis virus V-HTS Virtual high-throughput screening VPT Virtual phenotypic testing WNV West Nile virus YFV Yellow fever virus 1 Introduction 1.1 Modern Drug Discovery and Development (MDDD) Process Contrary to traditional trial-and-error based drug design which involves testing of chemicals on tissues, organs or whole animals, the modern drug discovery process exploits knowledge about drug targets, and therefore, is referred to as rational drug design. Modern drug discovery is a lengthy, costly and complex process usually consisting of three stages, namely, discovery research, pre-clinical development and clinical development (Figure 1). A brief overview of different drug discovery and development phases is given below. 1) Drug Target Identification A drug target is a key biomolecule involved in a particular functional (meta- bolic or signaling) pathway that is specific to a pathological condition or to the survival or infectivity of a pathogen. The revolution in automation and information technologies has made the whole genome sequencing and proteome characterisation a routine task, and consequently has opened windows to new worlds of potential drug targets. 2) Target Validation The relevance of the target in a disease process or pathogenicity is a difficult task to prove and which has no general approach. A plausible relevance of a target maybe demonstrated by knock-out or knock-in animal models, site- directed mutagenesis studies [paper II], use of ribozymes, or neutralising antibodies, along with many other approaches. 3) Lead Discovery A ‘lead’ is a chemical compound that has pharmacological or biological activity, and has the potential to be optimised to a drug candidate. Large libraries (106-107) of diverse substances may be tested on drug tar- get using real (usually in vitro) or virtual (in silico) high-throughput screen- ing [1]. Generating millions of compounds and screening in the wet-lab is often practically unfeasible. Alternate approaches requiring fewer com- pounds include statistical molecular design (SMD) [paper V], structure- based design (SBD) [papers II, III], fragment-based design (FBD), pharma- cophore-based design (PBD), quantitative structure- activity/property rela- 11 tionship (QSAR/QSPR) and proteochemometrics (PCM) [paper I] and are methods of great importance that can be utilized in drug discovery processes.
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