As doenças tropicais têm sido alvo de constantes pesquisas nos últimos anos, em diversos centros em todo o mundo. A procura por novos tratamentos eficazes contra estas desordens estimula a identificação de novos alvos moleculares. A esquistossomose é uma doença parasitária crônica, que afeta cerca de 200 milhões de indivíduos em todo o mundo, causada pelo platelminto trematodo do gênero Schistosoma. No Brasil, a única espécie encontrada é o Schistosoma mansoni. Neste trabalho de mestrado, a enzima purina nucleosídeo fosforilase (PNP, EC 2.4.2.1) do parasita Schistosoma mansoni foi selecionada como alvo molecular para a identificação de novos inibidores e sua caracterização cinética através de potência biológica, seletividade e mecanismo de ação. Adicionalmente, dois complexos cristalográficos foram obtidos na presença de inibidores potentes da PNP, tornando-se assim parâmetros para a modelagem molecular empregando a técnica de docagem. Os estudos computacionais permitiram boa compreensão das interações intermoleculares, fornecendo as bases para um estudo detalhado das relações entre a estrutura e atividade destes compostos. Esse conjunto de informações será de grande utilidade nas etapas de planejamento de novas moléculas bioativas mais potentes e seletivas, candidatas a agentes quimioterápicos contra a esquistossomose. / Tropical diseases have been a target of constant research for several years. The search for new efficient treatments against these parasitic diseases has been stimulating the identification of new molecular targets and new drug candidates. Schistosomiasis is a chronic parasitic disease, which affects about 200 million people in the world, caused by a trematode worm of Schistosoma genus. In Brazil, Schistosoma mansoni is the only specie found. In this Master´s dissertation, the enzyme purine nucleoside phosphorylase (PNP, EC 2.4.2.1) from S. mansoni was selected as a molecular target for the identification and kinetic characterization, including potency, affinity and mechanism of action, of new inhibitors from two chemical classes. In addition, two complexes enzyme-inhibitor were characterized by X-ray diffraction, and were useful for molecular modeling studies employing the docking method. These provided important insights about the main intermolecular interactions responsible for the process of molecular recognition and enzyme inhibition. The information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of S. mansoni PNP having increased potency and affinity.

Li, Hongjian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (p. 132-156). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Motivation --- p.2 / Chapter 1.2 --- Objective --- p.2 / Chapter 1.3 --- Method --- p.3 / Chapter 1.4 --- Outline --- p.4 / Chapter 2 --- Background --- p.7 / Chapter 2.1 --- Overview of the Pharmaceutical Industry --- p.7 / Chapter 2.2 --- The Process of Modern Drug Discovery --- p.10 / Chapter 2.2.1 --- Development of an Innovative Idea --- p.10 / Chapter 2.2.2 --- Establishment of a Project Team --- p.11 / Chapter 2.2.3 --- Target Identification --- p.11 / Chapter 2.2.4 --- Hit Identification --- p.12 / Chapter 2.2.5 --- Lead Identification --- p.13 / Chapter 2.2.6 --- Lead Optimization --- p.14 / Chapter 2.2.7 --- Clinical Trials --- p.14 / Chapter 2.3 --- Drug Discovery via Computational Means --- p.15 / Chapter 2.3.1 --- Structure-Based Virtual Screening --- p.16 / Chapter 2.3.2 --- Computational Synthesis of Potent Ligands --- p.20 / Chapter 2.3.3 --- General-Purpose Computing on GPU --- p.23 / Chapter 3 --- Approximate Matching of DNA Patterns --- p.26 / Chapter 3.1 --- Problem Definition --- p.27 / Chapter 3.2 --- Motivation --- p.28 / Chapter 3.3 --- Background --- p.30 / Chapter 3.4 --- Method --- p.32 / Chapter 3.4.1 --- Binary Representation --- p.32 / Chapter 3.4.2 --- Agrep Algorithm --- p.32 / Chapter 3.4.3 --- CUDA Implementation --- p.34 / Chapter 3.5 --- Experiments and Results --- p.39 / Chapter 3.6 --- Discussion --- p.44 / Chapter 3.7 --- Availability --- p.45 / Chapter 3.8 --- Conclusion --- p.47 / Chapter 4 --- Structure-Based Virtual Screening --- p.50 / Chapter 4.1 --- Problem Definition --- p.51 / Chapter 4.2 --- Motivation --- p.52 / Chapter 4.3 --- Medicinal Background --- p.52 / Chapter 4.4 --- Computational Background --- p.59 / Chapter 4.4.1 --- Scoring Function --- p.59 / Chapter 4.4.2 --- Optimization Algorithm --- p.65 / Chapter 4.5 --- Method --- p.68 / Chapter 4.5.1 --- Scoring Function --- p.69 / Chapter 4.5.2 --- Inactive Torsions --- p.72 / Chapter 4.5.3 --- Optimization Algorithm --- p.73 / Chapter 4.5.4 --- C++ Implementation Tricks --- p.74 / Chapter 4.6 --- Data --- p.75 / Chapter 4.6.1 --- Proteins --- p.75 / Chapter 4.6.2 --- Ligands --- p.76 / Chapter 4.7 --- Experiments and Results --- p.77 / Chapter 4.7.1 --- Program Validation --- p.77 / Chapter 4.7.2 --- Virtual Screening --- p.81 / Chapter 4.8 --- Discussion --- p.89 / Chapter 4.9 --- Availability --- p.90 / Chapter 4.10 --- Conclusion --- p.91 / Chapter 5 --- Computational Synthesis of Ligands --- p.92 / Chapter 5.1 --- Problem Definition --- p.93 / Chapter 5.2 --- Motivation --- p.93 / Chapter 5.3 --- Background --- p.94 / Chapter 5.4 --- Method --- p.97 / Chapter 5.4.1 --- Selection --- p.99 / Chapter 5.4.2 --- Mutation --- p.102 / Chapter 5.4.3 --- Crossover --- p.102 / Chapter 5.4.4 --- Split --- p.103 / Chapter 5.4.5 --- Merging --- p.104 / Chapter 5.4.6 --- Drug Likeness Testing --- p.104 / Chapter 5.5 --- Data --- p.105 / Chapter 5.5.1 --- Proteins --- p.105 / Chapter 5.5.2 --- Initial Ligands --- p.107 / Chapter 5.5.3 --- Fragments --- p.107 / Chapter 5.6 --- Experiments and Results --- p.109 / Chapter 5.6.1 --- Binding Conformation --- p.112 / Chapter 5.6.2 --- Free Energy and Molecule Weight --- p.115 / Chapter 5.6.3 --- Execution Time --- p.116 / Chapter 5.6.4 --- Support for Phosphorus --- p.116 / Chapter 5.7 --- Discussion --- p.120 / Chapter 5.8 --- Availability --- p.123 / Chapter 5.9 --- Conclusion --- p.123 / Chapter 5.10 --- Personal Contribution --- p.124 / Chapter 6 --- Conclusion --- p.125 / Chapter 6.1 --- Conclusion --- p.125 / Chapter 6.2 --- Future Work --- p.128 / Chapter A --- Publications --- p.130 / Chapter A.1 --- Conference Papers --- p.130 / Chapter A.2 --- Journal Papers --- p.131 / Bibliography --- p.132

This dissertation describes the design, synthesis and biological evaluation of novel CDK5/p25 small molecule inhibitors. Cyclin dependent kinase 5 (CDK5) is a proline directed serine/threonine kinase which plays an important role in the pathology of Alzheimer's disease (AD). CDK5/p25 has been implicated in hyperphosphorylation of tau protein which forms neurofibrillary tangles (NFTs), a contributing factor to the pathology of Alzheimer's disease (AD). Based on the deposited X-ray crystal structure of CDK5/p25 with a non-selective CDK inhibitor R-Roscovitine (PDB ID: 1UNL), eight series of novel compounds with a benzimidazole core were designed, synthesized and tested as inhibitors of CDK5/p25. An efficient synthesis of trisubtituted benzimidazoles was developed to explore the SAR at the 1-, 4-, and 6- positions of the benzimidazole core. X-ray crystal structure verification of an intermediate confirmed selective alkylation of the N-1 position of the benzimidazole scaffold. Synthesis of N-1, N-4, C6-O, C6-N, C6-C and C-2 substituted benzimidazoles were achieved via Mitsunobu coupling, Suzuki Miyaura coupling, Buchwald coupling and reductive alkylation strategies. Aminothiazole scaffolds are an established class of CDK inhibitors including CDK5. A molecular hybridization technique was applied to the design of a series of 2-, 5- disubstituted aminothiazoles incorporating structural features of both the Meriolins, natural product CDK inhibitors, and known aminothiazole scaffolds. Synthetic techniques employed included aryl lithiation, deoxygenation and acylation. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Medicinal Chemistry / PhD / Dissertation

Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles. In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions. I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding. Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction. Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

This dissertation describes the design, synthesis and biological evaluation of novel CDK5/p25 small molecule inhibitors. Cyclin dependent kinase 5 (CDK5) is a proline directed serine/threonine kinase which plays an important role in the pathology of Alzheimer's disease (AD). CDK5/p25 has been implicated in hyperphosphorylation of tau protein which forms neurofibrillary tangles (NFTs), a contributing factor to the pathology of Alzheimer's disease (AD). Based on the deposited X-ray crystal structure of CDK5/p25 with a non-selective CDK inhibitor R-Roscovitine (PDB ID: 1UNL), eight series of novel compounds with a benzimidazole core were designed, synthesized and tested as inhibitors of CDK5/p25. An efficient synthesis of trisubtituted benzimidazoles was developed to explore the SAR at the 1-, 4-, and 6- positions of the benzimidazole core. X-ray crystal structure verification of an intermediate confirmed selective alkylation of the N-1 position of the benzimidazole scaffold. Synthesis of N-1, N-4, C6-O, C6-N, C6-C and C-2 substituted benzimidazoles were achieved via Mitsunobu coupling, Suzuki Miyaura coupling, Buchwald coupling and reductive alkylation strategies. Aminothiazole scaffolds are an established class of CDK inhibitors including CDK5. A molecular hybridization technique was applied to the design of a series of 2-, 5- disubstituted aminothiazoles incorporating structural features of both the Meriolins, natural product CDK inhibitors, and known aminothiazole scaffolds. Synthetic techniques employed included aryl lithiation, deoxygenation and acylation. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Le choix des molécules à tester joue un rôle essentiel dans le succès d'un criblage destiné à identifier de nouveaux composés bioactifs. Les deux étapes importantes dans la préparation des molécules pour le criblage sont le choix de l'espace chimique à considérer et la sélection des molécules pertinentes dans cet espace.<br />Idéalement la préparation des composés destinés au criblage devrait se faire grâce à un logiciel dédié à cette problématique. Il n'existe cependant aucun logiciel qui soit complètement adapté. Nous avons donc entrepris le développement d'un logiciel de ce type : ScreeningAssistant.<br />Ce logiciel s'appuie sur un système de gestion de bases de données et permet de créer et de maintenir à jour des chimiothèques de plusieurs millions de molécules uniques provenant de fournisseurs différents. Il permet également de filtrer les structures, en éliminant les molécules potentiellement problématiques ou avec des probabilités d'activités faibles, et de sélectionner un ensemble de composés divers. <br />Ce logiciel a été utilisé pour l'analyse d'une base de 5 millions de références provenant de 38 fournisseurs de produits chimiques. La proportion de composés uniques, originaux, « drug-like », « lead-like », et divers ont été comparés. La diversité a été étudiée en utilisant des notions différentes, et un score de diversité globale, prenant en compte la diversité suivant les différents critères, a été proposé.<br />Différentes applications de sélection de composés pour le criblage sont présentées. Ces applications utilisent le programme ScreeningAssistant et d'autres algorithmes développés pour résoudre certains problèmes particuliers.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-107).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 6 uppsatser.

The research described in this dissertation is focused on the knowledge-based, often in silico assisted design, targeted synthesis, and biological evaluation of small molecules of interest for two translational medicinal chemistry projects. The first project (Part 1) is aimed at the identification of blood brain barrier (BBB) penetrable dual specificity tyrosine phosphorylation regulated kinase-1A (DYRK1A) inhibitors as a potential disease modifying approach to mitigate cognitive deficits associated with Alzheimer's neurodegeneration. Two major series with potent activity against DYRK1A were identified in addition to a number of other chemotype sub-series that also exhibit somewhat promising activity. Extensive profiling of active analogs revealed interesting biological activity and selectivity, which led to the identification of two analogs for in vivo studies and revealed new opportunities for further investigation into other kinase targets implicated in neurodegeneration and polypharmacological approaches. The second project (Part 2) is focused on the development of compounds that inhibit PGE₂ production, while not affecting cyclooxygenase (COX) activity, as a novel approach to treat cancer. Compounds were designed with the intention of inhibiting microsomal prostaglandin E₂ synthase-1 (mPGES-1); however, biological evaluation revealed phenotypically active compounds in a cell based assay with an unknown mechanism of action. Further profiling revealed promising anticancer activity in xenograft mouse models. In addition, PGE₂ has been implicated in an immune evasion mechanism of F. tularensis, a strain of bacteria that remains an exploitable threat in biowarfare, thus a small number of analogs were evaluated in a cell model of F. tularensis infection stimulated PGE₂ production.

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