Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-13T11:59:49Z No. of bitstreams: 1 arquivototal.pdf: 7758959 bytes, checksum: db745d41b196978192ebc789e25f442b (MD5) / Made available in DSpace on 2017-09-13T11:59:49Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 7758959 bytes, checksum: db745d41b196978192ebc789e25f442b (MD5) Previous issue date: 2016-02-26 / The use of plants for medicinal purposes is one of the oldest forms of medical practice of mankind, emphasizing the alkaloids because they present rich structural and pharmacological properties extensive variety. The drug design is aided by computer based strategies based on linkers or target. When developing new compounds, the structure-based techniques, such as docking, can be applied to study of certain receptor and its corresponding ligand, evaluating bindingprotein interactions. Whereas in the ligand-based methods, a database of known ligands is used, looking for ways to evaluate parameters (molecular descriptors) that can assist in the development of compounds with higher power. This study aimed to perform in silico studies to investigate drug-target interactions with alkaloids derived from natural products and their analogues with relevant pharmacological activity. Different molecular descriptors and methodologies were used in the studies developed. In chapter 2, the interaction of alkaloid bisindolic caulerpine (CLP) was evaluated with the enzyme involved in Alzheimer's disease (AD) monoamine oxidase B (MAO-B), and a database with 109 analogs. It was possible to observe a chemical parameter of inhibition of PLC analogues where the replacement of the radicals must be asymmetric with different polarity. The studies based on the linker and the structure associated with the classification drug-like chemical skeleton suggest that the PLC has potential use in the treatment of AD. In chapter 3, 8 alkaloids isolated Cissampelos sympodialis and 101 derivatives, had their inhibitory potential against enzyme (BACE, GSK-3β and MAO-A) involved in degenerative diseases assessed by in silico methods. consensual analysis showed affinity alkaloids bisbenzilisoquinolinics by BACE, incluindos the roraimine natural alkaloids and simpodialine-β-N-oxide, supporting interest in investigating this skeleton as an antagonist of this enzyme. In Chapter 4 we evaluated the multi-target potential of 148 aphorphinics alkaloids Annonaceae against Leishmania donovani. Six were selected enzymes of this neglected disease for theoretical study, which was associated with experimental four alkaloids available data and integrating the bank, which had pIC50 value inferior to 5.26. The xyloguyelline alkaloid was named as a potential multi-agent target, demonstrating activity against 5 of 6 enzymes evaluated, likely to activity of over 60%. fragment descriptors were used to create model-based binder in a parallel approach with molecular docking to predict the cytotoxic and against topoisomerase II activity azaphenantrene alkaloids in chapter 5. The cytotoxic activity of this skeleton alkaloids are well described in the literature, molecules having activity against several tumor cell lines. The IMB 6 analog and 23 IMB showed interesting activity and selectivity, with MolDock energy similar to liriodenine composed characterized by potent anti-tumor action, but with high toxicity. Important structural information is provided by spectroscopy nuclear magnetic resonance (NMR), and Chapter 6 aimed to discuss the importance of this technique for generating molecular descriptors. Studies that applied successfully in drug design NMR descriptors assisted by computer are described and several QSAR and QSPR having as support data chemical shifts. / A utilização de plantas com fins medicinais é uma das mais antigas formas de prática medicinal da humanidade, enfatizando os alcaloides, por apresentarem rica variedade estrutural e extensa propriedade farmacológica. O desenho de drogas auxiliado pelo computador é fundamentado em estratégias baseadas nos ligantes ou no alvo. No desenvolvimento de novos compostos, técnicas baseadas na estrutura, como o docking, podem ser aplicadas no estudo de um determinado receptor e seu respectivo ligante, avaliando as interações ligante-proteína. Ao passo que nos métodos baseados no ligante, um banco de ligantes conhecidos é utilizado, buscando modos de avaliar parâmetros (descritores moleculares) que possam auxiliar no desenvolvimento de compostos com maior potência. Este estudo teve como objetivo realizar estudos in silico para investigar interações fármaco-alvo com alcaloides oriundos de produtos naturais, e respectivos análogos, com relevante atividade farmacológica. Diferentes descritores moleculares e metodologias foram utilizadas nos estudos desenvolvidos. No capítulo 2, foi avaliado a interação do alcaloide bisindolico caulerpina (CLP) com a enzima envolvida na doença de Alzheimer (DA) monoamina oxidase B (MAO-B), além de um banco com 109 análogos. Foi possível observar um parâmetro químico de inibição dos análogos da CLP, onde a substituição dos radicais deve ser assimétrica com polaridade distinta. Os estudos dos baseados no ligante e na estrutura, associado à classificação drug-like, sugerem que o esqueleto químico da CLP tem potencial uso no tratamento da DA. No capítulo 3, 8 alcaloides isolados de Cissampelos sympodialis e 101 derivados, tiveram seu potencial inibitório contra enzimas (BACE, GSK-3β e MAO-A) envolvidas em doenças degenerativas avaliado por metodologias in silico. Análise consensual demonstrou afinidade de alcaloides bisbenzilisoquinolínicos pela BACE, incluindos os alcaloides naturais roraimina e simpodialina- β-N-oxide, suportando interesse em investigar este esqueleto como antagonista desta enzima. No capítulo 4 foi avaliado o potencial multi-target de 148 alcaloides aporfinicos de Annonaceae contra Leishmania donovani. Foram utilizadas seis enzimas desta doença negligenciada para o estudo teórico, que foi associado com dados experimentais de quatro alcaloides disponíveis e que integram o banco, que apresentaram valor pIC50 inferior a 5.26. O alcaloide xyloguyellina foi apontado como potencial agente multitarget, demonstrando atividade contra 5 das 6 enzimas avaliadas, com probabilidade de atividade superior a 60%. Descritores de fragmento foram utilizados para criar modelo baseado no ligante em uma abordagem paralela com docking molecular, para predizer a atividade citotóxica e contra topoisomerase II de azafenantreno alcaloides, no capítulo 5. A atividade citotóxica deste esqueleto de alcaloides está bem descrita na literatura, com diversas moléculas apresentando atividade contra linhagens de células tumorais. Os análogos IMB 6 e IMB 23 apresentaram interessante atividade e com seletividade, apresentando energia MolDock similar à liriodenina, composto caracterizado por potente ação antitumoral, porém com elevada toxicidade. Importantes informações estruturais são fornecidas pela espectroscopia de ressonância magnética nuclear (RMN), sendo o capítulo 6 destinado a discorrer sobre a importância desta técnica para geração de descritores moleculares. Estudos que aplicaram com sucesso descritores RMN em design de drogas assistida pelo computador encontram-se descritos, além de diversos estudos de QSAR e QSPR tendo como amparo dados de deslocamentos químicos.

Os receptores dos hormônios da tireóide (TRs) são fatores de transcrição envolvidos na diferenciação celular, metabolismo e funções fisiológicas da maioria dos tecidos. Muitos estudos mostram que diversos efeitos farmacológicos mediados pelos TRs podem ser benéficos na farmacoterapia, especialmente aqueles mediados pelo TR que podem ser úteis em condições médicas importantes como obesidade, hipercolesterolemia e diabetes. Além disso, a descoberta que o TR é a isoforma predominante no coração, mediando a maioria dos efeitos cardiovasculares prejudiciais, estimulou a pesquisa por ligantes seletivos para o TR que poderiam ser utilizados em quadros clínicos importantes com perfil de segurança aceitável. Foi realizado um estudo das relações quantitativas entre a estrutura e atividade (QSAR) de um conjunto de compostos com atividade biológica descrita para TR e TR, que gerou modelos de Holograma QSAR com elevada consistência interna e externa, apresentando bom poder de correlação e predição das propriedades biológicas. Também foi realizado um minucioso estudo de triagem virtual, que propiciou a seleção de 7 compostos que foram adquiridos para terem suas atividades biológicas avaliadas. Ensaios de transfecção e gene repórter foram estabelecidos e utilizados na avaliação da atividade biológica dos compostos selecionados pelo ensaio virtual. Finalmente, um ensaio utilizando ressonância plasmônica de superfície (SPR) foi desenvolvido e utilizado para avaliar a atividade agonista desses compostos, e que pode ser útil para avaliar a atividade de novos ligantes. A técnica de SPR também foi empregada em um cuidadoso estudo da interação do TR com seus correguladores, que incluiu estudos cinéticos e termodinâmicos, propiciando a determinação das taxas cinéticas e parâmetros termodinâmicos para a interação do complexo TR-T3 com peptídeos derivados de dois de seus correguladores. Os resultados obtidos são relevantes e devem ser considerados no planejamento de futuros experimentos utilizando o LBD de TR e agonistas. / The thyroid hormone receptors (TRs) are transcriptional factors involved in cell differentiation, development, metabolism and physiological function of most tissues. Many lines of evidence show that several pharmacological actions of TRs might be beneficial in medical therapy, specially those mediated by TR that target important medical conditions like obesity, hypercholesterolemia and diabetes. Additionally, the findings that TR is the predominant isoform in the heart and mediates most of the TRs deleterious cardiovascular effects, stimulated the research for selective TR ligands which could address important medical needs with an acceptable safety profile. In this PhD thesis, studies of the quantitative structure-activity relationships (QSAR) of a dataset of compounds with reported biologic activity for both TR and TR were performed, and statistically significant Hologram QSAR models with good predictive ability for untested compounds were created. In parallel, a careful virtual screening procedure was executed, leading to the selection of 7 compounds which were purchased for the evaluation of their biological activities. Cell transfection and reporter gene assays were developed, validated and used to evaluate the biological activities of these compounds. Finally, a surface plasmon resonance (SPR) assay was developed and used to assess the agonistic activity of these compounds. The SPR technique was also employed in a careful study of the interaction between the ligand binding domain of TR and peptides derived from its coregulators, which included the determination of the kinetic and thermodynamic parameters for this interaction. The results suggest that flexibility plays an important role in the interaction between the receptor and its coregulators, and point out important aspects of experimental design that should be addressed when using TR LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR ligands.

Made available in DSpace on 2014-07-29T16:11:54Z (GMT). No. of bitstreams: 1 MARCELO DO NASCIMENTO capitulos.pdf: 46847 bytes, checksum: 9184c68b4afbd4f8c18e081f4c9274be (MD5) Previous issue date: 2009-06-15 / In the field of a research line that seeks the planning, the synthesis and the pharmacologycal evaluation of new candidates to prototypes of anti-inflammatory drugs, we will describe in this project the planning of derived new pyrazolics (LQFM 002-003), originally drawn starting from the nerolidilcatecol (24) and arylsulfonilpiperazines, (25) that present profile inhibition of the enzyme sPLA2. The compounds (E)-N-(3,7-dimethylocta-2,6-dienyl)-1,3-dimethtyl-1-H-pyrazol-5-amine (LQFM 002) and (E)-N-(3,7-dimethylocta-2,6-dienyl)-1,3-dimethyl-4-((4-methylpiperazin-1-il) methyl)-1H-pyrazol-5-amine (LQFM 003), were submitted to pharmacologycal rehearsals in vitro, seeking to evaluate the enzymatic inhibition activity of the sPLA2. For the prototype (LQFM 002) the inhibition halos were 14,43 ± 6,28%, 16,68 ± 2,45%, 23,61 ± 2,62%, 37,06 ± 3,25%, in the doses of 250, 500, 1000 and 2000 μg/mL respectively. For the compound (LQFM 003), the halos were 1,85 ± 1,38%, 9,29 ± 3,33%, 7,82 ± 3,32%, 13,21 ± 3,22%, respectively. Subsequently the rehearsal in vivo was accomplished to evaluate the profile of cellular migration. Being also analyzed the concentration of plasmatic protein by the methodology of the Evans of blue once the inflammatory process was induced. The compound (LQFM 002) presented inhibition on cellular migration of 50,46 ± 14,34% in the peritonit test, 68,7 ± 2,65% in the pleurisy test and reduction of 40,1 ± 6,40% of the plasmatics proteins for the method of coloration of the Evans of blue. The compound (LQFM 003) presented 25,89 ± 5,39% inhibition, in the doses of 50 mg/Kg, characterizing, significant anti-inflammatory profile for the prototype (LQFM 002). Parallel to the pharmacologycal synthetic work and, we carried out theoretical studies through the application of molecular dynamics. The parameters of the enzyme sPLA2 was more effective through the applications of the water box's, once the smaller state energy observed was of -175000kcal/mol. At the end of this project, we can conclude that the structural planning applied in the project was validated through the applications of the pharmacologycal rehearsals, once both molecules were recognized by the enzyme sPLA2 and they presented anti-inflammatory activity in the rehearsal of cellular migration. In addition, the applied synthetic methodology for obtaining of the prototypes pyrazolics (LQFM 002-003) studied was satisfatory. / No âmbito de uma linha de pesquisa que visa o planejamento, a síntese e a avaliação farmacológica de novos candidatos a protótipos de fármacos anti-inflamatórios, descreveremos neste trabalho o planejamento de novos derivados pirazólicos (LQFM 002-003), originalmente desenhados a partir do nerolidilcatecol (24) e arilsulfonilpiperazínicos (25), que apresentam perfil inibitório da enzima sPLA2. Os compostos (E)-N-(3,7-dimetilocta-2,6-dienil)-1,3-dimetil-1-H-pirazol-5-amina (LQFM 002) e (E)-N-(3,7-dimetilocta-2,6-dienil)-1,3-dimetil-4-((4-metilpiperazina-1-il) metil)-1H-pirazol-5-amina (LQFM 003), foram submetidos a ensaios farmacológicos in vitro, visando avaliar a atividade de inibição enzimática da sPLA2. Para o protótipo LQFM 002 os halos de inibição foram 14,43 ± 6,28%, 16,68 ± 2,45 %, 23,61 ± 2,62 %, 37,06 ± 3,25 %, nas doses de 250, 500, 1000 e 2000 μg/mL respectivamente. Para o composto LQFM 003, os halos foram 1,85 ± 1,38 %, 9,29 ± 3,33 %, 7,82 ± 3,32 %, 13,21 ± 3,22 %, respectivamente. Posteriormente foram realizados os ensaios in vivo o qual avalia o perfil de migração celular para cavidade peritoneal e pleural. Sendo também analisada a concentração de proteína plasmática pela metodologia do azul de Evans quando induzido o processo inflamatório. O composto LQFM 002 apresentou inibição sobre a migração celular de 50,46 ± 14,34 % no teste de peritonite, 68,7 ± 2,65 % no teste de pleurisia e redução de 40,1 ± 6,40 % das proteínas plasmáticas pelo método de coloração do azul de Evans. O composto LQFM 003 apresentou inibição de 25,89 ± 5,39%, nas doses de 50 mg/Kg, caracterizando, desta forma, perfil anti-inflamatório significativo para o protótipo (LQFM 002). Paralelamente ao trabalho sintético e farmacológico, foi realizado estudos teóricos através do emprego de dinâmica molecular. A parametrização da enzima sPLA2 foi mais efetiva através do emprego de caixa de água, uma vez que o estado de menor energia observado foi de -175000kcal/mol. Ao término deste trabalho, podemos concluir que o planejamento estrutural empregado no mesmo foi validado através dos ensaios farmacológicos empregados, uma vez que ambas as moléculas foram reconhecidas pela enzima sPLA2 e apresentaram atividade anti-inflamatória no ensaio de migração celular. Ademais, a metodologia sintética empregada se mostrou satisfatória para a obtenção dos protótipos pirazólicos (LQFM 002 - 003) estudados.

Venous thromboembolism that cause blood clotting in blood vessels, prevent blood circulation, depending on changes in one or more of the coagulation factors II, VII, IX and X. Patients who have had a blood clot or cardiovascular diseases are treated with oral anti-vitamin K (Warfarin®) to reducing and prevent relapse. Warfarin is also used as a preventive treatment before the disease. An overdose of Warfarin® may cause bleeding-complications and low dose cause blood clotting. The dosage of the drug is controlled by measuring prothrombin in plasma. The aim of this study was to investigate if prothrombin-complex value changes due to re-spinning and re-analysis after six hours. Fitty whole blood samples from warfarin-treated patients were divided into three subgroups, those with protrombinkomplex-values of 2-4 (n=20), >4 (n=15) and <2 (n=15). The samples were centrifugated and measured (Method A), re-centrifugated and measured (Method B) or re-analysed after six hours (Method C). All results were compared in a Bland-Altman plot as follows: Method B vs. Method A and Method C vs. Method A. The scatter graph yielded a strong correlation between Method A and Method B (R2=0.9984) and Method A and Methods C (R2=0.9977). The results from t-test showed a significance level (p<0.001) for both analyses (statistical significance=p<0.05). In this study we showed that prothrombin complex value ware stable after re-centrifugation and re-measurement after six hours. Statistical calculations yielded a strong correlation between the methods (A, B, C), and there was no significance difference between the methods.

Le projet de recherche a été réalisé en collaboration avec Professeur Jean-François Masson du Département de Chimie de l'Université de Montréal. The research project was made in collaboration with Professor Jean-François Masson from the Chemistry department of University of Montreal. / L'objectif du projet de recherche est de développer une méthode de criblage d’inhibiteurs basée sur une technologie émergente, soit un dispositif portatif utilisant la résonance des plasmons de surface (SPR). La cible du criblage est la dihydrofolate réductase R67 (DHFR R67), une enzyme qui confère une résistance bactérienne à l'antibiotique triméthoprime. Ici, l'enzyme cible est immobilisée sur une surface d'or mince avec des propriétés plasmoniques (optiques) spécifiques qui varient en fonction de la masse des molécules se liant à cette surface. Cette technique permet de suivre les événements de liaison de molécules à la DHFR R67 immobilisée, et ainsi peut permettre l'identification d'inhibiteurs potentiels. Cependant, la masse molaire des inhibiteurs typiquement utilisés lors de criblages préliminaires (i.e. 500-1000 g/mol) est trop faible pour générer un signal SPR détectable. Afin de contrer cette lacune, ce mémoire a pour objet de développer un essai compétitif indirect qui mettra en jeu des molécules de masse supérieure. D’abord, une nanoparticule d'or portant un analogue de substrat se liera à la DHFR R67 immobilisée à la surface d’or, générant ainsi un signal SPR important en raison de la masse molaire élevée de la nanoparticule. Ensuite, lors du criblage d'inhibiteurs potentiels, les nanoparticules liées seront déplacées de l'enzyme cible si la molécule criblée fournit une affinité suffisante. Ainsi, il sera possible de suivre indirectement la liaison d'un inhibiteur à la cible. Ce projet vise donc à tester et à valider l'approche de criblage SPR appliquée à la DHFR R67. / The objective of the research project is to develop a method for inhibitor screening based on a portable Surface Plasmon Resonance (SPR) device, an emerging technology. The target is R67 dihydrofolate reductase (R67 DHFR), an enzyme that confers bacterial resistance to the antibiotic trimethoprim. Here, the target enzyme is linked to a thin gold surface having specific plasmonic (optical) properties that vary as a function of the mass of bound molecules. This allows monitoring binding to the surface-linked R67 DHFR, and thus permits identification of inhibitors. However, the mass of the low-affinity inhibitors typically identified in early stages of screening (i.e. 500-1000 g/mol) is too low to produce a significant SPR signal. To address this shortcoming, a competitive assay will be developed: a gold nanoparticle carrying a substrate analog will bind the surface-immobilized R67 DHFR, resulting in a strong SPR signal due to its high mass. Then, upon screening for potential inhibitors, the bound nanoparticle will be displaced from the target enzyme if a molecule provides sufficient affinity. By those means, it will be possible to indirectly monitor the binding of an inhibitor to the target. This goal of this project is to test and validate the SPR screening approach applied to R67 DHFR.

Le portentol est un polypropionate qui possède une structure spirocyclique très originale. Cette molécule, dont il n'existe encore aucune synthèse totale, a été isolée du lichen Rocella portentosa à la fin des années 1960. Dans le but d'accéder au squelette du portentol, nous avons développé une méthodologie visant à synthétiser des spirotétrahydropyranes fonctionnalisés via une cyclisation de Prins. Au cours de ce travail, un phénomène de dédoublement cinétique dynamique a aussi été mis en évidence. La stachybotrine C, isolée à partir de la bactérie Stachybotrys parvispora, possède des propriétés neurotrophiques et neuroprotectrices très intéressantes. Deux voies de synthèse ont été envisagées pour préparer ce produit naturel. La première voie repose sur une réaction d'hydroarylation d'un éther propargylique catalysée à l'or, suivie d'une oxydation régiosélective du chromène résultant. La seconde voie fait intervenir une étape clé d'époxydation/cyclisation d'un alkénylphénol obtenu suite à un réarrangement de Claisen. Ces travaux nous ont permis d'accomplir la synthèse totale de la stachybotrine C, de réviser sa structure et de déterminer sa configuration absolue. / Portentol is a polypropionate with an unusual spirocylic structure. This natural product, which has never been synthesized, has been isolated from the lichen Rocella portentosa. To access the spiranic moiety of the portentol, we have developed a methodology to synthesize spirotetrahydropyranes through a Prins cyclization. Through the study of the scope of this reaction, an usual dynamic kinetic resolution has been highlighted. Stachybotrin C, isolated from the bacteria Stachybotrys parvispora, exhibits interesting neuritogenic and neuroprotective properties. To prepare this natural product, two synthetic routes have been investigated. The first one is based on a gold-catalyzed hydroarylation of a propargyllic ether followed by a regioselective oxidation of the resulting chromene. The second pathway involves as a key step an epoxydation/cyclisation of a phenol prepared via a Claisen rearrangment. We accomplished the synthesis of stachybotrin C, revised its structure and established its absolute configuration.

<p> </p> <p>Infectious diseases caused by bacteria, fungi, and plasmodium parasites are a huge global health problem which ultimately leads to millions of deaths annually. The emergence of strains that exhibit resistance to nearly every class of antimicrobial agents, and the inability to keep up with these resistance trends has brought to the fore the need for new therapeutic agents (antibacterial, antifungal, and antimalarial) with novel scaffolds and functionalities capable of targeting microbial resistance. A novel class of compounds featuring an aryl isonitrile moiety has been discovered that exhibits potent inhibitory activity against several clinically relevant strains of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Synthesis, structure-activity relationship (SAR) studies, and biological investigations have led to lead molecules that exhibit anti-MRSA inhibitory activity as low as 1 – 2 µM. The most potent compounds have also been shown to have low toxicity against mammalian cells and exhibit <i>in vivo</i> efficacy in MRSA skin and thigh infection mouse models.</p> <p>The novel aryl isonitriles have also been evaluated for antifungal activity. This study examines the SAR of aryl isonitrile compounds and showed the isonitriles as compounds that exhibit broad spectrum antifungal activity against species of <i>Candida</i> and <i>Cryptococcus</i>. The most potent derivatives are capable of inhibiting growth of these pathogens at concentrations as low as 0.5 µM. Notably, the most active compounds exhibit excellent safety profile and are non-toxic to mammalian cells up to 256 µM.</p> <p>Beyond the antibacterial and antifungal activities, structure-antimalarial relationship analysis of over 40 novel aryl isonitrile compounds has established the importance of the isonitrile functionality as an important moiety for antimalarial activity. Of the many isonitrile compounds exhibiting potent antimalarial activity, two have emerged as leads with activity comparable to that of Artemisinin. The SAR details presented in this study will prove essential for the development new aryl isonitrile analogues to advance them to the next step in the antimalarial drug discovery process.</p> <p>17-nor-Excelsinidine, a zwitterion monoterpene indole alkaloid isolated from <i>Alstonia scholaris</i> is a subject of synthetic scrutiny. This is primarily due to its intriguing chemical structure which includes a bridged bicyclic ammonium moiety, and its anti-adenovirus and anti-HSV activity. Herein we describe a six-step total synthesis of (±)-17-nor-Excelsinidine from tryptamine. Key to the success of this synthesis is the use of palladium-catalyzed carbonylative heck lactamization methodology which built the 6, 7-membered ring lactam in one step. The resulting pentacyclic product, beyond facilitating the easy access to (±)-17-nor-Excelsinidine, could also serve as a precursor to other related indole alkaloids.</p> <br> <p> </p> <p></p>

The deleterious effects of MPO make it a new target for medicinal research. The aim of our study is to find promising inhibitors of MPO for using them as starting point of new anti-inflammatory drugs. Depending on previous researches on MPO inhibitors, we selected 5-fluorotryptamine as starting compounds. Using docking experiments, we designed a series of compounds derived from 5-fluorotryptamine. Two modifications were proposed: <p>& / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.

Small interfering RNAs (siRNAs) are a promising class of drugs for treating genetically-defined diseases. Therapeutic siRNAs enable specific modulation of gene expression, but require chemical architecture that facilitates efficient in vivodelivery. siRNAs are informational drugs, therefore specificity for a target gene is defined by nucleotide sequence. Thus, developing a chemical scaffold that efficiently delivers siRNA to a particular tissue provides an opportunity to target any disease-associated gene in that tissue. The goal of this project was to develop a chemical scaffold that supports efficient siRNA delivery to the brain for the treatment of neurodegenerative diseases, specifically Huntington’s disease (HD). HD is an autosomal dominant neurodegenerative disorder that affects 3 out of every 100,000 people worldwide. This disorder is caused by an expansion of CAG repeats in the huntingtin gene that results in significant atrophy in the striatum and cortex of the brain. Silencing of the huntingtin gene is considered a viable treatment option for HD. This project: 1) identified a hyper-functional sequence for siRNA targeting the huntingtin gene, 2) developed a fully chemically modified architecture for the siRNA sequence, and 3) identified a new structure for siRNA central nervous system (CNS) delivery—Divalent-siRNA (Di-siRNA). Di-siRNAs, which are composed of two fully chemically-stabilized, phosphorothioate-containing siRNAs connected by a linker, support potent and sustained gene modulation in the CNS of mice and non-human primates. In mice, Di-siRNAs induced potent silencing of huntingtin mRNA and protein throughout the brain one month after a single intracerebroventricular injection. Silencing persisted for at least six months, with the degree of gene silencing correlating to guide strand tissue accumulation levels. In Cynomolgus macaques, a bolus injection exhibited significant distribution and robust silencing throughout the brain and spinal cord without detectable toxicity. This new siRNA scaffold opens the CNS for RNAi-based gene modulation, creating a path towards developing treatments for genetically-defined neurological disorders.