Today, current drug discovery and lead generation efforts focus on high throughput screening of large chemical libraries as the primary source of lead candidates. A lack of investment in novel chemotype development by pharmaceutical companies over the last 15 years coupled with the concurrent merger of screening collections and the availability of generic compound libraries commercially have resulted in many discovery efforts that lack uniqueness and do not offer a strong patent position to operate. The need for better, more diverse, and more drug-like libraries is essential in order to feed high throughput screening efforts with molecules that probe new dimensions of chemical space and allow for the discovery of untapped intellectual property.This dissertation details a complete structure based study to design novel inhibitors of B-Raf and p38a MAP Kinase. A structural evaluation of the important and similar interactions necessary for DFG-out allosteric inhibitors to bind their respective targets was accomplished through the synthesis and evaluation of three known allosteric kinase inhibitors, Gleevec®, Nexavar®, and BIRB-796, and 8 additional DFG-out allosteric inhibitors that were developed directly from fragments of these successful scaffolds. The structural insight that was gained from the evaluation of known DFG-out allosteric inhibitors was then utilized to design novel inhibitors that incorporated two unique scaffolds based on two new [3+2] cycloaddition reactions.A pyrrolo-3,4-dicarboximide scaffold has been developed through the utilization of a novel tandem [3+2] cycloaddition then elimination reaction scheme. This scaffold, which contains three sites for variation, was then rationally incorporated into lead molecules using structure-based methods and in silico feedback for the production of dual DFG-out allosteric kinase inhibitors of p38a and B-Raf kinase. These inhibitors display micromolar to submicromolar enzymatic IC50's for both p38a and B-Raf kinase and low micromolar inhibition of cell growth in 4 separate cancer cell lines.We also explored new chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of specific allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile.

Made available in DSpace on 2015-04-11T13:54:29Z (GMT). No. of bitstreams: 1 Zanair Soares Vasconcelos.pdf: 2858178 bytes, checksum: 6b26a0905110117fcff488bcc6f386c1 (MD5) Previous issue date: 2013-08-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Malignant melanoma is one of the few cancers has been increasing in prevalence and mortality worldwide. It affects mainly white populations, however, all ethnic groups are affected to some extent. Increased understanding of the causes, progression, and in particular, genes that affect the development of cancer enable improvements in tools for detecting and treating cancer, however, despite significant advances in understanding the biology of melanoma, no changes in practices involved in both approaches and therapies to treat advanced disease. Thus, malignant melanoma has been intensively investigated due to its high metastatic potential. Many patients, when diagnosed with metastatic melanoma have already, which is the leading cause of death among them. This tumor is almost insensitive to standard regimens of chemotherapy, and the chemotherapeutic agents available are limited. Some mutations are known in cutaneous melanomas and N-Ras, Tp16 and Tp53, however, changes in B-Raf significantly exceed the frequency of the other mutations (40-50%). In this context, this project explored the possible mechanisms of action of the derivative benzotiazólico (E)-2-((2-(Benzo[d] thiazole-2-ila)hydrazono) methyl)-4-nitrophenol on cell viability, the activity clonogenic, motility and invasiveness in melanoma cell lines SK-Mel-19, SK-Mel-28 and SK-Mel-103 that have different characteristics from mutations in the genes responsible for the control of cellular homeostasis, as Tp53 and B-Raf. The results of tests performed showed an IC50 at 72hours between (4.87and>10 um) and a possible selective toxicity to the gene mutated B-Raf. Other tests showed induction of cell death by apoptosis, impaired motility and invasiveness of cells treated with SK-Mel benzotiazolic derivative (E)-2-((2-(Benzo[d]thiazole-2-Ila)hydrazono)methyl)-4-nitrophenol. The obtained results make this target compound for more research to use against cancer. / O melanoma maligno é um dos poucos cânceres que vem aumentando em prevalência, bem como taxa de mortalidade em todo o mundo. Afeta principalmente populações brancas, no entanto, todos os grupos étnicos são afetados em alguma proporção. Os avanços na compreensão das causas, progressão e, em particular, dos genes que afetam o desenvolvimento do câncer possibilitam melhorias nas ferramentas para a detecção e tratamento câncer, no entanto, apesar dos avanços significativos na compreensão da biologia do melanoma e das abordagens para tratar a doença em estágio avançado agora se voltarem para inibidores de genes envolvidos nessa doença, houve poucas mudanças aprovadas nas terapias utilizadas. Assim, o melanoma maligno vem sendo intensamente investigado devido ao seu elevado potencial metastático. Muitos pacientes, ao serem diagnosticados com melanoma já tem metástases, o que é a principal causa de morte entre eles. Este tipo de tumor é quase insensível ao regime padrão de quimioterapia, e os agentes quimioterapêuticos disponíveis são limitados. Algumas mutações são conhecidas nos melanomas cutâneos como N-Ras, Tp16 e Tp53, entretanto, mutações em B-Raf excedem significativamente a frequência de outras mutações (40-50%). Nesse contexto, tratamentos mais eficazes são necessários e na constante busca por novos compostos, os derivados benzotiazólicos mostram-se como uma importante alternativa, já que tem sido apontados como possíveis agentes antitumorais. Nesse sentido, este projeto explorou os possíveis mecanismos de ação do derivado benzotiazólico (E)-2-((2-(Benzo[d]tiazo-2-ila)hidrazono)metil)-4-nitrofenol sobre a viabilidade celular, a atividade clonogênica, a motilidade e a capacidade invasiva em linhagens celulares de melanoma SK-Mel-19, SK-Mel-28 e SK-Mel-103 que possuem diferentes características de mutações nos genes responsáveis pelo controle da homeostasia celular, como Tp53 e B-Raf. Os resultados dos ensaios executados revelaram um CI50 em 72 horas entre (4,87 e >10 μM) e uma possível toxicidade seletiva para o gene B-Raf mutado. Outros testes evidenciaram indução de morte celular por apoptose, diminuição da motilidade e da capacidade invasiva das células SK-Mel tratadas com derivado benzotiazólico (E)-2-((2-(Benzo[d]tiazo-2-ila)hidrazono)metil)-4-nitrofenol. Os resultados obtidos tornam este composto alvo de mais pesquisas para sua utilização contra o câncer.

The tuberous sclerosis-2 (Tsc-2) gene product, tuberin, functions as a renal tumor suppressor. Treatment of Eker (Tsc-2 EK/+) rats and primary renal epithelial cells derived from Tsc-2 EK/+ rats (QTRRE cells) with 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of heterozygosity at the Tsc-2 locus in kidney tumors and QTRRE cells. QTRRE cells are carcinogenic in athymic nude mice. Analysis of kidney tumors formed in Tsc-2 EK/+ + rats following 8-months of TGHQ treatment reveals increases in B-Raf, Raf-1, pERK, cyclin D1, p27Kip1, 4EBP1, p-4EBP1(Thr70), p-4EBP1(Ser65), and p-4EBP1(Thr37/46) protein expression. These data establish the involvement of mTOR and MAPK signaling cascades in tuberin null tumors. Similar increases in 4EBP1 and p4EBP1 are observed in renal tumor QTRRE-xenografts in nude mice. Concomitant with increases in expression of these proteins in TGHQ-induced renal tumors, similar changes are observed in QTRRE cells, which also exhibit high ERK, B-Raf and Raf-1 kinase activity; and increased expression of cyclin D1, p27, p-4EBP1 (Thr70), p-4EBP1 (Ser65), and p-4EBP1 (Thr37/46). Manipulation of the Raf/MEK/ERK kinase cascade in QTRRE cells, with kinase inhibitors and siRNA, indicates that Raf-1/MEK/ERK participates in crosstalk with 4EBP1 to regulate translation of cyclin D1.Cyclin D1 and p27 protein levels are increased in the cytoplasm in our RCC models. In normal HK-2 cells, p27 and cyclin D1 are localized to the nucleus. Due to the instability of the cyclin D1-CDK4 complex, p27 interaction is necessary for cyclin D1-CDK4 complex assembly and stabilization in the nucleus. Manipulation of p27 protein levels in QTRRE cells with phosphodiesterase inhibitors, dibutyryl cAMP, and the proteosome inhibitor MG132, all result in a parallel increase in p27 and cyclin D1. Furthermore, p27 siRNA and sorafenib treatment both cause a decrease in p27 and cyclin D1. Further manipulation of cAMP, Rap1B, and B-Raf proteins, revealed that cAMP/PKA/Rap1B/B-Raf activation and B-Raf//ERK MAPK inhibition both modulate p27 expression and compartmental localization in tuberous sclerosis renal cancer. Phosphodiesterase inhibitors play a role in regulating the expression, degradation, and cytoplasmic localization of p27. Therefore, cytoplasmic p27-cyclin D1 mislocalization and stabilization may have an oncogenic role in the cytosol and play a crucial role in tumor formation.

Primary sensory axons fail to regenerate into the spinal cord following dorsal root injury leading to permanent sensory deficits. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Current approaches for promoting DR regeneration across the DREZ have had some success, but sustained, long-distance regeneration, particularly of large-diameter myelinated axons, still remains a formidable challenge. Our lab has previously shown that induced expression of constitutively active B-RAF (kaBRAF) enhanced the regenerative competence of injured DRG neurons in adult mice. In this study, I investigated whether robust intraspinal regeneration can be achieved by selective expression of kaBRAF alone or in combination with deletion of the myelin-associated inhibitors or neuron-intrinsic growth suppressors (PTEN or SOCS3). To this end, I used LSL-kaBRAF: brn3a-CreERT2 transgenic mice in which kaBRAF can be induced selectively in sensory neurons. I have also bred LSL-kaBRAF: brn3a-CreERT2 mice with triple knock-out mice lacking Nogo, Mag and OMgp or mouse lines carrying floxed alleles of PTEN or SOCS3. Single, double, and triple conditional mice were subjected to cervical DR crush and AAV2-eGFP vectors were used to selectively label regenerating axons of large-diameter neurons. I compared the extent of regeneration at 3 weeks or 2 months after DR injury using conventional anatomical and behavioral analyses. I found that kaBRAF alone promoted axon regeneration across the DREZ but did not produce significant functional recovery by two months. Supplementary deletion of Nogo, MAG, and OMgp did not improve kaBRAF-induced regeneration. Deletion of PTEN or SOCS3 individually or in combination failed to promote axon regeneration across the DREZ. In marked contrast, simultaneous deletion of PTEN, but not SOCS3, dramatically enhanced kaBRAF-mediated regeneration enabling many more axons to penetrate the DREZ and grow deep into the spinal cord. This study shows that dual activation of BRAF-MEK-ERK and PI3K-Akt signaling is an effective strategy to stimulate robust intraspinal DR regeneration and may lead to recovery of sensory function after DR injury. / Biomedical Sciences

La polyarthrite rhumatoïde (PR) est le rhumatisme inflammatoire chronique le plus fréquent. La PR est une maladie génétique où il existe plusieurs allèles de susceptibilité HLA-DRB1. Les auto-anticorps anti-protéines citrullinées sont les plus spécifiques de la PR. Ils sont détectés par les tests anti-peptides cycliques citrullinés (anti-CCP). 1/L’objectif de notre premier travail était de montrer l’influence des allèles susceptibilité HLA-DR sur la présence d’anti-CCP dans notre population marseillaise de PR. Nous avons montré que les allèles de susceptibilité HLA-DR ne sont pas nécessaires à la présence des anticorps anti-CCP. Nous avons mis en évidence une association entre l’allèle HLA-DRB1*04:04 et la présence des anti-CCP.2/Environ un tiers des patients présentant une PR n’ont pas d’anticorps anti-CCP. Nous avons donc recherché des nouveaux auto-anticorps pour le diagnostic de la maladie.Les auto-anticorps dirigés contre le domaine catalytique de la protéine B-Raf (v raf murine sarcoma viral oncogen homologue B1) ont été identifiés par la technique des puces à protéines chez les patients PR. B-Raf est une sérine-thréonine kinase qui est impliquée dans la voie des MAP-kinases. Nous avons montré que les auto-anticorps anti-B-Raf activent B-Raf. Nous avons montré que le peptide p25 de B-Raf est spécifiquement reconnu par les auto-anticorps des PR. Les auto-anticorps anti-p25 identifient 21% des patients PR sans anticorps anti-CCP.3/En utilisant des puces à protéines, nous avons identifié 24 nouveaux auto-antigènes associés aux PR débutantes. Quatre de ces auto-antigènes ont été validés par ELISA : GABA(A) receptor associated protein like, zinc finger protein 706, tropomyosin 2 et WIBG (within BGCN homolog (Drosophila)). Les auto-anticorps anti-WIBG identifient exclusivement les PR.Ces nouveaux auto-antigènes pourront être utilisés dans le diagnostic des PR débutantes et des PR sans anticorps anti-CCP. / Rheumatoid arthritis (RA) is a chronic inflammatory disease with a prevalence of 0.5% wordwilde. HLA-DR genes are the strongest genetic prevalence in RA. The sera of RA patients contain many auto-antibodies. The most characteristic are directed at citrullinated proteins (ACPA). ACPA can be detected by commercially available enzyme-linked immune-absorbent assays using synthetic cyclic citrullinated peptides (CCP).1/In the first work we tested whether the presence of RA associated HLA-DRB1 alleles individually influences anti-CCP production in a population of RA from Marseille. We showed RA associated HLA-DR alleles are not mandatory for the production of anti-CCP. HLA-DRB1*04:04 was the most strongly associated with the presence of anti-CCP in RA sera. 2/ Anti-CCP antibodies are detected in 65% of RA patients. We wanted to detect new auto-antibodies for the diagnosis of RA.By screening protein arrays we found that B-Raf (v raf murine sarcoma viral oncogen homologue B1) is a major non-citrullinated auto-antigen recognized by 35% of RA patients’sera. B-Raf encodes a serine threonine-kinase involved in the MAPK signaling pathway. We showed that anti-B-Raf auto-antibodies activate the in vitro phosphorylation of MEK1 mediated by B-Raf.We found that one peptide of B-Raf, p25, is specifically recognized by auto-antibodies from RA patients. Of interest, anti-p25 auto-antibodies are detected in 21% of anti-CCP negative RA patients.3/We identified 24 new auto-antigens associated with RA patients with disease duration less than one year using 8000 human protein arrays. We identified four auto-antigens recognized almost uniquely by sera of early RA patients: GABA(A) receptor associated protein like, zinc finger protein 706, tropomyosin 2 and WIBG (within BGCN homolog (Drosophila)). These reactivities were confirmed by ELISA on purified proteins. Auto-antibodies to anti-WIBG identify exclusively RA patients’sera. These new auto-antigens could be used for the diagnosis of anti-CCP negative RA patients and in early RA.

Muitos pacientes submetidos à tireoidectomia por Carcinoma Papilífero da Tireoide (CPT) têm doença linfonodal subclínica no momento da cirurgia. A mutação BRAF T17799A (V600E) é um evento comum no CPT e alguns estudos demonstram correlação entre a mutação e características de maior agressividade tumoral, incluindo a presença de metástases linfonodais. O esvaziamento eletivo do compartimento central ganha aceitação, uma vez que alguns estudos evidenciam que a presença de metástases linfonodais aumenta o risco de recidiva e mortalidade. Devido ao grande potencial de complicações do esvaziamento do compartimento central, o objetivo deste trabalho foi avaliar a associação entre a presença da mutação BRAF T17799A (V600E), a presença de metástases linfonodais e fatores clínicos e histopatológicos de pior prognóstico. Métodos: 51 casos consecutivos de pacientes com CPT foram submetidos à tireoidectomia total e ao esvaziamento eletivo ou terapêutico do compartimento central. Em todos os pacientes foi pesquisada a mutação BRAF T17799A (V600E) no tecido tireoidiano com Carcinoma Papilífero de Tireoide. Resultados: Cinquenta e quatro por cento (54,9%) dos pacientes apresentaram metástases linfonodais. Seis pacientes apresentaram metástases laterais confirmadas por punção aspirativa por agulha fina no pré-operatório e 22 pacientes (43%) apresentaram metástases não detectadas no pré ou no intra operatório A mutação BRAF T17799A (V600E) foi encontrada em 15 pacientes portadores de CPT (29,4%). A presença da mutação não teve associação estatisticamente significante para sexo, idade, tamanho do tumor, extensão extratireoidiana, multicentricidade, embolização angiolinfática e metástases linfonodais. As metástases linfonodais se associaram à multifocalidade (p = 0,005) e invasão angiolinfática (p = 0,003) na análise univariada. Conclusão: A presença da mutação BRAF T17799A (V600E) não se associou à metástases linfonodais em nosso estudo. A multifocalidade e a detecção de invasão angiolinfática no CPT foram os fatores mais importantes na predição de metástases linfonodais / Background: Many patients undergoing thyroidectomy for Papillary Thyroid Carcinoma (PTC) have subclinical node disease at the time of surgery. The BRAF T17799A (V600E) mutation is a common event in PTC and some studies have demonstrated a correlation between the mutation and aggressive characteristics including lymph node metastasis. Prophylactic Central Node Dissection (CND) is gaining acceptance in the treatment of PTC as studies have shown nodal disease increases local recurrence and may alter mortality. Given the potential complications of CND, the aim of this study was to determine the correlation among BRAF mutation, lymph node metastasis and clinical and histopathological factors of worse prognosis. Methods: A total of 51 consecutive cases of patients with PTC underwent total thyroidectomy and routine prophylactic (CND) or therapeutic neck dissection when metastases were found. All patients were tested for the BRAF mutation. Results: Overall, positive lymph nodes were found in Fifty four per cent9% of patients. Six patients had lateral metastases confirmed by fine needle aspirative cytology and 22 patients (43%) had occult metastases. The BRAF mutation was found in 15 patients (29.4%). BRAF was not correlated with sex, age, size of tumor, multifocality, extrathyroid extension or lymph node metastases. Lymph node metastases were correlated with multifocality (p = 0.005) and angiolymphatic invasion (p = 0.003) in univariate. Conclusions: The BRAF mutation was not correlated with lymph node metastases in our study. Multifocality and angiolymphatic invasion were important factors for predicting lymph node metastases

Muitos pacientes submetidos à tireoidectomia por Carcinoma Papilífero da Tireoide (CPT) têm doença linfonodal subclínica no momento da cirurgia. A mutação BRAF T17799A (V600E) é um evento comum no CPT e alguns estudos demonstram correlação entre a mutação e características de maior agressividade tumoral, incluindo a presença de metástases linfonodais. O esvaziamento eletivo do compartimento central ganha aceitação, uma vez que alguns estudos evidenciam que a presença de metástases linfonodais aumenta o risco de recidiva e mortalidade. Devido ao grande potencial de complicações do esvaziamento do compartimento central, o objetivo deste trabalho foi avaliar a associação entre a presença da mutação BRAF T17799A (V600E), a presença de metástases linfonodais e fatores clínicos e histopatológicos de pior prognóstico. Métodos: 51 casos consecutivos de pacientes com CPT foram submetidos à tireoidectomia total e ao esvaziamento eletivo ou terapêutico do compartimento central. Em todos os pacientes foi pesquisada a mutação BRAF T17799A (V600E) no tecido tireoidiano com Carcinoma Papilífero de Tireoide. Resultados: Cinquenta e quatro por cento (54,9%) dos pacientes apresentaram metástases linfonodais. Seis pacientes apresentaram metástases laterais confirmadas por punção aspirativa por agulha fina no pré-operatório e 22 pacientes (43%) apresentaram metástases não detectadas no pré ou no intra operatório A mutação BRAF T17799A (V600E) foi encontrada em 15 pacientes portadores de CPT (29,4%). A presença da mutação não teve associação estatisticamente significante para sexo, idade, tamanho do tumor, extensão extratireoidiana, multicentricidade, embolização angiolinfática e metástases linfonodais. As metástases linfonodais se associaram à multifocalidade (p = 0,005) e invasão angiolinfática (p = 0,003) na análise univariada. Conclusão: A presença da mutação BRAF T17799A (V600E) não se associou à metástases linfonodais em nosso estudo. A multifocalidade e a detecção de invasão angiolinfática no CPT foram os fatores mais importantes na predição de metástases linfonodais / Background: Many patients undergoing thyroidectomy for Papillary Thyroid Carcinoma (PTC) have subclinical node disease at the time of surgery. The BRAF T17799A (V600E) mutation is a common event in PTC and some studies have demonstrated a correlation between the mutation and aggressive characteristics including lymph node metastasis. Prophylactic Central Node Dissection (CND) is gaining acceptance in the treatment of PTC as studies have shown nodal disease increases local recurrence and may alter mortality. Given the potential complications of CND, the aim of this study was to determine the correlation among BRAF mutation, lymph node metastasis and clinical and histopathological factors of worse prognosis. Methods: A total of 51 consecutive cases of patients with PTC underwent total thyroidectomy and routine prophylactic (CND) or therapeutic neck dissection when metastases were found. All patients were tested for the BRAF mutation. Results: Overall, positive lymph nodes were found in Fifty four per cent9% of patients. Six patients had lateral metastases confirmed by fine needle aspirative cytology and 22 patients (43%) had occult metastases. The BRAF mutation was found in 15 patients (29.4%). BRAF was not correlated with sex, age, size of tumor, multifocality, extrathyroid extension or lymph node metastases. Lymph node metastases were correlated with multifocality (p = 0.005) and angiolymphatic invasion (p = 0.003) in univariate. Conclusions: The BRAF mutation was not correlated with lymph node metastases in our study. Multifocality and angiolymphatic invasion were important factors for predicting lymph node metastases

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Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

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