Design of new ecto-5'nucleotidase (cd73) inhibitors as potential antitumor

No Brasil, o câncer de bexiga é a oitava neoplasia mais incidente entre os homens. Estudos anteriores mostraram que a inibição da ecto-5’-nucleotidase (CD73), levou a uma redução significativa da proliferação de células de câncer de bexiga T24, além disso, outros estudos em células uroteliais de camundongo mostraram que a enzima CD73 é superexpressa somente em células malignas, tornando-se também um possível alvo seletivo.Nesta tese foram utilizadas uma série de ferramentas virtuais para elucidar os mecanismos envolvidos na captação de ligantes, bem como os bolsões disponíveis para interação na ecto-5’-nucleotidase. Os movimentos de abertura de fechamento, além do posicionamento peculiar de duas fenilalaninas (Phe500 e Phe417) fazem desta enzima uma máquina de capturar AMP. A quimioteca do LaSOM forneceu duas coleções de compostos para o estudo: cumarinas e dihidropirimidinonas (DHPMs). Desta forma, uma série de 152 cumarinas foram triadas virtualmente por métodos previamente validados onde foram selecionados o LaSOM 366 e o LaSOM 191 como ligantes, o que não foi confirmado no ensaio enzimático nas condições empregadas. A coleção de DHPMs investigada revelou o LaSOM 335 com inibição in vitro contra células de câncer de bexiga T24 (IC50=10,7μM). A análise in silico mostrou afinidade com a CD73 e as principais interações foram entre os fragmentos 3-nitrobenzeno e o resíduo Arg395, anel dihiropirimidinona com as fenilalaninas Phe500 e Phe417, e o anel aromático em N1 com o bolsão hidrofóbico do sítio ortostérico, que estabiliza o complexo formado. Estes estudos in silico foram ratificados com o ensaio enzimático, no qual o LaSOM 335 apresentou IC50=136,5μM. Soma-se que esta DHPM apresentou inibição do gene let-60 na cepa MT4244 de Caernobidiythis elegans, este gene é associado à codificação do gene Ras e a sua inibição diminue a adesão e migração do tumor. A análise do perfil toxicológico através de modelos in vivo (C. elegans) e in silico mostrou um composto com baixa toxicidade, concordando com dados anteriores do nosso grupo de pesquisa com as DHPMs. O conjunto de dados revelam que o LaSOM 335 é um candidato a potencial protótipo antitumoral. A possibilidade de atuar por no mínimo dois mecanismos de ação distintos devem ser investigados, principalmente a ação inibitória sobre a CD73.In Brazil, bladder cancer is the eighth most frequent neoplasm among men. Previous studies showed that the ecto-5'-nucleotidase (CD73) inhibition led to a significant reduction in the T24 bladder cancer cells proliferation, furthermore, other studies in mouse urothelial cells showed that the CD73 enzyme is overexpressed only in malignant cells, becoming also a possible selective target. In this work, a series of virtual tools were used to elucidate the mechanisms involved in the ligands uptake, as well as the available pockets for interaction in the ecto-5'-nucleotidase. The opening and closing movements, and the peculiar positioning of two phenylalanines (Phe500 and Phe417) make this enzyme a perfect machine to catch AMP molecules. The LaSOM library provided two scaffold of compounds for the study: coumarins and dihydropyrimidinones (DHPMs). Thus, a series of 152 coumarins were virtually screened by previously validated methods where LaSOM 366 and LaSOM 191 were selected as ligands. On the other hand, the activity was not confirmed in the enzymatic assay, under the used conditions. The investigated DHPMs collection revealed LaSOM 335 with in vitro inhibition against T24 bladder cancer cells (IC50=10.7μM). The in silico analysis showed affinity with CD73 and the LaSOM 335. The LaSOM 335 main interactions were between the 3-nitrobenzene fragments and the Arg395 residue, the dihyropyrimidinone ring and the phenylalanines Phe500 and Phe417, and the aromatic ring in N1 and the hydrophobic orthosteric site pocket, which stabilizes the complex formed. These in silico studies were confirmed with the enzymatic assay, in which LaSOM 335 presented IC50=136.5μM. In addition, this DHPM showed inhibition of the let-60 gene in the MT4244 strain of C. elegans. The let-60 gene is associated with the Ras gene coding. The Ras gene sinalization inhibition decreases tumor adhesion and migration. Analysis of the toxicological profile through in vivo (C. elegans) and in silico models showed a compound with low toxicity, corroborating previous data from our research group with DHPMs. The dataset reveals that LaSOM 335 is a candidate for a potential antitumor prototype. The possibility of acting by at least two distinct mechanisms of action should be investigated, mainly the inhibitory action on CD73

Estudo in silico de novas diidropirimidin-2-tionas com ação inibitória da enzima ecto-5'-nucleotidase

Os tumores cerebrais primários mais comuns são os gliomas. Os gliomas representam 31% de todos os tumores cerebrais diagnosticados nos Estados Unidos sendo 81% destes tumores malignos. O glioblastoma (GBM) é o tumor mais comum entre os gliomas, sua taxa de incidência varia de 0,59 a 3,69 a cada 100.000 pessoas, dependendo da idade e do país. A ecto-5’-nucleotidase regula os níveis extracelulares de AMP e adenosina, a qual tem sido amplamente descrita como fator indutor de proliferação celular. Estudos demonstraram que a atividade da enzima ecto-5’-nucleotidase foi aumentada em linhagens de células de glioma, quando comparada aos astrócitos. Também, o tratamento com 1 μM de APCP, um inibidor competitivo da ecto-5'-nucleotidase, causou uma redução significativa de 30% na proliferação das células de glioma. Através de estudos de modelagem molecular foi possível planejar três compostos com perfil farmacológico adaptado para o tratamento do glioblastoma. As moléculas LaSOM 281, LaSOM 282 e LaSOM 287, apresentaram um baixo risco toxicológico, capacidade de atravessar a barreira hematoencefálica e afinidade ao alvo pela predição realizada. Os compostos planejados foram sintetizados com bom rendimento e grau de pureza. No entanto os testes de atividade enzimática realizados para a enzima ecto-5’-nucleotidase das células de glioma, mostraram que os três compostos sintetizados não apresentaram ação inibitória, possivelmente por sua pouca solubilidade em meio aquoso.The most common primary brain tumors are gliomas. Gliomas represent 31% of all brain tumors diagnosed in the United States and 81% of these tumors are malignant. Glioblastoma (GBM) is the most common tumor among gliomas, with an incidence rate ranging from 0.59 to 3.69 per 100,000 people, depending on the age and country. The ecto-5'-nucleotidase regulates the extracellular levels of AMP and adenosine, which has been widely described as a cell proliferation inducing factor. Studies have shown that the activity of the enzyme ecto-5'-nucleotidase was increased in glioma cell lines when compared to astrocytes. Also, treatment with 1 μM APCP, a competitive inhibitor of ecto-5'-nucleotidase, caused a significant 30% reduction in glioma cell proliferation. Through molecular modeling studies it was possible to plan three compounds with pharmacological profile adapted for the treatment of glioblastoma. The molecules LaSOM 281, LaSOM 282 and LaSOM 287 showed a low toxicological risk, ability to cross the blood brain barrier and affinity to the target by the prediction. The planned compounds were synthesized in good yield and purity. However, the enzymatic activity tests performed for the ecto-5'-nucleotidase enzyme of glioma cells showed that the three compounds did not present an inhibitory action, possibly due to their low solubility in aqueous medium

Estudo in silico de novas diidropirimidin-2-tionas com ação inibitória da enzima ecto-5'-nucleotidase

Os tumores cerebrais primários mais comuns são os gliomas. Os gliomas representam 31% de todos os tumores cerebrais diagnosticados nos Estados Unidos sendo 81% destes tumores malignos. O glioblastoma (GBM) é o tumor mais comum entre os gliomas, sua taxa de incidência varia de 0,59 a 3,69 a cada 100.000 pessoas, dependendo da idade e do país. A ecto-5’-nucleotidase regula os níveis extracelulares de AMP e adenosina, a qual tem sido amplamente descrita como fator indutor de proliferação celular. Estudos demonstraram que a atividade da enzima ecto-5’-nucleotidase foi aumentada em linhagens de células de glioma, quando comparada aos astrócitos. Também, o tratamento com 1 μM de APCP, um inibidor competitivo da ecto-5'-nucleotidase, causou uma redução significativa de 30% na proliferação das células de glioma. Através de estudos de modelagem molecular foi possível planejar três compostos com perfil farmacológico adaptado para o tratamento do glioblastoma. As moléculas LaSOM 281, LaSOM 282 e LaSOM 287, apresentaram um baixo risco toxicológico, capacidade de atravessar a barreira hematoencefálica e afinidade ao alvo pela predição realizada. Os compostos planejados foram sintetizados com bom rendimento e grau de pureza. No entanto os testes de atividade enzimática realizados para a enzima ecto-5’-nucleotidase das células de glioma, mostraram que os três compostos sintetizados não apresentaram ação inibitória, possivelmente por sua pouca solubilidade em meio aquoso.The most common primary brain tumors are gliomas. Gliomas represent 31% of all brain tumors diagnosed in the United States and 81% of these tumors are malignant. Glioblastoma (GBM) is the most common tumor among gliomas, with an incidence rate ranging from 0.59 to 3.69 per 100,000 people, depending on the age and country. The ecto-5'-nucleotidase regulates the extracellular levels of AMP and adenosine, which has been widely described as a cell proliferation inducing factor. Studies have shown that the activity of the enzyme ecto-5'-nucleotidase was increased in glioma cell lines when compared to astrocytes. Also, treatment with 1 μM APCP, a competitive inhibitor of ecto-5'-nucleotidase, caused a significant 30% reduction in glioma cell proliferation. Through molecular modeling studies it was possible to plan three compounds with pharmacological profile adapted for the treatment of glioblastoma. The molecules LaSOM 281, LaSOM 282 and LaSOM 287 showed a low toxicological risk, ability to cross the blood brain barrier and affinity to the target by the prediction. The planned compounds were synthesized in good yield and purity. However, the enzymatic activity tests performed for the ecto-5'-nucleotidase enzyme of glioma cells showed that the three compounds did not present an inhibitory action, possibly due to their low solubility in aqueous medium

ShiftCrypt: a web server to understand and biophysically align proteins through their NMR chemical shift values

Nuclear magnetic resonance (NMR) spectroscopy data provides valuable information on the behaviour of proteins in solution. The primary data to determine when studying proteins are the per-atom NMR chemical shifts, which reflect the local environment of atoms and provide insights into amino acid residue dynamics and conformation. Within an amino acid residue, chemical shifts present multi-dimensional and complexly cross-correlated information, making them difficult to analyse. The ShiftCrypt method, based on neural network auto-encoder architecture, compresses the per-amino acid chemical shift information in a single, interpretable, amino acid-type independent value that reflects the biophysical state of a residue. We here present the ShiftCrypt web server, which makes the method readily available. The server accepts chemical shifts input files in the NMR Exchange Format (NEF) or NMR-STAR format, executes ShiftCrypt and visualises the results, which are also accessible via an API. It also enables the "biophysically-based" pairwise alignment of two proteins based on their ShiftCrypt values. This approach uses Dynamic Time Warping and can optionally include their amino acid code information, and has applications in, for example, the alignment of disordered regions. The server uses a token-based system to ensure the anonymity of the users and results. The web server is available at www.bio2byte.be/shiftcrypt.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

ShiftCrypt: a web server to understand and biophysically align proteins through their NMR chemical shift values

Nuclear magnetic resonance (NMR) spectroscopy data provides valuable information on the behaviour of proteins in solution. The primary data to determine when studying proteins are the per-atom NMR chemical shifts, which reflect the local environment of atoms and provide insights into amino acid residue dynamics and conformation. Within an amino acid residue, chemical shifts present multi-dimensional and complexly cross-correlated information, making them difficult to analyse. The ShiftCrypt method, based on neural network auto-encoder architecture, compresses the per-amino acid chemical shift information in a single, interpretable, amino acid-type independent value that reflects the biophysical state of a residue. We here present the ShiftCrypt web server, which makes the method readily available. The server accepts chemical shifts input files in the NMR Exchange Format (NEF) or NMR-STAR format, executes ShiftCrypt and visualises the results, which are also accessible via an API. It also enables the "biophysically-based" pairwise alignment of two proteins based on their ShiftCrypt values. This approach uses Dynamic Time Warping and can optionally include their amino acid code information, and has applications in, for example, the alignment of disordered regions. The server uses a token-based system to ensure the anonymity of the users and results. The web server is available at www.bio2byte.be/shiftcrypt.status: publishe

alanwilter/acpype: Release 2023.10.25

OFFICIAL: AnteChamber PYthon Parser interfac

alanwilter/acpype: Release 2023.10.27

OFFICIAL: AnteChamber PYthon Parser interfac