Estudo estrutural e comparativo entre Sirtuínas 2 de diferentes espécies de Leishmania / Structural and comparative study between Sirtuins 2 of different species of Leishmania by means of Comparative Modeling and Molecular Dynamic

As leishmanioses são doenças negligenciadas que afetam mais de um bilhão e meio de pessoas ao redor do mundo, principalmente nos países em desenvolvimento, provocando grandes impactos socioeconômicos. Os fármacos disponíveis para o tratamento dessas doenças são ineficazes e apresentam graves efeitos adversos. O processo de pesquisa de novos fármacos envolve, entre outras coisas, a seleção de alvos bioquímicos essenciais para a sobrevivência e desenvolvimento do agente causador. Neste sentido, a Sirtuína 2, uma enzima epigenética com atividade hidrolase essencial para a sobrevivência dos parasitas do gênero Leishmania se apresenta como um alvo validado na busca de novos fármacos contra essas parasitoses. O planejamento de fármacos baseado na estrutura do receptor requer o conhecimento da estrutura tridimensional da proteína alvo. Desta forma, a elucidação estrutural e um estudo minucioso das Sirtuínas das várias espécies do gênero Leishmania apresenta-se como uma importante abordagem na aplicação desta estratégia na busca por agentes quimioterápicos. Até o momento, na família Trypanosomatidae, a única estrutura tridimensional resolvida experimentalmente de uma enzima Sirtuína 2 é a da espécie L. infantum. Assim, este trabalho aplicou a abordagem de Modelagem Comparativa utilizando o software Modeller na construção de modelos da Sir2rp1 das espécies L. infantum, L. major e L. braziliensis, cujas sequências de aminoácidos foram extraídas do banco de dados UNIProt. Os modelos construídos foram validados por meio da função de escore DOPE do Modeller e dos servidores PROCHECK, MolProbity e QMEAN, avaliando sua qualidade estereoquímica e seu enovelamento. Os ligantes naturais da enzima foram sobrepostos nos modelos construídos por alinhamento estrutural utilizando o software PyMol e os complexos validados foram submetidos a simulações de Dinâmica Molecular através do pacote GROMACS. Os complexos refinados foram então analisados por meio dos softwares PyMol e LigPlotPlus e dos pacotes GROMACS e gmx_MMPBSA, e foram estudados os sítios de ligação dos substratos e os resíduos de aminoácidos relevantes envolvidos em sua ligação e reconhecimento. A Modelagem Comparativa da Sirtuína 2 humana e seus homólogos das espécies L. infantum, L. major e L. braziliensis, as simulações de Dinâmica Molecular realizadas com os modelos enzimáticos construídos e validados complexados com seus ligantes naturais, os cálculos de energia de interação entre os modelos e seus substratos e o estudo estrutural comparativo realizado entre eles nos fornecem uma base teórica para a busca de novos inibidores da Sirtuína 2 que sejam mais seletivos e potentes contra as enzimas parasitárias, abrindo caminho para o desenvolvimento de candidatos a fármacos leishmanicidas mais seguros e eficazes

Leishmaniasis are neglected diseases that affect more than one and a half billion people around the world, mainly in developing countries, causing major socioeconomic impacts. The drugs available for the treatment of these diseases are ineffective and have serious adverse effects. The process of researching new drugs involves, among other things, the selection of biochemical targets essential for the survival and development of the causative agent. In this sense, Sirtuin 2, an epigenetic enzyme with hydrolase activity essential for the survival of parasites of the Leishmania genus, presents itself as a validated target in the search for new drugs against these parasites. Structure-Based Drug Design requires knowledge of the three-dimensional structure of the target protein. In this way, structural elucidation and a detailed study of Sirtuins from various species of the genus Leishmania presents itself as an important approach in the application of this strategy in the search for chemotherapeutic agents. To date, in the Trypanosomatidae family, the only experimentally resolved three-dimensional structure of a Sirtuin 2 enzyme is that of the species L. infantum. Thus, this work applied the Comparative Modeling approach using the Modeller software in the construction of Sir2rp1 models of the species L. infantum, L. major and L. braziliensis, whose amino acid sequences were retrieved from the UNIProt database. The constructed models were validated using Modeller's DOPE score function and the PROCHECK, MolProbity and QMEAN servers, evaluating their stereochemical quality and folding. The enzyme's natural ligands were superimposed on the built models by structural alignment using the PyMol software and the validated complexes were subjected to Molecular Dynamics simulations using the GROMACS package. The refined complexes were then analyzed using the PyMol and LigPlotPlus softwares and the GROMACS and gmx_MMPBSA packages, and the substrate binding sites and relevant amino acid residues involved in their binding and recognition were studied. The Comparative Modeling of human Sirtuin 2 and its homologues from the species L. infantum, L. major and L. braziliensis, the Molecular Dynamics simulations carried out with the constructed and validated enzymatic models complexed with their natural ligands, the interaction energy calculations between the models and their substrates and the comparative structural study carried out between them provide us with a theoretical basis for the search for new Sirtuin 2 inhibitors that are more selective and potent against the parasitic enzymes, paving the way for the development of safer and more effective leishmanicidal drug candidates

Triagem biológica de inibidores de sirtuína 2 (TcSir2rp1) candidatos a antichagásicos / Biological screening of sirtuin 2 inhibitors (TcSir2rp1) antichagasic candidates

A doença de Chagas é causada pelo Trypanosoma cruzi, e atualmente, acomete entre 6 a 7 milhões de pessoas em todo o mundo. A quimioterapia disponível para seu o tratamento se baseia apenas em dois fármacos, nifurtimox e benznidazol, com mais de 50 anos de descoberto. Estes fármacos apresentam eficácia limitada, pois são pouco efetivos na fase crônica e apresentam alta toxicidade, resultando em efeitos adversos graves. Esse panorama mostra a necessidade de novas abordagens terapêuticas contra essa doença. Nesse sentido, a inibição de vias bioquímicas essencias para o parasita se mostram como uma boa sugestão para identificação de compostos promissores candidatos a novos agentes quimioterápicos. A sirtuína 2 (Sir2) são enzimas reguladoras que participam de mecanismos epigenéticos em tripanossomatídeos, e no T. cruzi possuem um papel fundamental em todos os seus estágios evolutivos, devido a este fato, se apresentam como um alvo promissor na busca por novos fármacos contra a doença de Chagas. Neste sentido propomos a busca de inibidores da Sir2 proteína 1 do T. cruzi (TcSir2rp1) que é geneticamente validada como alvo farmacológico, por meio da estratégia de triagem biológica. Realizou-se a expressão da enzima recombinante por biologia molecular em um sistema de transformação utilizando cepa de Escherichia coli Artic Express (DE3). Foi feita a purificação e a confirmação da obtenção da proteína recombinante se deu por gel SDS-PAGE. Após a obtenção da enzima os parâmetros cinéticos foram determinados por experimentos de fluorimetria. A triagem foi realizada para um conjunto de 82 compostos, previamente sintetizados pelo nosso grupo de pesquisa, como inibidores da TcSir2p1 em dose única de 100 µM. Os ensaios foram realizados em triplicata e em experimentos independentes. Dentre os 82 compostos testados, 20 apresentaram inibições maior que 50% contra a enzima TcSir2rp1, na dose de 100 µM. Dentre estes, se destacaram 3 compostos derivados de chalconas, para os quais foi determinada a potência. O composto 1 foi o que mais potente, apresentando valor de IC50 de 11,65 µM, já os compostos 3 e 5 foram menos potentes (IC50= 38,50 µM e 19,85 µM, respectivamente). Diante dos resultados obtidos, pode-se concluir que a estratégia de triagem biológica é promissora na identificação de inibidores da TcSir2p1 candidatos a agentes anti- T. cruzi

Chagas disease is caused by Trypanosoma cruzi, and currently affects 6 to 7 million people worldwide. The chemotherapy available for its treatment is based on only two drugs, nifurtimox and benznidazole, with more than 50 years of discovery. These drugs have limited efficacy, as they are ineffective in the chronic phase and have high toxicity, resulting in serious adverse effects. This panorama shows the need for new therapeutic approaches against this disease. In this sense, the inhibition of essential biochemical pathways for the parasite proves to be a good suggestion for the identification of promising compounds candidates for new chemotherapeutic agents. Sirtuin 2 (Sir2) are regulatory enzymes that participate in epigenetic mechanisms in trypanosomatids, and in T. cruzi they have a fundamental role in all their evolutionary stages, due to this fact, they present themselves as a promising target in the search for new drugs against Chagas disease. In this sense, we propose the search for inhibitors of Sir2 protein 1 of T. cruzi (TcSir2rp1) which is genetically validated as a pharmacological target, through the biological screening strategy. The expression of the recombinant enzyme was performed by molecular biology in a transformation system using strain of Escherichia coli Artic Express (DE3). Purification was performed and confirmation of obtaining the recombinant protein was performed by SDS-PAGE gel. After obtaining the enzyme, the kinetic parameters were determined by fluorimetry experiments. Screening was performed for a set of 82 compounds, previously synthesized by our research group, as TcSir2p1 inhibitors in a single dose of 100 µM. Assays were performed in triplicate and in independent experiments. Among the 82 compounds tested, 20 showed inhibitions greater than 50% against the enzyme TcSir2rp1, at a dose of 100 µM. Among these, 3 compounds derived from chalcones stood out, for which the potency was determined. Compound 1 was the most potent, with an IC50 value of 11.65 µM, while compounds 3 and 5 were less potent (IC50= 38.50 µM and 19.88 µM, respectively). In view of the results obtained, it can be concluded that the biological screening strategy is promising in the identification of TcSir2p1 inhibitors candidates for anti-T. cruzi agents

Expression of IGFBP-rP1 in ovarian and breast cancers in association with diabetes mellitus status

@#Introduction: Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is an important component of the IGF system that regulates insulin resistance-related to tumour development. The aim of this study is to investigate the expression of IGFBP-rP1 among female cancer patients who are known or not known to have Type 2 Diabetes Mellitus (T2DM). Materials and Methods: Using a cross-sectional design, cases of ovarian and breast cancer with clinical status of T2DM were selected over a 10-year period in Hospital Universiti Sains Malaysia. Immunohistochemical staining for IGFBP-rP1 was performed on paraffin-embedded tissues and the results were correlated with the patient’s demographic and clinicopathological data. Results: A total of 152 breast cancer patients were recruited into the current study with 33.5% (51/152) patients were positive T2DM. Most of the breast cancer patients with T2DM were IGFBP-rP1-negative (66.7%, 34/51). The IGFBP-rP1 expression was significantly difference between breast cancer subjects with and without T2DM (p<0.001). There was no significant association of IGFBP-rP1 expression with data on the demographic and clinicopathological profiles of patients with breast cancer. Meanwhile, positive IGFBP-rP1 expression was evident in 44 out of 108 (40.74%) ovarian cancer cases. Among these cases, 36 were T2DM. In contrast to breast cancer cases, IGFBP-rP1 was mostly expressed among ovarian cancer patients with T2DM (66.7%, 24/36, p < 0.001). However, the -positive expression was not significantly associated with any sociodemographic and clinicopathological features of ovarian cancers. Conclusions: Majority of breast cancer patients with T2DM did not express IGFBP-rP1. In contrast, majority of the ovarian cancer patients with T2DM expressed IGFBP-rP1.

Effects of long non-coding RNA RP1-90L14.1 on the biological behaviors of cancer prostate LNCaP cells and its regulating mechanisms / 中华男科学杂志

Objective@#To investigate the effects of long non-coding RNA RP1-90L14.1 on the proliferation, migration and invasion of prostate cancer LNCaP cells and the expressions of GRIN2A and BACE2.@*METHODS@#Using RT-PCR, we detected the expression of RP1-90L14.1 in LNCaP and LNCaP-AI cells, transiently transfected the RP1-90L14.1 overexpression plasmid (the RP1-90L14.1 group) and vector plasmid (the LNCaP-NC group) into the LNCaP cells, and cultured the two groups of cells with ordinary medium and phenol red-free activated carbon adsorption medium (PRF-ACA). Then we examined the proliferation, migration and invasiveness of the cells by CCK-8 and Transwell, and determined the mRNA and protein expressions of GRIN2A and BACE2 by RT-PCR and Western blot.@*RESULTS@#The expression of RP1-90L14.1 was significantly higher in the LNCaP-AI than in the LNCaP cells (8.49 ± 0.43 vs 2.53 ± 0.95, P < 0.05), and so was that of LNCaP-RP1-90L14.1 in the RP1-90L14.1 than in the LNCaP-NC group after transfection (0.71 ± 0.22 vs 0.02 ± 0.01, P < 0.05). The optical densities (OD) of the cells were 51.95% and 50.69% higher in the RP1-90L14.1 than in the LNCaP-NC group after 72 hours of culture with ordinary medium and phenol red-free ACA (1.22 ± 0.08 vs 0.08 ± 0.05, P < 0.05; 0.79 ± 0.02 vs 0.53 ± 0.05, P < 0.05), and 51.72% and 60.23% higher in the former than in the latter after 96 hours (1.72 ± 0.07 vs 1.13 ± 0.05, P < 0.05; 1.18 ± 0.05 vs 0.73 ± 0.08, P < 0.05). The numbers of the migrating cells cultured with common medium and PRF-ACA were markedly higher in the RP1-90L14.1 than in the LNCaP-NC group after transfection (682.0 ± 42.7 vs 422.0 ± 37.1, P < 0.05; 419.0 ± 42.9 vs 251.0 ± 25.9, P < 0.05), and so were those of the invading cells (507.0 ± 22.2 vs 274.0 ± 19.6, P < 0.05; 352.0 ± 14.1 vs 216.0 ± 14.3, P < 0.05). Statistically significant differences were observed between the RP1-90L14.1 and LNCaP-NC groups in the mRNA and protein expressions of GRIN2A (5.13 ± 0.89 vs 2.09 ± 0.54, P < 0.05; 5.88 ± 0.29 vs 2.03 ± 0.22, P < 0.05) and BACE2 (5.82 ± 0.50 vs 2.53 ± 0.30, P < 0.05; 4.89 ± 0.19 vs 3.37 ± 0.13, P < 0.05).@*CONCLUSIONS@#　lncRNA RP1-90L14.1 may play important roles in the proliferation, migration and invasiveness of prostate cancer cells. RP1-90L14.1 can promote the expressions of GRIN2A and BACE2 and may have an endogenous competitive relation with GRIN2A and BACE2.

Induction of apoptosis in mouse spleen cells by Ginsenoside Rp1 / 대한수의학회지

Ginsenoside Rp1 is one of ginseng saponins with chemotherapeutic activity. In this study, we investigated the effects of Rp1 on spleen cells. Spleen is a major immune organ consisted of crucial immune cells, such as T lymphocytes, B lymphocytes, natural killer cells, and some antigen-presenting cells. Although the anti-tumor potential of Rp1 was studied, the effects of Rp1 on immune cells have not investigated yet. A viability assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), flow cytometric analysis, Western blot analysis were used to detect cellular changes on Rp1-treated spleen cells. MTT assay showed that Rp1 decreased the viability of spleen cells. To further investigate the effects of Rp1 on activated spleen cells, we treated lipopolysaccharide (LPS) as a representative inflammatory agent and Rp1 on spleen cells in a combination. The surface expression levels of activation markers for lymphocytes, CD25 and CD69 were measured. Apoptotic analysis revealed the cytotoxic effects of Rp1 on both naive and activated cells, and the expression pattern of some apoptosis-related proteins was correlated to apoptotic events of cells. Taken together, ginsenoside Rp1 increases the cellular death of spleen cells and also inhibits the LPS-induced activation of spleen cells.

Mutations analysis of RP1 gene in 110 Chinese with retinitis pigmentosa / 中华实验眼科杂志

Background Retinitis pigmentosa (RP) is a monogenic inheritance and blinding disease of fundus oculi.There is not an effective therapeutic method now.Objective This work was to identify the mutations of RP1 gene in Chinese RP patients in Ningxia area and to explore the potential interactions in the pathogenesis of RP.Methods The periphery blood of 3-5 ml was collected from 110 individuals with RP(35 ADRP and 75SRP)and 100 normal controls in Ningxia area.Polymerase chain reaction (PCR) and direct DNA sequencing were used to screening the sequence alterations in the entire coding region and splice sites of RP1 gene.Multivariate analysis and two web-based programs( PolyPhen and SIFT) were used to analyze the results.Results Eleven mutation locus were detected in the exon 4 of RP1 gene including two novel sequence variants:p.Lys1152Lys without a higher mutation rate in comparison with normal control group(x2 =9.12 P＜0.01 ),but c.* 247A＞C with a higher mutation rate in comparison with normal control group(x2 =12.77,P＜0.01 ) and c.* 247A＞C mutation was thought to be correlated with RP( r=1.11,P＜0.05 ).The other ten mutation locus were reported as single nucleotide polymorphisms (SNP).The mutation rate of p.Gln1725Gln was found to be higher in the RP patients than the normal controls (x2 =42.09,P＜0.01 ),but no the significant correlation was seen between the pathogenesis of RP and mutation of p.Gln1725Gln(r=1.74,P＞0.05).p.Lys1152Lys mutation was found in only 1 patient.Three SNPs( p.Arg872His,Ala1670Thr,Ser1691Pro) were always occurred in the same 83 RP patient and the relevance ratio was higher than controls ( P＜0.01 ).The age of night blindness on patients with concurrent three mutations was (30.54± 13.68 ) years,and the best corrected visual acuity (BCVA) was 0.50 ± 0.38.The age of night blindness on patients without concurrent three mutations was(21.06± 16.24) years,and the BCVA was 0.40 ±0.33 and were higher than controls ( t =2.11,P ＜ 0.05 ).Conclusions In this study,the prevalence of RP1 mutations among the RP patients in Ningxia population was lower than other populations (＜ 1％ ).The alliance of SNPs (p.Arg872His、p.Ala1670Thr、p.Ser1691Pro) may play a protective role on RP patients and reduce the frequency of mutatiaon in RP1 gene.

The expression of insulin-like growth factor-binding protein related protein 1 (IGFBP-rP1) in children with acute leukemia and its clinical significance / 肿瘤

Objective:To explore the expression of insulin-like growth factor-binding protein related protein 1(IGFBP-rP1) gene in children with acute leukemia and its potential significance. Methods:Real-time fluorescence quantitative PCR (RFQ-PCR) method was used for detecting IGFBP-rP1 mRNA expression in bone marrow (BM) cells of 168 children with acute leukemia. The results were compared with those of 30 non-leukemia children in control group. Meanwhile the relationship between IGFBP-rP1 expression level and clinical prognosis was analyzed according to clinical prognostic factors of children acute leukemia. Results:Expression level of IGFBP-rP1 in initial acute leukemia children was significantly higher than that of non leukemia children (P<0.01). It was higher in acute myeloid leukemia (AML) than in acute lymphoblastic leukemia (ALL)(P =0.013). The transcription level of IGFBP-rP1 mRNA in patients who had complete remission (CR) were lowest, which was nearly the same as non-leukemia childish patients. It increased again when leukemia relapsed, which was significantly higher than that in CR. However, as far as ALL was concerned, IGFBP-rP1 expression levels had no significant difference between newly-diagnosed, complete remission, and recurrent groups.Conclusion:IGFBP-rP1 may be involved in the initiation and development of childish leukemia. It has the potential to become a new target for AML treatment.

Studies with RP1 deletion plasmids: Incompatibility properties, plasmid curing and the spontaneous loss of resistance.

Four deletion plasmids, pHH301, pHH302, pHH303 and pHH401, obtained from RP1 DNA-transformed bacterial clones, were shown to be incompatible with three Ρ plasmids in Escherichia coli K12 strains. Kinetic experiments and colony tests were used to verify the position of these R plasmids. Pseudomonas aeruginosa and Ε. coli strains, harbouring deletion plasmids, could be cured by using two mutagens, acriflavine and mitomycin C, which affect a percentage of the cell population. The deletion plasmid-positive strains could also be induced at an elevated temperature to spontaneously loose their plasmids.