Estudo da regulação das DNA Estudo da regulação das DNA topoisomerases durante o desenvolvimento de resistência ao etoposídeo e do papel antitumoral de novos compostos em leucemias agudas / DNA topoisomerases's regulation study during resistance development toetoposide and antitumor role of new compounds in acute leukemias

Entre as neoplasias hematológicas, as leucemias agudas configuram o maior número de mortes a cada ano. A quimioterapia para estas neoplasias envolve inibidores de topoisomerase, como as antraciclinas, associados a outros fármacos. Entretanto, alguns pacientes não respondem ao tratamento devido ao desenvolvimento do fenótipo de resistência a múltiplas drogas (MDR), considerada a principal causa de refratariedade e falha no tratamento. Devido à alta taxa de proliferação celular, os tumores super expressam as DNA topoisomerases I e IIα humana (hTopo I e IIα), tornando essas enzimas bons alvos para o desenvolvimento de novos fármacos. Neste contexto, a procura por novos compostos capazes de aumentar a taxa de sobrevida global em pacientes com leucemias agudas e que sejam eficazes em células com fenótipo MDR se faz necessária. Os objetivos deste trabalho foram: a)desenvolver linhagens celulares de leucemias agudas resistentes ao etoposido (VP-16); b)caracterizar o fenótipo de resistência, mediado por alterações nas enzimas hTopo I e IIα; c)avaliar o mecanismo de ação dos novos compostos LQBs (LQB-118, -192, -223, -266, -268 e -326) e ácido pomólico (PA), como potenciais inibidores de hTopo I e/ou IIα; e d)investigar a atividade antitumoral dos compostos mais promissores nas linhagens de leucemias agudas resistentes em comparação às parentais. Foi demonstrado que dentre os compostos LQBs avaliados apenas LQB-118 e LQB-223 foram efetivos e específicos em inibir hTopoIIα. PA demonstrou ser um composto dual inibindo hTopo I e IIα. Os três compostos ativos não intercalam no DNA e atuam como inibidores catalíticos, não apresentando afinidade de interação ao sítio de ligação entre o DNA e camptotecina ou VP-16. Os estudos de modelagem molecular também sugeriram que LQB-118 e LQB-223 apresentam alta afinidade de ligação à região ATPase de hTopoIIα...

Acute leukemias represent the largest number of annual deaths from hematologic malignancy. The chemotherapy for these neoplasms involves topoisomerase inhibitors, such as anthracyclines, associated with other drugs. However, some patients do not respond to this treatment scheme because of the development of multiple drug resistance (MDR) phenotype.MDR phenotype is the main cause of refractoriness and treatment failure in acute leukemias. Due to the high rate of cell proliferation, tumors overexpress human DNA topoisomerases I and IIα (hTopo I and IIα), leading these enzymes as good targets for the development of new anticancer drugs. In this context, the searching for novel compounds capable of increase theoverall survival rate in patients with acute leukemias and be effective on cells with MDR phenotype is urgent. The objectives of this research are: a) todevelop acute leukemia cell linesresistant to etoposide (VP-16); b) to characterize the resistance phenotype mediated by changes on hTopo I and IIα; c) to evaluate themechanism of action of new compounds LQBs(LQB-118, -192, -223, -266, -268 and -326) and pomolic acid (PA), as potential inhibitors of hTopo I and/or IIα; and d) to investigate the antitumor activity of the most promising compounds on parental or resistant acute leukemia cell lines. It was demonstrated that among the LQBs evaluated only LQB-118 and LQB-223 were effective and specific to hTopo IIα and that PA inhibited both hTopo I and IIα. They did not intercalate into DNA and acted as catalytic inhibitors with poor affinity to interact with camptothecin or VP-16 biding site. The molecular modeling studies also suggested that LQB-118 and LQB-223 presented a high affinity to bindto ATPase region of hTopo IIα. Acute lymphoid CEM-R and myeloid leukemia U937-R cell lines were developed by exposition to increasing concentrations of VP-16...

New molecular biologist perspective and insight: DNA topoisomerases production by recombinant DNA technology for medical laboratory application and pharmaceutical industry

DNA topoisomerases are essential enzymes that control the topological state of DNA replication during mitosis. These enzymes are classified based on their mechanisms and physical properties. During mitosis, superhelical DNA must be unwound or relaxed by DNA topoisomerases prior to a decoding step by DNA processing enzymes, such as DNA polymerase and RNA polymerase. By blocking the reaction of resealing the breaks in the DNA ultimately can result in cellular death. Compounds that inhibit the catalytic function of these enzymes can serve as potential anticancer agents. DNA topoisomerases are found in nature and used as high quality and well-validated targets for the screening of potential anticancer agents. Our current work focuses on determining potential anticancer agents from natural resources using DNA topoisomerases as the screening targets. Large scale production of these enzymes using recombinant DNA technology in our academic laboratory is utilised to avoid dependence on expensive commercially available enzymes. The in-house produced enzymes can also be used to enhance our research in the field of molecular medicine by providing an enzyme source that can be used to screen potential anticancer agents, and for other newly developed diagnostic and medical research projects in the near future as well as a step in moving our efforts into the industrial sector.

Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil

The objective of this study was to identify mutations in the Quinolone Resistance Determining sources Regions (QRDR) of the gyrA, gyrB, parC, and parE genes and to determine if any of the qnr variants or the aac(6')-Ib-cr variant were present in strains of Salmonella spp. isolated in Brazil. A total of 126 Salmonella spp. strains from epidemic (n = 114) and poultry (n = 12) origin were evaluated. One hundred and twelve strains (88.8%) were resistant to nalidixic acid (NAL) and 29 (23.01%) showed a reduced susceptibility to ciprofloxacin (Cip). The mutations identified were substitutions limited to the QRDR of the gyrA gene in the codons for Serine 83, Aspartate 87 and Alanine 131. The sensitivity to NAL seems to be a good phenotypic indication of distinguishing mutated and nonmutated strains in the QRDR, however the double mutation in gyrA did not cause resistance to ciprofloxacin. The qnrA1 and qnrB19 genes were detected, respectively, in one epidemic strain of S. Enteritidis and one strain of S. Corvallis of poultry origin. Despite previous detection of qnr genes in Brazil, this is the first report of qnr gene detection in Salmonella, and also the first detection of qnrB19 gene in this country. The results alert for the continuous monitoring of quinolone resistance determinants in order to minimize the emergence and selection of Salmonella spp. strains showing reduced susceptibility or resistance to quinolones.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Estudo computacional da interação de compostos naftoquinônicos e a enzima DNA topoisomerase IB humana por modelagem comparativa, docking e dinâmica molecular / A computational study of the interaction of compounds naftoquinônicos and the enzyme DNA topoisomerase IB human by comparative modeling, docking and molecular dynamics

As DNA topoisomerases constituem um família de enzimas que desempenham um importante papel nos processos de replicação e atuam na regulação da topologia do DNA, inserindo uma quebra temporária em uma ou em ambas as fitas do DNA. A topoisomerases IB (TopoIB) produz quebras numa cadeia do DNA e permite o giro da cadeia quebrada sobre a cadeia intacta. A TopoIB conserva a energia do rompimento da ligação fosfodiéster, estocando-a na forma de ligação covalente que ocorre entre ela e os grupamentos fosfatos, no ponto de clivagem. Depois ela utiliza essa energia para restaurar a ligação fosfodiéster e selar a quebra. Algumas topoisomerases I podem relaxar super enovelamentos positivos e negativos no DNA. Estas enzimas são potenciais alvos para drogas citotóxicas. Alguns compostos já foram descritos como capazes de interferir na atividade de TopoI. Estes inibidores, chamados poisons, como a camptotecina e seus derivados, são efetivas drogas anticâncer. Outros, como os derivados de naftoquinonas são conhecidos como inibidores catalíticos, já foram descritos como atuantes sobre TopoI por mecanismos diferentes. Dentre estes compostos, o lapachol, uma naftoquinona natural, e alguns de seus derivados sintéticos como a (alfa)- e a (beta)-lapachona, já foram associados à inibição de topoisomerases. Desta forma, o objetivo principal deste trabalho foi o de estudar computacionalmente a interação entre alguns compostos derivados de naftoquinonas e a enzima DNA TopoIB humana, utilizando as técnicas de docking vii molecular e simulações de dinâmica molecular. Os compostos aqui estudados foram analisados quanto ao sítio mais provável de interação com a enzima, por docking molecular. Utilizando a mesma metodologia, foi possível descartar a atuação dos 75 compostos avaliados como poisons. Em geral, os compostos aqui estudados indicam que derivados de naftoquinonas são interessantes para o desenvolvimento de drogas anticancerígenas e antibióticas.