Aplicação de ferramentas de quimioinformática na comparação da estrutura tridimensional da dihidrofolato redutase expressa em diferentes organismos / Application of cheminformatics tools for comparing dihydrofolate reductase's tridimensional structure expressed by different organisms

A quimioinformática, definida como o emprego de técnicas informáticas na solução de problemas da química, evolui em conjunto com o desenvolvimento de ferramentas computacionais e é de grande relevância para o planejamento racional de fármacos ao otimizar etapas do desenvolvimento de novas moléculas e economizar recursos e tempo. Dentre as técnicas disponíveis destacam-se o planejamento de fármacos baseado na estrutura e no ligante, que quando combinadas auxiliam na identificação e otimização de moléculas ativas frente a alvos farmacológicos. A Dihidrofolato Redutase (DHFR) é uma importante enzima da via dos folatos que catalisa a redução do dihidrofolato em tetrahidrofolato, utilizando NADPH como cofator, reação essencial para a replicação celular, visto que este ciclo resulta na síntese de precursores das bases nitrogenadas que compõem o DNA, consequentemente, inibidores de DHFR são utilizados no tratamento de infecções bacterianas e alguns tipos de câncer. Trypanosoma cruzi, protozoário causador da doença de chagas, é um dos organismos que expressam a DHFR, além do próprio Homo sapiens. Analisaram-se ligantes conhecidos e as estruturas da proteína expressa pelos dois organismos, visando identificar pontos de divergência que possam ser explorados no planejamento de moléculas seletivas para o tratamento da doença de Chagas. Os 6 modelos cristalográficos de T. cruzi e 2 de H. sapiens foram obtidos do banco de dados de proteínas (PDB) após aplicação de filtros de qualidade. Foram analisadas as sequências de aminoácidos dos modelos, com o uso do Cluster Ômega, sua estrutura tridimensional com os programas Pymol e Chimera X, além da análise das cavidades proteicas com o CavityPlus, que também gerou os farmacóforos de ambos alvos. A análise de estrutura primária identificou mutações em três aminoácidos nos cristais do parasita, que podem ser explicados por diferentes caminhos evolutivos de grupos segregados, embora nenhuma mutação observada esteja em regiões de sítio ativo. A análise dos modelos permitiu que fossem identificados os 25 aminoácidos que estão a menos de 5 Å de distância dos ligantes de T. cruzi, sendo 5 aminoácidos responsáveis por interações de hidrogênio com pelo menos um dos ligantes analisados. Destes, 18 se repetem na proteína humana ou são substituídos por outro aminoácido que mantém a mesma interação. Quanto às diferenças observadas, destacam-se a asparagina 44 substituída por uma prolina na proteína humana e a prolina 92, substituída por uma lisina. A análise de cavidades identificou três cavidades em cada proteína, embora somente as cavidades correspondentes ao sítio ativo sejam druggables. A cavidade da proteína humana é maior e mais alongada, além de apresentar o aspecto de um túnel, enquanto a cavidade da proteína parasita é mais aberta, tal abertura permite que ligantes com o anel benzeno meta substituídos explorem uma região existente na cavidade de T. cruzi que é fechada na humana. O farmacóforo de ambas proteínas foi identificado, apresentando diferenças no tamanho e angulação que também podem ser explorados no planejamento de fármacos seletivos

Chemoinformatic, defined as the use of informatic techniques to solve chemical problems, has evolved together with new computational tools and it is quite important for rational drug designing, by optimizing different steps on the development pipeline of new molecules, saving resources and time. From all the available tools, structure and ligand based drug design shall be highlighted, when combined, they support the identification and optimization of active molecules from pharmaceutical targets. Dihydrofolate reductase (DHFR) is an important enzyme of the folate pathway that catalyzes the reduction of dihydrofolate to tetrahydrofolate, by using NADPH as cofactor. This reaction is essential for cell replication, as this pathway results in the synthesis of nucleobases that build the DNA. That's the reason why DHFR inhibitors are used for treating bacterial infections and some types of cancer. Trypanosoma cruzi, a protozoa that causes Chagas disease, is one of the organisms that express DHFR, besides Homo sapiens itself. This work analyzed known ligands and the structure of the protein expressed by both organisms, aiming to identify divergence points that could be explored for designing selective drugs for Chagas disease treatment. The 6 proteins crystallographic models from T. cruzi and 2 from H. sapiens were obtained from protein data bank (PDB) after the application of quality filters. The amino acid sequence of each model was analyzed by Clustal Omega, its tridimensional structure by Pymol and Chimera X and the cavity analysis by CavityPlus, that also generated the pharmacophore from both targets. The primary structure analysis identified mutations on three amino acids on the parasite christal, which may be explained by different evolutive paths from segregated groups, although none of the observed mutations are on the active site region. The model's analysis allowed the identification of 24 amino acids that are closer than 5 Å from the T. cruzi ligands, 5 of them responsible for hydrogen interactions on at least one of the ligands analyzed. 18 of them are repeated on the human protein or are replaced by another amino acid that preserves the same interaction. As by the differences observed that shall be highlighted, asparagine 44 is replaced by a proline on the human protein, and proline 92 by a lysin. The cavity analysis identified three cavities on each protein, although only the cavities of the active site are druggables. The human protein cavity is bigger and longer, besides it looks like its a tunnel, when the parasite protein is open, that opening allows ligands with benzene ring meta substituted to explore the existing regions of the T. cruzi protein that is closed on the human protein. Lastly, the pharmacophore from both proteins was identified, it shows differences on size and angulation that also could be explored in the designing of selective drugs

Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.

Dual and selective inhibitors of pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Leishmania chagasi.

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).

Targeting pteridine reductase 1 and dihydrofolate reductase: the old is a new trend for leishmaniasis drug discovery.

Leishmaniasis is one of the major neglected tropical diseases in the world and it is considered endemic in 88 countries. This disease is transmitted by a Leishmania spp. infected sandfly and it may lead to cutaneous or systemic manifestations. The preconized treatment has low efficacy and there are cases of resistance to some drugs. Therefore, the search for new efficient molecular targets that can lead to the preparation of new drugs must be pursued. This review aims to evaluate both Leishmania enzymes PTR1 and DHFR-TS as potential drug targets, highlight their inhibitors and to discuss critically the use of chemoinformatics to elucidate interactions and propose new molecules against these enzymes.

Crystal structures of the closed form of Mycobacterium tuberculosis dihydrofolate reductase in complex with dihydrofolate and antifolates.

Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M. tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through Kd measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest Kd) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Overexpression of dihydrofolate reductase is a factor of poor survival in acute lymphoblastic leukemia.

Dihydrofolate reductase (DHFR) has an important function in DNA synthesis and is a target of methotrexate, which is a crucial treatment option for acute lymphoblastic leukemia (ALL). However, the number of studies conducted to date on DHFR expression in childhood ALL is limited. The aim of the present study was to determine whether the expression of DHFR is associated with survival in childhood ALL. The expression of DHFR in 96 children with ALL and 100 control individuals was determined using reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the expression of DHFR mRNA in children with ALL was significantly increased (P<0.001), compared with that in the control group. In addition, increased levels of DHFR mRNA were observed in patients with B-cell lineage, compared with patients with T-cell lineage ALL (P<0.05). The Kaplan-Meier estimator analysis revealed that children with ALL who exhibited increased levels of DHFR mRNA had a decreased overall survival time (P<0.05). It was observed that certain patient prognostic features (including age, sex, white blood cell count and high DHFR expression), are associated with poor survival (log-rank test, P<0.05). Therefore, the results of the present study indicated that DHFR upregulation is a factor for poor survival in ALL.

Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase.

The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP+ and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.

Analysis of Coxiela burnetti dihydrofolate reductase via in silico docking with inhibitors and molecular dynamics simulation.

Coxiella burnetii is a gram-negative bacterium able to infect several eukaryotic cells, mainly monocytes and macrophages. It is found widely in nature with ticks, birds, and mammals as major hosts. C. burnetii is also the biological warfare agent that causes Q fever, a disease that has no vaccine or proven chemotherapy available. Considering the current geopolitical context, this fact reinforces the need for discovering new treatments and molecular targets for drug design against C. burnetii. Among the main molecular targets against bacterial diseases reported, the enzyme dihydrofolate reductase (DHFR) has been investigated for several infectious diseases. In the present work, we applied molecular modeling techniques to evaluate the interactions of known DHFR inhibitors in the active sites of human and C. burnetii DHFR (HssDHFR and CbDHFR) in order to investigate their potential as selective inhibitors of CbDHFR. Results showed that most of the ligands studied compete for the binding site of the substrate more effectively than the reference drug trimethoprim. Also the most promising compounds were proposed as leads for the drug design of potential CbDHFR inhibitors.

Dihydrofolate reductase 19-bp deletion polymorphism modifies the association of folate status with memory in a cross-sectional multi-ethnic study of adults.

BACKGROUND: Folate status has been positively associated with cognitive function in many studies; however, some studies have observed associations of poor cognitive outcomes with high folate. In search of an explanation, we hypothesized that the association of folate with cognition would be modified by the interaction of high-folate status with a common 19-bp deletion polymorphism in the dihydrofolate reductase (DHFR) gene. To our knowledge, the cognitive effects of this gene have not been studied previously. OBJECTIVE: We examined the association between cognitive outcomes with the 19-bp deletion DHFR polymorphism, folate status, and their interaction with high or normal plasma folate. DESIGN: This was a pooled cross-sectional study of the following 2 Boston-based cohorts of community living adults: the Boston Puerto Rican Health Study and the Nutrition, Aging, and Memory in Elders study. Individuals were genotyped for the DHFR 19-bp deletion genotype, and plasma folate status was determined. Cognitive outcomes included the Mini-Mental State Examination, Center for Epidemiologic Studies Depression Scale, and factor scores for the domains of memory, executive function, and attention from a set of cognitive tests. RESULTS: The prevalence of the homozygous deletion (del/del) genotype was 23%. In a multivariable analysis, high folate status (>17.8 ng/mL) was associated with better memory scores than was normal-folate status (fourth-fifth quintiles compared with first-third quintiles: ß ± SE = -0.22 ± 0.06, P < 0.01). Carriers of the DHFR del/del genotype had worse memory scores (ß ± SE = -0.24 ± 0.10, P < 0.05) and worse executive scores (ß = -0.19, P < 0.05) than did those with the del/ins and ins/ins genotypes. Finally, we observed an interaction such that carriers of the del/del genotype with high folate had significantly worse memory scores than those of both noncarriers with high-folate and del/del carriers with normal-folate (ß-interaction = 0.26 ± 0.13, P < 0.05). CONCLUSIONS: This study identifies a putative gene-nutrient interaction that, if confirmed, would predict that a sizable minority carrying the del/del genotype might not benefit from high-folate status and could see a worsening of memory. An understanding of how genetic variation affects responses to high-folate exposure will help weigh risks and benefits of folate supplementation for individuals and public health.

The 19-bp deletion polymorphism of dihydrofolate reductase (DHFR) and nonsyndromic cleft lip with or without cleft palate: evidence for a protective role

Objective Nonsyndromic cleft lip with or without cleft palate (NS-CL/P) are among the most common congenital birth defects worldwide. Several lines of evidence point to the involvement of folate, as well as folate metabolizing enzymes in risk reduction of orofacial clefts. Dihydrofolate reductase (DHFR) enzyme participates in the metabolic cycle of folate and has a crucial role in DNA synthesis, a fundamental feature of gestation and development. A functional polymorphic 19-bp deletion within intron-1 of DHFR has been associated with the risk of common congenital malformations. The present study aimed to evaluate the possible association between DHFR 19-bp deletion polymorphism and susceptibility to NS-CL/P in an Iranian population. Material and Methods The current study recruited 100 NS-CL/P patients and 100 healthy controls. DHFR 19-bp deletion was determined using an allele specific-PCR method. Results We observed the DHFR 19-bp homozygous deletion genotype (D/D) vs. homozygous wild genotype (WW) was more frequent in controls than in NS-CL/P patients (25% vs. 13%), being associated with a reduced risk of NS-CL/P in both codominant (OR=0.33, P=0.027) and recessive (OR=0.45, P=0.046) tested inheritance models. We also stratified the cleft patients and reanalyzed the data. The association trend for CL+CL/P group compared to the controls revealed that the DD genotype in both codominant (OR=0.30, P=0.032) and recessive models (OR=0.35, P=0.031) was associated with a reduced risk of CL+CL/P. Conclusions Our results for the first time suggested the DHFR 19-bp D/D genotype may confer a reduced risk of NS-CL/P and might act as a protective factor against NS-CL/P in the Iranian subjects. .

Design, synthesis and biological evaluation of quinazoline derivatives as anti-trypanosomatid and anti-plasmodial agents.

In this paper, the design, synthesis and biological evaluation of a set of quinazoline-2,4,6-triamine derivatives (1-9) as trypanocidal, antileishmanial and antiplasmodial agents are explained. The compounds were rationalized basing on docking studies of the dihydrofolate reductase (DHFR from Trypanosoma cruzi, Leishmania major and Plasmodium vivax) and pteridin reductase (PTR from T. cruzi and L. major) structures. All compounds were in vitro screened against both bloodstream trypomastigotes of T. cruzi (NINOA and INC-5 strains) and promatigotes of Leishmania mexicana (MHOM/BZ/61/M379 strain), and also for cytotoxicity using Vero cell line. Against T. cruzi, three compounds (5, 6 and 8) were the most effective showing a better activity profile than nifurtimox and benznidazole (reference drugs). Against L. mexicana, four compounds (5, 6, 8, and 9) exhibited the highest activity, even than glucantime (reference drug). In the cytotoxicity assay, protozoa were more susceptible than Vero cells. In vivo Plasmodium berghei assay (ANKA strain), the compounds 1, 5, 6 and 8 showed a more comparable activity than chloroquine and pyrimethamine (reference drugs) when they were administrated by the oral route. The antiprotozoal activity of these substances, endowed with redox properties, represented a good starting point for a medicinal chemistry program aiming for chemotherapy of Chagas' disease, leishmaniosis and malaria.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99 percent). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5 percent), followed by the wild-type A383/A553 (17.5 percent) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Mutaciones puntuales en los genes dhfr y dhps de Plasmodium falciparum de tres regiones endémicas para malaria en Colombia / Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum from three endemic malaria regions in Colombia

Introducción. La acumulación progresiva de mutaciones en los genes dhfr y dhps lleva al parásito Plasmodium falciparum a evadir la acción de la sulfadoxina-pirimetamina, situación que aumenta el nivel de resistencia del parásito a estos medicamentos y conlleva a la aparición de fallas del tratamiento. Objetivos. Determinar la frecuencia de mutaciones en los genes dhfr y dhps de P. falciparum asociadas con resistencia a sulfadoxina-pirimetamina, en muestras de pacientes de tres zonas endémicas de Colombia: La Carpa, Guaviare; Casuarito, Vichada; Tierralta y Puerto Libertador, Córdoba. Materiales y métodos. Se incluyeron 40 muestras de pacientes con malaria no complicada por P. falciparum. Los alelos 108, 59 y 164 del gen dhfr se analizaron mediante PCR específica de alelo y los alelos 51 del gen dhfr y 436, 437 y 540 del gen dhps por PCR y restricción enzimática. Resultados. En el gen dhfr encontramos en todas las muestras las mutaciones asparagina 108 e isoleucina 51. No se detectaron alelos mutantes en los codones 59 y 164 del gen dhfr, ni en el codón 436 del gen dhps. La mutación glicina 437 estuvo presente en 36 muestras y el alelo silvestre alanina en tres de Tierralta y una de La Carpa. La mutación ácido glutámico 540 sólo se halló en Casuarito. Conclusiones. En las poblaciones de P. falciparum analizadas prevalecen los alelos asparagina 108, isoleucina 51 y glicina 437, lo que indica un efecto acumulativo de mutaciones y la necesidad de vigilar la aparición de nuevos alelos mutantes que puedan conducir a la pérdida total de la eficacia de la sulfadoxina-pirimetamina.

Introduction. Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure. Objectives. To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba. Materials and methods. Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism. Results. The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito. Conclusions. The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.

Actividad inhibttoria sobre la enzima dihidrofoato reductasa de extractos de eesponjas marinas del golfo de urabá / Inhibitory actviity of some marine sponge extrcts from urabá gulf on dihydroffolate reductase enzyme

La enzima dihidrofolato reductasa está implicada en la producción de la base pririmidínica timidina, componente esencial de la estructura del ADN. Por tanto, cualquier sustancia que la inhiba tiene como efecto la inhibición de la síntesis del ADN, y es potencialmente útil para el tratamiento de varios tipos de cáncer como leucemias linfoblásticas. En este trabajo se determina el grado de inhibición que los extractos etanólicos obtenidos de las esponjas marinas colombianas Svenzea zeai, Amphimedon compressa, Ircinia campana, Aplysina archeri, Xestospongia proxima y Xestospongia muta, presentan sobre la enzima purificada de origen humano dihidrofolato reductasa. Los resultados muestran que la mayoría de los extractos de estas esponjas inhiben esta enzima. Estos resultados se comparan con los del medicamento usado contra el cáncer, Metotrexate®, el cual se utiliza como control de inhibición de los ensayos y se observa que algunas de las esponjas tienen mayor inhibición que este medicamento.

Dihydrofolate reductase is an enzyme involved in the production of pyrimidinic base timidin, a structural component of DNA, therefore whatever substance that inhibit this enzyme inhibit the DNA synthesis as a consequence and it can be potentially useful as a treatment of several types of cancer like lymphoblastic leukemias. In this work we determinate the inhibition grade that the ethanol extracts from Colombian marine sponges: Svenzea zeai, Amphimedon compressa, Ircinia campana, Aplysina archeri, Xestospongia proxima y Xestospongia muta, over the human purified enzyme dihydrofolate reductase. The results shown that most of marine sponge extracts inhibite the enzyme. Results are compared with methotrexate® a medicament used against cancer which is used as a control for the bioassays. Results demonstrate that some of the analyzed extracts have more inhibition than the control metotrexate®.

Resistência à sulfadoxina-pirimetamina em Maputo, Moçambique: presença de mutações nos genes dhfr e dhps do Plasmodium falciparum / Sulfadoxine-pyrimethamine resistance in Maputo, Mozambique: presence of mutations in the dhfr and dhps genes of Plasmodium falciparum

Foram analisadas a freqüência e distribuição de mutações nos genes dihidrofolato redutase e dihidropteroato sintetase do Plasmodium falciparum, usando a metodologia de reação em cadeia da polimerase e polimorfismos de hidrólise por enzimas de restrição, em amostras de sangue infectado proveniente de crianças moçambicanas, residentes em Maputo. A análise foi feita antes e 7 dias após o tratamento com sulfadoxina-pirimetamina (S/P). Os resultados mostraram a ocorrência de mutações pontuais nos genes estudados e a presença de combinações de três alelos em dhfr (51Ile, 59Arg e 108Asn) e do quintúplo mutante (dhfr 51Ile, 59Arg, 108Asn e dhps 437Gly, 540Glu), ambas situações associadas à falha terapêutica no sétimo dia após tratamento com S/P. Esses achados mostram a importância de se estudar a resistência à S/P em Moçambique, e como os marcadores moleculares de resistência aos antimaláricos podem fornecer dados importantes para a política nacional de controlo da malária.

The frequency and distribution of mutations in Plasmodium falciparum, dihydrofolate reductase and dihydropteroate synthase genes were analyzed, using the polymerase chain reaction and restriction fragment length polymorphism methodology, in infected blood samples from Mozambican children living in Maputo, before and seven days after treatment with sulfadoxine/pyrimethamine (S/P). The results showed the occurrence of point mutations in the genes studied and the presence of combinations of three alleles in dhfr (51Ile, 59Arg and 108Asn) and "quintuple" mutant (dhfr 51Ile, 59Arg, 108Asn and dhps 437Gly, 540Glu). Both of these situations were associated with seven-day therapeutic failure, following treatment with S/P. These findings show the importance of studying S/P resistance in Mozambique, and how molecular markers for antimalarial resistance can provide important data for national malaria control policy.