RESUMO
The study reports heterologous expression in Pichia pastoris of active neuraminidase derived from avian influenza virus A/Viet Nam/DT-036/2005(H5N1). A gene encoding the neuraminidase N1 head domain (residues 63-449) was fused directly in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal in pPICZ(A vector. Recombinant N1 neuraminidase was expressed in P. pastoris as a 72kDa secreted, soluble protein. Glycopeptidase F treatment generated a 45kDa product, indicating that the secreted recombinant N1 neuraminidase is an N-linked glycoprotein. Kinetic studies and inhibition tests with oseltamivir carboxylate demonstrated that the recombinant N1 neuraminidase has similar K(m) and K(i) values to those of the viral N1 neuraminidase. This yeast-based heterologous expression system provided functionally active recombinant N1 neuraminidase that should be useful in anti-influenza drug screening, and also as a potential protein-based vaccine.
Assuntos
Virus da Influenza A Subtipo H5N1/enzimologia , Neuraminidase/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Expressão Gênica , Genes Virais , Vetores Genéticos , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Cinética , Dados de Sequência Molecular , Neuraminidase/isolamento & purificação , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Transformação GenéticaRESUMO
Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.
Assuntos
Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Engenharia de Proteínas/métodos , Tetra-Hidrofolato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Antagonistas do Ácido Fólico/farmacologia , Biblioteca Gênica , Genes Reporter/genética , Teste de Complementação Genética/métodos , Proteínas de Fluorescência Verde/genética , Interações Hidrofóbicas e Hidrofílicas , Cinética , Mutagênese Sítio-Dirigida , Mutação , Proguanil , Estrutura Quaternária de Proteína , Pirimetamina/farmacologia , Proteínas Recombinantes de Fusão/genética , Solubilidade , Tetra-Hidrofolato Desidrogenase/biossíntese , Triazinas/farmacologiaRESUMO
Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti-malarial activities against drug-resistant P. falciparum bearing the mutant enzymes.
Assuntos
Antimaláricos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , Animais , Sítios de Ligação , Resistência a Medicamentos/fisiologia , Estrutura Molecular , Complexos Multienzimáticos/química , Mutação , Ligação Proteica , Conformação Proteica , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/químicaRESUMO
Nucleophilic additions of lithium keto and ester enolates and mono- and bifunctional Grignard reagents to artemisitene provided C-16-derived artemisinin monomers, dimers, trimers, and tetramers whose antimalarial and cytotoxic activities have been evaluated.
Assuntos
Antimaláricos/síntese química , Antineoplásicos/síntese química , Artemisininas , Sesquiterpenos/química , Sesquiterpenos/síntese química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular , Chlorocebus aethiops , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Plasmodium falciparum/efeitos dos fármacos , Polímeros , Sesquiterpenos/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Artemisinin-type compounds are used for the treatment of uncomplicated and severe forms of malaria. They reduce parasitaemia more rapidly than any other antimalarial compound known, and are effective against multidrug-resistant parasites. However, uncertainties remain as to how they act on the parasite and cause toxicity. In this review, we summarize current ideas.
Assuntos
Antimaláricos/farmacologia , Artemisininas , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/química , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Sesquiterpenos/química , Sesquiterpenos/farmacologiaRESUMO
The Ala16Val+Ser108Thr (A16V+S108T) mutant of the Plasmodium falciparum dihydrofolate reductase (DHFR) is a key mutant responsible for cycloguanil-resistant malaria due to steric interaction between Val-16 and one of the C-2 methyl groups of cycloguanil. 4,6-Diamino-1,2-dihydrotriazines have been prepared, in which both methyl groups of cycloguanil are replaced by H or by H and an alkyl or phenyl group, and their inhibition constants against wild-type and mutant DHFR determined. The S108T mutation is considered to decrease cycloguanil binding further through the effect on the orientation of the p-chlorophenyl group. By moving the p-chloro-substituent to the m-position in the chlorophenyl group, the activity against the A16V+S108T mutant enzyme is improved, and this effect is reinforced by the p-chloro substituent in the 3, 4-dichlorophenyl group. A lead compound has been found with inhibitory activity similar to that of cycloguanil against the wild-type DHFR and about 120-fold more effective than cycloguanil against the A16V+S108T mutant enzyme. The activity of this compound against P. falciparum clone (T9/94 RC17) which harbors the A16V+S108T DHFR is about 85-fold greater than cycloguanil.
Assuntos
Antimaláricos/síntese química , Antagonistas do Ácido Fólico/síntese química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Triazinas/síntese química , Triazinas/farmacologia , Substituição de Aminoácidos , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Resistência a Medicamentos , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Mutação , Proguanil , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/genética , Triazinas/químicaRESUMO
The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.
Assuntos
Resistência a Medicamentos , Antagonistas do Ácido Fólico/farmacologia , Pirimetamina/farmacologia , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Triazinas/farmacologia , Sequência de Aminoácidos , Antagonistas do Ácido Fólico/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Proguanil , Pirimetamina/química , Homologia de Sequência de Aminoácidos , Tetra-Hidrofolato Desidrogenase/química , Triazinas/químicaRESUMO
Plasmodium falciparum infecting alpha-thalassemic erythrocytes (Hb H or Hb H/Hb Constant Spring) is resistant to artemisinin derivatives. Similar resistance, albeit at a much lower level, is shown by the parasite infecting beta-thalassemia/Hb E erythrocytes. The resistance is due to host-specific factors, one of which is the higher uptake of the drugs by thalassemic erythrocytes than normal erythrocytes, due to binding with Hb H. In addition to higher drug binding, incubation of artemisinin with alpha-thalassemic erythrocytes resulted in preferential inactivation of the drug. Both thalassemic and normal erythrocytes have the capability to inactivate the drug. Addition of serum can protect against inactivation by normal erythrocytes, but not by thalassemic erythrocytes. Incubation with either the hemolysate or the membrane fraction from these erythrocytes also resulted in preferential inactivation of the drug. The drug was also inactivated by purified Hb H. It is concluded that the ineffectiveness of artemisinin derivatives against P. falciparum infecting thalassemic erythrocytes is due partly to competition of the host cell components for binding with the drugs, and partly to inactivation of the drugs by the cell components.
Assuntos
Antimaláricos/farmacologia , Artemisininas , Eritrócitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Sesquiterpenos/farmacologia , Talassemia/sangue , Animais , Antimaláricos/antagonistas & inibidores , Antimaláricos/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Resistência a Medicamentos , Hemoglobina H/metabolismo , Humanos , Técnicas In Vitro , Substâncias Protetoras/farmacologia , Albumina Sérica/farmacologia , Sesquiterpenos/antagonistas & inibidores , Sesquiterpenos/metabolismo , Talassemia/metabolismoRESUMO
The need for new antimalarials comes from the widespread resistance to those in current use. New antimalarial targets are required to allow the discovery of chemically diverse, effective drugs. The search for such new targets and new drug chemotypes will likely be helped by the advent of functional genomics and structure-based drug design. After validation of the putative targets as those capable of providing effective and safe drugs, targets can be used as the basis for screening compounds in order to identify new leads, which, in turn, will qualify for lead optimization work. The combined use of combinatorial chemistry--to generate large numbers of structurally diverse compounds--and of high throughput screening systems--to speed up the testing of compounds--hopefully will help to optimize the process. Potential chemotherapeutic targets in the malaria parasite can be broadly classified into three categories: those involved in processes occurring in the digestive vacuole, enzymes involved in macromolecular and metabolite synthesis, and those responsible for membrane processes and signalling. The processes occurring in the digestive vacuole include haemoglobin digestion, redox processes and free radical formation, and reactions accompanying haem release followed by its polymerization into haemozoin. Many enzymes in macromolecular and metabolite synthesis are promising potential targets, some of which have been established in other microorganisms, although not yet validated for Plasmodium, with very few exceptions (such as dihydrofolate reductase). Proteins responsible for membrane processes, including trafficking and drug transport and signalling, are potentially important also to identify compounds to be used in combination with antimalarial drugs to combat resistance.
Assuntos
Antimaláricos/farmacologia , Tecnologia Farmacêutica , Membrana Eritrocítica/fisiologia , Glicólise , Hemoglobinas/metabolismo , Humanos , Malária/tratamento farmacológico , Ácidos Nucleicos/metabolismo , Transdução de SinaisRESUMO
Activity-guided investigation of Artemisia indica Willd. has led to isolation of exiguaflavone A, exiguaflavone B, maackiain, and 2-(2, 4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran. Exiguaflavones A and B exhibit in vitro antimalarial activities of 4.60 x 10(-6) and 7.05 x 10(-6) g/mL, respectively, against Plasmodium falciparum.
Assuntos
Antimaláricos/isolamento & purificação , Artemisia/química , Benzofuranos/isolamento & purificação , Flavonoides/isolamento & purificação , Plantas Medicinais , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Benzofuranos/química , Benzofuranos/farmacologia , Cromatografia Líquida de Alta Pressão , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Flavonoides/química , Flavonoides/farmacologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Plasmodium falciparum/efeitos dos fármacos , Espectrofotometria UltravioletaRESUMO
Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.
Assuntos
Antimaláricos/farmacologia , Desenho de Fármacos , Antagonistas do Ácido Fólico/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Pirimetamina/análogos & derivados , Pirimetamina/farmacologia , Animais , Resistência a Medicamentos , Malária/tratamento farmacológico , Masculino , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/farmacologia , Mutação , Plasmodium berghei , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Pirimetamina/síntese química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/farmacologia , Timidilato Sintase/genética , Timidilato Sintase/farmacologiaRESUMO
Dihydroartemisinin and other artemisinin derivatives are relatively ineffective against Plasmodium falciparum infecting alpha-thalassemic erythrocytes, namely hemoglobin (Hb) H or HbH/Hb Constant Spring erythrocytes, as compared with those infecting genetically normal erythrocytes. The variant erythrocytes accumulate radiolabeled dihydroartemisinin to a much higher extent than the normal ones, and the accumulated drug was retained after extensive washing, in contrast to the drug in normal erythrocytes which was mostly removed. At initial drug concentration of 1 mM, most (82-88%) of the drug was found in the cytosol fraction of both variant and normal erythrocytes. Binding of the drug to hemoglobins accounted for 40-70% of the total uptake. Hb H accounted for 10.9 +/- 2.7% and 12.4 +/- 6.2% of total protein in HbH and HbH/Hb Constant Spring erythrocytes. HbH bound with 28.7 +/- 6.7% of the drug, whereas HbH/Hb Constant Spring erythrocytes bound with 21.8 +/- 8.3% of the drug. Binding experiments showed that Hb H had 5-7 times the drug-binding capacity of Hb A. For Hb H, the maximum binding capacity (Bmax) = 1.67 +/- 0.17 mol/mol Hb, and the dissociation constant (Kd) = 66 +/- 17 microM, and for Hb A, Bmax = 0.74 +/- 0.18 mol/mol Hb and Kd = 224 +/- 15 microM. It is concluded that preferential binding of dihydroartemisinin to Hb H over Hb A accounts partly for the higher accumulation capacity of the alpha-thalassemic erythrocytes, which leads to its antimalarial ineffectiveness.
Assuntos
Antimaláricos/metabolismo , Artemisininas , Eritrócitos/metabolismo , Hemoglobina H/metabolismo , Sesquiterpenos/metabolismo , Talassemia alfa/sangue , Animais , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacosRESUMO
The codon for serine residue 108 of the Plasmodium falciparum dihydrofolate reductase gene was replaced with those for the other 19 amino acids. Except for the Lys108 mutant, which was not expressed, all other substitutions yielded DHFR mutants which were expressed in Escherichia coli as inactive inclusion bodies. Nine of the mutants--Asn108, Thr108, Gly108, Ala108, Gln108, Cys108, Val108, Leu108, and Met108--yielded active DHFR upon refolding of the protein from the inclusion bodies. The remaining mutants--IIe108, Arg108, Pro108, Asp108, His108, Tyr108, Phe108, Trp108, and Glu108--did not exhibit detectable DHFR activity on refolding. The Asn108 mutant had almost unperturbed kinetic parameters but conferred resistance to pyrimethamine and cycloguanil; other active mutants showed poorer DHFR activity. We purified and characterized four mutants which produced highest DHFR activity, i.e., the Gln108, Gly108, Cys108, and Ala108 mutants. These mutant enzymes had kcat/K(m) values ranging from 7 to 22% of the wild-type enzyme. While DHFRs from Gly108, Cys108, and Ala108 mutants were as susceptible to pyrimethamine and cycloguanil as the wild type, the Gln108 mutation conferred high resistance to both inhibitors. Our data suggest that residue 108 is important for antifolate binding, and that the Ser108 to Asn108 mutation was selected in nature because of (i) the need for only a single base change, (ii) its good activity, and (iii) its resistance to antifolates.
Assuntos
Asparagina/genética , Antagonistas do Ácido Fólico/farmacologia , Mutação , Plasmodium falciparum/enzimologia , Tetra-Hidrofolato Desidrogenase/genética , Animais , Resistência a Medicamentos/genética , Genes de Protozoários , Genes Sintéticos , Plasmodium falciparum/genética , Proguanil , Pirimetamina/farmacologia , Proteínas Recombinantes/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Triazinas/farmacologiaRESUMO
Single and multiple mutations at residues 16, 51, 59, 108, and 164 of Plasmodium falciparum dihydrofolate reductase (pfDHFR) have been linked to antifolate resistance in malaria. We prepared and characterized all seven of the pfDHFR mutants found in nature, as well as six mutants not observed in nature. Mutations involving residues 51, 59, 108, or 164 conferred cross resistance to both the antifolates pyrimethamine and cycloguanil, whereas mutation of residue 16 specifically conferred resistance to cycloguanil. The antifolate resistance of enzyme mutants found in nature correlated with in vivo antifolate resistance; however, mutants not found in nature were either poorly resistant or had insufficient catalytic activity to support DNA synthesis. Thus, specific combinations of multiple mutations at target residues were selected in nature to optimize resistance. Further, the resistance of multiple mutants was more than the sum of the component single mutations, indicating that residues were selected for their synergistic as well as intrinsic effects on resistance. Pathways inferred for the evolution of pyrimethamine-resistant mutants suggested that all multiple mutants emerged from stepwise selection of the single mutant, S108N. Thus, we propose that drugs targeted to both the wild-type pfDHFR and S108N mutant would have a low propensity for developing resistance, and hence could provide effective antimalarial agents.
Assuntos
Antimaláricos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Animais , Evolução Biológica , Resistência a Medicamentos/genética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Plasmodium falciparum/efeitos dos fármacos , Mutação Puntual , Proguanil , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , TriazinasRESUMO
The antimalarial activity of a number of artemisinin derivatives, both newly synthesized and currently used as drugs, against Plasmodium falciparum in culture shows a correlation with their affinity of binding with ferroprotoporphyrin IX, as measured from the spectral change of the latter. The new C-16-functionalized artemisinin derivatives were obtained through a novel one-pot synthesis of artemisitene (2) from naturally abundant artemisinin (1), followed by Michael addition with nucleophiles. The correlation points to the biological significance of the interaction of these derivatives with ferroprotoporphyrin IX and may provide a basis for primary screening of peroxidic antimalarials of similar structures.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Artemisininas , Heme/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Sesquiterpenos/farmacologia , Animais , Células Cultivadas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sesquiterpenos/síntese química , Sesquiterpenos/metabolismoRESUMO
In the culture of red cells with Plasmodium falciparum, erythrocytes from both Thai patients and subjects (patient's parents) with hereditary ovalocytosis have a protective effect against malarial infection. High percentage of ovalocyte (75-100%) was found in patients whereas their parents had lower percentage (25-50%). Invasion index (II) and multiplication ratio (MR) of P. falciparum in these abnormal red cells from the patients were significantly decreased as compared to those in normal red cells (patients: II = 1.52 +/- 0.91, MR = 8.83 +/- 6.73; normal subjects: II = 4.45 +/- 1.51, MR = 25.23 +/- 6.25). This suggests that the red cells from these patients had significant degree of malaria protection. The significant protection was also shown in red cells from the parent group (II = 1.86 +/- 0.81, MR = 15.69 +/- 3.50). Although the parents had lower ovalocyte percentage, degree of protection against malaria parasite was as effective as those found in patients with high ovalocytic red cells. This has been confirmed by statistical analysis showing nonsignificant difference in II value between the two groups. In contrast, red cells of both groups had poor deformability (deformability index, DI) as compared to the normal group. No statistically different DI values were demonstrated between the two. This indicates that poorly deformable red cells, not their ovalocytic shape, make a significant contribution to limitation of malaria parasite invasion. The MR values in patients were less than those found in the parent group but statistical analysis showed no significant difference. Reduced MR values were found with increased numbers of microcytic, hyperchromic and hypochromic red cells in patients.
Assuntos
Eliptocitose Hereditária/sangue , Deformação Eritrocítica/fisiologia , Malária Falciparum/sangue , Humanos , Imunidade Inata , Tailândia , Fatores de TempoRESUMO
Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil. The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designed based on E. coli codon preference with unique restriction sites evenly placed throughout the coding sequence. The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain. Each of these domains contained numerous unique restriction sites to facilitate mutagenesis. The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence. The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E. coli about 10-fold more efficiently than from the wild-type sequence. The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study. The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria.
Assuntos
Genes de Protozoários , Genes Sintéticos , Complexos Multienzimáticos/genética , Plasmodium falciparum/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Animais , Composição de Bases , Códon , DNA de Protozoário/síntese química , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Mutação , Plasmodium falciparum/enzimologia , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismoRESUMO
Activity-guided investigation of Cyperus rotundus tubers led to the isolation of patchoulenone, caryophyllene alpha-oxide, 10,12-peroxycalamenene and 4,7-dimethyl-1-tetralone. The antimalarial activities of these compounds are in the range of EC50 10(-4)-10(-6) M, with the novel endoperoxide sesquiterpene, 10,12-peroxycalamenene, exhibiting the strongest effect at EC50 2.33 x 10(-6) M.
Assuntos
Antimaláricos/química , Plantas Medicinais , Sesquiterpenos/química , Animais , Antimaláricos/isolamento & purificação , Antimaláricos/toxicidade , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Rotação Ocular , Raízes de Plantas , Plasmodium falciparum/efeitos dos fármacos , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/toxicidadeRESUMO
Various derivatives of artemisinin covalently linked to iron chelators were synthesized, and their antimalarial activities were evaluated. Although results show no indication that the presence of an iron chelator in the vicinity of artemisinin potentiates its action, the linked compounds prepared still retain comparable activities to that of artemisinin.