RESUMO
Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.
Assuntos
Anti-Helmínticos/farmacologia , Antiprotozoários/farmacologia , Brugia Malayi/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Helminto/antagonistas & inibidores , Leishmania major/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anti-Helmínticos/química , Antiprotozoários/química , Brugia Malayi/enzimologia , Brugia Malayi/genética , Brugia Malayi/crescimento & desenvolvimento , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Ensaios de Triagem em Larga Escala , Leishmania major/enzimologia , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de SequênciaRESUMO
Glutathione-S-transferase also referred as GST is one of the major detoxification enzymes in parasitic helminths. The crucial role played by GST in various chronic infections has been well reported. The dependence of nematodes on detoxification enzymes to maintain their survival within the host established the crucial role of GST in filariasis and other related diseases. Hence, this well-established role of GST in filariasis along with its greater nonhomology with its human counterpart makes it an important therapeutic drug target. Here in this study, we have tried to explore the inhibitory potential of some of the well-reported natural ant-filarial compounds against the GST from Wuchereria bancrofti (W.bancrofti) and Brugia malayi (B.malayi). In silico virtual screening, approach was used to screen the selected natural compounds against GST from W.bancrofti and B.malayi. On the basis of our results, here we are reporting some of the natural compounds which were found to be very effective against GSTs. Along with we have also revealed the characteristic of the active site of BmGST and WbGST and the role of important active site residues involve in the binding of natural compounds within the active site of GSTs. This information will oped doors for using natural compounds as anti-filarial therapy and will also be helpful for future drug discovery.
Assuntos
Anti-Helmínticos/análise , Anti-Helmínticos/farmacologia , Produtos Biológicos/análise , Produtos Biológicos/farmacologia , Brugia Malayi/enzimologia , Avaliação Pré-Clínica de Medicamentos , Glutationa Transferase/antagonistas & inibidores , Wuchereria bancrofti/enzimologia , Alcaloides/química , Alcaloides/farmacologia , Animais , Benzodioxóis/química , Benzodioxóis/farmacologia , Brugia Malayi/efeitos dos fármacos , Capsaicina/química , Capsaicina/farmacologia , Domínio Catalítico , Curcumina/química , Curcumina/farmacologia , Glutationa Transferase/metabolismo , Simulação de Acoplamento Molecular , Piperidinas/química , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/química , Alcamidas Poli-Insaturadas/farmacologia , Estricnina/química , Estricnina/farmacologia , Wuchereria bancrofti/efeitos dos fármacosRESUMO
Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design.
Assuntos
Biologia Computacional , Ácido Fólico/metabolismo , Animais , Anti-Helmínticos/uso terapêutico , Brugia Malayi/enzimologia , Filariose/tratamento farmacológico , Filariose/parasitologia , Humanos , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Lymphatic filariasis (LF) is a vector borne infectious disease caused by the nematode Wuchereria bancrofti, Brugia malayi, and Brugia timori. Over 120 million people are affected by LF in the world, of which two-thirds are in Asia. The infection restricts the normal flow of lymph from the infected area resulting in swelling of the extremities and causing permanent disability. As the available drugs for the treatment of LF are becoming ineffective due to the development of resistance, there is an urgent need to find new leads for drug development. In this study, asparaginyl-tRNA synthetase (AsnRS; PDB ID: 2XGT) essential for the protein bio-synthesis in the filarial nematode was used to carry out virtual screening (VS) of plant constituents from traditional Chinese medicine (TCM) database. Docking as well as E-pharmacophore based VS were carried out to identify the hits. The top scoring hits, Agri 1 (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one-3-O-beta-D-glucopyranoside) and Agri 2 (5,7-dihydroxy-2-propylchromone 7-O-beta-D-glucopyranoside), constituents of Agrimonia pilosa, were selected for molecular dynamics (MD) simulation study for 10 ns. MD simulation showed that both the glycosides Agri 1 and Agri 2 were forming stable interactions with the target protein. Moreover, docking and MD simulation of the lead A (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one; Mol. Wt.: 304.25; CLogP: 3.07) and lead B (5,7-dihydroxy-2-propylchromone; Mol. Wt.: 220.22; CLogP: 3.02), the aglycones of Agri 1 and Agri 2, respectively, were carried out with the target AsnRS. The in silico investigations of the aglycones suggest that the lead B could be a suitable fragment-like lead molecule for anti-filarial drug discovery.
Assuntos
Aspartato-tRNA Ligase/antagonistas & inibidores , Brugia Malayi/efeitos dos fármacos , Bases de Dados de Produtos Farmacêuticos , Medicamentos de Ervas Chinesas/farmacologia , Filariose Linfática/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Filaricidas/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Aminoacil-RNA de Transferência/antagonistas & inibidores , Wuchereria bancrofti/efeitos dos fármacos , Animais , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Sítios de Ligação , Brugia Malayi/enzimologia , Desenho Assistido por Computador , Desenho de Fármacos , Medicamentos de Ervas Chinesas/química , Filariose Linfática/diagnóstico , Filariose Linfática/parasitologia , Inibidores Enzimáticos/química , Filaricidas/química , Humanos , Ligantes , Estrutura Molecular , Terapia de Alvo Molecular , Ligação Proteica , Conformação Proteica , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Relação Estrutura-Atividade , Wuchereria bancrofti/enzimologiaRESUMO
Glutathione-S-transferase(s) (GST) enzyme from Brugia malayi has been exploited as a target in lymphatic filariasis therapeutics. An active GST is a homodimer of a 208 residue long monomer consisting of two domains, a smaller α/ß domain and a larger α domain. The components of the glutathione (GSH) system, mainly GST enzymes, are critical antioxidant and detoxification system responsible for the long-term existence of filarial worms in mammalian host; hence they are major chemotherapeutic targets in filarial species. In the present study, 58 phytochemicals from 10 plants, predicted and reported to have potential nematicidal activity and ADMET satisfaction, have been docked to GST enzyme of B. malayi to assess their binding affinity and consequently their inhibitory activity. A comparative study has been made with commonly employed chemotherapeutic GST inhibitors such as cibacron-blue, butylated hydroxyanisole, hexyl glutathione and ethacrynic acid. In vitro effects of potential drug like compound from in silico results have been done for validation of docking studies. In vitro assay revealed efficacy in GST inhibition in the following compounds: linalool (97.50%), alpha-pinene (90.00%), strychnine (87.49%), vanillin (84.99%), piperine (79.99%), isoeugenol (62.49%), curcumin (57.49%), beta-caryophyllene (39.50%), cinnamic acid (27.49%), capsaicin (19.99%), citronellol (19.99%) and geraniol (17.49%). An online database ( www.spicebioinfo.res.in/gstleadbase ) has been developed, which will serve as a useful repository of information on GST inhibitors for future development of drugs against filarial nematodes. These findings thus suggest that the above phytochemicals could be potentially developed as lead molecules for targeting GST of lymphatic filarial parasites.
Assuntos
Brugia Malayi/efeitos dos fármacos , Brugia Malayi/enzimologia , Filariose/tratamento farmacológico , Glutationa Transferase/antagonistas & inibidores , Extratos Vegetais/química , Sequência de Aminoácidos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Glutationa Transferase/metabolismo , Modelos Químicos , Extratos Vegetais/farmacologia , Estrutura Secundária de Proteína , Alinhamento de Sequência , EspeciariasRESUMO
Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-ß-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 mM. The assay sensitivity was enhanced by the thiol agents, DTT and L-Cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.
Assuntos
Aminoacil-tRNA Sintetases/genética , Asparagina/análogos & derivados , Brugia Malayi/genética , Ensaios de Triagem em Larga Escala , Extratos Vegetais/análise , Edição de RNA/fisiologia , Staphylococcus epidermidis/genética , Aminoácidos/fisiologia , Aminoacil-tRNA Sintetases/análise , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/fisiologia , Animais , Asparagina/análise , Asparagina/química , Asparagina/genética , Asparagina/fisiologia , Aspartato-tRNA Ligase/análise , Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/fisiologia , Brugia Malayi/enzimologia , Brugia Malayi/metabolismo , Corantes , Cisteína/metabolismo , Descoberta de Drogas , RNA de Transferência/genética , RNA de Transferência/fisiologia , Aminoacil-RNA de Transferência/análise , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/fisiologia , Corantes de Rosanilina , Staphylococcus epidermidis/enzimologia , Staphylococcus epidermidis/metabolismoRESUMO
A gene encoding a copper/zinc superoxide dismutase (Cu/ Zn-SOD) of a filarial nematode, Brugia malayi, has been isolated and the biochemical properties of a functionally expressed recombinant enzyme were investigated. The cloned complementary DNA contained a single open reading frame of 477 bp encoding 158 amino acids (aa), which conserved metal-binding residues as well as residues specific for Cu/Zn-SODs. Comparison of the deduced aa sequence of the enzyme with that of other helminthes species, including filarial worms, exhibited high degree of similarities (49-98%). Recombinant enzyme of 32 kDa had an isoelectric point of 6.6 and was shown to consist of 2 subunits linked by interchain disulfide bonds. Enzyme activity of the recombinant protein was inhibited by potassium cyanide and hydrogen peroxide but not by sodium azide. It showed a wide range of pH optima, i.e., 7.0-11.0 and was highly resistant to heat inactivation.
Assuntos
Brugia Malayi/enzimologia , Superóxido Dismutase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Brugia Malayi/genética , DNA Complementar/química , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Gerbillinae , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , RNA de Helmintos/genética , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/química , Superóxido Dismutase/metabolismoRESUMO
Prior studies have demonstrated that transglutaminase (TGase) from the human filarial parasite Brugia malayi is critical for the growth and development of the larval stages. In this report, we describe the cloning and partial characterization of a cDNA encoding the B. malayi TGase (BmTGase). Using RT-PCR and RACE-PCR, the cDNA was amplified from adult worm mRNA. BmTGase is 1,881 bp long and codes for a protein with a predicted molecular mass of 54 kDa. Amino acid sequence analysis of BmTGase revealed significant homology to the protein disulfide isomerase (PDI), particularly, to the PDI-related protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. The activity of recombinant B. malayi TGase enzyme (rBmTG) was found to be calcium-dependent and could be inhibited by EDTA. ELISA studies showed that approximately 88% of 48 sera from healthy Indian patients living in a bancroftian filariasis endemic area were reactive with rBmTG. In contrast, only 33% of sera from patients with clinical filariasis were reactive to rBmTG. Non-endemic sera were uniformly non-reactive. Additional studies are needed to elucidate the role, if any, of B. malayi TGase in protective immunity to filariasis.
Assuntos
Brugia Malayi/enzimologia , Clonagem Molecular , Análise de Sequência de DNA , Transglutaminases/genética , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Brugia Malayi/genética , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Filariose/imunologia , Filariose/parasitologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transglutaminases/imunologia , Transglutaminases/isolamento & purificação , Transglutaminases/metabolismoRESUMO
Brugia malayi L3 molt to the L4 stage in serum-free cultures supplemented with arachidonic, linoleic, or linolenic acids and the basidiomycetous yeast Rhodotorula minuta. These fatty acids are capable of entering the eicosanoid pathway of arachidonate metabolism, the pathway responsible for generating a number of biologically active mediators, including prostaglandins, leukotrienes, and lipoxins. To determine whether this pathway was required for L3 development, we added dual inhibitors of cyclooxygenase and lipoxygenase to in vitro cultures containing B. malayi L3. These compounds significantly inhibited L3 molting. To evaluate whether 1 or both of these pathways of arachidonate metabolism were involved in molting, we tested drugs inhibiting either cyclooxygenase or lipoxygenase. Lipoxygenase inhibitors blocked L3 molting, whereas cyclooxygenase inhibitors did not. To assess whether enzymes operating downstream of lipoxygenase were also involved in L3 molting, we added inhibitors of enzymes involved in leukotriene synthesis and found they were also capable of preventing development. We tested the same inhibitor panel on Dirofilaria immitis L3. A single lipoxygenase inhibitor and inhibitors of 2 different enzymes operating downstream of lipoxygenase disrupted D. immitis development. These results demonstrate that a lipoxygenase pathway product is required for molting of the infective stage larvae of filarial parasites.