RESUMEN
The enzyme aspartate semialdehyde dehydrogenase (ASDH) plays a pivotal role in the amino acid biosynthesis pathway, making it an attractive target for the development of new antimicrobial drugs due to its absence in humans. This study aims to investigate the presence of ASDH in the filarial parasite Wolbachia endosymbiont of Brugia malayi (WBm) using both in vitro and in silico approaches. The size exclusion chromatography (SEC) and Native-PAGE analysis demonstrate that WBm-ASDH undergoes pH-dependent oligomerization and dimerization. To gain a deeper understanding of this phenomenon, the modelled monomer and dimer structures were subjected to pH-dependent dynamics simulations in various conditions. The results reveal that residues Val240, Gln161, Thr159, Tyr160, and Trp316 form strong hydrogen bond contacts in the intersurface area to maintain the structure in the dimeric form. Furthermore, the binding of NADP+ induces conformational changes, leading to an open or closed conformation in the structure. Importantly, the binding of NADP+ does not disturb either the dimerization or oligomerization of the protein, a finding confirmed through both in vitro and in silico analysis. These findings shed light on the structural characteristics of WBm-ASDH and offer valuable insights for the development of new inhibitors specific to WBm, thereby contributing to the development of potential therapies for filarial parasitic infections.
Asunto(s)
Aspartato-Semialdehído Deshidrogenasa , Brugia Malayi , Multimerización de Proteína , Wolbachia , Brugia Malayi/enzimología , Brugia Malayi/microbiología , Concentración de Iones de Hidrógeno , Animales , Aspartato-Semialdehído Deshidrogenasa/metabolismo , Aspartato-Semialdehído Deshidrogenasa/química , Aspartato-Semialdehído Deshidrogenasa/genética , Wolbachia/enzimología , Simulación de Dinámica Molecular , Simulación por Computador , Simbiosis , NADP/metabolismoRESUMEN
Aspartate kinase (AK), an enzyme from the Wolbachia endosymbiont of Brugia malayi (WBm), plays a pivotal role in the bacterial cell wall and amino acid biosynthesis, rendering it an attractive candidate for therapeutic intervention. Allosteric inhibition of aspartate kinase is a prevalent mode of regulation across microorganisms and plants, often modulated by end products such as lysine, threonine, methionine, or meso-diaminopimelate. The intricate and diverse nature of microbial allosteric regulation underscores the need for rigorous investigation. This study employs a combined experimental and computational approach to decipher the allosteric regulation of WBmAK. Molecular Dynamics (MD) simulations elucidate that ATP (cofactor) and ASP (substrate) binding induce a closed conformation, promoting enzymatic activity. In contrast, the binding of lysine (allosteric inhibitor) leads to enzyme inactivation and an open conformation. The enzymatic assay demonstrates the optimal activity of WBmAK at 28 °C and a pH of 8.0. Notably, the allosteric inhibition study highlights lysine as a more potent inhibitor compared to threonine. Importantly, this investigation sheds light on the allosteric mechanism governing WBmAK and imparts novel insights into structure-based drug discovery, paving the way for the development of effective inhibitors against filarial pathogens.
Asunto(s)
Aspartato Quinasa , Brugia Malayi , Simulación de Dinámica Molecular , Wolbachia , Brugia Malayi/enzimología , Brugia Malayi/microbiología , Regulación Alostérica , Animales , Aspartato Quinasa/metabolismo , Aspartato Quinasa/genética , Aspartato Quinasa/química , Simbiosis , Adenosina Trifosfato/metabolismo , Lisina/química , Lisina/metabolismoRESUMEN
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.
Asunto(s)
Antihelmínticos/farmacología , Antiprotozoarios/farmacología , Brugia Malayi/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas del Helminto/antagonistas & inhibidores , Leishmania major/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antihelmínticos/química , Antiprotozoarios/química , Brugia Malayi/enzimología , Brugia Malayi/genética , Brugia Malayi/crecimiento & desarrollo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Ensayos Analíticos de Alto Rendimiento , Leishmania major/enzimología , Leishmania major/genética , Leishmania major/crecimiento & desarrollo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Alineación de SecuenciaRESUMEN
Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans.
Asunto(s)
Brugia Malayi/efectos de los fármacos , Brugia Malayi/enzimología , Glucuronosiltransferasa/metabolismo , Albendazol/farmacología , Animales , Antígenos Helmínticos/sangre , Brugia Malayi/inmunología , Brugia Malayi/metabolismo , Quimioterapia Combinada , Filariasis Linfática/tratamiento farmacológico , Filariasis Linfática/prevención & control , Femenino , Filaricidas/farmacología , Glucuronosiltransferasa/antagonistas & inhibidores , Glucuronosiltransferasa/genética , Humanos , Inmunoglobulina E/sangre , Intestinos/enzimología , Microfilarias/efectos de los fármacos , Movimiento , Probenecid/farmacología , ARN Interferente Pequeño , Sulfinpirazona/farmacologíaRESUMEN
Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45â¯kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca2+on the activity of rBmPGK was studied at different concentration of CaM (0.01-5.0⯵M) and calcium chloride (0.01-100⯵M). The rBmPGK was activated up to 85% in the presence of CaM at 1⯵M and 10⯵M concentration of CaCl2. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90⯱â¯10) % in ScPGK activity was observed in the presence of CaM and CaCl2 at 1.0⯵M and 1.0â¯mM respectively, further increase in the conc. of CaCl2, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain. On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca2+-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.
Asunto(s)
Brugia Malayi/enzimología , Calmodulina/metabolismo , Fosfoglicerato Quinasa/química , Setaria (Nematodo)/enzimología , Animales , Calcio/metabolismo , Bovinos , Unión Proteica , Dominios ProteicosRESUMEN
Background: Lymphatic filariasis (LF) is a global health problem, with a peculiar nature of parasite-specific immunosuppression that promotes long-term pathology and disability. Immune modulation in the host by parasitic antigens is an integral part of this disease. The current study attempts to dissect the immune responses of aminoacyl-tRNA synthetases (AARS) with emphasis on Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS), since it is one among the highly expressed excretory/secretory proteins expressed in all stages of the parasite life cycle, whereas its role in filarial pathology has not been elaborately studied. Methods and Results: In this study, recombinant BmAsnRS (rBmAsnRS) immunological effects were studied in semipermissive filarial animal model Balb/c mice and on clinically defined human samples for LF. In mice study, humoral responses showed considerable titer levels with IgG2a isotype followed by IgG2b and IgG1. Immunoreactivity studies with clinical samples showed significant humoral responses especially in endemic normal with marked levels of IgG1 and IgG2 followed by IgG3. The cell-mediated immune response, evaluated by splenocytes and peripheral blood mononuclear cells proliferation, did not yield significant difference when compared with control groups. Cytokine profiling and qRT-PCR analysis of mice samples immunized with rBmAsnRS showed elevated levels of IFN-γ, IL-10, inhibitory factor-cytotoxic T lymphocyte-associated protein-A (CTLA-4) and Treg cell marker-Forkhead Box P3 (FoxP3). Conclusions: These observations suggest that rBmAsnRS has immunomodulatory effects with modified Th2 response along with suppressed cellular proliferation indicating the essence of this molecule for immune evasion by the parasite.
Asunto(s)
Antígenos Helmínticos/inmunología , Aspartato-ARNt Ligasa/inmunología , Brugia Malayi/inmunología , Filariasis Linfática/inmunología , Filariasis Linfática/parasitología , Interacciones Huésped-Parásitos/inmunología , Aminoacil-ARN de Transferencia/inmunología , Animales , Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/aislamiento & purificación , Brugia Malayi/enzimología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Filariasis Linfática/diagnóstico , Femenino , Humanos , Inmunoglobulina G/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos BALB C , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/aislamiento & purificación , Proteínas Recombinantes , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Lymphatic filariasis is a debilitating vector borne parasitic disease that infects human lymphatic system by nematode Brugia malayi. Currently available anti-filarial drugs are effective only on the larval stages of parasite. So far, no effective drugs are available for humans to treat filarial infections. In this regard, aspartate semialdehyde dehydrogenase (ASDase) in lysine biosynthetic pathway from Wolbachia endosymbiont Brugia malayi represents an attractive therapeutic target for the development of novel anti-filarial agents. In this present study, molecular modeling combined with molecular dynamics simulations and structure-based virtual screening were performed to identify potent lead molecules against ASDase. Based on Glide score, toxicity profile, binding affinity and mode of interactions with the ASDase, five potent lead molecules were selected. The molecular docking and dynamics results revealed that the amino acid residues Arg103, Asn133, Cys134, Gln161, Ser164, Lys218, Arg239, His246, and Asn321 plays a crucial role in effective binding of Top leads into the active site of ASDase. The stability of the ASDase-lead complexes was confirmed by running the 30 ns molecular dynamics simulations. The pharmacokinetic properties of the identified lead molecules are in the acceptable range. Furthermore, density functional theory and binding free energy calculations were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-filarial agents to combat the pathogenecity of Brugia malayi.
Asunto(s)
Antihelmínticos/química , Aspartato-Semialdehído Deshidrogenasa/química , Brugia Malayi/enzimología , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Animales , Antihelmínticos/farmacología , Aspartato-Semialdehído Deshidrogenasa/antagonistas & inhibidores , Sitios de Unión , Dominio Catalítico , Fenómenos Químicos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Unión ProteicaRESUMEN
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.
Asunto(s)
Antihelmínticos/análisis , Antihelmínticos/farmacología , Productos Biológicos/análisis , Productos Biológicos/farmacología , Brugia Malayi/enzimología , Evaluación Preclínica de Medicamentos , Glutatión Transferasa/antagonistas & inhibidores , Wuchereria bancrofti/enzimología , Alcaloides/química , Alcaloides/farmacología , Animales , Benzodioxoles/química , Benzodioxoles/farmacología , Brugia Malayi/efectos de los fármacos , Capsaicina/química , Capsaicina/farmacología , Dominio Catalítico , Curcumina/química , Curcumina/farmacología , Glutatión Transferasa/metabolismo , Simulación del Acoplamiento Molecular , Piperidinas/química , Piperidinas/farmacología , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/farmacología , Estricnina/química , Estricnina/farmacología , Wuchereria bancrofti/efectos de los fármacosRESUMEN
Filariasis is a tropical disease caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill Brugia malayi nematodes and to inhibit Brugia malayi DHFR (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in WbDHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the KM for dihydrofolate (3.7 ± 2 µM), kcat (7.4 ± 0.6 s-1), and pH dependence of activity were determined. IC50 values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for WbDHFR and BmDHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of WbDHFR, with an KI of 1.2 ± 0.2 nM and 2.1 ± 0.5 nM, respectively, suggesting that repurposing of known antifolate compound may be an effective strategy to treating filariasis. Most compounds showed similar inhibition profiles toward both enzymes, suggesting that the two enzymes have important similarities in their active site environments and can be targeted with the same compound, once a successful inhibitor is identified.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Wuchereria bancrofti/enzimología , Secuencia de Aminoácidos , Animales , Brugia Malayi/enzimología , Brugia Malayi/genética , Clonación Molecular , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Alineación de Secuencia , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/aislamiento & purificación , Wuchereria bancrofti/genéticaRESUMEN
Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS-PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be â¼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Km values for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Ki value 1.88 µM. Albendazole also inhibited PGK competitively with Ki value 35.39 µM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.
Asunto(s)
Brugia Malayi , Clonación Molecular , Expresión Génica , Proteínas del Helminto , Sistemas de Lectura Abierta , Fosfoglicerato Quinasa , Animales , Brugia Malayi/enzimología , Brugia Malayi/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Fosfoglicerato Quinasa/biosíntesis , Fosfoglicerato Quinasa/química , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl d-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (Ki) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The Ki of 9a with the B. malayi T6PP ortholog is 5.3 ± 0.6 µM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl d-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Glucosiltransferasas/antagonistas & inhibidores , Monosacáridos/farmacología , Fosfatos de Azúcar/metabolismo , Trehalosa/análogos & derivados , Animales , Brugia Malayi/enzimología , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Mycobacterium tuberculosis/enzimología , Salmonella typhimurium/enzimología , Shigella boydii/enzimología , Trehalosa/metabolismoRESUMEN
BACKGROUND: Galactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp. This sugar maintains the integrity of parasite surface and is essential for virulence. UDP-Galactopyranose mutase (bmugm) plays a key role in Galf biosynthesis by catalyzing conversion of UDP-Galactopyranose into UDP-galactofuranose and knockout studies of the gene in Leishmania major, Mycobacterium and Aspergillus fumigatus displayed attenuated virulence while RNA interference study in C. elegans exhibited detrimental effects. Presence of UGM in several prokaryotic and eukaryotic microbial pathogens and its absence in higher eukaryotes renders it an attractive drug target. In the present study, RNA interference studies have been carried out to validate bmugm as an antifilarial drug target. METHODS: RNA interference studies using two different sequences of siRNAs targeting bmugm were carried out. The in vitro gene silencing of adult B. malayi parasites was undertaken to observe the effects on parasites. Infective larvae were also exposed to siRNAs and their in vivo development in jirds was observed. RESULTS: The in vitro gene silencing induced by siRNA1 and 2 individually as well as together knocked down the bmugm gene expression causing impaired viability of the exposed worms along with extremely reduced motility, abridged microfilarial release and adversely effected embryogenesis. The combinatorial in vitro gene silencing revealed marginally better results than both the siRNAs individually. Thus, infective larvae were treated with siRNA combination which showed downregulation of bmugm mRNA expression resulting into sluggish larval movements and/or death. The siRNA-treated actively motile larvae when inoculated intraperitoneally into jirds demonstrated highly reduced transformation of these larvae into adult worms with detrimental effects on embryogenesis. The effects of gene silencing were long-lasting as the adult worms developed from siRNA-treated larvae showed noticeable knockdown in the target gene expression. CONCLUSIONS: The validation studies undertaken here conclude that bmugm is essential for the proper development and survival of the parasite and support its candidature as an antifilarial drug target.
Asunto(s)
Brugia Malayi/embriología , Brugia Malayi/enzimología , Técnicas de Silenciamiento del Gen , Transferasas Intramoleculares/metabolismo , Interferencia de ARN , Animales , Brugia Malayi/genética , Transferasas Intramoleculares/genética , Larva/crecimiento & desarrollo , Análisis de SupervivenciaRESUMEN
The present work deals with investigating the role of ionic interactions in the native conformation of BmGK by altering pH and salt concentration as well as by disruption of inter-subunit region. The study on structural and functional properties of BmGK as a function of pH showed that the secondary and tertiary elements of the protein were disturbed at low pH with loss of its native oligomerization and functional activity. High concentration of NaCl also changed the native conformation of BmGK with dissociation of its dimeric form. We also mutated dimeric interface of BmGK and identified intersubunit residues, Arg105 and Glu140, essential for dimer stability as double mutation at both positions hinders dimerization. The quaternary structure is found to be essential for full enzymatic activity and stability. In vitro results were supported by in silico molecular dynamics simulation studies through conformational stability analysis. Thus, the work carried out points toward new approach of targeting dimeric interface of BmGK in lieu of its similar active site region to its counterpart human enzyme. This may lead to the design of inhibitors targeted to key parasitic enzyme (BmGK) specifically.
Asunto(s)
Brugia Malayi/enzimología , Guanilato-Quinasas/química , Guanilato-Quinasas/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas/efectos de los fármacos , Guanilato-Quinasas/genética , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Concentración Osmolar , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Cloruro de Sodio/farmacologíaRESUMEN
Thymidylate kinase (TMK) is a key enzyme for the synthesis of DNA, making it an important target for the development of anticancer, antibacterial, and antiparasitic drugs. TMK homologs exhibit significant variations in sequence, residue conformation, substrate specificity, and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly, and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on TMK from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three-dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode, and substrate recognition in TMKs and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors.
Asunto(s)
Proteínas Arqueales/química , Brugia Malayi/química , Proteínas del Helminto/química , Nucleósido-Fosfato Quinasa/química , Nucleótidos de Purina/química , Nucleótidos de Pirimidina/química , Sulfolobus/química , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/metabolismo , Sitios de Unión , Brugia Malayi/enzimología , Cristalografía por Rayos X , Proteínas del Helminto/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Nucleósido-Fosfato Quinasa/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Nucleótidos de Purina/metabolismo , Nucleótidos de Pirimidina/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Sulfolobus/enzimología , TermodinámicaRESUMEN
The trehalose biosynthetic pathway is of great interest for the development of novel therapeutics because trehalose is an essential disaccharide in many pathogens but is neither required nor synthesized in mammalian hosts. As such, trehalose-6-phosphate phosphatase (TPP), a key enzyme in trehalose biosynthesis, is likely an attractive target for novel chemotherapeutics. Based on a survey of genomes from a panel of parasitic nematodes and bacterial organisms and by way of a structure-based amino acid sequence alignment, we derive the topological structure of monoenzyme TPPs and classify them into 3 groups. Comparison of the functional roles of amino acid residues located in the active site for TPPs belonging to different groups reveal nuanced variations. Because current literature on this enzyme family shows a tendency to infer functional roles for individual amino acid residues, we investigated the roles of the strictly conserved aspartate tetrad in TPPs of the nematode Brugia malayi by using a conservative mutation approach. In contrast to aspartate-213, the residue inferred to carry out the nucleophilic attack on the substrate, we found that aspartate-215 and aspartate-428 of BmTPP are involved in the chemistry steps of enzymatic hydrolysis of the substrate. Therefore, we suggest that homology-based inference of functionally important amino acids by sequence comparison for monoenzyme TPPs should only be carried out for each of the 3 groups.-Cross, M., Lepage, R., Rajan, S., Biberacher, S., Young, N. D., Kim, B.-N., Coster, M. J., Gasser, R. B., Kim, J.-S., Hofmann, A. Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.
Asunto(s)
Brugia Malayi/enzimología , Secuencia Conservada , Proteínas del Helminto/química , Monoéster Fosfórico Hidrolasas/química , Animales , Ácido Aspártico/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Mycobacterium/enzimología , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
Guanylate kinase is one of the key enzymes in nucleotide biosynthesis. The study highlights the structural and functional properties of Brugia malayi Guanylate kinase (BmGK) in the presence of chemical denaturants. An inactive, partially unfolded, dimeric intermediate was observed at 1-2M urea while GdnCl unfolding showed monomer molten globule like intermediate at 0.8-1.0M. The results also illustrate the protective role of substrates in maintaining the integrity of the enzyme. The thermo stability of protein was found to be significantly enhanced in the presence of the substrates. Furthermore, binding of the substrates, GMP and ATP to BmGK changed its GdnCl induced unfolding pattern. Docking and molecular dynamic simulation performed for native BmGK, BmGK bound to GMP and GMP+ATP showed change in the fluctuation in the region between 130 and 150 residues. Arg134 lost its interaction with GMP and Arg145 interaction shifted to ATP after 40ns simulation upon binding of ATP to BmGK-GMP complex. We, thus, propose the importance of specific rearrangements contributed by binding of substrates which participate in the overall stability of the protein. The work here emphasizes on detailed biophysical characterization of BmGK along with the significant role of substrates in modulating the structural and functional properties of BmGK.
Asunto(s)
Adenosina Trifosfato/química , Brugia Malayi/química , Guanosina Monofosfato/química , Guanilato-Quinasas/química , Proteínas del Helminto/química , Animales , Brugia Malayi/enzimología , Reactivos de Enlaces Cruzados/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutaral/química , Guanidina/química , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Urea/químicaRESUMEN
Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a â¼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 µM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 µM for trimethoprim, 109 ± 34 µM for pyrimethamine, 154 ± 46 µM for 2,4-diaminoquinazoline, 771 ± 44 µM for cycloguanil, and >20,000 µM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR.
Asunto(s)
Brugia Malayi/genética , Expresión Génica , Proteínas del Helminto , Tetrahidrofolato Deshidrogenasa , Animales , Brugia Malayi/enzimología , Catálisis , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Tetrahidrofolato Deshidrogenasa/biosíntesis , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/aislamiento & purificaciónRESUMEN
Lymphatic filariasis, a vector-borne neglected tropical disease affects millions of population in tropical and subtropical countries. Vaccine unavailability and emerging drug resistance against standard antifilarial drugs necessitate search of novel drug targets for developing alternate drugs. Recently, UDP-galactopyranose mutases (UGM) have emerged as a promising drug target playing an important role in parasite virulence and survival. This study deals with the cloning and characterization of Brugia malayi UGM and further exploring its antifilarial drug target potential. The recombinant protein was actively involved in conversion of UDP-galactopyranose (substrate) to UDP-galactofuranose (product) revealing Km and Vmax to be â¼51.15 µM and â¼1.27 µM/min, respectively. The purified protein appeared to be decameric in native state and its 3D homology modeling using Aspergillus fumigatus UGM enzyme as template revealed conservation of active site residues. Two specific prokaryotic inhibitors (compounds A and B) of the enzyme inhibited B. malayi UGM enzymatic activity competitively depicting Ki values â¼22.68 and â¼23.0 µM, respectively. These compounds were also active in vitro and in vivo against B. malayi The findings suggest that B. malayi UGM could be a potential antifilarial therapeutic drug target.
Asunto(s)
Brugia Malayi/enzimología , Transferasas Intramoleculares/metabolismo , Secuencia de Aminoácidos , Animales , Antihelmínticos/química , Antihelmínticos/farmacología , Antihelmínticos/uso terapéutico , Brugia Malayi/clasificación , Brugia Malayi/efectos de los fármacos , Brugia Malayi/genética , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Filariasis/tratamiento farmacológico , Filariasis/parasitología , Expresión Génica , Humanos , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/química , Transferasas Intramoleculares/genética , Ligandos , Modelos Moleculares , Pruebas de Sensibilidad Parasitaria , Filogenia , Conformación Proteica , Multimerización de Proteína , Análisis de Secuencia de ADNRESUMEN
Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme.
Asunto(s)
Brugia Malayi/enzimología , Filariasis Linfática/enzimología , Nucleósido-Fosfato Quinasa/química , Proteínas Recombinantes/química , Animales , Brugia Malayi/patogenicidad , Dominio Catalítico , Dimerización , Filariasis Linfática/tratamiento farmacológico , Filariasis Linfática/parasitología , Humanos , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Nucleótidos de Timina/químicaRESUMEN
OBJECTIVES: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. MATERIALS AND METHODS: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. RESULTS: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). CONCLUSIONS: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.