Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).A enzima purina nucleosídeo fosforilase do parasita Schistosoma mansoni (SmPNP) é um alvo molecular atrativo para o desenvolvimento de candidatos a novos fármacos para o tratamento da esquistossomose, doença tropical negligenciada que afeta mais de 200 milhões de pessoas em todo mundo. No presente trabalho, estudos de cinética enzimática foram conduzidos para a determinação da potência e do mecanismo de inibição de uma série de inibidores da enzima SmPNP. Além das investigações bioquímicas, estudos cristalográficos e de modelagem molecular revelaram importantes bases moleculares para a afinidade de ligação frente à enzima alvo, levando ao desenvolvimento de relações entre a estrutura e atividade (SAR).(FAPESP) São Paulo Research FoundationFundação de Amparo à Pesquisa do Estado da Bahia (FAPESB)(CNPq) National Council for Scientific and Technological Developmen

Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid

Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date. After incubation for 15 days at 37 °C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation in a biologically relevant environment, opening new avenues of investigation into the molecular mechanisms of LA.CNPqFAPERJCAPE

The Structure Of The Giant Haemoglobin From Glossoscolex Paulistus.

The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.711257-127

Immunization with purine salvation pathway recombinant enzymes induces the production of anti- Schistosoma mansoni immunoglobulines

Schistosomiasis, a disease caused by the trematodes of the Schistosoma genus, affects about 240 million people worldwide. The Praziquantel (PZQ) is the drug used to treat this disease. However, reports of resistant strains reinforce the need to develop a new schistosomicidal drug. The study of new drugs and vaccines that can contribute to the control of this pathology becomes urgent. A new approach can be held by the study of the following Schistosoma mansoni enzymes: purine nucleoside phosphorilase 1 (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT) and adenylate kinase 1 (ADK). The parasite, incapable of synthetizing purine nucleotides through the de novo pathway, has multiple mechanisms to incorporate purine bases through the purine salvage pathway. Our goal was to assess, through immunoenzimatic assay (ELISA-indirect), the production of total IgG, IgE and IgG2a in the plasma after immunization with the PNP, HGPRT and ADK enzymes, using the S. mansoni cercariae - infected murine model. Our results showed that the immunization in Balb/c mice with the enzymes mentioned above induced production of the immunoglobulines at the 48th and 85th days, post-infection. Thereby, new assays must be made for a better assessment on how these enzymes modulate an immune response.CNPqFAPES

Evaluation of immunization with purine salvation pathway recombinant enzymes in Schistosoma mansoni worms and eggs in murine schistosomiasis

According to the World Health Organization (WHO), on tropical and subtropical areas, schistosomiasis is the second parasitic disease of greater prevalence, in terms of morbidity and mortality, surpassed only by malaria. The Praziquantel (PZQ) is used for the treatment of this disease. However, reports of resistant strains reinforce the need to develop a new schistosomicidal drug. The infection by the parasite induces an inflammatory reaction of long duration due to the presence of adult worms living in the mesenteric venous system. The parasite lays eggs in small vessels of the submucosa of the intestines. These eggs are transported by the blood flow to the liver and they cause a granulomatous inflammatory reaction. A new approach can be held by the study of the following Schistosoma mansoni enzymes: purine nucleoside phosphorilase 1 (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT) and adenylate kinase (ADK). The parasite, incapable of synthetizing purine nucleotides through the de novo pathway, has multiple mechanisms to incorporate purine bases through the purine salvage pathway. In our results, we suggest that the immunization in Balb/c mice with the mentioned recombinant enzymes was capable of inducing a specific immune response, favoring the reduction of both the parasite load and number of eggs per gram of feces. The acquired data show that these enzymes can be considered as new targets to immunotherapy against schistosomiasis mansoni.CNPqFAPES

Purine nucleoside fosforilase from Schistosoma Mansoni: crystal structure, knetics, studies and ligands search

O parasita Schistosoma mansoni não possui a via de síntese de bases púricas e depende integralmente da via de salvação de purinas para o seu requerimento de purinas. Uma das enzimas participantes desta via é a Purina Nucleosídeo Fosforilase (PNP) (E.C. 2.4.2.1). A PNP catalisa a fosforólise reversível de nucleosídeos de purina para gerar a base correspondente e ribose-1-fosfato. No Projeto Genoma de Schistosoma mansoni, o gene para esta enzima foi identificado.O cDNA para a PNP de S. mansoni (SmPNP), possui 1055pb e codifica para uma proteína de 287 aminoácidos, que possui 49% de identidade quando comparada a PNP de eritrócitos humana ou de Baco bovino. O gene foi clonado no vetor de expressão pMAL C2G, e expresso na forma de uma proteína de fusão, com MBP (80mg/L). Após a purificação da proteína de fusão, a clivagem proteolítica das duas proteínas foi realizada utilizando-se o Factor Xa. A SmPNP foi então purificada utilizando uma coluna de troca catiônica. Foram determinadas as constantes catalíticas para a fosforólise de inosina pela SmPNP, as quais são 3?M para o KM e 222 s-1para o kcat. O valor para o KM é O menor já descrito para uma PNP de baixa massa molecular. Foram obtidos cristais da SmPNP utilizando 18-24 % de PEG 1500, 20% de glicerol em 32mM de tampão acetato de sódio pH 4,9-5,O. Quando utilizado o aditivo NDSB195 na cristalização da SmPNP a solução de cristalização foi 28-30% de PEG 1500, 20% de glicerol em 32mM de tampão acetato de sódio pH 4,9-5,O. Cinco conjuntos de dados de difração de raios X foram obtidos tanto na presença como na ausência de ligantes. A resolução deste conjuntos de dados variou de 2,75? a 1,75 ?. Todas estas estruturas foram resolvidas pelo método da substituição molecular, sendo que na primeira estrutura resolvida (a 2,75?) foi utilizado a estrutura da PNP bovina como modelo de busca, e na resolução das estruturas subsequentes foi utilizado a estrutura da SmPNP como modelo de busca. A estrutura da SmPNP a 1,75? de resolução foi obtida a partir de um cristal crescido na presença de NDSB 195, e após sua resolução foi encontrado este composto ligado em todos os sítios ativos da SmPNP via a sítio de ligação do fosfato. Foram também obtidas as estruturas da SmPNP na forma apo a 1,9? de resolução e em complexo com fosfato a 2,0? de resolução. Todas as estruturas foram utilizadas na comparação com outras PNPs de baixa massa molecular. Utilizando a estrutura da SmPNP a 1,75? de resolução, foi realizada uma busca por compostos (Virtual Screening) que ligassem a SmPNP. Nesta busca foi utilizando o programa GOLD, utilizando uma base de dados de cerca de 36000 compostos com peso molecular inferior a 280 Da. Vinte e dois compostos foram selecionados com base no escore de docking e pelas interações (pontes de hidrogênio) com os resíduos chave do sítio ativo. Utilizando PNP bovina e a mesma abordagem de docking foram selecionados 19 compostos. Estes compostos foram ensaiados contra a SmPNP e 12 compostos se mostraram capazes de inibir a SmPNP. Um complexo entre a SmPNP e o composto AT2169 (oriundo do VS) foi obtido e refinado. Esta abordagem foi validada utilizando ligantes conhecidos das PNPs.The parasite Schistosoma mansoni, unlike its mammalian hosts, lacks the \"de novo\" pathway for purine biosynthesis and depends on the salvage pathways for its purine requirements. One component of this pathway is Purine Nucleoside Phosphorylase (PNP) (E.C. 2.4.2.1). PNP catalyzes the reversible phosphorolysis of purine nucleosides to generate the corresponding purine base and ribose 1-phosphate. In the Schistosoma mansoni Genome Project the gene for this enzyme was isolated. The SmPNP cDNA was sequenced, corresponding to 1055 bases that code for a 287 amino acid protein with 49% sequence identity to its human erythrocyte homologue. The SmPNP gene was cloned into the pMAL C2G expression vector and the recombinant fusion protein was produced and purified using an amylose affinity column (approx. 80mg/mL). The fusion protein is composed of a Maltose Binding Protein adjoined to the SmPNP. After cleavage with Factor Xa, the cleavage product was purified using a cation exchange column. The KM and kcat of SmPNP for inosine phosphorolisis was determined to be 3?M and 222 s-1 respectively. This corresponds to the lowest value for KM yet described for a low molecular mass PNP. SmPNP crystals were obtained by the hanging drop method, using 18-24% PEG 1500, 20% glycerol in the presence of 32mM sodium acetate buffer (pH 4.9-5.0). When NDSB195 was used as an additive in the SmPNP crystallization the solution used was 28-30% PEG 1500, 20% glycerol and 32mM sodium acetate buffer (pH 4.9-5.O).Five datasets were obtained in the presence and absence of ligands, varying in resolution from 2,75 to 1,75?. All structures were solved by the molecular replacement method. The first structure (at 2,75?) was solved using the bovine PNP as the search model and in the remaining structures the search model was the SmPNP structure itself. The 1,75? structure was obtained from a crystal grow in the presence of the additive NDSB195, and after its determination NDSB195 was observed bound to the SmPNP active site via its phosphate binding site. Structures were also obtained for the apo SmPNP at 1,9? and its complex with phosphate at 2,0? resolution. All structures were used in the comparison with other low molecular mass PNPs. The SmPNP structure at 1,75A resolution was used in the search small molecule ligands, which potentially bind to SmPNP via virtual screening. For this purpose the program Gold together with a database of 36000 compounds with MW lower than 280Da was used. As a result 22 compounds were selected using the docking score and the H-bonding interaction with the key residues of the SmPNP active site. Using the bovine PNP and same docking approach 19 compounds were selected. These 41 compounds were assayed against SmPNP enzyme and 12 compounds were active against the SmPNP. One complex between SmPNP and one of these compound (AT2 169) was obtained and refined. This virtual screening approach was validated using known ligands of PNPs including inosine, guanosine, immucilins, etc

Structural and kinetic studies of Schistosoma mansoni adenylate kinases

The human parasite Schistosoma mansoni is totally dependent on the purine salvage pathway in order to supply large quantities of purine precursors for its energy and DNA biosynthetic needs. Adenylate kinase (ADK) is responsible for the conversion of AMP (produced by the adenosine kinase reaction) into ADP, which is subsequently converted into ATP by nucleoside diphosphate kinase (NDPK). ADK and NDPK are the most active enzymes of the pathway, probably reflecting an evolutionary adaptation due to the intense use of the branch in which they participate. However, notwithstanding their importance very little information has been accumulated found regarding these enzymes. In this work two adenylate kinases from S. mansoni were cloned and heterologously expressed in Escherichia coil. The purified products were utilized in activity assays, and displayed kinetic parameters similar to the corresponding human orthologous proteins. The cytosolic S. mansoni ADK was crystallized and its structure solved allowing us to detect a difference in the nucleotide binding site when compared with the human ortholog. (C) 2012 Elsevier B.V. All rights reserved.FAPESPFAPESP [1998/14138-2, 06/60280-3]CNPq [550514/2011-2]CNP

Bacterial selenocysteine synthase structure revealed by single-particle cryoEM

The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNASec). In bacteria, Sec is synthesized from Ser-tRNA[Ser]Sec by Selenocysteine Synthase (SelA), which is a pivotal enzyme in the biosynthesis of Sec. The structural characterization of bacterial SelA is of paramount importance to decipher its catalytic mechanism and its role in the regulation of the Sec-synthesis pathway. Here, we present a comprehensive single-particle cryo-electron microscopy (SPA cryoEM) structure of the bacterial SelA with an overall resolution of 2.69 Å. Using recombinant Escherichia coli SelA, we purified and prepared samples for single-particle cryoEM. The structural insights from SelA, combined with previous in vivo and in vitro knowledge, underscore the indispensable role of decamerization in SelA's function. Moreover, our structural analysis corroborates previous results that show that SelA adopts a pentamer of dimers configuration, and the active site architecture, substrate binding pocket, and key K295 catalytic residue are identified and described in detail. The differences in protein architecture and substrate coordination between the bacterial enzyme and its counterparts offer compelling structural evidence supporting the independent molecular evolution of the bacterial and archaea/eukarya Ser-Sec biosynthesis present in the natural world

Crystal Structure of <i>Schistosoma mansoni</i> Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway - Fig 4

<p>A. Composite omit map contoured at 1σ for the region containing the SS bond in <i>Sm</i>MTAP. B. Stick model for the same region showed in A. This disulphide bond is formed by the residues Cys 233 and Cys 242 only in some Apo mutant structures. This SS bond helps maintain the gate loop in open conformation and could be involved in the nucleoside accessibility of the active site.</p

Ligplus⁺ plots for the sulphate molecule.

<p><b>A</b>. wt <i>Sm</i>MTAP. <b>B</b>. Double mutant S12T/N87T and their consequence in sulphate/phosphate binding, where 3 new H-bonds are formed.</p