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1.
Arch Microbiol ; 203(2): 799-808, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33051772

RESUMO

The green sulfur bacterium, Chlorobaculum tepidum, is an anaerobic photoautotroph that performs anoxygenic photosynthesis. Although genes encoding rubredoxin (Rd) and a putative flavodiiron protein (FDP) were reported in the genome, a gene encoding putative NADH-Rd oxidoreductase is not identified. In this work, we expressed and purified the recombinant Rd and FDP and confirmed dioxygen reductase activity in the presence of ferredoxin-NAD(P)+ oxidoreductase (FNR). FNR from C. tepidum and Bacillus subtilis catalyzed the reduction of Rd at rates comparable to those reported for NADH-Rd oxidoreductases. Also, we observed substrate inhibition at high concentrations of NADPH similar to that observed with ferredoxins. In the presence of NADPH, B. subtilis FNR and Rd, FDP promoted dioxygen reduction at rates comparable to those reported for other bacterial FDPs. Taken together, our results suggest that Rd and FDP participate in the reduction of dioxygen in C. tepidum and that FNR can promote the reduction of Rd in this bacterium.


Assuntos
Chlorobi/química , Chlorobi/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Rubredoxinas/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredução , Enxofre/metabolismo
2.
Chemistry ; 23(57): 14345-14357, 2017 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-28967982

RESUMO

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18-56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2-2.6 Šresolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

3.
Antimicrob Agents Chemother ; 60(8): 4453-63, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27161627

RESUMO

Dihydropteroate synthase (DHPS) is a known sulfa drug target in malaria treatment, existing as a bifunctional enzyme together with hydroxymethyldihydropterin pyrophosphokinase (HPPK). Polymorphisms in key residues of Plasmodium falciparum DHPS (PfDHPS) have been characterized and linked to sulfa drug resistance in malaria. Genetic sequencing of P. vivax dhps (Pvdhps) from clinical isolates has shown several polymorphisms at the positions equivalent to those in the Pfdhps genes conferring sulfa drug resistance, suggesting a mechanism for sulfa drug resistance in P. vivax similar to that seen in P. falciparum To characterize the role of polymorphisms in the PvDHPS in sulfa drug resistance, various mutants of recombinant PvHPPK-DHPS enzymes were expressed and characterized. Moreover, due to the lack of a continuous in vitro culture system for P. vivax parasites, a surrogate P. berghei model expressing Pvhppk-dhps genes was established to demonstrate the relationship between sequence polymorphisms and sulfa drug susceptibility and to test the activities of PvDHPS inhibitors on the transgenic parasites. Both enzyme activity and transgenic parasite growth were sensitive to sulfadoxine to different degrees, depending on the number of mutations that accumulated in DHPS. Ki values and 50% effective doses were higher for mutant PvDHPS enzymes than the wild-type enzymes. Altogether, the study provides the first evidence of sulfa drug resistance at the molecular level in P. vivax Furthermore, the enzyme inhibition assay and the in vivo screening system can be useful tools for screening new compounds for their activities against PvDHPS.


Assuntos
Di-Hidropteroato Sintase/genética , Polimorfismo Genético/genética , Animais , Difosfotransferases/genética , Escherichia coli/metabolismo , Cinética , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/patogenicidade , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/patogenicidade , Sulfadoxina/farmacologia
4.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 6): 1517-27, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24914963

RESUMO

Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Šresolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme.


Assuntos
Glicina Hidroximetiltransferase/química , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Animais , Cristalização , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Difração de Raios X
5.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 12): 3177-86, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25478836

RESUMO

Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of L- and D-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with L-serine and in a ternary complex with D-serine (D-Ser) and (6R)-5-formyltetrahydrofolate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT-Gly-(6S)-5FTHF from other organisms. This suggested that the presence of D-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of D-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125-Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT.


Assuntos
Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/metabolismo , Malária Vivax/parasitologia , Plasmodium vivax/enzimologia , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Plasmodium vivax/química , Plasmodium vivax/metabolismo , Ligação Proteica , Serina/química , Serina/metabolismo , Tetra-Hidrofolatos/química , Tetra-Hidrofolatos/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-38749208

RESUMO

Pigmentation genes expressed in skin, body muscle and tail of Thai-flag compared with Blue, White and Red varieties of Siamese fighting fish Betta splendens were identified. In total, 22,919 new unigenes were found. Pearson correlation and PCA analysis revealed that expression profiles of genes in muscle, skin and tail across solid color variety were similar. In contrast, those in skin and red tail part of Thai-flag were closely related but they showed different expression profiles with the white tail part. Moreover, 21,347-64,965 SNPs were identified in exonic regions of identified genes. In total, 28,899 genes were differentially expressed between paired comparisons of libraries where 13,907 genes (48.12 %) were upregulated and 14,992 genes (51.88 %) were downregulated. DEGs between paired libraries were 106-5775 genes relative to the compared libraries (56-2982 and 50-2782 for upregulated and downregulated DEGs). Interestingly, 432 pigmentation genes of B. splendens were found. Of these, 297 DEGs showed differential expression between varieties. Many DEGs in melanogenesis (Bsmcr1r, Bsmcr5r, and Bsslc2a15b), tyrosine metabolism (Bstyr, Bstyrp1b and Bsdct), stripe repressor (BsAsip1 and BsAsip2b), pteridine (Bsgch2) and carotenoid (BsBco2) biosynthesis were downregulated in the Thai-flag compared with solid color varieties. Expression of Bsbco1l, Bsfrem2b, Bskcnj13, Bszic2a and Bspah in skin, muscle and tail of Thai-flag, Blue, Red and White varieties was analyzed by qRT-PCR and revealed differential expression between fish varieties and showed anatomical tissue-preferred expression patterns in the same fish variety. The information could be applied to assist genetic-based development of new B. splendens varieties in the future.


Assuntos
Pigmentação , Animais , Proteínas de Peixes/genética , Peixes/genética , Perfilação da Expressão Gênica , Músculos/metabolismo , Pigmentação/genética , Polimorfismo de Nucleotídeo Único , Pele/metabolismo , Pigmentação da Pele/genética , Cauda , Tailândia , Transcriptoma
7.
Gene ; 848: 146900, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36126819

RESUMO

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.


Assuntos
Braquiúros , Receptores de Lipoproteínas , Sequência de Aminoácidos , Animais , Braquiúros/genética , Braquiúros/metabolismo , Dissulfetos/metabolismo , Ecdisona/metabolismo , Ecdisteroides , Muda/genética , Prolina , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lipoproteínas/metabolismo , Secretina/metabolismo
8.
Parasitol Int ; 67(6): 787-792, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30114522

RESUMO

Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P. knowlesi dihydrofolate reductase-thymidylate synthase enzyme (PkDHFR-TS) and its sensitivity to antifolates. The putative Pkdhfr gene was PCR amplified from field isolates collected from the Southern Thailand. Molecular analysis showed 11 polymorphisms in the dhfr domain of the bifunctional dhfr-ts gene. Of these, 1 polymorphism was a non-synonymous substitution (R34L) that had previously been reported but not associated with antifolate resistance. The recombinant PkDHFR-TS enzyme was found to be sensitive to standard antifolates-pyrimethamine and cycloguanil-as well as P218, a registered candidate drug currently first in human clinical trial. Results suggest that antifolates class of compounds should be effective against P. knowlesi infection.


Assuntos
Antimaláricos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Plasmodium knowlesi/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Sequência de Bases , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Plasmodium knowlesi/genética , Proguanil/farmacologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirimetamina/farmacologia , Alinhamento de Sequência , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Triazinas/farmacologia
9.
ChemMedChem ; 13(9): 931-943, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29655285

RESUMO

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2-indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14-76 nm) and cellular potencies in the low nanomolar range (165-334 nm) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co-crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2-2.4 Šresolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non-spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor-bound ligands is discussed.


Assuntos
Inibidores Enzimáticos/farmacologia , Glicina Hidroximetiltransferase/antagonistas & inibidores , Indenos/farmacologia , Oxindóis/farmacologia , Plasmodium/efeitos dos fármacos , Compostos de Espiro/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicina Hidroximetiltransferase/metabolismo , Humanos , Indenos/síntese química , Indenos/química , Ligantes , Modelos Moleculares , Estrutura Molecular , Oxindóis/síntese química , Oxindóis/química , Testes de Sensibilidade Parasitária , Plasmodium/enzimologia , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-Atividade
10.
J Med Chem ; 60(12): 4840-4860, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28537728

RESUMO

Target-based approaches toward new antimalarial treatments are highly valuable to prevent resistance development. We report several series of pyrazolopyran-based inhibitors targeting the enzyme serine hydroxymethyltransferase (SHMT), designed to improve microsomal metabolic stability and to identify suitable candidates for in vivo efficacy evaluation. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target assays and PfNF54 strains in cell-based assays with values in the low nanomolar range (3.2-55 nM). A set of carboxylate derivatives demonstrated markedly improved in vitro metabolic stability (t1/2 > 2 h). A selected ligand showed significant in vivo efficacy with 73% of parasitemia reduction in a mouse model. Five new cocrystal structures with PvSHMT were solved at 2.3-2.6 Å resolution, revealing a unique water-mediated interaction with Tyr63 at the end of the para-aminobenzoate channel. They also displayed the high degree of conformational flexibility of the Cys364-loop lining this channel.


Assuntos
Antimaláricos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicina Hidroximetiltransferase/antagonistas & inibidores , Animais , Antimaláricos/química , Proteínas de Arabidopsis/antagonistas & inibidores , Técnicas de Química Sintética , Cristalografia por Raios X , Cisteína/química , Estabilidade de Medicamentos , Inibidores Enzimáticos/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Meia-Vida , Ligantes , Malária Falciparum/tratamento farmacológico , Camundongos SCID , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/patogenicidade , Plasmodium vivax/enzimologia , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/farmacologia
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