RESUMEN
Brucella spp. are pathogenic intracellular Gram-negative bacteria adapted to life within cells of several mammals, including humans. These bacteria are the causative agent of brucellosis, one of the zoonotic infections with the highest incidence in the world and for which a human vaccine is still unavailable. Current therapeutic treatments against brucellosis are based on the combination of two or more antibiotics for prolonged periods, which may lead to antibiotic resistance in the population. Riboflavin (vitamin B2) is biosynthesized by microorganisms and plants but mammals, including humans, must obtain it from dietary sources. Owing to the absence of the riboflavin biosynthetic enzymes in animals, this pathway is nowadays regarded as a rich resource of targets for the development of new antimicrobial agents. In this work, we describe a high-throughput screening approach to identify inhibitors of the enzymatic activity of riboflavin synthase, the last enzyme in this pathway. We also provide evidence for their subsequent validation as potential drug candidates in an in vitro brucellosis infection model. From an initial set of 44 000 highly diverse low molecular weight compounds with drug-like properties, we were able to identify ten molecules with 50% inhibitory concentrations in the low micromolar range. Further Brucella culture and intramacrophagic replication experiments showed that the most effective bactericidal compounds share a 2-Phenylamidazo[2,1-b][1,3]benzothiazole chemical scaffold. Altogether, these findings set up the basis for the subsequent lead optimization process and represent a promising advancement in the pursuit of novel and effective antimicrobial compounds against brucellosis.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Brucella abortus/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Brucella abortus/enzimología , Línea Celular , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Ratones , Unión Proteica , Riboflavina Sintasa/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Nucleósidos/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Conformación Molecular , Simulación de Dinámica Molecular , Nucleósidos/síntesis química , Nucleósidos/química , Relación Estructura-ActividadRESUMEN
Riboflavin (vitamin B2) is the direct precursor of redox enzyme cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential for multiple cell physiology. The riboflavin biosynthetic pathway is regarded as a rich resource for therapeutic targets for broad spectrum antibiotics. Enzymatic pathways, regulatory factors of the riboflavin biosynthesis, and relevant drug discovery are summarized in this review. The novel riboswitch regulatory mechanism of riboflavin metabolism is also described. A compendium of chemical modulators of riboflavin biosynthesis and regulatory networks is listed and such demonstrates the promise of riboflavin biosynthesis and regulatory mechanisms as potential therapeutic targets for novel antibiotic drug discovery.
Asunto(s)
Antiinfecciosos/farmacología , Riboflavina Sintasa/metabolismo , Riboflavina/biosíntesis , Secuencia de Aminoácidos , Antiinfecciosos/química , Antituberculosos/química , Antituberculosos/farmacología , Datos de Secuencia Molecular , Riboflavina/metabolismo , Riboflavina Sintasa/antagonistas & inhibidores , Riboflavina Sintasa/química , Alineación de SecuenciaRESUMEN
A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli riboflavin synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a k(is) of 8.7 microM vs. M. tuberculosis riboflavin synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 microM vs. M. tuberculosis replicating phenotype and 48.9 microM vs. M. tuberculosis nonreplicating phenotype. The HTS hit compound and its analogues provide the first examples of riboflavin synthase inhibitors with antibiotic activity.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Hidrocarburos Fluorados/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Pirazoles/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Sitios de Unión , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Relación Estructura-Actividad , Agua/químicaRESUMEN
The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Ácido Oxámico/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Bacillus subtilis/enzimología , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Cinética , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Ácido Oxámico/análogos & derivados , Ácido Oxámico/síntesis química , Ácido Oxámico/química , Schizosaccharomyces/enzimología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin. To obtain structural and mechanistic probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a sulfur analogue of the pyrimidine substrate of the lumazine synthase-catalyzed reaction and product of the riboflavin synthase-catalyzed reaction was designed. Facile syntheses of the S-nucleoside 5-amino-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione hydrochloride (15) and its nitro precursor 5-nitro-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione (14) are described. These compounds were tested against lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. Compounds 14 and 15 were found to be inhibitors of both riboflavin synthase and lumazine synthase. Compound 14 is an inhibitor of Bacillus subtilis lumazine synthase (Ki 26 microM), Schizosaccharomyces pombe lumazine synthase (Ki 2.0 microM), Mycobacterium tuberculosis lumazine synthase (Ki 11 microM), Escherichia coli riboflavin synthase (Ki 2.7 microM), and Mycobacterium tuberculosis riboflavin synthase (Ki 0.56 muM), while compound 15 is an inhibitor of B. subtilis lumazine synthase (Ki 2.6 microM), S. pombe lumazine synthase (Ki 0.16 microM), M. tuberculosis lumazine synthase (Ki 31 microM), E. coli riboflavin synthase (Ki 47 microM), and M. tuberculosis riboflavin synthase (Ki 2.5 microM).
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Complejos Multienzimáticos/antagonistas & inhibidores , Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Bacillus subtilis/enzimología , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/enzimología , Nucleósidos de Pirimidina/metabolismo , Riboflavina Sintasa/química , Riboflavina Sintasa/metabolismo , Especificidad por SustratoRESUMEN
The last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine synthase and riboflavin synthase. To obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of Bacillus subtilislumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In phosphate-free buffer, the phosphate moieties of the inhibitors were found to contribute positively to their binding to Mycobacterium tuberculosis lumazine synthase, resulting in very potent inhibitors with Ki values in the low nanomolar range. The additional carbonyl in the dioxolumazine system versus the purinetrione system was found to make a positive contribution to its binding to E. coli riboflavin synthase.
Asunto(s)
Antituberculosos/farmacología , Bacillus subtilis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Pteridinas/síntesis química , Riboflavina Sintasa/antagonistas & inhibidores , Antituberculosos/síntesis química , Antituberculosos/química , Bacillus subtilis/enzimología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Pteridinas/química , Pteridinas/farmacologíaRESUMEN
A synthetic gene specifying the catalytic domain of the Arabidopsis thaliana riboflavin synthase was expressed with high efficiency in a recombinant Escherichia coli strain. The recombinant pseudomature protein was shown to convert 6,7-dimethyl-8-ribityllumazine into riboflavin at a rate of 0.027 s-1 at 25 degrees C. The protein sediments at a rate of 3.9 S. Sedimentation equilibrium analysis afforded a molecular mass of 67.5 kDa, indicating a homotrimeric structure, analogous to the riboflavin synthases of Eubacteria and fungi. The protein binds its product riboflavin with relatively high affinity (Kd =1.1 microM). Product inhibition results in a characteristic sigmoidal velocity versus substrate concentration relationship. Characterization of the enzyme/product complex by circular dichroism and UV absorbance spectroscopy revealed a shift of the absorption maxima of riboflavin from 370 and 445 to 399 and 465 nm, respectively. Complete or partial sequences for riboflavin synthase orthologs were analyzed from 11 plant species. In each case for which the complete plant gene sequence was available, the catalytic domain was preceded by a sequence of 1-72 amino acid residues believed to function as plastid targeting signals. Comparison of all available riboflavin synthase sequences indicates that hypothetical gene duplication conducive to the two-domain architecture occurred very early in evolution.
Asunto(s)
Arabidopsis/enzimología , Riboflavina Sintasa/antagonistas & inhibidores , Riboflavina/química , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Dicroismo Circular , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Pteridinas/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Riboflavina Sintasa/química , Riboflavina Sintasa/genética , Homología de Secuencia de Aminoácido , Espectrofotometría UltravioletaRESUMEN
Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, a vitamin that is involved in many critical biochemical reactions that are essential for the maintenance of life. To obtain inhibitors and structural probes that could be useful in studying the structures of bound reaction intermediates, the ribitylamino N-H moiety of the lumazine synthase substrate was replaced by CH(2) and N-CH(3) groups. The CH(2) replacement unexpectedly and completely abolished the affinity for lumazine synthase, thus revealing a critical, yet unexplained, role of the ribitylamino N-H moiety in conferring affinity for the enzyme. In contrast, the N-CH(3) replacement resulted in an inhibitor of both lumazine synthase and riboflavin synthase. Replacement of the ribitylamino N-H moiety with epimeric C-F moieties led to inhibition of lumazine synthase and riboflavin synthase when combined with the replacement of the 5-amino group with a nitro substituent.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/síntesis química , Inhibidores Enzimáticos/farmacología , Metilación , Modelos Moleculares , Conformación Molecular , Nucleósidos de Pirimidina/farmacología , Riboflavina/biosíntesis , Riboflavina Sintasa/antagonistas & inhibidores , Riboflavina Sintasa/químicaRESUMEN
Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport processes. To obtain structural probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a series of ribitylpurinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents were synthesized. Since the purinetrione ring system and the ribityl hydroxyl groups can be alkylated, the synthesis required the generation of these two moieties in protected form before the desired alkylation reaction could be carried out. These substances were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site. All of the compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of 3-(1,3,7,9-tetrahydro-9-D-ribityl-2,6,8-trioxopurin-7-yl)propane 1-phosphate provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. Interestingly, the enzyme kinetics of these new compounds in comparison with the parent purinetrione demonstrated unexpectedly that the phosphate and phosphonate substituents contributed negatively to the binding. A possible explanation for these effects on lumazine synthase would be that the inorganic phosphate in the assay buffer competes with the substituted purinetriones for binding to the enzyme. This would be consistent with the observed increase in K(m) of the 3,4-dihydroxybutanone-4-phosphate substrate from 5.2 microM in Tris buffer or from 6.7 microM in MOPS buffer to 50 microM in phosphate buffer when tested on Bacillus subtilis lumazine synthase. However, when tested in Tris buffer vs Mycobacterium tuberculosis lumazine synthase, three of the phosphate inhibitors displayed inhibition constants in the 4-5 nM range, indicating that they are much more potent than the parent purinetrione. Under these conditions, the phosphate moieties of the inhibitors do contribute positively to their binding. The alpha,alpha-difluorophosphonate analogue, which is expected to have enhanced metabolic stability relative to the phosphates, was also found to be an inhibitor of Mycobacterium tuberculosis lumazine synthase with a K(i) of 60 nM.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Purinas/síntesis química , Purinas/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Bacillus subtilis/enzimología , Unión Competitiva , Diseño de Fármacos , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Cinética , Modelos Moleculares , Complejos Multienzimáticos/genética , Organofosfatos/química , Organofosfatos/farmacología , Organofosfonatos/química , Organofosfonatos/farmacología , Purinas/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribitol/análogos & derivados , Ribitol/farmacología , Riboflavina Sintasa/genéticaRESUMEN
Several analogues of a hypothetical intermediate in the reaction catalyzed by lumazine synthase were synthesized and tested as inhibitors of both Bacillus subtilis lumazine synthase and Escherichia coli riboflavin synthase. The new compounds were designed by replacement of a two-carbon fragment of several 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors with an amide linkage that was envisioned as an analogue of a Schiff base moiety of a hypothetical intermediate in the enzyme-catalyzed reaction. The incorporation of the amide group led to an unexpected reversal in selectivity for inhibition of lumazine synthase vs riboflavin synthase. Whereas the parent 5-phosphonoalkyl-6-D-ribitylaminopyrimidinediones were lumazine synthase inhibitors and did not inhibit riboflavin synthase, the amide-containing derivatives inhibited riboflavin synthase and were only very weak or inactive as lumazine synthase inhibitors. Molecular modeling of inhibitor-lumazine synthase complexes did not reveal a structural basis for these unexpected findings. However, molecular modeling of one of the inhibitors with E. coli riboflavin synthase demonstrated that the active site of the enzyme could readily accommodate two ligand molecules.
Asunto(s)
Amidas/química , Inhibidores Enzimáticos/síntesis química , Complejos Multienzimáticos/antagonistas & inhibidores , Pirimidinonas/síntesis química , Riboflavina Sintasa/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Complejos Multienzimáticos/química , Conformación Proteica , Pteridinas , Riboflavina Sintasa/química , Relación Estructura-ActividadRESUMEN
A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase. The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3'-carboxypropyl compound 22. Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity. In the phosphonoxyalkyl series, the 3'-phosphonoxypropyl compound 33 was more potent than the 4'-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3'-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2'-carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Complejos Multienzimáticos/antagonistas & inhibidores , Pteridinas/síntesis química , Ribitol/análogos & derivados , Riboflavina Sintasa/antagonistas & inhibidores , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Cinética , Modelos Moleculares , Conformación Molecular , Complejos Multienzimáticos/química , Conformación Proteica , Pteridinas/química , Pteridinas/farmacología , Ribitol/síntesis química , Ribitol/química , Ribitol/farmacología , Riboflavina Sintasa/química , Relación Estructura-ActividadRESUMEN
Reduction of 5-nitro-6-D-ribitylaminouracil (9) afforded 5-amino-6-D-ribitylaminouracil (1), which reacted with ethyl chloroformate to yield 5-ethylcarbamoyl-6-D-ribitylaminouracil (12). The latter compound was cyclized to 9-D-ribityl-1,3,7-trihydropurine-2,6,8-trione (13), which was found to be a relatively potent inhibitor of both Escherichia coli riboflavin synthase (K(i) 0.61 microM) and Bacillus subtilis lumazine synthase (K(i) 46 microM). Molecular modeling of the lumazine synthase-inhibitor complex indicated the possibility for hydrogen bonding between the Lys135 epsilon-amino group of the enzyme and both the 8-keto group and the 4'-hydroxyl group of the ligand. A bisubstrate analogue of the riboflavin synthase-catalyzed reaction, 1,4-bis[1-(9-D-ribityl-1,3,7-trihydropurine-2,6,8-trionyl)]butane (18), was also synthesized using a similar route and was found to be inactive as an inhibitor of both riboflavin synthase and lumazine synthase.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Complejos Multienzimáticos/antagonistas & inhibidores , Purinas/síntesis química , Ribitol/síntesis química , Riboflavina Sintasa/antagonistas & inhibidores , Bacillus subtilis/química , Cristalografía por Rayos X , Escherichia coli/química , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Proteínas Recombinantes/química , Ribitol/análogos & derivadosRESUMEN
A novel inhibitor of lumazine synthase, the penultimate enzyme in the biosynthesis of riboflavin, has been synthesized. The inhibitor was designed by computer graphics molecular modeling using a hypothetical structure of the enzyme-inhibitor complex. The new compound is relatively potent when compared with the known inhibitors, and displays a KI of 109 microM.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Timidina/análogos & derivados , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/enzimología , Sitios de Unión , Cápside/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Organofosfonatos/química , Riboflavina Sintasa/metabolismo , Programas Informáticos , Timidina/síntesis química , Timidina/química , Timidina/farmacologíaRESUMEN
2,6-Dioxo-(1H,3H)-9-N-ribitylpurine (6) and 2,6-dioxo-(1H,3H)-8-aza-9-N-ribitylpurine (7) have been synthesized and evaluated as inhibitors of lumazine synthase and riboflavin synthase. Reaction of 5-amino-6-ribitylaminouracil hydrochloride (8) with diethoxymethyl acetate (9) afforded the purine 6, while diazotization of 8 afforded the 8-aza purine 7. Compounds 6 and 7 were evaluated against lumazine synthase of Bacillus subtilis and riboflavin synthase of Escherichia coli. Both 6 and 7 were better inhibitors of lumazine synthase than riboflavin synthase. The 8-azapurine 7 had a lower KI (0.33 and 0.39 mM) than the purine 6 (0.47 and 0.54 mM) when evaluated with lumazine synthase and riboflavin synthase, respectively.