RESUMEN
The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis have been identified as attractive targets with novel modes of action for the development of herbicides for crop protection and agents against infectious diseases. This pathway is present in many pathogenic organisms and plants, but absent in mammals. By using high-throughput screening, we identified highly halogenated marine natural products, the pseudilins, to be inhibitors of the third enzyme, IspD, in the pathway. Their activity against the IspD enzymes from Arabidopsis thaliana and Plasmodium vivax was determined in photometric and NMR-based assays. Cocrystal structures revealed that pseudilins bind to an allosteric pocket by using both divalent metal ion coordination and halogen bonding. The allosteric mode of action for preventing cosubstrate (CTP) binding at the active site was elucidated. Pseudilins show herbicidal activity in plant assays and antiplasmodial activity in cell-based assays.
Asunto(s)
Productos Biológicos/metabolismo , Ácido Mevalónico/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Protozoarias/metabolismo , Alcaloides/química , Alcaloides/metabolismo , Regulación Alostérica , Sitio Alostérico , Arabidopsis/enzimología , Sitios de Unión , Productos Biológicos/química , Halogenación , Herbicidas/química , Herbicidas/metabolismo , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas de Plantas/antagonistas & inhibidores , Plasmodium vivax/enzimología , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidoresRESUMEN
Enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are attractive targets for the development of herbicides and drugs against infectious diseases. While this pathway is essential for many pathogens and plants, mammals do not depend on it for the synthesis of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway, is unique in that it has an allosteric regulatory site. We elucidated the binding mode of phenylisoxazoles, a new class of allosteric inhibitors. Allosteric inhibition is effected by large conformational changes of a loop region proximal to the active site. We investigated the different roles of residues in this loop by mutation studies and identified repulsive interactions with Asp291 and Asp292 to be responsible for inhibition. Crystallographic data and the response of mutant enzymes to three different classes of allosteric inhibitors provide an in-depth understanding of the allosteric mechanism. The obtained mutant enzymes show selective resistance to allosteric inhibitors and provide conceptually valuable information for future engineering of herbicide-resistant crops. We found that the isoprenoid precursors IPP and DMAPP are natural inhibitors of Arabidopsis thaliana IspD; however, they do not seem to bind to the allosteric site.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Arabidopsis , Proteínas de Escherichia coli/antagonistas & inhibidores , Isoxazoles/química , Ligandos , Complejos Multienzimáticos/antagonistas & inhibidores , Oxidorreductasas/antagonistas & inhibidores , Sitio Alostérico , Arabidopsis/enzimología , Sitios de Unión , Inhibidores Enzimáticos/farmacología , Hemiterpenos/química , Hemiterpenos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/química , Indoles/farmacología , Isoxazoles/farmacología , Modelos Moleculares , Estructura Molecular , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacologíaRESUMEN
The emergence and spread of multidrug-resistant pathogens are widely believed to endanger human health. New drug targets and lead compounds exempt from cross-resistance with existing drugs are urgently needed. We report on the synthesis and properties of "reverse" thia analogs of fosmidomycin, which inhibit the first committed enzyme of a metabolic pathway that is essential for the causative agents of tuberculosis and malaria but is absent in the human host. Notably, IspC displays a high level of enantioselectivity for an α-substituted fosmidomycin derivative.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Antiinfecciosos/farmacología , Descubrimiento de Drogas/métodos , Fosfomicina/análogos & derivados , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Fosfomicina/síntesis química , Fosfomicina/química , Fosfomicina/farmacología , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Homología de Secuencia de Aminoácido , EstereoisomerismoRESUMEN
Specific inhibition of enzymes of the non-mevalonate pathway is a promising strategy for the development of novel antiplasmodial drugs. α-Aryl-substituted ß-oxa isosteres of fosmidomycin with a reverse orientation of the hydroxamic acid group were synthesized and evaluated for their inhibitory activity against recombinant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) of Plasmodium falciparum and for their in vitro antiplasmodial activity against chloroquine-sensitive and resistant strains of P. falciparum . The most active derivative inhibits IspC protein of P. falciparum (PfIspC) with an IC(50) value of 12 nM and shows potent in vitro antiplasmodial activity. In addition, lipophilic ester prodrugs demonstrated improved P. falciparum growth inhibition in vitro.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfomicina/análogos & derivados , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/química , Antiprotozoarios/síntesis química , Antiprotozoarios/metabolismo , Técnicas de Química Sintética , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Fosfomicina/síntesis química , Fosfomicina/química , Fosfomicina/metabolismo , Fosfomicina/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Profármacos/síntesis química , Profármacos/metabolismo , Conformación ProteicaRESUMEN
Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray analysis. Pilot experiments have demonstrated in vivo potential.