RESUMEN
Exploration of triclosan analogs has led to novel diaryl ureas with significant potency against in vitro cultures of drug-resistant and drug-sensitive strains of the human malaria parasite Plasmodium falciparum. Compound 18 demonstrated EC(50) values of 37 and 55 nM versus in vitro cultured parasite strains and promising in vivo efficacy in a Plasmodium berghei antimalarial mouse model, with >50% survival at day 31 post-treatment when administered subcutaneously at 256 mg/kg. This series of compounds provides a chemical scaffold of novel architecture, as validated by cheminformatics analysis, to pursue antimalarial drug discovery efforts.
Asunto(s)
Antimaláricos/farmacología , Derivados del Benceno/farmacología , Malaria Falciparum/tratamiento farmacológico , Urea/análogos & derivados , Urea/farmacología , Animales , Antimaláricos/química , Derivados del Benceno/química , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Malaria Falciparum/parasitología , RatonesRESUMEN
Drug resistance in Mycobacterium tuberculosis has become a serious global health threat, which is now complicated by the emergence of extensively drug-resistant strains. New drugs that are active against drug-resistant tuberculosis (TB) are needed. We chose to search for new inhibitors of the enoyl-acyl carrier protein (ACP) reductase InhA, the target of the first-line TB drug isoniazid (also known as isonicotinoic acid hydrazide [INH]). A subset of a chemical library, composed of 300 compounds inhibiting Plasmodium falciparum enoyl reductase, was tested against M. tuberculosis. Four compounds were found to inhibit M. tuberculosis growth with MICs ranging from 1 µM to 10 µM. Testing of these compounds against M. tuberculosis in vitro revealed that only two compounds (CD39 and CD117) were bactericidal against drug-susceptible and drug-resistant M. tuberculosis. These two compounds were also bactericidal against M. tuberculosis incubated under anaerobic conditions. Furthermore, CD39 and CD117 exhibited increased bactericidal activity when used in combination with INH or rifampin, but CD39 was shown to be toxic to eukaryotic cells. The compounds inhibit InhA as well the fatty acid synthase type I, and CD117 was found to also inhibit tuberculostearic acid synthesis. This study provides the TB drug development community with two chemical scaffolds that are suitable for structure-activity relationship study to improve on their cytotoxicities and bactericidal activities in vitro and in vivo.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Aerobiosis , Anaerobiosis , Animales , Antituberculosos/química , Proteínas Bacterianas/metabolismo , Catalasa/metabolismo , Células Cultivadas , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Ácidos Esteáricos/metabolismo , Relación Estructura-Actividad , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
The synthesis and proteolytic inhibitor function of two new tetrapeptides, methoxysuccinyl-Ala-Ala-Pro-Phe-chloromethyl ketone (MeOSuc-AAPF-CH(2)Cl) and methoxysuccinyl-Ala-Pro-Ala-Phe-chloromethyl ketone (MeOSuc-APAF-CH(2)Cl) are described. The efficacy of these two new analogs in inhibiting the proteolytic activity of proteinase K has been compared with the previously-documented proteainase K inhibitor, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MeOSuc-AAPV-CH(2)Cl). An examination of inhibitory activity using a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay in the presence of proteinase K reveals that the AAPF inhibitor (MeOSuc-AAPF-CH(2)Cl) at a concentration of 0.05mM allows a signal to be obtained for an exogenous target ("Xeno RNA") at 30cycles (i.e. Ct=30), whereas the MeOSuc-AAPV-CH(2)Cl control requires a 10-fold higher concentration (0.5mM) to produce the same Ct. Interestingly, the other new analog, with the rearranged amino acid sequence APAF (MeO Suc-APAF-CH(2)Cl), provides no proteinase K inhibition under the same experimental conditions. These results suggest that when P1 is phenylalanine, alanine at P2 and proline at P3 is not tolerated as a good proteinase K inhibitor. A plausible explanation for the higher efficiency of MeOSuc-AAPF-CH(2)Cl over control is proposed based on the molecular modeling studies.
Asunto(s)
Clorometilcetonas de Aminoácidos/síntesis química , Clorometilcetonas de Aminoácidos/farmacología , Endopeptidasa K/antagonistas & inhibidores , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Endopeptidasa K/metabolismoRESUMEN
The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.
Asunto(s)
Antimaláricos/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Triclosán/química , Animales , Antimaláricos/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Resistencia a Medicamentos/efectos de los fármacos , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Triclosán/análogos & derivados , Triclosán/metabolismoRESUMEN
There is a dire need for novel therapeutics to treat the virulent malarial parasite, Plasmodium falciparum. Recently, the X-ray crystal structure of enoyl-acyl carrier protein reductase (ENR) in complex with triclosan has been determined and provides an opportunity for the rational design of novel inhibitors targeting the active site of ENR. Here, we report the discovery of several compounds by virtual screening and their experimental validation as high potency PfENR inhibitors.
Asunto(s)
Antimaláricos/farmacología , Diseño de Fármacos , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Animales , Antimaláricos/química , Sitios de Unión , Células CACO-2 , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Enlace de Hidrógeno , Cinética , Malaria/tratamiento farmacológico , Modelos Moleculares , Triclosán/química , Triclosán/farmacologíaRESUMEN
2'-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC50<500 nM) against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and modest (IC50=1-20 microM) potency against purified PfENR enzyme. Compared to triclosan, this survey of 2'-substituted derivatives has afforded gains in excess of 20- and 30-fold versus the 3D7 and Dd2 strains of parasite, respectively.
Asunto(s)
Antimaláricos/síntesis química , Antimaláricos/farmacología , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Éteres/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Triclosán/química , Animales , Cristalografía por Rayos X , Farmacorresistencia Microbiana , Relación Estructura-Actividad , Triclosán/análogos & derivadosRESUMEN
A structure-based approach has been taken to develop 4'-substituted analogs of triclosan that target the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit nanomolar potency against purified PfENR enzyme and modest (2-10microM) potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite. X-ray crystal structures of nitro 29, aniline 30, methylamide 37, and urea 46 demonstrate the presence of hydrogen-bonding interactions with residues in the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular parasites.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Triclosán/análogos & derivados , Animales , Antimaláricos/síntesis química , Cristalografía por Rayos X , Estructura Molecular , Plasmodium falciparum/efectos de los fármacos , Triclosán/químicaRESUMEN
Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.
Asunto(s)
Dinitrobencenos/farmacología , Indoles/farmacología , Malaria/tratamiento farmacológico , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Piperazinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Pirimidinas/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antiinfecciosos Locales/metabolismo , Antiinfecciosos Locales/farmacología , Proteínas Bacterianas , Dinitrobencenos/química , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Indoles/química , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Piperazinas/química , Plasmodium falciparum/enzimología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pirimidinas/química , Relación Estructura-Actividad , Triclosán/metabolismo , Triclosán/farmacologíaRESUMEN
Structural genomics, the large-scale determination of protein structures, promises to provide a broad structural foundation for drug discovery. The tuberculosis (TB) Structural Genomics Consortium is devoted to encouraging, coordinating, and facilitating the determination of structures of proteins from Mycobacterium tuberculosis and hopes to determine 400 TB protein structures over 5 years. The Consortium has determined structures of 28 proteins from TB to date. These protein structures are already providing a basis for drug discovery efforts.
Asunto(s)
Aciltransferasas , Antibacterianos/farmacología , Antígenos Bacterianos , Proteínas Bacterianas/química , Diseño de Fármacos , Genómica , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efectos de los fármacos , Aldehído-Liasas/química , Sistema Enzimático del Citocromo P-450/química , Glutamato-Amoníaco Ligasa/química , Metiltransferasas/química , Mio-Inositol-1-Fosfato Sintasa/química , Oxidorreductasas/química , Proteína Disulfuro Isomerasas/químicaRESUMEN
The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.