RESUMEN
Alzheimer's disease (AD) remains a significant burden on society. In the search for new AD drugs, modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are of particular interest, as loss of synaptic AMPARs has been linked to AD learning and memory deficits. Previously reported fluorine-containing BPAM121, an AMPA positive allosteric modulator (pam) with high activity, low toxicity, and slow metabolism, was considered to be a perfect 18 F-labeled candidate for positron emission tomography (PET) AD diagnostic investigations. For the preclinical use of this compound, an automated synthesis avoiding human radiation exposure was developed. The detailed production of [18 F]BPAM121 in relatively high quantity using a commercial FASTlab synthesizer from GE Healthcare coupled with a full set of quality controls is presented, along with procedures for the synthesis of the tosylated precursor and the fluorinated reference. To evaluate the clinical usefulness of [18 F]BPAM121 as a potential AD diagnostic, some inâ vivo studies in mice were then realized, alongside blocking and competition studies.
Asunto(s)
Radioisótopos de Flúor/química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/síntesis química , Radiofármacos/farmacología , Receptores AMPA/efectos de los fármacos , Tiadiazinas/síntesis química , Tiadiazinas/farmacología , Regulación Alostérica , Enfermedad de Alzheimer/diagnóstico por imagen , Animales , Automatización , Evaluación Preclínica de Medicamentos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores AMPA/metabolismoRESUMEN
Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson's Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70. Next, twelve MAL1-271 derivatives were synthesized and examined in a refined α-synuclein aggregation model as well as in an assay that monitors maturation of a disease-causing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant, which is also linked to Hsp70 function. Compared to the control, MAL1-271 significantly increased the number of cells lacking α-synuclein inclusions and increased the steady-state levels of the CFTR mutant. We also found that a nitrile-containing MAL1-271 analog exhibited similar effects in both assays. None of the derivatives exhibited cellular toxicity at concentrations up to 100⯵m, nor were cellular stress response pathways induced. These data serve as a gateway for the continued development of a new class of Hsp70 agonists with efficacy in these and potentially other disease models.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Activadores de Enzimas/farmacología , Ésteres/farmacología , Proteínas HSP70 de Choque Térmico/agonistas , Multimerización de Proteína/efectos de los fármacos , Pirimidinonas/farmacología , Línea Celular Tumoral , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activadores de Enzimas/síntesis química , Activadores de Enzimas/química , Activadores de Enzimas/toxicidad , Ésteres/síntesis química , Ésteres/química , Ésteres/toxicidad , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Estructura Molecular , Pliegue de Proteína/efectos de los fármacos , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/toxicidad , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , alfa-Sinucleína/agonistas , alfa-Sinucleína/metabolismoRESUMEN
Human polyomaviruses are generally latent but can be reactivated in patients whose immune systems are suppressed. Unfortunately, current therapeutics for diseases associated with polyomaviruses are non-specific, have undefined mechanisms of action, or exacerbate the disease. We previously reported on a class of dihydropyrimidinones that specifically target a polyomavirus-encoded protein, T antigen, and/or inhibit a cellular chaperone, Hsp70, that is required for virus replication. To improve the antiviral activity of the existing class of compounds, we performed Biginelli and modified multi-component reactions to obtain new 3,4-dihydropyrimidin-2(1H)-ones and -thiones for biological evaluation. We also compared how substituents at the N-1 versus N-3 position in the pyrimidine affect activity. We discovered that AMT580-043, a N-3 alkylated dihydropyrimidin-2(1H)-thione, inhibits the replication of a disease-causing polyomavirus in cell culture more potently than an existing drug, cidofovir.
Asunto(s)
Antivirales/farmacología , Poliomavirus/efectos de los fármacos , Pirimidinonas/farmacología , Animales , Antivirales/química , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Pirimidinonas/química , Relación Estructura-ActividadRESUMEN
The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.
Asunto(s)
Antranilato Sintasa/antagonistas & inhibidores , Antranilato Sintasa/química , Mycobacterium tuberculosis/enzimología , Triptófano/metabolismo , Tuberculosis/microbiología , Antranilato Sintasa/metabolismo , Vías Biosintéticas , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Polyomavirus infections are common and relatively benign in the general human population but can become pathogenic in immunosuppressed patients. Because most treatments for polyomavirusassociated diseases nonspecifically target DNA replication, existing treatments for polyomavirus infection possess undesirable side effects. However, all polyomaviruses express Large Tumor Antigen (T Ag), which is unique to this virus family and may serve as a therapeutic target. Previous screening of pyrimidinonepeptoid hybrid compounds identified MAL2-11B and a MAL2-11B tetrazole derivative as inhibitors of viral replication and T Ag ATPase activity (IC50 of ~20-50 µM. To improve upon this scaffold and to develop a structureactivity relationship for this new class of antiviral agents, several iterative series of MAL2-11B derivatives were synthesized. The replacement of a flexible methylene chain linker with a benzyl group or, alternatively, the addition of an ortho-methyl substituent on the biphenyl side chain in MAL2-11B yielded an IC50 of 50 µM, which retained antiviral activity. After combining both structural motifs, a new lead compound was identified that inhibited T Ag ATPase activity with an IC50 of 50 µM. We suggest that the knowledge gained from the structureactivity relationship and a further refinement cycle of the MAL2-11B scaffold will provide a specific, novel therapeutic treatment option for polyomavirus infections and their associated diseases.
Asunto(s)
Antígenos Virales de Tumores/química , Antivirales/síntesis química , Virus 40 de los Simios/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Antígenos Virales de Tumores/metabolismo , Antivirales/farmacología , Antivirales/toxicidad , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Peptoides/química , Poliomavirus/efectos de los fármacos , Unión Proteica , Pirimidinonas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Hidroxibenzoatos/química , Liasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Bibliotecas de Moléculas Pequeñas/síntesis química , Acrilatos/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Ésteres , Cinética , Liasas/metabolismo , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efectos de los fármacos , Unión Proteica , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.
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Dominio Catalítico , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Liasas/antagonistas & inhibidores , Liasas/metabolismo , Mycobacterium tuberculosis/enzimología , Ácido Corísmico/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Liasas/química , Modelos Moleculares , Unión Proteica , Piruvatos/química , Piruvatos/metabolismo , Piruvatos/farmacología , Soluciones , TemperaturaRESUMEN
The TB Structural Genomics Consortium is a worldwide organization of collaborators whose mission is the comprehensive structural determination and analyses of Mycobacterium tuberculosis proteins to ultimately aid in tuberculosis diagnosis and treatment. Congruent to the overall vision, Consortium members have additionally established an integrated facilities core to streamline M. tuberculosis structural biology and developed bioinformatics resources for data mining. This review aims to share the latest Consortium developments with the TB community, including recent structures of proteins that play significant roles within M. tuberculosis. Atomic resolution details may unravel mechanistic insights and reveal unique and novel protein features, as well as important protein-protein and protein-ligand interactions, which ultimately lead to a better understanding of M. tuberculosis biology and may be exploited for rational, structure-based therapeutics design.
Asunto(s)
Genómica/métodos , Cooperación Internacional , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/química , Cristalografía por Rayos X , Bases de Datos de Proteínas , Diseño de Fármacos , Genoma Bacteriano , Genómica/tendencias , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismoRESUMEN
Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate-utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate-based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI-catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3-dihydroxybenzoate scaffold, proved to be low-micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol-pyruvyl side chain found in chorismate and isochorismate.