RESUMEN
Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8-trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF-expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.
Asunto(s)
Oxidorreductasas Intramoleculares , Factores Inhibidores de la Migración de Macrófagos , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/inmunología , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Alelos , Repeticiones de Microsatélite , Regiones Promotoras Genéticas , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Ratones , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismoRESUMEN
BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in multiple inflammatory and non-inflammatory diseases, including liver injury induced by acetaminophen (APAP) overdose. Multiple small molecule inhibitors of MIF have been described, including the clinically available anti-rheumatic drug T-614 (iguratimod); however, this drug's mode of inhibition has not been fully investigated. METHODS: We conducted in vitro testing including kinetic analysis and protein crystallography to elucidate the interactions between MIF and T-614. We also performed in vivo experiments testing the efficacy of T-614 in a murine model of acetaminophen toxicity. We analyzed survival in lethal APAP overdose with and without T-614 and using two different dosing schedules of T-614. We also examined MIF and MIF inhibition effects on hepatic hydrogen peroxide (H2O2) as a surrogate of oxidative stress in non-lethal APAP overdose. RESULTS: Kinetic analysis was consistent with a non-competitive type of inhibition and an inhibition constant (Ki) value of 16 µM. Crystallographic analysis revealed that T-614 binds outside of the tautomerase active site of the MIF trimer, with only the mesyl group of the molecule entering the active site pocket. T-614 improved survival in lethal APAP overdose when given prophylactically, but this protection was not observed when the drug was administered late (6 h after APAP). T-614 also decreased hepatic hydrogen peroxide concentrations during non-lethal APAP overdose in a MIF-dependent fashion. CONCLUSIONS: T-614 is an allosteric inhibitor of MIF that prevented death and decreased hepatic hydrogen peroxide concentrations when given prophylactically in a murine model of acetaminophen overdose. Further studies are needed to elucidate the mechanistic role of MIF in APAP toxicity.
Asunto(s)
Benzopiranos , Enfermedad Hepática Inducida por Sustancias y Drogas , Cromonas , Factores Inhibidores de la Migración de Macrófagos , Sulfonamidas , Ratones , Animales , Acetaminofén/efectos adversos , Peróxido de Hidrógeno/metabolismo , Modelos Animales de Enfermedad , Cinética , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Estrés Oxidativo , Hígado/metabolismoRESUMEN
Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase-like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos , Animales , Humanos , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/química , Proteínas de Plantas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Saccharomyces cerevisiae/metabolismo , Neutrófilos/metabolismo , Mamíferos/metabolismo , Oxidorreductasas Intramoleculares/genéticaRESUMEN
Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight.
RESUMEN
The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.
Asunto(s)
Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitio Alostérico/fisiología , Secuencia de Aminoácidos/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Diferenciación de Linfocitos B/metabolismo , Sitios de Unión/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Citocinas/inmunología , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Oxidorreductasas Intramoleculares/fisiología , Factores Inhibidores de la Migración de Macrófagos/fisiología , Unión Proteica/genética , Relación Estructura-ActividadRESUMEN
Sirtuins are NAD+ dependent histone deacetylases (HDAC) that play a pivotal role in neuroprotection and cellular senescence. SIRT1-7 are different homologs from sirtuins. They play a prominent role in many aspects of physiology and regulate crucial proteins. Modulation of sirtuins can thus be utilized as a therapeutic target for metabolic disorders. Neurological diseases have distinct clinical manifestations but are mainly age-associated and due to loss of protein homeostasis. Sirtuins mediate several life extension pathways and brain functions that may allow therapeutic intervention for age-related diseases. There is compelling evidence to support the fact that SIRT1 and SIRT2 are shuttled between the nucleus and cytoplasm and perform context-dependent functions in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). In this review, we highlight the regulation of SIRT1 and SIRT2 in various neurological diseases. This study explores the various modulators that regulate the activity of SIRT1 and SIRT2, which may further assist in the treatment of neurodegenerative disease. Moreover, we analyze the structure and function of various small molecules that have potential significance in modulating sirtuins, as well as the technologies that advance the targeted therapy of neurodegenerative disease.
RESUMEN
Sirtuins (SIRTs), class III HDAC (Histone deacetylase) family proteins, are associated with cancer, diabetes, and other age-related disorders. SIRT1 and SIRT2 are established therapeutic drug targets by regulating its function either by activators or inhibitors. Compounds containing indole moiety are potential lead molecules inhibiting SIRT1 and SIRT2 activity. In the current study, we have successfully synthesized 22 indole derivatives in association with an additional triazole moiety that provide better anchoring of the ligands in the binding cavity of SIRT1 and SIRT2. In-vitro binding and deacetylation assays were carried out to characterize their inhibitory effects against SIRT1 and SIRT2. We found four derivatives, 6l, 6m, 6n, and 6o to be specific for SIRT1 inhibition; three derivatives, 6a, 6d and 6k, specific for SIRT2 inhibition; and two derivatives, 6s and 6t, which inhibit both SIRT1 and SIRT2. In-silico validation for the selected compounds was carried out to study the nature of binding of the ligands with the neighboring residues in the binding site of SIRT1. These derivatives open up newer avenues to explore specific inhibitors of SIRT1 and SIRT2 with therapeutic implications for human diseases.
Asunto(s)
Diseño de Fármacos , Inhibidores de Histona Desacetilasas/farmacología , Indoles/farmacología , Simulación del Acoplamiento Molecular , Sirtuina 1/antagonistas & inhibidores , Sirtuina 2/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Indoles/síntesis química , Indoles/química , Estructura Molecular , Sirtuina 1/metabolismo , Sirtuina 2/metabolismo , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Formation of Cu, Zn superoxide dismutase 1 (SOD1) protein inclusions within motor neurons is one of the principal characteristics of SOD1-related amyotrophic lateral sclerosis (ALS). A hypothesis as to the nature of SOD1 aggregation implicates oxidative damage to a solvent-exposed tryptophan as causative. Here, we chart the discovery of a phenanthridinone based compound (Lig9) from the NCI Diversity Set III by rational methods by in silico screening and crystallographic validation. The crystal structure of the complex with SOD1, refined to 2.5 Å, revealed that Lig9 binds the SOD1 ß-barrel in the ß-strand 2 and 3 region which is known to scaffold SOD1 fibrillation. The phenanthridinone moiety makes a substantial π-π interaction with Trp32 of SOD1. The compound possesses a significant binding affinity for SOD1 and inhibits oxidation of Trp32; a critical residue for SOD1 aggregation. Thus, Lig9 is a good candidate from which to develop a new library of SOD1 aggregation inhibitors through protection of Trp32 oxidation. Communicated by Ramaswamy H. Sarma.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Descubrimiento de Drogas , Modelos Moleculares , Oxidación-Reducción/efectos de los fármacos , Superóxido Dismutasa-1/antagonistas & inhibidores , Triptófano/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/patología , Bases de Datos Farmacéuticas , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Cu/Zn superoxide dismutase-1 (SOD1) mutations are causative for a subset of amyotrophic lateral sclerosis (ALS) cases. These mutations lead to structural instability, aggregation and ultimately motor neuron death. We have determined crystal structures of SOD1 in complex with a naphthalene-catechol-linked compound which binds with low micro-molar affinity to a site important for oxidative damage-induced aggregation. SOD1 Trp32 oxidation is indeed significantly inhibited by ligand binding. Our work shows how compound linking can be applied successfully to ligand interactions on the SOD1 surface to generate relatively good binding strength. The ligand, positioned in a region important for SOD1 fibrillation, offers the possibility that it, or a similar compound, could prevent the abnormal self-association that drives SOD1 toxicity in ALS.
Asunto(s)
Superóxido Dismutasa-1/metabolismo , Sitios de Unión , Catecoles/metabolismo , Cristalografía por Rayos X , Dimerización , Humanos , Ligandos , Mutación , Naftalenos/metabolismo , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/genética , Resonancia por Plasmón de Superficie , Triptófano/metabolismoRESUMEN
Bromodomain containing proteins recognize the level of histone acetylation and regulate epigenetically controlled processes like gene transcription and chromatin modification. The BET (bromodomain and extra-terminal) family proteins, which are transcriptional co-regulators, have been implicated in the pathogenesis of cancer, neurodegenerative disorders, and defects in embryonic stem cell differentiation. Inhibitors selectively targeting the BET bromodomains can pave the path for new drug discovery against several forms of major diseases. By a rational structure-based approach, we have identified a new inhibitor (NSC127133) of the second bromodomain (BD2) of the BET family protein BRD2 using the NCI Diversity Set III library. A high-resolution crystal structure of the BRD2-BD2 in complex with this compound and in apo- form is refined to 0.91 and 0.94 Å, respectively. The compound, which is a phenanthridinone derivative, binds well to the acetyl-lysine binding pocket of BD2 and displays significant hydrophobic and hydrophilic interactions. Moreover, the atomic resolution data obtained in this study allowed us to visualize certain structural features of BD2 which remained unobserved so far. We propose that the discovered compound may be a potential molecule to develop a new library for inhibiting the BRD2-BD2 function.
Asunto(s)
Fenantrenos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Acetilación , Antineoplásicos/química , Antineoplásicos/farmacología , Cristalografía por Rayos X , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Histonas/metabolismo , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Fenantrenos/química , Dominios Proteicos , Resonancia por Plasmón de Superficie , Factores de Transcripción , Interfaz Usuario-ComputadorRESUMEN
The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through their bromodomains (BDs) and help in regulating gene expression. BDs are chromatin 'readers': by interacting with acetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulate gene expression and repression. Extensive efforts have been employed by scientific communities worldwide to identify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylated histone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinity but also have high specificity to BET BDs. These developments make BET family proteins an important therapeutic targets for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review and discuss the structural biology of BET family BDs and their applications in major diseases.