RESUMEN
Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.
RESUMEN
Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Diseño de Fármacos , Éteres/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Compuestos de Anilina/química , Antineoplásicos/química , Línea Celular Tumoral , Éteres/química , Humanos , L-Lactato Deshidrogenasa/químicaRESUMEN
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Colestasis Intrahepática/tratamiento farmacológico , Colestasis Intrahepática/genética , Eliminación de Gen , Liposomas/química , Sistema de Administración de Fármacos con Nanopartículas/química , Nanopartículas/química , Fenotipo , ARN Mensajero/administración & dosificación , Subfamilia B de Transportador de Casetes de Unión a ATP/administración & dosificación , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Colestasis Intrahepática/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Homocigoto , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Mensajero/genética , Transfección , Resultado del Tratamiento , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.
Asunto(s)
Terapia de Reemplazo Enzimático/métodos , Acidemia Propiónica/terapia , ARN Mensajero/uso terapéutico , Animales , Modelos Animales de Enfermedad , Glutamatos/uso terapéutico , Humanos , Cinética , Lípidos/química , Hígado/enzimología , Metilmalonil-CoA Descarboxilasa/química , Metilmalonil-CoA Descarboxilasa/genética , Metilmalonil-CoA Descarboxilasa/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/enzimología , Nanopartículas/administración & dosificación , Nanopartículas/química , Acidemia Propiónica/genética , Acidemia Propiónica/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , ARN Mensajero/administración & dosificación , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Isolated methylmalonic acidemia/aciduria (MMA) is an ultra-rare, serious, inherited metabolic disorder with significant morbidity and mortality. Exogenously delivered mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, is a potential therapy to produce functional MUT enzyme in liver. METHODS: Two 12-week repeat-dose studies were conducted to evaluate the efficacy and safety of intravenously-administered hMUT mRNA encapsulated in lipid nanoparticles in two murine models of MMA. FINDINGS: In MMA hypomorphic mice, hMUT mRNA treatment resulted in dose-dependent and reproducible biomarker responses after each dose. Enzymatically-active MUT protein was produced in liver in a dose-dependent manner. hMUT mRNA was well-tolerated with no adverse effects, as indicated by the lack of clinical observations, minimal changes in clinical chemistry parameters, and histopathology examination across all tissues. In severe MMA mice, hMUT mRNA led to substantially improved survival and growth and ameliorated biochemical abnormalities, all of which are cardinal clinical manifestations in severely affected patients. INTERPRETATION: These data demonstrate durable functional benefit of hMUT mRNA and support development of this new class of therapy for a devastating, pediatric disorder. FUND: This work was funded by Moderna, Inc.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/terapia , Hígado/metabolismo , Metilmalonil-CoA Mutasa/farmacología , ARN Mensajero/farmacología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/patología , Animales , Niño , Modelos Animales de Enfermedad , Humanos , Lípidos/genética , Hígado/efectos de los fármacos , Hígado/patología , Metilmalonil-CoA Mutasa/genética , Ratones , ARN Mensajero/genéticaRESUMEN
Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.
Asunto(s)
Citrulinemia/tratamiento farmacológico , Citrulinemia/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ARN Mensajero/uso terapéutico , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Glucosafosfato Deshidrogenasa/genética , Células HeLa , Células Hep G2 , Humanos , Lípidos/química , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Nanopartículas/química , Sistemas de Lectura Abierta/genética , ARN Mensajero/síntesis química , ARN Mensajero/química , ARN Mensajero/genética , Transfección , Resultado del TratamientoRESUMEN
Methylmalonic acidemia/aciduria (MMA) is a genetically heterogeneous group of inherited metabolic disorders biochemically characterized by the accumulation of methylmalonic acid. Isolated MMA is primarily caused by the deficiency of methylmalonyl-CoA mutase (MMA mut; EC 5.4.99.2). A systematic literature review and a meta-analysis were undertaken to assess and compile published epidemiological data on MMA with a focus on the MMA mut subtype (OMIM #251000). Of the 1114 identified records, 227 papers were assessed for eligibility in full text, 48 articles reported on disease epidemiology, and 39 articles were included into the quantitative synthesis. Implementation of newborn screening in various countries has allowed for the estimation of birth prevalence of MMA and its isolated form. Meta-analysis pooled point estimates of MMA (all types) detection rates were 0.79, 1.12, 1.22 and 6.04 per 100,000 newborns in Asia-Pacific, Europe, North America and the Middle East and North Africa (MENA) regions, respectively. The detection rate of isolated MMA was < 1 per 100,000 newborns in all regions with the exception of MENA where it approached 6 per 100,000 newborns. Few studies published data on the epidemiology of MMA mut, therefore no meta-analysis could have been performed on this subtype. Most of the identified papers reported birth prevalence estimates below 1 per 100,000 newborns for MMA mut. The systematic literature review clearly demonstrates that MMA and its subtypes are ultra-rare disorders.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/epidemiología , Metilmalonil-CoA Mutasa/genética , Errores Innatos del Metabolismo de los Aminoácidos/genética , Femenino , Humanos , Recién Nacido , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/epidemiología , Errores Innatos del Metabolismo/genética , Metilmalonil-CoA Mutasa/deficiencia , Tamizaje NeonatalRESUMEN
Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.
Asunto(s)
Enfermedad de Fabry/genética , Enfermedad de Fabry/terapia , ARN Mensajero/uso terapéutico , alfa-Galactosidasa/genética , Animales , Modelos Animales de Enfermedad , Endocitosis , Terapia de Reemplazo Enzimático , Terapia Genética , Humanos , Lípidos/química , Lisosomas/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/farmacocinética , Distribución Tisular , Trihexosilceramidas/metabolismoRESUMEN
Propionic acidemia (PA, OMIM #606054) is a serious, life-threatening, inherited, metabolic disorder caused by the deficiency of the mitochondrial enzyme propionyl-coenzyme A (CoA) carboxylase (EC 6.4.1.3). The primary objective of this study was to conduct a systematic literature review and meta-analysis on the epidemiology of PA. The literature search was performed covering Medline, Embase, Cochrane Database of Systematic Reviews, CRD Database, Academic Search Complete, CINAHL and PROSPERO databases. Websites of rare disease organizations were also searched for eligible studies. Of the 2338 identified records, 188 articles were assessed for eligibility in full text, 43 articles reported on disease epidemiology, and 31 studies were included into the quantitative synthesis. Due to the rarity of PA, broadly targeted population-based prevalence studies are not available. Nonetheless, implementation of newborn screening programs has allowed the estimation of the birth prevalence data of PA across multiple geographic regions. The pooled point estimates indicated detection rates of 0.29; 0.33; 0.33 and 4.24 in the Asia-Pacific, Europe, North America and the Middle East and North Africa (MENA) regions, respectively. Our systematic literature review and meta-analysis confirm that PA is an ultra-rare disorder, with similar detection rates across all regions with the exception of the MENA region where the disease, similar to other inherited metabolic disorders, is more frequent.
Asunto(s)
Acidemia Propiónica/diagnóstico , Acidemia Propiónica/epidemiología , Humanos , Recién Nacido , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/epidemiología , Tamizaje Neonatal/métodosRESUMEN
Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.
Asunto(s)
Terapia Genética , Hidroximetilbilano Sintasa/genética , Porfiria Intermitente Aguda/terapia , ARN Mensajero/administración & dosificación , Animales , Modelos Animales de Enfermedad , Femenino , Haploinsuficiencia/genética , Hemo/genética , Hemo/metabolismo , Hepatocitos/efectos de los fármacos , Humanos , Hidroximetilbilano Sintasa/uso terapéutico , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Porfiria Intermitente Aguda/genética , Porfiria Intermitente Aguda/patología , ARN Mensajero/genéticaRESUMEN
Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/terapia , Terapia Genética/métodos , Metilmalonil-CoA Mutasa/genética , Nanopartículas/administración & dosificación , ARN Mensajero/genética , Administración Intravenosa , Animales , Femenino , Humanos , Lípidos/química , Hígado/metabolismo , Masculino , Metilmalonil-CoA Mutasa/metabolismo , Ratones , Nanopartículas/química , ARN Mensajero/metabolismoRESUMEN
We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.
Asunto(s)
Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Pirazoles/farmacología , Tiazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Membranas Artificiales , Ratones , Microsomas Hepáticos/efectos de los fármacos , Permeabilidad , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacocinética , Ratas , Solubilidad , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/química , Tiazoles/farmacocinéticaRESUMEN
Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.
Asunto(s)
Mutación Missense , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Humanos , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Secundaria de ProteínaRESUMEN
The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.
Asunto(s)
Antivirales/química , Virus de la Hepatitis B/fisiología , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/efectos de los fármacos , Antivirales/metabolismo , Antivirales/farmacología , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
Tankyrase activity has been linked to the regulation of intracellular axin levels, which have been shown to be crucial for the Wnt pathway. Deregulated Wnt signaling is important for the genesis of many diseases including cancer. We describe herein the discovery and development of a new series of tankyrase inhibitors. These pyranopyridones are highly active in various cell-based assays. A fragment/structure based optimization strategy led to a compound with good pharmacokinetic properties that is suitable for in vivo studies and further development.
RESUMEN
The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.
Asunto(s)
Aspartato Aminotransferasas/química , Giardia lamblia/química , Leishmania major/química , Trypanosoma brucei brucei/química , Cristalización , Giardia lamblia/enzimología , Leishmania major/enzimología , Estructura Secundaria de Proteína , Trypanosoma brucei brucei/enzimologíaRESUMEN
MEK inhibitors are clinically active in BRAF(V600E) melanomas but only marginally so in KRAS mutant tumors. Here, we found that MEK inhibitors suppress ERK signaling more potently in BRAF(V600E), than in KRAS mutant tumors. To understand this, we performed an RNAi screen in a KRAS mutant model and found that CRAF knockdown enhanced MEK inhibition. MEK activated by CRAF was less susceptible to MEK inhibitors than when activated by BRAF(V600E). MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting such complexes enhanced inhibition of CRAF-dependent ERK signaling. Newer MEK inhibitors target MEK catalytic activity and also impair its reactivation by CRAF, either by disrupting RAF-MEK complexes or by interacting with Ser 222 to prevent MEK phosphorylation by RAF.
Asunto(s)
Resistencia a Antineoplásicos/genética , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/genética , Factor 3 Asociado a Receptor de TNF/genética , Proteínas ras/genética , Animales , Benzamidas/farmacología , Línea Celular , Cumarinas/farmacología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Indoles/farmacología , MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 1/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/genética , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras) , Piridonas/farmacología , Pirimidinonas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Sulfonamidas/farmacología , Resonancia por Plasmón de Superficie , Factor 3 Asociado a Receptor de TNF/metabolismo , Vemurafenib , Quinasas raf/metabolismoRESUMEN
Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridazinas/síntesis química , Piridazinas/farmacología , Bazo/enzimología , Amidas/síntesis química , Amidas/farmacología , Animales , Biología Computacional , Simulación por Computador , Diseño de Fármacos , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Conformación Molecular , Mutagénesis/efectos de los fármacos , Pruebas de Mutagenicidad , Inhibidores de Proteínas Quinasas/farmacocinética , Piridazinas/farmacocinética , Ratas , Bazo/efectos de los fármacos , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
INTRODUCTION: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy. METHODS: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA). RESULTS: A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation. CONCLUSIONS: Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.