RESUMEN
Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.
Asunto(s)
Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Lactamas/farmacología , Animales , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Lactamas/química , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 µM lactate IC50 in a MiaPaca2 cell line. Optimization of the series led to 29, a potent cell active molecule (MiaPaca2 IC50 = 0.67 µM) that also possessed good exposure when dosed orally to mice.
RESUMEN
Modification of the δ-sultam ring of RORc inverse agonist 2 led to the discovery of more polar oxa-sultam 65. The less lipophilic inverse agonist (65) displayed high potency in a biochemical assay, which translated into inhibition of IL-17 production in human peripheral blood mononuclear cells. The successful reduction of lipophilicity of this new analog gave rise to additional improvements in ROR selectivity and aqueous kinetic solubility, as well as reduction in plasma protein binding, while maintaining high cellular permeability.
Asunto(s)
Lípidos/química , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Descubrimiento de Drogas , Agonismo Inverso de Drogas , Naftalenosulfonatos/químicaRESUMEN
A palladium(0)-catalyzed rearrangement of piperidones and piperidines bearing a spirocyclopropane ring was developed. The ring expansion reaction led to a variety of functionalized caprolactam and azepane products in good to excellent yields. Experimental and computational mechanistic studies revealed an initial oxidative addition of the distal carbon-carbon bond of a cyclopropane ring to the palladium(0) catalyst and the relief of ring strain as a driving force for product formation.
RESUMEN
A high-throughput screen of the Genentech/Roche compound collection using a retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) biochemical assay revealed a N-sulfonyl-tetrahydroquinoline hit. Herein, we describe the hit-to-lead optimization and structure-activity relationships of these tetrahydroquinoline RORc inverse agonists. Through iterative synthesis and analog design, we identified compounds with improved biochemical RORc inverse agonist activity and RORc cellular potencies. These improved N-sulfonyl-tetrahydroquinoline compounds also exhibited selectivity for RORc over other nuclear receptors.
Asunto(s)
Agonismo Inverso de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Quinolinas/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the previously reported tertiary sulfonamide 1 as a starting point, we engineered structural modifications that significantly improved human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile. The most advanced δ-sultam compound, GNE-3500 (27, 1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone), possessed favorable RORc cellular potency with 75-fold selectivity for RORc over other ROR family members and >200-fold selectivity over 25 additional nuclear receptors in a cell assay panel. The favorable potency, selectivity, in vitro ADME properties, in vivo PK, and dose-dependent inhibition of IL-17 in a PK/PD model support the evaluation of 27 in preclinical studies.
Asunto(s)
Óxidos S-Cíclicos/administración & dosificación , Óxidos S-Cíclicos/farmacología , Descubrimiento de Drogas , Agonismo Inverso de Drogas , Leucocitos Mononucleares/efectos de los fármacos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Sulfonamidas/química , Tiazinas/administración & dosificación , Tiazinas/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Leucocitos Mononucleares/citología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Relación Estructura-ActividadRESUMEN
The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.
Asunto(s)
Imidazoles/química , Imidazoles/farmacología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Piridinas/química , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Línea Celular , Células Cultivadas , Descubrimiento de Drogas , Células HEK293 , Humanos , Imidazoles/metabolismo , Imidazoles/farmacocinética , Interleucina-17/inmunología , Hígado/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Piridinas/metabolismo , Piridinas/farmacocinética , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity. Compounds behaving as agonists in our biochemical assay also gave rise to an increased production of IL-17 in human PBMCs whereas inverse agonists led to significant suppression of IL-17 under the same assay conditions. The most potent inverse agonist compound showed >180-fold selectivity over the ROR isoforms as well as all other nuclear receptors that were profiled.
RESUMEN
We report that chiral 3-substituted γ-sultam α-carbanions undergo diastereoselective alkylation reactions with alkyl halides to predominantly produce trans-3,5-disubstituted γ-sultam products. Quantum mechanical calculations provided a stereoelectronic rationale for the observed diastereoselectivity.
Asunto(s)
Naftalenosulfonatos/síntesis química , Teoría Cuántica , Alquilación , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , Naftalenosulfonatos/química , EstereoisomerismoRESUMEN
Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (410 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Perros , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Células de Riñón Canino Madin DarbyRESUMEN
T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-ß signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.
Asunto(s)
Interleucina-17/biosíntesis , Biosíntesis de Proteínas , Células Th17/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Estabilidad de Enzimas , Femenino , Inflamación/genética , Inflamación/patología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transducción de Señal , Especificidad por Sustrato , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/biosíntesis , Proteasas Ubiquitina-Específicas/deficiencia , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC50=30 nM).
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Pironas/síntesis química , Pironas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The identification of a new series of RORc inverse agonists is described. Comprehensive structure-activity relationship studies of this reversed sulfonamide series identified potent RORc inverse agonists in biochemical and cellular assays which were also selective against a panel of nuclear receptors. Our work has contributed a compound that may serve as a useful in vitro tool to delineate the complex biological pathways involved in signalling through RORc. An X-ray co-crystal structure of an analogue with RORc has also provided useful insights into the binding interactions of the new series.
Asunto(s)
Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Sulfonamidas/química , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Citocinas/biosíntesis , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Simulación de Dinámica Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/toxicidadRESUMEN
A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 µM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 µM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).
Asunto(s)
Ciclohexanonas/farmacología , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Compuestos de Sulfhidrilo/farmacología , Administración Oral , Animales , Ciclohexanonas/administración & dosificación , Ciclohexanonas/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Ratas , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/administración & dosificación , Compuestos de Sulfhidrilo/químicaRESUMEN
Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma-protein unbound fraction and improvements in cellular permeability and aqueous solubility.
Asunto(s)
Proteínas Sanguíneas/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Sulfonamidas/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Cristalografía por Rayos X , Perros , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células de Riñón Canino Madin Darby , Modelos Moleculares , Estructura Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Ratas , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Palladium(0)-catalyzed conditions for the α-arylation of sultams with aryl and heteroaryl iodides have been developed. Arylation of 3-substituted 1,3-propanesultams gave rise to high yields and high diastereomeric ratios, leading to the thermodynamically favored cis product. The arylation was broadly applicable to various electron-rich and electron-poor (hetero)aromatic iodides.
Asunto(s)
Hidrocarburos Yodados/química , Paladio/química , Sulfonamidas/síntesis química , Catálisis , Espectroscopía de Resonancia Magnética , Estructura Molecular , Sulfonamidas/químicaRESUMEN
Screening a nuclear receptor compound subset in a RORc biochemical binding assay revealed a benzylic tertiary sulfonamide hit. Herein, we describe the identification of compounds with improved RORc biochemical inverse agonist activity and cellular potencies. These improved compounds also possessed appreciable selectivity for RORc over other nuclear receptors.
Asunto(s)
Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Sulfonamidas/química , Sulfonamidas/farmacología , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Relación Estructura-ActividadRESUMEN
As the biology surrounding the nuclear receptor retinoic acid receptor-related orphan receptor-gamma (RORγ or RORc) continues to evolve, significant effort has been invested in discovering modulators of this potentially important target for the treatment of metabolic and immunological diseases. Several major pharmaceutical and biotechnology companies have disclosed RORc inhibitors or partnered with other players in the field. In this perspective, we discuss both the biology and the underlying structural biology of RORc, and summarize the RORc modulators disclosed in the scientific and patent literature.
Asunto(s)
Agonismo Inverso de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Animales , Humanos , Modelos Moleculares , Estructura Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismoRESUMEN
The structure-activity relationships of T0901317 analogs were explored as RORc inverse agonists using the principles of property- and structure-based drug design. An X-ray co-crystal structure of T0901317 and RORc was obtained and provided molecular insight into why T0901317 functioned as an inverse agonist of RORc; whereas, the same ligand functioned as an agonist of FXR, LXR, and PXR. The structural data was also used to design inhibitors with improved RORc biochemical and cellular activities. The improved inhibitors possessed enhanced selectivity profiles (rationalized using the X-ray crystallographic data) against other nuclear receptors.
Asunto(s)
Diseño de Fármacos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Propanoles/química , Sulfonamidas/química , Sulfonamidas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Agonismo Inverso de Drogas , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/metabolismo , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Simulación de Dinámica Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismoRESUMEN
A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships.