RESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Lactamas/farmacologia , Animais , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , L-Lactato Desidrogenase/metabolismo , Lactamas/química , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
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.
Assuntos
Lipídeos/química , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Descoberta de Drogas , Agonismo Inverso de Drogas , Naftalenossulfonatos/químicaRESUMO
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.
RESUMO
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.
Assuntos
Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Quinolinas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Óxidos S-Cíclicos/administração & dosagem , Óxidos S-Cíclicos/farmacologia , Descoberta de Drogas , Agonismo Inverso de Drogas , Leucócitos Mononucleares/efeitos dos fármacos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Sulfonamidas/química , Tiazinas/administração & dosagem , Tiazinas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Linhagem Celular , Células Cultivadas , Descoberta de Drogas , Células HEK293 , Humanos , Imidazóis/metabolismo , Imidazóis/farmacocinética , Interleucina-17/imunologia , Fígado/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Piridinas/metabolismo , Piridinas/farmacocinética , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
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.
Assuntos
Naftalenossulfonatos/síntese química , Teoria Quântica , Alquilação , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Naftalenossulfonatos/química , EstereoisomerismoRESUMO
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.
Assuntos
Interleucina-17/biossíntese , Biossíntese de Proteínas , Células Th17/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Estabilidade Enzimática , Feminino , Inflamação/genética , Inflamação/patologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transdução de Sinais , Especificidade por Substrato , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/biossíntese , Proteases Específicas de Ubiquitina/deficiência , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , L-Lactato Desidrogenase/antagonistas & inibidores , Animais , Cristalografia por Raios X , Cães , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , L-Lactato Desidrogenase/metabolismo , Células Madin Darby de Rim CaninoRESUMO
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).
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Pironas/síntese química , Pironas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Humanos , L-Lactato Desidrogenase/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Sulfonamidas/química , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Citocinas/biossíntese , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Simulação de Dinâmica Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/toxicidadeRESUMO
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%).
Assuntos
Cicloexanonas/farmacologia , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Administração Oral , Animais , Cicloexanonas/administração & dosagem , Cicloexanonas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , L-Lactato Desidrogenase/metabolismo , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Compostos de Sulfidrila/administração & dosagem , Compostos de Sulfidrila/químicaRESUMO
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.
Assuntos
Proteínas Sanguíneas/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Sulfonamidas/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células Madin Darby de Rim Canino , Modelos Moleculares , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ratos , Solubilidade , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
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.
Assuntos
Hidrocarbonetos Iodados/química , Paládio/química , Sulfonamidas/síntese química , Catálise , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sulfonamidas/químicaRESUMO
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.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Sulfonamidas/química , Sulfonamidas/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Animais , Humanos , Modelos Moleculares , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
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.
Assuntos
Desenho de Fármacos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Propanóis/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Agonismo Inverso de Drogas , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Simulação de Dinâmica Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismoRESUMO
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.