RESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].
RESUMO
Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.
RESUMO
Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Hepcidinas/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Inibidores de Serina Proteinase/química , Benzotiazóis/química , Sítios de Ligação , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Proteínas Ligadas por GPI/metabolismo , Proteína da Hemocromatose/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Peptidomiméticos , Proteólise/efeitos dos fármacos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and â¼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.
Assuntos
Antimaláricos/farmacologia , Transporte Biológico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Plasmodium falciparum/efeitos dos fármacos , Purinas/metabolismo , Antimaláricos/química , Eritrócitos/efeitos dos fármacos , Técnicas de Inativação de Genes , Células Hep G2/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Malária/parasitologia , Malária Falciparum/parasitologia , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/efeitos dos fármacos , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Transcriptoma , Leveduras/efeitos dos fármacosRESUMO
RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.
Assuntos
Benzotiazóis/farmacologia , Fosfatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Animais , Benzotiazóis/química , Benzotiazóis/farmacocinética , Benzotiazóis/uso terapêutico , Colite/tratamento farmacológico , Cães , Descoberta de Drogas , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Fosfatos/química , Fosfatos/farmacocinética , Fosfatos/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/uso terapêutico , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Suínos , Porco MiniaturaRESUMO
RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.
RESUMO
RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Disponibilidade Biológica , Linhagem Celular , Doença Crônica , Desenho de Fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Esclerose Múltipla/tratamento farmacológico , Pirazóis/farmacocinética , Ratos , Retinose Pigmentar/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 µM).
RESUMO
RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Colite Ulcerativa/tratamento farmacológico , Inflamação/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Animais , Benzazepinas/química , Benzazepinas/farmacologia , Colite Ulcerativa/imunologia , Citocinas/imunologia , Cães , Haplorrinos , Humanos , Inflamação/imunologia , Camundongos , Simulação de Acoplamento Molecular , Coelhos , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Suínos , Porco Miniatura , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic ß-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Regiões Promotoras Genéticas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , gama-Globinas/genética , Animais , Linhagem Celular , Vaga-Lumes/genética , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/genética , Renilla/genética , Transfecção/métodosRESUMO
RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.
Assuntos
Aminoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Sulfonas/farmacologia , Aminoquinolinas/sangue , Aminoquinolinas/química , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Relação Estrutura-Atividade , Sulfonas/sangue , Sulfonas/químicaRESUMO
The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.
Assuntos
DNA/química , Isoxazóis/farmacologia , Oxazepinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Oxazepinas/síntese química , Oxazepinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Células U937RESUMO
Receptor interacting protein 2 (RIP2) is an intracellular kinase and key signaling partner for the pattern recognition receptors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2). As such, RIP2 represents an attractive target to probe the role of these pathways in disease. In an effort to design potent and selective inhibitors of RIP2 we established a crystallographic system and determined the structure of the RIP2 kinase domain in an apo form and also in complex with multiple inhibitors including AMP-PCP (ß,γ-Methyleneadenosine 5'-triphosphate, a non-hydrolysable adenosine triphosphate mimic) and structurally diverse ATP competitive chemotypes identified via a high-throughput screening campaign. These structures represent the first set of diverse RIP2-inhibitor co-crystal structures and demonstrate that the protein possesses the ability to adopt multiple DFG-in as well as DFG-out and C-helix out conformations. These structures reveal key protein-inhibitor structural insights and serve as the foundation for establishing a robust structure-based drug design effort to identify both potent and highly selective inhibitors of RIP2 kinase.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Inibidores de Proteínas Quinases/química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Cinética , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismoRESUMO
Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.
Assuntos
Apoptose , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Técnicas de Introdução de Genes , Células HT29 , Humanos , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Necrose/enzimologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidoresRESUMO
Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.