RESUMO
RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between ß2 and ß3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.
Assuntos
Proteína Adaptadora de Sinalização NOD2/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.
Assuntos
Compostos de Fenilureia/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Piridinas/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Compostos de Fenilureia/síntese química , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Piridinas/síntese química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/químicaRESUMO
In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.
Assuntos
Lisina , Sacaropina Desidrogenases , Erros Inatos do Metabolismo dos Aminoácidos , Animais , Encefalopatias Metabólicas , Cisteína , Etilmaleimida , Glutaril-CoA Desidrogenase/deficiência , Humanos , Lisina/metabolismo , Camundongos , Sacaropina Desidrogenases/química , Sacaropina Desidrogenases/metabolismoRESUMO
According to the World Health Organization (WHO), 422 million people are suffering from diabetes worldwide. Current diabetes therapies are focused on optimizing blood glucose control to prevent long-term diabetes complications. Unfortunately, current therapies have failed to achieve glycemic targets in the majority of people with diabetes. In this context, regeneration of functional insulin-producing human ß-cells in people with diabetes through the use of DYRK1A inhibitor drugs has recently received special attention. Several small molecule DYRK1A inhibitors have been identified that induce human ß-cell proliferation in vitro and in vivo. Furthermore, DYRK1A inhibitors have also been shown to synergize ß-cell proliferation with other classes of drugs, such as TGFß inhibitors and GLP-1 receptor agonists. In this perspective, we review the status of DYRK1A as a therapeutic target for ß-cell proliferation and provide perspectives on technical and scientific challenges for future translational development.
Assuntos
Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Células Secretoras de Insulina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Diabetes Mellitus/metabolismo , Descoberta de Drogas , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases DyrkRESUMO
Both genetic predisposition and environmental factors appear to play a role in inflammatory bowel disease (IBD) development. Genetic studies in humans have linked the interleukin (IL)-23 signaling pathway with IBD, but the environmental factors contributing to disease have remained elusive. Here, we show that the azo dyes Red 40 and Yellow 6, the most abundant food colorants in the world, can trigger an IBD-like colitis in mice conditionally expressing IL-23, or in two additional animal models in which IL-23 expression was augmented. Increased IL-23 expression led to generation of activated CD4+ T cells that expressed interferon-γ and transferred disease to mice exposed to Red 40. Colitis induction was dependent on the commensal microbiota promoting the azo reduction of Red 40 and generation of a metabolite, 1-amino-2-naphthol-6-sulfonate sodium salt. Together these findings suggest that specific food colorants represent novel risk factors for development of colitis in mice with increased IL-23 signaling.
Assuntos
Bactérias/metabolismo , Colite , Corantes de Alimentos/metabolismo , Interleucina-23/genética , Mucosa Intestinal/microbiologia , Animais , Colite/genética , Colite/metabolismo , Colite/microbiologia , Modelos Animais de Doenças , Corantes de Alimentos/efeitos adversos , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Interferon gama/genética , Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , SimbioseRESUMO
Receptor-interacting protein kinase 2 (RIPK2) is a key mediator of nucleotide-binding oligomerization domain (NOD) cell signaling that has been implicated in various chronic inflammatory conditions. A new class of RIPK2 kinase/NOD signaling inhibitors based on a 3,5-diphenyl-2-aminopyridine scaffold was developed. Several co-crystal structures of RIPK2â¢inhibitor complexes were analyzed to provide insights into inhibitor selectivity versus the structurally related activin receptor-like kinase 2 (ALK2) demonstrating that the inhibitor sits deeper in the hydrophobic binding pocket of RIPK2 perturbing the orientation of the DFG motif. In addition, the structure-activity relationship study revealed that in addition to anchoring to the hinge and DFG via the 2-aminopyridine and 3-phenylsulfonamide, respectively, appropriate occupancy of the region between the gatekeeper and the αC-helix provided by substituents in the 4- and 5-positions of the 3-phenylsulfonamide were necessary to achieve potent NOD cell signaling inhibition. For example, compound 18t (e.g. CSLP37) displayed potent biochemical RIPK2 kinase inhibition (IC50 = 16 ± 5 nM), >20-fold selectivity versus ALK2 and potent NOD cell signaling inhibition (IC50 = 26 ± 4 nM) in the HEKBlue assay. Finally, in vitro ADME and pharmacokinetic characterization of 18t further supports the prospects of the 3,5-diphenyl-2-aminopyridine scaffold for the generation of in vivo pharmacology probes of RIPK2 kinase and NOD cell signaling functions.
Assuntos
Aminopiridinas/química , Proteínas Adaptadoras de Sinalização NOD/química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Transdução de Sinais/efeitos dos fármacos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Inflamação , Relação Estrutura-AtividadeRESUMO
DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.
Assuntos
Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Tiamina Pirofosfato/química , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Estrutura Molecular , Mutação de Sentido IncorretoRESUMO
INTRODUCTION: Inosine-5'-monophosphate dehydrogenase (IMPDH) is an enzyme involved in the de novo biosynthesis of guanine nucleotides. To date human IMPDH inhibitors have been approved for prevention of organ transplant rejection and as anti-viral agents. More recently, the use of IMPDH inhibitors for other indications including cancer and pathogenic microorganisms has been pursued. Areas covered: IMPDH inhibitors disclosed primarily in the patent and scientific literature from 2002 to the present are discussed. Several interesting chemotypes that have not been pursued by patent protection are also highlighted. Expert opinion: Progress has been made in the development of IMPDH inhibitors, particularly compounds that are structurally distinct from mycophenolic acid and nucleoside-based inhibitors. However, clinical progression has been hampered primarily by a limited understanding of the enzyme's role in disease pathophysiology. Finally, most of the IMPDH inhibitors developed over the past fourteen years fall within a relatively narrow set of chemotypes. This provides opportunities for expanding IMPDH inhibitor chemical space to further evaluate this class of molecular targets.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Humanos , IMP Desidrogenase/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Patentes como AssuntoRESUMO
RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.
Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Feminino , Células HEK293 , Humanos , Imidazóis/química , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Piridazinas/química , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Especificidade por SubstratoRESUMO
RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.
Assuntos
Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Imidazóis/química , Imidazóis/farmacologia , Inflamação/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/química , Piridazinas/química , Piridazinas/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Células Sf9 , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
Cryptosporidium inosine 5'-monophosphate dehydrogenase (CpIMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]benzopyrano[4,3-c]pyrazole derivative was identified as a moderately potent (IC50 = 1.5 µM) inhibitor of CpIMPDH. We report a SAR study for this compound series resulting in 8k (IC50 = 20 ± 4 nM). In addition, an X-ray crystal structure of CpIMPDH·IMP·8k is also presented.