ABSTRACT
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.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , RNA-Binding Proteins/antagonists & inhibitors , Animals , Biological Availability , Cell Line , Chronic Disease , Drug Design , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Enzyme Inhibitors/pharmacokinetics , Haplorhini , High-Throughput Screening Assays , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Multiple Sclerosis/drug therapy , Pyrazoles/pharmacokinetics , Rats , Retinitis Pigmentosa/drug therapy , Structure-Activity RelationshipABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.
Subject(s)
Carcinoma, Pancreatic Ductal/immunology , Immune Tolerance/immunology , Macrophages/immunology , Pancreatic Neoplasms/immunology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Humans , Immune Tolerance/genetics , L Cells , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , STAT1 Transcription Factor/metabolism , Th1 Cells/cytology , Th17 Cells/cytologyABSTRACT
C-methylation of aromatic small molecules by C-methyltransferases (C-MTs) is an important biological transformation that involves C-C bond formation using S-adenosyl-l-methionine (SAM) as the methyl donor. Here, two advances in the mechanistic understanding of C-methylation of the 8-position of coumarin substrates catalyzed by the C-MT NovO from Streptomyces spheroides are described. First, a crystal structure of NovO reveals the Arg116-Asn117 and His120-Arg121 motifs are essential for coumarin substrate binding. Second, the active-site His120 is responsible for deprotonation of the phenolic 7-hydroxyl group on the coumarin substrate, activating the rate-determining methyl transfer step from SAM. This work expands our mechanistic knowledge of C-MTs, which could be used in the downstream development of engineered biocatalysts for small molecule C-alkylations.
Subject(s)
Coumarins/metabolism , Catalysis , Crystallography, X-Ray , Methylation , Methyltransferases/metabolism , Molecular Structure , Novobiocin/biosynthesis , Novobiocin/chemistry , Streptomyces/metabolism , Substrate SpecificityABSTRACT
A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.
