ABSTRACT
Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.
Subject(s)
Genetic Diseases, Inborn/genetics , Genetic Predisposition to Disease , Protein Phosphatase 2/genetics , Proteomics , TOR Serine-Threonine Kinases/genetics , Autistic Disorder/genetics , Autistic Disorder/pathology , CRISPR-Cas Systems/genetics , Genetic Diseases, Inborn/pathology , Humans , Intellectual Disability/genetics , Intellectual Disability/pathology , Megalencephaly/genetics , Megalencephaly/pathology , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Proto-Oncogene Proteins c-akt/geneticsABSTRACT
Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.
Subject(s)
Antineoplastic Agents/pharmacology , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/antagonists & inhibitors , Piperidines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Hepatocytes/drug effects , Humans , Jurkat Cells , Microsomes/drug effects , Molecular Structure , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , Piperidines/chemical synthesis , Piperidines/chemistry , Proteolysis/drug effects , Rats , Structure-Activity RelationshipABSTRACT
MAP-activated protein kinase 2 (MK2) plays an important role in the regulation of innate immune response as well as in cell survival upon DNA damage. Despite its potential for the treatment of inflammation and cancer, to date no MK2 low molecular weight inhibitors have reached the clinic, mainly due to inadequate absorption, distribution, metabolism, and excretion (ADME) properties. We describe here an approach based on specifically placed fluorine within a recently described pyrrole-based MK2 inhibitor scaffold for manipulation of its physicochemical and ADME properties. While preserving target potency, the novel fluoro-derivatives showed greatly improved permeability as well as enhanced solubility and reduced in vivo clearance leading to significantly increased oral exposure.
ABSTRACT
Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/chemistry , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Administration, Oral , Amino Acids/chemical synthesis , Amino Acids/chemistry , Amino Acids/pharmacology , Animals , Binding Sites , Crystallography, X-Ray , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Isoquinolines/chemistry , Ketones/chemical synthesis , Ketones/chemistry , Ketones/pharmacology , Lactams/chemical synthesis , Lactams/chemistry , Lactams/pharmacology , Mice , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Pyrroles/chemistry , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolismABSTRACT
New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFalpha release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFalpha release in vivo.
Subject(s)
Drug Discovery , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/chemistry , Cell Line, Tumor , Cells, Cultured , Crystallography, X-Ray , Drug Discovery/methods , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Protein Conformation , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/metabolism , Pyrazoles/pharmacologyABSTRACT
Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidinones/chemical synthesis , Pyrimidinones/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Arthritis, Rheumatoid/drug therapy , Combinatorial Chemistry Techniques , Cytokines/metabolism , Dose-Response Relationship, Drug , Drug Design , HSP27 Heat-Shock Proteins/antagonists & inhibitors , HSP27 Heat-Shock Proteins/metabolism , Humans , Leukocytes, Mononuclear/drug effects , Lipopolysaccharides/pharmacology , Molecular Structure , Monocytes/drug effects , Phosphorylation , Pyrimidinones/chemistry , Pyrroles/chemistry , Tumor Necrosis Factor-alpha/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Cinnamides as novel CCR1 antagonist chemotypes are described with high affinity to human and rodent receptors. A1B1 and A4B7 showed oral activity in the mouse collagen induced arthritis.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Experimental/drug therapy , Receptors, Chemokine/antagonists & inhibitors , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Humans , Mice , Rats , Receptors, CCR1 , Structure-Activity RelationshipABSTRACT
A class of potent, selective adenosine A(3) receptor antagonists was obtained via optimisation of the screening hit N-[4-(4-methoxyphenyl)-thiazol-2-yl]-acetamide. Structural modifications of this hit revealed very quickly that a 5-(pyridin-4-yl) substituent on the 2-aminothiazole ring was optimal for high potency at the adenosine A(3) receptor. Structure activity relationship studies led to both potent and selective A(3) receptor antagonists, including N-[5-pyridin-4-yl-4-(3,4,5-trimethoxyphenyl)-thiazol-2-yl]-acetamide, a highly potent aden-osine A(3) receptor antagonist with greater than 100- fold selectivity against the related adenosine receptors. As well as demonstrating selective in vitro binding on the human A(3) adenosine receptor, this compound was also shown to selectively block the rat A(3) receptor in vivo. This important new compound can be readily synthesised in four steps from commercially available starting materials.
Subject(s)
Acetamides/chemistry , Adenosine A3 Receptor Antagonists , Thiazoles/chemistry , Acetamides/chemical synthesis , Acetamides/pharmacology , Animals , Drug Design , Humans , Rats , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/pharmacologyABSTRACT
A series of potent p38 inhibitors based on the dihydroquinazoline scaffold was synthesized using a novel Pd-catalyzed cyclization reaction of aryl-benzyl ureas. Optimization of this compound class led to compound 20, which inhibits p38alpha in vitro with IC(50)=14 nM and is active in the mouse TNFalpha-release model.
