ABSTRACT
Chimeric antigen receptor (CAR)-modified T cells are endowed with novel antigen specificity and are most often administered to patients without an engineered mechanism to control the CAR T cells once infused. "Suicide switches" such as the small molecule-controlled, inducible caspase-9 (iCas9) system afford the ability to selectively eliminate engineered T cells; however, these approaches are designed for all-or-none, irreversible termination of an ongoing immune response. In order to permit reversible and adjustable modulation, we have created a CAR that is capable of on-demand downregulation by fusing the CAR to a previously developed ligand-induced degradation (LID) domain. Addition of a small molecule ligand triggers exposure of a cryptic degron within the LID domain, resulting in proteasomal degradation of the CAR-LID fusion protein and loss of CAR on the surface of T cells. This fusion construct allowed for reversible and "tunable" inhibition of CAR T cell activity in vitro. Delivery of the triggering molecule in CAR-LID-treated tumor-bearing mice temporarily reduced CAR activity through modulation of CAR surface expression. The ability to more flexibly modulate CAR T cell expression through a small molecule provides a platform for controlling possible adverse side effects, as well as preclinical investigations of CAR T cell biology.
Subject(s)
Morpholines/chemistry , Neoplasms/therapy , Receptors, Chimeric Antigen/metabolism , Recombinant Fusion Proteins/chemistry , Small Molecule Libraries/administration & dosage , T-Lymphocytes/transplantation , Animals , Cell Line, Tumor , Cell Proliferation , Female , Humans , Immunotherapy, Adoptive , Ligands , Mice , Neoplasm Transplantation , Neoplasms/immunology , Proteasome Endopeptidase Complex/metabolism , Protein Domains , Proteolysis , Receptors, Chimeric Antigen/chemistry , Recombinant Fusion Proteins/metabolism , Small Molecule Libraries/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of ERK1/2, converting it into an active kinase that regulates the expression of numerous genes and cellular processes. Inhibitors of MEK1/2 have demonstrated preclinical and clinical efficacy in certain cancers and types of cardiomyopathy. We report the synthesis of a novel, allosteric, macrocyclic MEK1/2 inhibitor that potently inhibits ERK1/2 activity in cultured cells and tissues of mice after systemic administration. Mice with dilated cardiomyopathy caused by a lamin A/C gene mutation have abnormally increased cardiac ERK1/2 activity. In these mice, this novel MEK1/2 inhibitor is well tolerated, improves left ventricular systolic function, decreases left ventricular fibrosis, has beneficial effects on skeletal muscle structure and pathology and prolongs survival. The novel MEK1/2 inhibitor described herein may therefore find clinical utility in the treatment of this rare cardiomyopathy, other types of cardiomyopathy and cancers in humans.
Subject(s)
Cardiomyopathy, Dilated/drug therapy , Disease Models, Animal , Lamin Type A/genetics , Macrocyclic Compounds/pharmacology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Cardiomyopathy, Dilated/genetics , Dose-Response Relationship, Drug , Macrocyclic Compounds/administration & dosage , Macrocyclic Compounds/chemistry , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinase 1/metabolism , Molecular Structure , Mutation , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The peptide hormone ghrelin is the endogenous ligand for the type 1a growth hormone secretagogue receptor (GHS-R1a) and the only currently known circulating appetite stimulant. GHS-R1a antagonism has therefore been proposed as a potential approach for obesity treatment. More recently, ghrelin has been recognized to also play a role in controlling glucose-induced insulin secretion, which suggests another possible benefit for a GHS-R1a antagonist, namely, the role as an insulin secretagogue with potential value for diabetes treatment. In our laboratories, piperidine-substituted quinazolinone derivatives were identified as a new class of small-molecule GHS-R1a antagonists. Starting from an agonist with poor oral bioavailability, optimization led to potent, selective, and orally bioavailable antagonists. In vivo efficacy evaluation of selected compounds revealed suppression of food intake and body weight reduction as well as glucose-lowering effects mediated by glucose-dependent insulin secretion.
Subject(s)
Diabetes Mellitus/drug therapy , Obesity/drug therapy , Quinazolinones/chemical synthesis , Receptors, Ghrelin/antagonists & inhibitors , Administration, Oral , Animals , Binding, Competitive , Blood Glucose/analysis , Cell Line , Eating/drug effects , Glucose Tolerance Test , Humans , Male , Mice , Mice, Inbred C57BL , Quinazolinones/chemistry , Quinazolinones/pharmacology , Radioligand Assay , Rats , Rats, Wistar , Stereoisomerism , Structure-Activity Relationship , Weight Loss/drug effectsABSTRACT
Bis-aryl ureas have been disclosed previously as a potent class of Raf kinase inhibitors. Modifications in the amide portion led to an improvement in aqueous solubility, an important characteristic for an oral drug. Based on this finding, we hypothesize that this portion of the molecule is directed towards the solvent in Raf-1.
