ABSTRACT
Signaling pathways are frequently activated through signal-receiving membrane proteins, and the discovery of small molecules targeting these receptors may yield insights into their biology. However, due to their intrinsic properties, membrane protein targets often cannot be identified by means of established approaches, in particular affinity-based proteomics, calling for the exploration of new methods. Here, we report the identification of indophagolin as representative member of an indoline-based class of autophagy inhibitors through a target-agnostic phenotypic assay. Thermal proteome profiling and subsequent biochemical validation identified the purinergic receptor P2X4 as a target of indophagolin, and subsequent investigations suggest that indophagolin targets further purinergic receptors. These results demonstrate that thermal proteome profiling may enable the de novo identification of membrane-bound receptors as cellular targets of bioactive small molecules.
Subject(s)
Autophagy/drug effects , Proteome/genetics , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X4/metabolism , Temperature , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/pathology , Dose-Response Relationship, Drug , Female , Gene Expression Profiling , Humans , Male , Molecular Structure , Purinergic P2X Receptor Antagonists/chemistry , Receptors, Purinergic P2X4/genetics , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP-like chemical space and biological target space. These limitations can be overcome by combining NP-centered strategies with fragment-based compound design through combination of NP-derived fragments to afford structurally unprecedented "pseudo-natural products" (pseudo-NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo-NPs that combine biosynthetically unrelated indole- and morphan-alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT-1 and GLUT-3. Glupin suppresses glycolysis, reduces the levels of glucose-derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT-1 and GLUT-3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.
Subject(s)
Biological Products/pharmacology , Cell Proliferation , Glucose Transporter Type 1/antagonists & inhibitors , Glucose Transporter Type 3/antagonists & inhibitors , Glucose/metabolism , Morphinans/chemical synthesis , Neoplasms/drug therapy , Biological Transport , Cell Cycle , Glycolysis , Humans , Tumor Cells, CulturedABSTRACT
Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.
Subject(s)
Autophagy/drug effects , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Autophagosomes/drug effects , Drug Discovery , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Pyrimidines/chemistry , Sirolimus/pharmacology , Structure-Activity RelationshipABSTRACT
A series of cyclopenta[c]pyridine aldosterone synthase (AS) inhibitors were conveniently accessed using batch or continuous flow Kondrat'eva reactions. Preparation of the analogous cyclohexa[c]pyridines led to the identification of a potent and more selective AS inhibitor. The structure-activity-relationship (SAR) in this new series was rationalized using binding mode models in the crystal structure of AS.
Subject(s)
Cytochrome P-450 CYP11B2/antagonists & inhibitors , Cytochrome P-450 Enzyme Inhibitors/chemical synthesis , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Chemistry Techniques, Synthetic , Cytochrome P-450 CYP11B2/chemistry , Cytochrome P-450 Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Protein Conformation , Pyridines/chemistry , Structure-Activity RelationshipABSTRACT
A Rebek imide receptor with an acetylene-linked phenyl ring complexes 2,6-di(isobutyramido)pyridine in (CDCl2 )2 via triple H-bonding and π-π-stacking interactions, and the influence of para-substituents on both rings was investigated by (1) Hâ
NMR binding titrations. When the phenyl ring was extended to biphenyl and the C(4)-pyridine substituent varied, interaction energies increased in the order CH3 CH2 â
â
â
phenyl
