ym-254890 and FR900359

Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (G

Topics: Amino Acid Sequence; Animals; Depsipeptides; Drug Discovery; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Proteins; Humans; Models, Molecular; Peptides, Cyclic; Protein Conformation; Protein Isoforms; Sequence Alignment; Signal Transduction

G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gα

Topics: Depsipeptides; GTP-Binding Protein alpha Subunits, Gq-G11; HEK293 Cells; Humans; Kinetics; Models, Molecular; Peptides, Cyclic; Protein Binding

Specific inhibition of G proteins holds a great pharmacological promise to, e.g., target oncogenic G

Topics: Depsipeptides; GTP-Binding Proteins; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Peptides, Cyclic; Protein Conformation

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.

Topics: Animals; CRISPR-Cas Systems; Depsipeptides; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Mice; Peptides, Cyclic; Protein Engineering

G proteins are key mediators in the signaling of G protein-coupled receptors and involved in a plethora of important physiological processes. The natural product cyclic depsipeptides YM-254890 and FR900359 are the only known selective inhibitors of the G

Topics: Amino Acid Sequence; Animals; Binding Sites; CHO Cells; Cricetulus; Crystallography, X-Ray; Depsipeptides; GTP-Binding Protein alpha Subunits, Gq-G11; Models, Molecular; Peptides, Cyclic; Protein Conformation; Structure-Activity Relationship

G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the G

Topics: Animals; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Depsipeptides; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gq-G11; HEK293 Cells; Humans; Mutagenesis; Peptides, Cyclic; Protein Structure, Tertiary; Receptors, G-Protein-Coupled; Signal Transduction; Structure-Activity Relationship