State-selective Modulation of Heterotrimeric Gαs Signaling with Macrocyclic Peptides

Abstract

The G protein-coupled receptor (GPCR) cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable receptors and enzymes with the exception of the stimulatory G protein α subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high affinity guanine nucleotides and the lack of other drug binding sites. We 5 explored a chemical library of 10 12 cyclic peptides in order to expand the chemical search for inhibitors of this enzyme class. We identified a macrocyclic peptide, GsIN-1, that antagonizes the GTP-bound active state of Gαs with high G protein specificity and nucleotide-binding-state selectivity. A 1.57 Å co-crystal structure reveals that GsIN-1 binds to a novel switch II / α3 pocket in Gαs and directly blocks adenylyl cyclase activation. GsIN-1 inhibits isoproterenol-stimulated 10 Gαs activation through binding to the crystallographically defined pocket. The discovery of GsIN-1 provides path for the development of state-dependent GTPase inhibitors. 15 20 One sentence summary: Targeting the previously undruggable ON-state of the GTPase Gαs with a macrocyclic peptide inhibitor.

β-arrestin-independent endosomal cAMP signaling by a polypeptide hormone GPCR

“…Real-time intracellular cAMP was measured using the Green Up cADDis cAMP biosensor (Montana Molecular), as described previously 36 . Briefly, cells were treated with cADDis BacMam and plated into 96-well plates (Corning 3340).…”

“…For the β-arr1/2 DKO531 cell line, ARRB1 and ARRB2 modifications were done simultaneously, while for the β-arr1/2 DKO836 cell line, modifications were done sequentially. cADDis microplate cAMP assay Real-time intracellular cAMP was measured using the Green Up cADDis cAMP biosensor (Montana Molecular), as described previously 36 . Briefly, cells were treated with cADDis BacMam and plated into 96-well plates (Corning 3340).…”

Strategies towards Targeting Gαi/s Proteins: Scanning of Protein‐Protein Interaction Sites To Overcome Inaccessibility

“…High‐throughput techniques, but also virtual design, have been increasingly applied to identify or design novel modulators. Structure‐activity relationships derived from crystal structures of complexes or molecular modeling and docking were frequently performed, too [188,215–217] …”

“…The peptides cycGiBP ( 10 ), [233] cycPRP‐1 ( 11 ), cycPRP‐3 ( 12 ), [234] and Gα SUPR ( 13 ) [235] are Gαi1⋅GDP selective. GsNI‐1 ( 14 ) [217] is Gαs⋅GTP selective.…”

“…Recently, in a modified mRNA display approach, the Gαs⋅GTP‐selective GsIN‐1 ( 14 , Figure 8) was identified using a Random nonstandard Peptide Integrated Discovery (RaPID) system, which also addresses Switch II/α3 and inhibits Gαs [217] …”