A Novel Gαq/11-selective Inhibitor

Abstract

YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks G␣ q/11 -coupled ADP receptor P2Y1-mediated Ca 2؉ mobilization. Here we report that YM-254890 is a selective G␣ q/11 inhibitor. YM-254890 blocked Ca 2؉ mobilization mediated by several G␣ q/11 -coupled receptors but not by G␣ i -or G␣ 15 -coupled receptor, indicating that phospholipase C␤ activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by G␣ q R183C, which is constitutively active in a receptor-dependent manner because of its reduced k cat of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by G␣ q Q209L, which is GTPase-deficient (activated) G␣ q . These suggested that the acting point of YM-254890 is receptor-G␣ q interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by G␣ qi5 , which interacts with G␣ i -coupled receptor and possesses the effector function of G␣ q , and (ii) had no effect on the K d value of high affinity [ These data indicate that YM-254890 blocks the exchange of GDP for GTP in G␣ q/11 activation. This novel G␣ q/11 -selective inhibitor is a promising and powerful tool for studying G␣ q/11 protein activation, G␣ q/11 -coupled receptor signaling, and G␣ q/11 -mediated biological events. G protein-coupled receptors (GPCR)1 and heterotrimeric G proteins, consisting of G␣, ␤, and ␥, transduce extracellular stimuli, such as hormones, neurotransmitters, chemokines, and other local mediators, into appropriate intracellular responses (1, 2). The activation of G␣ proteins is related to conformational change by guanine nucleotide interaction. The GPCRs, activated by the agonist, induced exchanges of GDP for GTP on the coupled G␣ subunit. The resultant G␣-GTP complex dissociates from the G␤␥ subunit and activates its downstream effectors, which in turn regulate various functions such as gene transcription, mitogenesis, metabolism, muscle contractile state, and ion channel regulation. The GTPase activity of ␣-subunit turns off effector signals by hydrolyzing G␣-GTP to G␣-GDP, which re-associates with G␤␥.There are over 20 G␣ subunits classified into subfamilies by its sequence homology and the downstream signal. These include the major four families, G␣ q/11 , G␣ s , G␣ i/o , and G␣ 12/13 . Main effector molecules of G␣ q/11 , G␣ s , G␣ i/o , and G␣ 12/13 are thought to be phospholipase C (PLC), adenylyl cyclase (activation), adenylyl cyclase (inhibition), and small GTPase families, respectively (3). Transient intracellular Ca 2ϩ mobilization is led by PLC␤ activation via the G␣ q/11 , G␣ 15/16 , or G␤␥ subunit with G␣ i . PLC␤ hydrolyzes phosphatidylinositol bisphosphate in the plasma membrane, and the generated inositol 1,4,5-trisphosphate (IP 3 ) activates ...

Structural basis for the specific inhibition of heterotrimeric G q protein by a small molecule

“…S2, we used ectopically expressed Gα proteins in lysates from mammalian cells, because both Gα 14 and Gα 16 proteins were difficult to purify. The results of a previous study (5) and those of our studies indicate that YM-254890 selectively inhibits Gα q , Gα 11 , and Gα 14 among mammalian Gα members.…”

“…Previously, it was reported that YM-254890 blocks agonist-induced GTPγS binding to G q∕11 in crude cell membranes (5). This finding suggests the possibility that YM-254890 directly binds to Gα q and inhibits the GDP/GTP exchange.…”

“…QS3666 as an inhibitor of ADPinduced platelet aggregation (4). YM-254890 strongly inhibits intracellular calcium ion mobilization and serum response element (SRE)-mediated transcription stimulated by several receptors coupled to G q , but not those coupled to G i , G s , or G 15 (5). YM-254890 also exhibited antithrombotic and thrombolytic effects in an electrically induced carotid artery thrombosis model in rats (6).…”

Leukotriene B4 Augments and Restores FcγRs-dependent Phagocytosis in Macrophages

“…Where indicated, LTB 4 (final concentration of 100 nM) (Cayman Chemicals, Ann Arbor, MI) was added along with opsonized zymosan particles. For the inhibition assay, BMM were pretreated with the following reagents and times prior to incubation with zymosan particles: CP105696 (1 M, 30 min), PP2 (5 M, 30 min) (Calbiochem), Piceatannol (20 M, 30 min) (Calbiochem), LY294002 (50 M, 16 h) (Calbiochem), Rac1 inhibitor (NSC23766; 200 M, 16 h) (Calbiochem), pertussis toxin (PTX; 100 ng/ml, 16 h) (List Biological Laboratories, Campbell, CA), and YM-254890 (10 M, 30 min), a kind gift from Dr. J. Takasaki, Astellas Pharmaceutical (22).…”

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

“…For example, BIM-46174 and BIM-46187 apparently interact with most G␣ subunits (11,12), and some selectivity for G␣ q has been reported for BIM-46187 (13). In contrast, YM-254890 (14), and related natural products such as FR900359 (15,16), specifically inhibit G␣ subunits of the G q family (17,18), and a crystal structure of a G protein heterotrimer bound to YM-254890 illustrates that this molecule prevents GDP release and heterotrimer dissociation (19). Unfortunately, synthesis of cyclic depsipeptides is difficult, and YM-254890 remains unavailable from commercial sources.…”