Discovery of small molecule Gαq/11 protein inhibitors against uveal melanoma

Constitutively activated G proteins caused by specific mutations mediate the development of multiple malignancies. The mutated Gαq/11 are perceived as oncogenic drivers in the vast majority of uveal melanoma (UM) cases, making directly targeting Gαq/11 to be a promising strategy for combating UM. Herein, we report the optimization of imidazopiperazine derivatives as Gαq/11 inhibitors, and identified GQ262 with improved Gαq/11 inhibitory activity and drug-like properties. GQ262 efficiently blocked UM cell proliferation and migration in vitro. Analysis of the apoptosis-related proteins, extracellular signal-regulated kinase (ERK), and yes-associated protein (YAP) demonstrated that GQ262 distinctly induced UM cells apoptosis and disrupted the downstream effectors by targeting Gαq/11 directly. Significantly, GQ262 showed outstanding antitumor efficacy in vivo with good safety at the testing dose. Collectively, our findings along with the favorable pharmacokinetics of GQ262 revealed that directly targeting Gαq/11 may be an efficient strategy against uveal melanoma.


Introduction
Heterotrimeric G proteins are the families of guanine nucleotidebinding proteins. The involvement of G proteins in transmembrane signal transduction was first discovered approximately 40 years ago 1 . Heterotrimeric G proteins consist of 3 non-identical subunits (a, b, and g) in the order of diminishing mass 1,2 . In mammals, 21 different Ga subunits encoded by 16 genes, 6 different Gb subunits encoded by 5 genes, and 12 different Gg subunits encoded by 12 genes have been identified 3e5 . G proteins are characteristically categorized into Gas, Gai, Gaq/11, and Ga12/13 with regard to the sequence similarity of Ga subunits 6 . The downstream effectors coupled to individual Ga subunits are also distinct: (i) Gas stimulates adenylyl cyclase (AC), which synthesizes the cAMP from ATP 7 ; (ii) Gai suppresses AC, which controls the intracellular cAMP levels 8 ; (iii) Gaq/11 activate phospholipase Cb (PLCb), which generates diacylglycerol (DAG) and inositol triphosphate (IP 3 ) 9e11 ; and (iv) Ga12/13, which are identified to directly interact with guanine nucleotide exchange factors (GEFs) for the GTPase Rho (RhoGEF) and induce the activation of Rho 2,5,12e14 .
G proteins, which in general are perceived as molecular switches, mediate signal transduction connecting the heptahelical G protein-coupled receptors (GPCRs) with various intracellular effectors 2,15 . Different from the transmembrane GPCRs, G proteins are situated in cells. The processes of signal transduction are initiated by extracellular stimuli binding to GPCRs, which induce the conformational change of GPCRs and activate G proteins 4 . Consequently, the G proteins cycle is triggered to further transmit the signals 2,16 . The signaling cycle could be interrupted by the mutation or covalent modification of Ga subunits 17 . Under certain circumstances, the intrinsic GTPase activity of Ga is blocked, which retains the Ga in a "switch-on" state and persistently activates the complicated downstream signaling networks 18,19 .
Of note, G proteins are served as the crucial nodes on the canonical GPCRs/G proteins/effectors axis, and involved in extensive human pathological and physiological processes 20e23 . In 2010, mutated Gaq (encoded by GNAQ) and Ga11 (encoded by GNA11) were unveiled to be associated with uveal melanoma (UM) 20,24e26 . The mutated G proteins impair the GTP hydrolysis and prolong the signaling activity 27e33 . The hotspots of Gaq/11 mutations flock around two residues, Q209 and R183. Hence, Gaq/11 harboring Q209 or R183 mutations are recognized as oncogenic drivers in the vast majority of UM cases 27 . Over the past decade, enormous efforts for UM treatment were devoted to regulate the complex downstream signaling of Gaq/11, including two key nodes MAPK and ERK 34e36 . Unfortunately, targeting either of the nodes did not exhibit satisfied therapeutic outcome in UM clinical trials 37e40 . A presumable explanation is that Gaq/11 could activate multiple and independent downstream signaling pathways, targeting a specific node could not achieve the optimal therapeutic results 41 . Thus, directly targeting the constitutively activated Gaq/11 maybe represent one potential application to overcome UM.
Up to now, very few reported Gaq/11 inhibitors are shown in Fig. 1, including YM-254890 (1) 42 , FR900359 (2) 43 , BIM-46187 (3) 44 , and GQ127 (4) 45 . The cyclic depsipeptide YM-254890 (1) powerfully inhibits Gaq/11 with the IC 50 value of 95 nmol/L on CHO cells that stably express the M 1 muscarinic receptor 42 . Analysis of the Gaqbg/YM-254890 crystal structure demonstrated that 1 blocks the GDP/GTP exchange through suppressing the GDP release 46 . FR900359 (2), which possesses subtle structural differences with YM-254890, exhibits inhibitory potency to Gaq/11 in low nanomolar range (IC 50 Z 32 nmol/L) and shares the same mode of action (MOA) with 1 42,47 . Although 1 and 2 showed exciting bioactivity, the development of these two compounds is immensely restricted due to the low abundance in nature and synthetic complexity. BIM-46187 (3) was reported as a small molecular Gaq/11 inhibitor in 2014 48 . 3 permits the GDP exit but prevents GTP entry, which is the possible mechanism of how 3 inhibits Gaq/11 48 . However, the large molecular weight and high toxicity of 3 complicate its development. Recently, our group performed structural optimization towards 3 with the purpose of improving the Gaq/11 inhibitory potency, simplifying the structure, and reducing the toxicity. The small molecule GQ127 (4) was obtained as an effective Gaq/11 inhibitor in vitro and in vivo without obvious toxicity, in comparison with 3 45 . Nevertheless, the electron-rich primary amine within 4 might be the issue for metabolic and oxidative stability, the relatively short elimination half-life (t 1/2 Z 1.2 h) and unsatisfactory Gaq/11 inhibitory potency encouraged us to pursue new small molecules with improved inhibitory potency and drug-like properties 45 . In this study, we performed structural optimization on compound 4 and successfully obtained GQ262 which showed improved Gaq/11 inhibitory potency. Moreover, the antitumor efficacy and mechanism of GQ262 were explored. The obtained data served as solid evidences for the good antitumor activity of GQ262 both in vitro and in vivo.

Synthetic design
In our previous study, we developed compound 4 as a new Gaq/11 inhibitor by the structural optimization of 3 45 . However, the Gaq/ 11 inhibitory activity, antitumor efficacy, and druggability of 4 are not satisfied. To further develop novel and potent Gaq/11 inhibitors with good druggability, our optimization began with modifying the phenyl group of 4 ( Fig. 2). Based on the application of bioisosterism in drug discovery, we utilized different aromatic heterocycles to replace the phenyl group. We also substituted the aromatic heterocycles with alkyls, cycloalkyls, and heterocycloalkyls to investigate the necessity of the rigid structure. When analyzing the structural characteristics of 4, we speculated that whether the flexibility of the amino acid fragment is related to the Gaq/11 inhibitory potency. Thus, the carbonyl group was reduced to increase the flexibility to explore the relationships between the flexibility and efficacy. In addition, the cyclohexylmethyl group was replaced by other alkyl groups to further investigate the structureeactivity relationships (SARs), by combining with the results of augmenting efficacy in our studies 45 . Ultimately, GQ220-GQ267 were synthesized for further evaluation of inhibitory effects on Gaq/11 and UM cells.

Chemistry
GQ220-GQ235 were prepared according to the synthetic route depicted in Scheme 1. The synthesis of imidazopyrazine analogs began with the commercially available N-tert-butoxycarbonyl-Lcyclohexylalanine methyl ester 5 which was treated with sodium borohydride in tetrahydrofuran (THF)/methanol, followed by treating with Dess-Martin periodinane to give intermediate 6 in 75% yield. 7 was obtained from 6 by DebuseRadziszewski reaction. Treating 7 with iodine and potassium hydroxide in N,Ndimethylformamide (DMF) gave 8. Then, nucleophilic substitution between 8 and ethyl bromoacetate was performed to deliver 9. Deprotection and intramolecular lactamization of 9 gave 10, followed by reducing the amide group by borane to give building block 11. Removing the iodine by treating 11 with sodium borohydride in methanol/THF resulted in 12. The key building blocks 13a-13p were prepared from 12 by Suzuki reaction. Finally, compounds GQ220-GQ235 were furnished by the condensation of N-tert-butoxycarbonyl-L-isoleucine with 13a-13p and removing the protecting groups.

Structureeactivity relationship studies
Inositol monophosphate (IP 1 ) accumulation assay was utilized to characterize Gaq/11 inhibitory activity on CHO-M 1 cells, which applied homogeneous time-resolved fluorescence (HTRF). In vitro antiproliferative potency was evaluated on UM cell lines MP41 with GNA11 Q209L mutation and 92.1 with GNAQ Q209L mutation by CCK-8 assay. Tables 1 and 2 summarized the results.
To augment the efficacy of compounds on Gaq/11 inhibition and tumor suppression, GQ220-GQ226 with aromatic heterocycles replacing the phenyl group of 4 were designed and synthesized using the concept of bioisosterism, as the SAR of the phenyl ring has never been carefully explored. As shown in Table 1, GQ220, GQ221 and GQ223 showed reduced activity on inhibiting Gaq/11 and UM cell lines. GQ222 and GQ224-GQ226 showed improved Gaq/11 inhibitory potency, but reduced activity on suppressing MP41 and 92.1 cell lines. Alkyls, cycloalkyls and heterocycloalkyls were also introduced with the purpose of providing flexibility for the molecules, and compounds GQ227-GQ235 were prepared and characterized (Table 1). However, most of the alkyl substituted compounds either suffered from losing Gaq/11 inhibitory potency, or showed weak antiproliferative ability towards MP41 and 92.1 cell lines, suggesting that the aromatic substitution is essential for maintaining the activity.
The amino acid fragment in 4 was proven to be essential for keeping the Gaq/11 inhibitory activity and reducing the cellular toxicity, we speculated whether increasing the molecular flexibility by reducing the amide group to tertiary amine could contribute to the inhibitory potency. Interestingly, reducing the amide group in 4 led to analog GQ236, which exhibited remarkable improvement both in inhibiting Gaq/11 and suppressing the proliferation of MP41 and 92.1 cells. The exciting results encouraged us to further explore the side chain of the amino acid part. Considering the natural and some unnatural amino acids with side chain diversity are abundant and easily available building blocks, we chose four natural and five unnatural amino acids with different alkyl branched chains and prepared GQ237-GQ245. As shown in Table 2, most compounds displayed improved Gaq/11 inhibitory activity at the concentration of 10 mmol/L in comparison to 3, except for GQ240 and GQ241. It is worth noting that the antiproliferative activity of these compounds on UM cells were also enhanced or maintained. Encouraged by these results, other natural amino acids with different side chains were employed to obtain GQ246-GQ259. Of note, all these compounds exhibited favorable Gaq/11 inhibitory activity (Table 2). Particularly, GQ248 and GQ251 showed better Gaq/11 inhibitory activity than 3 at 10 mmol/L. Similarly, most of the compounds potently inhibited the growth of MP41 or 92.1 cells at low micromolar concentrations in vitro, except for GQ248 and GQ251 showed uncompetitive efficacy in combating UM cell lines in comparison

Figure 2
Design and structural optimization strategies for GQ220-GQ267. The phenyl group (green) is modified to discuss the necessity of the rigid structure. Reduction of the carbonyl (red) and cyclization of the head fragment (blue) are to improve the potency and drug-like property. Replacing the cyclohexylmethyl fragment (pink) is to investigate the significance for inhibitory activity. with 3. The results unveiled that releasing the restricted conformation of the carbonyl group is beneficial for increasing the Gaq/ 11 and tumor inhibitory efficacy.
Of interest, proline which possesses a specific pyrrolidine segment among the natural amino acids was introduced into GQ260. As depicted in Table 2, GQ260 inhibited Gaq/11 with 53.4 AE 7.0% inhibition at 10 mmol/L, and suppressed MP41 and 92.1 cells proliferation with the IC 50 values of 6.7 AE 0.6 mmol/L and 11.6 AE 1.2 mmol/L. (S)-Piperidine-2-carboxylic acid containing the piperidine fragment, which is perceived as a bioisostere of pyrrolidine, was also introduced to prepare GQ261. Compared to GQ260, the antiproliferative activity of GQ261 was retained, despite the decreased Gaq/11 inhibitory efficacy ( Table  2). The results indicated that introducing the cyclic secondary amine motif into GQ260 and GQ261 could either improve the Gaq/11 inhibitory potency or enhance the antiproliferative activity of UM cells. Encouraged by these findings, and considering that the tertiary amine is potentially more metabolic and redox stable compared with the secondary amine, we thus further optimized the compounds by introducing a methyl group into the cyclic secondary amine, leading to analogs GQ262 and GQ263. To our delight, GQ262 potently inhibited Gaq/11 with 57.2 AE 1.9% suppression at 10 mmol/L, and disrupted MP41 cell proliferation with the IC 50 of 5.1 AE 0.6 mmol/L ( Table 2), suggesting that the cyclic tertiary amine motif is superior to secondary or primary amine.
Inspired by the above-mentioned SARs, we combined the prevogative R substituent phenyl group and R 1 substituent 1methylpyrrolidine with four different R 2 substituents (Ref. 45) which were beneficial for Gaq/11 inhibitory potency (Table 2). GQ264-GQ267 were designed and synthesized, but showed decreased inhibitory activity towards Gaq/11 and poor antitumor efficacy, compared with GQ262 (Table 2). These results indicated that the proper combination of the cyclohexylmethyl moiety with R substituent phenyl group and R 1 substituent 1-methylpyrrolidine is essential for maintaining or improving the activity of compounds for combating Gaq/11 and UM cells.
Hence, we summarized the SAR information of the synthesized imidazopyrazine derivatives in Fig. 3. The phenyl substituted R is important for maintaining the inhibitory activity towards Gaq/11 and UM cells. Increasing the molecular flexibility by reducing the amide to amine generally improves the Gaq/11 inhibitory potency and the anti-UM cell ability. Introducing the cyclic tertiary motif to R 1 not only acquires better Gaq/11 inhibitory efficacy, but also provides the possibility for modulating the metabolic stability. Besides, the proper combination of the R 1 and R 2 is essential for maintaining the Gaq/11 inhibitory efficacy and anti-UM cell ability.  3 SiH, rt, Ar.

Evaluation of the cytotoxicity of GQ262
The previous study revealed that part of the Gaq/11 inhibitory potency of 3 is derived from its cytotoxicity, we thus investigated the cytotoxicity of the newly designed compound GQ262 by treating the CHO-M 1 cells at different concentrations, and characterized the cell viability using CCK-8 assay. As shown in Fig. 4, 3 obviously inhibited the cell proliferation at the concentrations of 10 and 20 mmol/L in 2 h, but GQ262 did not impact the cell viability at indicated concentrations in 2 h, even when the concentration up to 20 mmol/L for 24 h (Fig. 4), indicating that GQ262 has satisfactory safety towards normal cells.

Effects of GQ262 on agonist-induced BRET signals and Ca 2þ release
To verify that GQ262 could trap Gaq/11 directly, we transfected AT 1 R, Gaq-RLuc8, unlabeled Gb1, and Gg2-Venus in HEK293 cells which were applied to determine the bioluminescence resonance energy transfer (BRET) alteration and Ca 2þ release. Pretreating HEK293-AT 1 R cells with 3 and GQ262 at different concentrations, followed by measuring the variable BRET signals between Ga and Gbg. As depicted in Fig. 5A-C, GQ262 displayed dose-dependently inhibitory efficacy on BRET signals. Also, the agonist-mediated Ca 2þ release was employed to evaluate the effects of GQ262 on blocking Gaq/11. When AngII binds to AT 1 R, the activated AT 1 R initiates Gaq/11, which intertwines and activates PLCb. As a consequence, Ca 2þ release is accelerated. We pretreated 3 and GQ262 with HEK293-AT 1 R cells at 100 mmol/L, and assessed the agonist-stimulated Ca 2þ release at different concentrations of AngII. As expected, GQ262 obviously suppressed the Ca 2þ release (Fig. 5D). All these results indicated that GQ262 directly acts on Gaq/11.

GQ262 inhibits the proliferation of UM cells by targeting Gaq/11
To further validate that GQ262 suppresses UM cell proliferation through directly targeting Gaq/11, we transfected the siRNA targeting GNA11 in MP41 cells and GNAQ in 92.1 cells to knock down the GNAQ/11 (Supporting Information Fig. S1). Then, we pre-treated the cells with GQ262. As depicted in Fig. 6A, GQ262 exhibited remarkable antiproliferative activity on MP41 (si#control) at 5 and 10 mmol/L, respectively. However, GQ262 could not inhibit MP41 cell proliferation (si#GNA11). Similarly, GQ262 had no inhibitory effect on proliferation of GNAQ knockdown 92.1 cells at 10 and 20 mmol/L (Fig. 6B), suggesting the antiproliferative potency of GQ262 on UM cells is achieved by blocking Gaq/11. In addition, the cellular thermal shift assay (CETSA) was employed to study whether GQ262 binds to Gaq/11 straightforwardly. As expected, the thermal denaturation temperature of Gaq/11 increased from 37 to 50 C after the incubation of the cell lysates with GQ262 ( Fig. 6C and D, Supporting Information Fig. S2), and the melting temperature was increased by 10 C, indicating that GQ262 indeed binds to Gaq/11. Taken together, these data demonstrated that GQ262 directly binds to Gaq/11.

GQ262 induces UM cell cycle arrest and apoptosis
To investigate the mechanism of GQ262 inhibiting UM cell proliferation, we performed the cell cycle analysis utilizing flow cytometry. As depicted in Fig. 7A and B, the cellular proportion elevated from 73.8% to 78.0% and 79.4% in G0/G1 phase after separately incubating MP41 cells with GQ262 at 3 and 10 mmol/L, and the cellular proportion in S and G2/M phases was clearly decreased. In agreement with MP41 cells, 92.1 cells were incubated with GQ262 at 10 and 20 mmol/L, the cellular proportion elevated from 70.1% to 77.1% and 83.5% in G0/G1 phase (Supporting Information Fig. S3). These results demonstrated that GQ262 significantly disrupts the cell cycle of MP41 and 92.1 cells at G0/G1 phase.
To obtain further insight into the antitumor mechanism of GQ262, we conducted an apoptosis assay utilizing flow cytometer. As depicted in Fig. 7C and D, GQ262 promoted the cell apoptosis which were 11.5% (3 mmol/L) and 16.0% (10 mmol/L), while 3     displayed the apoptotic value of 22.4% at 10 mmol/L. The cellular proportion augmented with the increasing concentrations of GQ262. Similarly, GQ262 effectively induced the apoptosis of 92.1 cells with the apoptotic rates of 9.34% (10 mmol/L) and 17.5% (20 mmol/L). In addition, we assessed the apoptosis related proteins, such as cysteinyl aspartate specific proteinase-7 (caspase-7), Bcl-2, and Mcl-1. As expected, GQ262 promoted the upregulation of cleaved caspase-7 and down-regulation of Bcl-2 and Mcl-1 (Fig. 7E, Supporting Information Fig. S4). These results suggested that GQ262 dose-dependently induces the UM cell apoptosis.

GQ262 suppresses UM colony formation
To better comprehend the antitumor capacity of GQ262, the colony survival assay was conducted to investigate the inhibition of clonogenicity on UM cell lines MP41 and 92.1 (Fig. 8). Corresponding to the antiproliferative potency, GQ262 obviously blocked the

GQ262 suppresses UM migration and invasion
UM cells have strong metastatic characteristics, which drive the invasion towards the distant organs 36 . Gaq/11 signaling is involved in UM cell migration, and suppressing Gaq/11 could eliminate the migratory and invasive capacities of UM cells 36 . To verify whether GQ262 could inhibit the cell migration and invasion, Boyden chamber Transwell experiment was performed on UM cells. As depicted in Fig. 9A-D, MP41 and 92.1 cells showed aggressive migration in the absence of GQ262. To our delight, GQ262 remarkably and dose-dependently inhibited the MP41 and 92.1 migration at 3, 10, and 20 mmol/L, compared to 3. In agreement with the effects on suppressing the migration, MP41 and 92.1 cells incubated with GQ262 exhibited poor invasion at 3, 10, and 20 mmol/L (Fig. 9E-H). Overall, the results revealed that GQ262 suppresses UM migration and invasion via blocking Gaq/11.

Metabolic stability of GQ262
Given the effective anticancer potency of GQ262 in vitro, we evaluated the metabolic stability of GQ262 in liver microsomes of human in vitro. As depicted in Table 3, GQ262 displayed  moderate metabolic stability with the half-time (t 1/2 ) of 57.5 min and the intrinsic clearance (CL int ) of 30.2 mL/min/kg, indicating that GQ262 possesses moderate in vitro metabolic stability and drug-like properties, which needs to be further optimized before clinical applications.

Anti-UM efficacy of GQ262 in vivo
Encouraged by the promising in vitro antitumor activity, we conducted MP41 xenograft model to further assess the in vivo anti-UM potency of GQ262. We administrated the nude BALB/ c nu/nu mice bearing tumor with GQ262 for 21 days. The doses were 5, 15, and 30 mg/kg/day. We determined the tumor volume every two days, as well as the body weight. To our delight, tumor growth inhibition (TGI) of GQ262 was up to 106.6% at 30 mg/kg ( Fig. 10A and B, Supporting Information Fig. S5). Meanwhile, GQ262 exhibited robust anticancer activity (TGI Z 82.8%, 15 mg/kg), in sharp contrast to 3 (TGI Z 55.3%, 15 mg/kg). GQ262 also displayed effectual tumor inhibitory potency at 5 mg/kg (TGI Z 55.2%, Fig. 10A and B), demonstrating that GQ262 is highly potent for combating MP41 xenograft model.
Importantly, body weight of the mice did not decline (Fig. 10D) and we did not observe manifest side effects during and after the treatment (Fig. 10E, Supporting Information Fig. S6). All these results strongly supported our hypothesis that directly inhibiting constitutively activated Gaq/11 is a powerful and effective approach for treating UM, other than inhibiting a specific node in the downstream signaling pathways.

GQ262 modulates ERK and YAP by targeting Gaq/11
ERK is a canonical effector in Gaq/11-PLCb signaling pathway, and participates in complex process of cell mitosis after phosphorylation 17e19 . Inhibiting Gaq/11 could block the phosphorylation of ERK, which then suppresses the growth of UM. To verify the effects of GQ262 on suppressing the ERK phosphorylation, we evaluated the expression level of phosphorylated ERK in MP41 xenograft tumor tissues (Fig. 11, Supporting Information Fig. S7). As expected, GQ262 indeed inhibited the phosphorylated ERK in tumor tissues at the testing dose. Yes-associated protein (YAP) is another primary mediator for tumorigenesis, and contributes to the development of UM 22 . Dephosphorylation of YAP is initiated by the activated Gaq/11, which promotes the proliferation of UM. Thereby, the   phosphorylated YAP expression was determined in MP41 xenograft tumor tissues (Fig. 11). As expected, GQ262 increased the accumulation of phosphorylated YAP at the testing dose. Altogether, these results unveiled that GQ262 effectively inhibits the downstream signaling networks of Gaq/11 by targeting Gaq/11 in UM.

Pharmacokinetic (PK) studies of GQ262
To better comprehend the druggability of GQ262, the male ICR mice were employed to assess the PK properties. The dose of intravenous injection (iv) was 5 mg/kg, and the dose of oral administration (po) was 15 mg/kg. In comparison to 4 with short t 1/2 (1.2 h) 45 , GQ262 showed increased elimination t 1/2 with the value of 1.8 h ( Table 4). As shown in Table 4, GQ262 attained the AUC 0-t of 1625 h$ng/mL with C max of 2471 ng/mL via iv. Also, GQ262 was rapidly distributed into tissue, with V ss value of 3.9 L/ kg, and showed the CL of 50.2 mL/min/kg following iv administration in mice. Besides, GQ262 given orally at 15 mg/kg exhibited favorable oral bioavailability (F ) of 59.5% and the mean residence time (MRT 0-t ) was 3.1 h ( Table 4, Supporting Information Fig. S8). All these data demonstrated that GQ262 possesses good PK properties.

Conclusions
In summary, we developed a novel Gaq/11 inhibitor GQ262 with high efficacy, good security, and augmented PK. GQ262 exhibited outstanding potency in combating UM both in vitro and in vivo, and we also verified the direct interaction between GQ262 and Gaq/11. Discovery of GQ262 provides a new and effective anti-UM drug candidate via directly targeting Gaq/11, however drug-like properties including the Gaq/11 inhibitory potency, metabolic stability, selectivity among normal cells and UM cells, and cardiotoxicity are needed to be further optimized, these works are currently under way in our lab.

Chemistry
All reagents used in this manuscript are commercially available. 1 H and 13 C NMR spectra were utilized to characterize chemical structures with a Bruker AVANCE at 400 and 100 MHz. Highresolution mass spectrometer was recorded utilizing Shimadzu LCMS-IT-TOF. Chemical reactions were monitored by utilizing TLC with 0.2 mm silica gel plates (HSGF254, Huanghai). All compounds are >95% pure by reverse-phase analytical HPLC. See supporting information for HPLC conditions, detailed chemical data, and 1 H and 13 C NMR spectra.

Cell culture
The CHO-M 1 cells were given to us by Xinmiao Liang (Dalian Institute of Chemical Physics, Chinese Academy of Sciences) as a generous gift. We cultured the CHO-M 1 cells in F12K media (zqxzbio), which was added to penicillin-streptomycin (1% (v/v), Gibco), G418 (0.25 mg/mL, Beyotime), and FBS (10% (v/v), Gibco). MP41 cells, one of UM cell lines, were given to us by Jingxuan Pan (Zhongshan Ophthalmic Center, Sun Yat-sen University) as a generous gift. 92.1 cells, another UM cell line, were bought from FuHeng BioLogy Co., Ltd. (Shanghai). We cultured MP41 and 92.1 cells in RPMI-1640 (Gibco), which was added to fetal bovine serum (10% (v/v), Gibco). These three cell lines were maintained in 95% air, 5% CO 2 and 37 C.

IP 1 assay
CHO-M 1 cells were collected and seeded in 384-well plates. The cell density reached 2 Â 10 4 cells/well. We added test compounds to pre-treat the cells at 37 C. After incubating for 1 h, carbachol (Sigma) containing LiCl (200 mmol/L) was prepared and added, which achieved the final concentration to 9 mmol/L. After incubating for 1 h, we added the detection solution (2.5% anti-IP 1 cryptate Tb conjugate (Cisbio) þ IP 1 conjugate and lysis buffer (Cisbio) þ 2.5% D-myo-IP 1 -d2 conjugate (Cisbio)) to the plates at room temperature. After incubating for 1 h, the plates were read on an HTRFâ compatible reader.

Cell proliferation inhibition assay
MP41 and 92.1 cells were collected and resuspended in RPMI-1640 (3 Â 10 3 cells/mL). Then we seeded the cells into 96-well plates and placed the plates overnight. After culturing, we added indicated concentrations of compounds to the plates. After incubating for 72 h, we added CCK-8 (10 mL/well). Then, we placed the plates in an incubator for 2e4 h and determined the absorption at 450 nm utilizing a microplate reader. We calculated the IC 50 values by utilizing GraphPad Prism 8.0 software. IC 50 values were shown as mean AE SEM.

BRET measurements
HEK293 cells, which expressed AT 1 receptor along with Gaq-RLuc8, unlabeled Gb1, and Gg2-Venus, were transfected transiently utilizing the standard protocol. In accordance with previously reported procedures 45 , we collected and seeded the cells into 96-well plates after transfecting for 24 h. The plates were placed in the incubator overnight. Then, 3 and GQ262 were added to pretreat the cells. After incubating for 1 h, agonist AngII was added, followed by we added coelenterazine (luciferase substrate). Flex Station 3 (Moleculardevices) was utilized to determine the BRET signals.

Intracellular calcium release measurements
HEK293 cells, which overexpressed AT 1 R, were utilized to measure calcium release. In accordance with previously reported procedures 45 , 1 mmol/L Fluo-4 AM, a calcium-sensitive molecular probe, were loaded into the cells at 37 C. After incubating for 1 h, we added 3 or GQ262 to plates to pre-treat the cells. After treating for 0.5 h, different concentrations of AngII were added. Flex Station 3 (Moleculardevices) was utilized to determine the excitation/emission (485 nm/525 nm).

Rescue assay
We collected and seeded MP41 cells into six-well plates, and placed the plates in the incubator overnight. Then, we utilized the DharmaFECT (Dharmacon, USA) to transfect siRNA targeting GNA11 (AGAUGAUGUUCUCCAGGUCGAAAGG) or control (AGAAAUGUAGUCUUGACCGCUGAGG) in MP41 cells and GNAQ (GCACAAUUAGUUCGAGAAGUU) or control (UUCUCCGAACGUGUCACGUTT) in 92.1 cells. After transfection for 48 h, we placed the cells in 96-well plates. The cell density reached 3000 cells/well. After culturing for 18e24 h, GQ262 was added to pre-treat the cells. After incubating for 72 h, we added CCK-8 (10 mL/well). Then, we placed the plates in the incubator for 2e4 h and determined the absorption at 450 nm utilizing a microplate reader.

Cellular thermal shift assay
We collected and seeded MP41 cells into six-well plates, and placed the plates in the incubator overnight. Then, GQ262 was added to pre-treat the cells. After incubating for 1 h, we collected the cells and conducted CETSA assay. Then, the cells were divided into 4 parts equally. We heated each sample under different temperature (37,40,45, and 50 C) for 3 min. After cooling at 4 C, the cells were left to snap frozen at liquid nitrogen for 3 min, then unfrozen at 55 C, and the process repeated one more time. Subsequently, we centrifuged cell lysates at 4 C (20,000Âg). After centrifuging for 15 min, levels of Gaq/11 were measured by western blot.

Cell cycle and apoptosis analysis
We collected and seeded MP41 cells into six-well plates and placed the plates in the incubator overnight. The cell density reached 3 Â 10 5 cells/well. Then, 3 and GQ262 were added to pretreat the cells for 48 h. For cell cycle, we washed and fixed the cells at 4 C by utilizing 70% ethanol. After fixing for 12 h, we stained the cells at 37 C by utilizing RNase (100 mg/mL) and PI (50 mg/mL) for 0.5 h. For apoptosis, we washed and collected the cells, which were then stained by utilizing 5 mL Annexin V-FITC. After staining for 15 min, we then stained the cells by utilizing 10 mL PI for 5 min. Flow cytometer was utilized to assess the samples (Beckman Coulter, USA).

Clonogenic assay
We collected and seeded MP41 cells into 12-well plates and placed the plates in the incubator overnight. The cell density reached 4000 cells/well. Next, 3 and GQ262 were added to treat the cells. RPMI-1640 and test compounds were replaced every three days. After incubating for 9 days, we fixed the cells utilizing 4% paraformaldehyde. After fixing for 15 min, crystal violet (Solarbio, China) was utilized to stain the clonogentic cells for 0.5 h. We counted the colony formation by utilizing a Colony-Counter.

Migration and invasion assay
We collected and seeded MP41 cells into 24-well Transwell upper chambers (1 Â 10 4 cells/well). The lower chamber was added RPMI-1640 containing 20% FBS. The plates were placed overnight in an incubator. Then, 3 and GQ262 were added to pre-treat the cells. After incubating for 48 h, we discarded the cells which situated in the upper chamber, then we stained the cells which situated in the lower chamber utilizing crystal violet (Solarbio, China). After staining for 0.5 h, migrative and invasive cells were observed and counted utilizing a microscope (magnification Â 40 and Â 100, Nikon).

Human liver microsome stability assay
We prepared potassium phosphate buffer (100 mmol/L, pH 7.4) containing 0.5 mg/mL liver microsome and 5 mmol/L MgCl 2 and added to 96-well plates. Next, GQ262 was incubated with the human liver microsome buffer at the concentration of 1 mmol/L. After incubating for 5 min (37 C), we added 1 mmol/L NADPH to the wells. 150 mL acetonitrile, which included internal standard, was added to quench the reactions at 0, 5, 15, 30, and 45 min. We shook the plates for 10 min (600 rpm). After shaking, the collected samples were centrifuged for 15 min (6000 rpm) and analyzed utilizing LC-MS/MS.

MP41 xenograft model
Nude BALB/c nu/nu mice (18e22 g, 6e8 weeks) were brought from Gempharmatech Co, Ltd. (Nanjing, SCXK(Guangdong) 2021-0029). The Research Ethics Committee of Sun Yat-sen University approved the animal procedures (Approval No. SYSU-IACUC-2021-000858). In accordance with previously reported procedures 45 , the mice with MP41 xenograft tumors were grouped randomly (n Z 6/group) and administrated for 21 consecutive days with vehicle, 3 (15 mg/kg/day), or GQ262 (5, 15, or 30 mg/kg/day). The route of administration was intraperitoneal injection (ip), and we monitored the body weight and tumor size every 2 days. On Day 22, we gained the tumor blocks. GraphPad Prism 8.0 software was utilized to analyze the data.

Pharmacokinetic studies
ICR mice were treated with GQ262 to study the pharmacokinetics. The dose of iv was 5 mg/kg, or the dose of po was 15 mg/ kg. After administration, we collected 30 mL blood samples at 0, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h. Then we centrifuged the samples at 6800Âg, 2e8 C. After centrifuging for 6 min, the samples were kept at À80 C and measured by LC-MS/MS. We calculated the pharmacokinetic parameters utilizing WinNonlin, version 7.0 (Pharsight, USA) and showed the data as mean AE SEM.

Statistical analysis
Each experiment was carried out in triplicate and repeated at least twice except for animal experiments. Data were shown as mean AE SEM. Significance between controls and treatments was assessed by Student's test utilizing GraphPad Prism 8.0 software. Animal experiments were analyzed utilizing the one-way ANOVA. The significant differences were considered at the level of * P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.