G Protein-Coupled Receptors: A Century of Research and Discovery.

GPCRs (G protein-coupled receptors), also known as 7 transmembrane domain receptors, are the largest receptor family in the human genome, with ≈800 members. GPCRs regulate nearly every aspect of human physiology and disease, thus serving as important drug targets in cardiovascular disease. Sharing a conserved structure comprised of 7 transmembrane α-helices, GPCRs couple to heterotrimeric G-proteins, GPCR kinases, and ß-arrestins, promoting downstream signaling through second messengers and other intracellular signaling pathways. GPCR drug development has led to important cardiovascular therapies, such as antagonists of ß-adrenergic and angiotensin II receptors for heart failure and hypertension, and agonists of the glucagon-like peptide-1 receptor for reducing adverse cardiovascular events and other emerging indications. There continues to be a major interest in GPCR drug development in cardiovascular and cardiometabolic disease, driven by advances in GPCR mechanistic studies and structure-based drug design. This review recounts the rich history of GPCR research, including the current state of clinically used GPCR drugs, and highlights newly discovered aspects of GPCR biology and promising directions for future investigation. As additional mechanisms for regulating GPCR signaling are uncovered, new strategies for targeting these ubiquitous receptors hold tremendous promise for the field of cardiovascular medicine.

Cabozantinib Plus Nivolumab in Patients with Non-Clear Cell Renal Cell Carcinoma: Updated Results from a Phase 2 Trial.

Treatment options are limited for patients with non-clear cell renal cell carcinoma (nccRCC). Patients with nccRCC experienced a favorable objective response rate (ORR) in a phase 2 trial of cabozantinib plus nivolumab. We now report updated efficacy and safety results at median follow-up of 34 mo for patients with papillary, unclassified, or translocation-associated RCC. Cabozantinib and nivolumab were administered at standard doses to patients with metastatic nccRCC that had progressed on zero or one line of systemic therapy. The primary endpoint was the ORR according to Response Evaluation Criteria in Solid Tumors v1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events. Forty patients were treated. At median follow-up of 34 mo for survivors, the ORR was 48% (95% confidence interval [CI] 31.5-63.9%). Median PFS was 13 mo (95% CI 7-16); the 12-mo and 24-mo PFS rates were 51% (95% CI 34-65%) and 23% (95% CI 11-37%), respectively. Median OS was 28 mo (95% CI 23-43); the 18-mo and 36-mo OS rates were 70% (95% CI 53-82%) and 44% (95% CI 28-60%), respectively. No new safety signals were seen with cabozantinib and nivolumab. This extended follow-up analysis demonstrates promising efficacy, and highlights the potential for sustained responses with cabozantinib plus nivolumab in patients with metastatic nccRCC.

Structure and dynamics determine G protein coupling specificity at a class A GPCR.

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The ß2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a new Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

G protein-coupled receptors: from radioligand binding to cellular signaling.

Radioligand binding techniques facilitated the identification and study of G-protein coupled receptors that now represent the largest class of targets for therapeutic drugs.

Combined RAF and MEK Inhibition to Treat Activated Non-V600 BRAF-Altered Advanced Cancers.

BACKGROUND: Cancers with non-V600 BRAF-activating alterations have no matched therapy. Preclinical data suggest that these tumors depend on ERK signaling; however, clinical response to MEK/ERK inhibitors has overall been low. We hypothesized that a narrow therapeutic index, driven by ERK inhibition in healthy (wild-type) tissues, limits the efficacy of these inhibitors. As these mutants signal as activated dimers, we further hypothesized that RAF inhibitors given concurrently would improve the therapeutic index by opposing ERK inhibition in normal tissues and not activate ERK in the already activated tumor. MATERIALS AND METHODS: Using cell lines and patient-derived xenografts, we evaluated the effect of RAF inhibition, alone and in combination with MEK/ERK inhibitors. We then undertook a phase I/II clinical trial of a higher dose of the MEK inhibitor binimetinib combined with the RAF inhibitor encorafenib in patients with advanced cancer with activating non-V600 BRAF alterations. RESULTS: RAF inhibition led to modest inhibition of signaling and growth in activated non-V600 BRAF preclinical models and allowed higher dose of MEK/ERK inhibitors in vivo for more profound tumor regression. Fifteen patients received binimetinib 60 mg twice daily plus encorafenib 450 mg daily (6 gastrointestinal primaries, 6 genitourinary primaries, 3 melanoma, and 2 lung cancer; 7 BRAF mutations and 8 BRAF fusions). Treatment was well tolerated without dose-limiting toxicities. One patient had a confirmed partial response, 8 had stable disease, and 6 had radiographic or clinical progression as best response. On-treatment biopsies revealed incomplete ERK pathway inhibition. CONCLUSION: Combined RAF and MEK inhibition does not sufficiently inhibit activated non-V600 BRAF-mutant tumors in patients.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by ß-arrestin.

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Allosteric modulator potentiates ß2AR agonist-promoted bronchoprotection in asthma models.

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled ß2-adrenergic receptor (ß2AR) agonists (ß2-agonists) promote - with limited efficacy - bronchodilation in asthma. All ß2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a ß2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on ß2AR-mediated bronchoprotection. Consistent with our findings using human ß2ARs, Cmpd-6 allosterically potentiated ß2-agonist binding to guinea pig ß2ARs and downstream signaling of ß2ARs. In contrast, Cmpd-6 had no such effect on murine ß2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated ß2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of ß2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and ß-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through ß-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT1R overexpression indicate that SII is a partial agonist for G protein signaling and lacks ß-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT1R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, ß-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT1R overexpression levels. Using inhibitors of G proteins, we demonstrated that both Gi and Gq/11 mediated overexpression-dependent calcium signaling by ß-arrestin-biased ligands. Regarding ß-arrestin-mediated cellular events, the ß-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT1R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.

MRI-Detectability of Clinically Significant Prostate Cancer Relates to Oncologic Outcomes After Prostatectomy.

INTRODUCTION/BACKGROUND: Magnetic resonance imaging (MRI) misses a proportion of "clinically significant" prostate cancers (csPC) as defined by histopathology criteria. The aim of this study was to analyze whether long-term oncologic outcomes differ between MRI-detectable and MRI-occult csPC. PATIENTS AND METHODS: Retrospective analysis of 1449 patients with pre-prostatectomy MRI and csPC on prostatectomy specimens (ie, Grade group ≥2 or extraprostatic spread) between 2001-2006. T2-weighted MRIs were classified according to the Prostate Imaging Reporting and Data System into MRI-occult (categories 1, 2), MRI-equivocal (category 3), and MRI-detectable (categories 4, 5). Cumulative incidence of biochemical recurrence (BCR), metastatic disease, and cancer-specific mortality, estimated with competing risk models. The median follow-up in survivors was 11.0 years (IQR: 8.9-13.1). RESULTS: In 188 (13%) cases, csPC was MRI-occult, 435 (30%) MRIs were equivocal, and 826 (57%) csPC were MRI-detectable. The 15-year cumulative incidence [95% CI] of BCR was 8.3% [2.2, 19.5] for MRI-occult cases, 17.4% [11.1, 24.8] for MRI-equivocal cases, and 43.3% [38.7, 47.8] for MRI-detectable cases (P < .001). The cumulative incidences of metastases were 0.61% [0.06, 3.1], 3.5% [1.5, 6.9], and 19.6% [15.4, 24.2] for MRI-occult, MRI-equivocal, and MRI-detectable cases, respectively (P < .001). There were no deaths from prostate cancer observed in patients with MRI-occult csPC, compared to an estimated 1.9% [0.54, 4.9], and 7.1 % [4.5, 10.6] for patients with MRI-equivocal and MRI-detectable cancer, respectively (P < .001). CONCLUSION: Oncologic outcomes after prostatectomy for csPC differ between MRI-occult and MRI-detectable lesions. Judging the clinical significance of a negative prostate MRI based on histopathologic surrogates alone might be misleading. MICROABSTRACT: Among 1449 patients with pre-prostatectomy MRI and clinically significant prostate cancer on prostatectomy histopathology, MRI-occult cancers (n = 188, 13%) were less likely to recur biochemically (8% vs. 43%, P < .001), metastasize (0.6% vs. 20%, P < .001), or lead to prostate cancer mortality (0% vs. 7%, P < .001) than MRI-detectable cancers (n = 826, 57%). MRI-occult cancers constitute a prognostically distinct subgroup among higher-grade prostate cancers.

GPCR-mediated ß-arrestin activation deconvoluted with single-molecule precision.

ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.

Phase II Trial of Cabozantinib Plus Nivolumab in Patients With Non-Clear-Cell Renal Cell Carcinoma and Genomic Correlates.

PURPOSE: To assess the efficacy and safety of cabozantinib plus nivolumab in a phase II trial in patients with non-clear-cell renal cell carcinoma (RCC). PATIENTS AND METHODS: Patients had advanced non-clear-cell renal carcinoma who underwent 0-1 prior systemic therapies excluding prior immune checkpoint inhibitors. Patients received cabozantinib 40 mg once daily plus nivolumab 240 mg once every 2 weeks or 480 mg once every 4 weeks. Cohort 1 enrolled patients with papillary, unclassified, or translocation-associated RCC; cohort 2 enrolled patients with chromophobe RCC. The primary end point was objective response rate (ORR) by RECIST 1.1; secondary end points included progression-free survival, overall survival, and safety. Next-generation sequencing results were correlated with response. RESULTS: A total of 47 patients were treated with a median follow-up of 13.1 months. Objective response rate for cohort 1 (n = 40) was 47.5% (95% CI, 31.5 to 63.9), with median progression-free survival of 12.5 months (95% CI, 6.3 to 16.4) and median overall survival of 28 months (95% CI, 16.3 to not evaluable). In cohort 2 (n = 7), no responses were observed; one patient had stable disease > 1 year. Grade 3/4 treatment-related adverse events were observed in 32% treated patients. Cabozantinib and nivolumab were discontinued because of toxicity in 13% and 17% of patients, respectively. Common mutations included NF2 and FH in cohort 1 and TP53 and PTEN in cohort 2. Objective responses were seen in 10/12 patients with either NF2 or FH mutations. CONCLUSION: Cabozantinib plus nivolumab showed promising efficacy in most non-clear-cell RCC variants tested in this trial, particularly those with prominent papillary features, whereas treatment effects were limited in chromophobe RCC. Genomic findings in non-clear-cell RCC variants warrant further study as predictors of response.

Sorafenib in Dupuytren and Ledderhose Disease.

Palmar and plantar fibromatosis are benign proliferative processes which present as a diffuse thickening or nodules of the hands and/or feet and may lead to flexion contractures, pain, and functional impairment known as Dupuytren and Ledderhose diseases, respectively. Current treatments are noncurative and associated with significant morbidity. Here, we report on the outcomes of 5 patients with advanced disease, no longer surgical candidates, treated with sorafenib. Sorafenib exhibited an expected safety profile. All 5 patients demonstrated objective responses as evaluated by a decrease in tumor size and/or tumor cellularity from baseline and all 5 patients reported subjective pain relief and/or functional improvement. Mechanistically, immunohistochemistry revealed patchy positivity for PDGFRß, a known target of sorafenib. The outcomes of these 5 patients suggest the safety and efficacy of a relatively well-tolerated oral agent in the treatment of Dupuytren and Ledderhose diseases and suggest the need for future controlled studies.

Translating science to medicine: The case for physician-scientists.

As the number of physician-scientists continues to decline, action must be taken to support them as they embark on their careers.

A Phase Ib/II Randomized Study of RO4929097, a Gamma-Secretase or Notch Inhibitor with or without Vismodegib, a Hedgehog Inhibitor, in Advanced Sarcoma.

PURPOSE: Because the Hedgehog and Notch pathways are often overexpressed in mesenchymal malignancies, we evaluated the efficacy of concurrent inhibition of Notch and Hedgehog signaling using the gamma-secretase inhibitor (GSI) RO4929097 and the smoothened antagonist vismodegib in unresectable or metastatic sarcoma. PATIENTS AND METHODS: In this investigator-initiated trial, phase Ib used standard 3+3 dose escalation in which patients first received vismodegib once daily for 21 days, followed by the combination of RO4929097 concurrently with vismodegib in 21-day cycles. In phase II, patients were randomized to RO4929097 alone or in combination with vismodegib. RESULTS: Nine patients were treated in phase Ib with no dose-limiting toxicities. RO4929097 at 15 mg daily in combination with 150 mg daily of vismodegib was declared the recommended phase II dose. Most adverse events were grade ≤ 2. In phase II (closed early due to discontinuation of RO4929097 evaluation), 34 patients were randomized to RO4929097 alone and 33 to RO4929097 plus vismodegib. RO4929097 did not interfere with the steady-state concentration of vismodegib, while vismodegib reduced the plasma concentration of RO492909. No patients had an objective response. Neither progression-free nor overall survival differed significantly between treatment arms. Paired tumor biopsies from a subset of patients demonstrated inhibition of cleaved Notch. CONCLUSIONS: The combination of RO4929097 plus vismodegib was generally well tolerated. Although accrual to this study was not completed, vismodegib did not meaningfully enhance the clinical efficacy of RO4929097 in an unplanned analysis. GSIs and GSIs plus vismodegib can inhibit intratumoral Notch and downstream phosphorylated Akt signaling.

Local Extent of Prostate Cancer at MRI versus Prostatectomy Histopathology: Associations with Long-term Oncologic Outcomes.

Background It is unknown how the imperfect accuracy of MRI for local staging of prostate cancer relates to oncologic outcomes. Purpose To analyze how staging discordances between MRI and histopathologic evaluation relate to recurrence and survival after radical prostatectomy. Materials and Methods Health Insurance Portability and Accountability Act-compliant retrospective analysis of preprostatectomy T2-weighted prostate MRI (January 2001 to December 2006). Extraprostatic extension and seminal vesicle invasion were assessed by using five-point Likert scales; scores of 4 or higher were classified as positive. Biochemical recurrence (BCR), metastases, and prostate cancer-specific mortality rates were estimated with Kaplan-Meier and Cox models. Results A total of 2160 patients (median age, 60 years; interquartile range, 55-64 years) were evaluated. Among patients with histopathologic extraprostatic (pT3) disease (683 of 2160; 32%), those with organ-confined disease at MRI (384 of 683; 56%) experienced better outcomes than those with concordant extraprostatic disease at MRI and pathologic analysis: 15-year risk for BCR, 30% (95% CI: 22, 40) versus 68% (95% CI: 60, 75); risk for metastases, 14% (95% CI: 8.4, 24) versus 32% (95% CI: 26, 39); risk for prostate cancer-specific mortality, 3% (95% CI: 1, 6) versus 15% (95% CI: 9.5, 23) (P < .001 for all comparisons). Among patients with histopathologic organ-confined disease (pT2) (1477 of 2160; 68%), those with extraprostatic disease at MRI (102 of 1477; 7%) were at higher risk for BCR (27% [95% CI: 19, 37] vs 10% [95% CI: 8, 14]; P < .001), metastases (19% [95% CI: 6, 48] vs 3% [95% CI: 1, 6]; P < .001), and prostate cancer-specific mortality (2% [95% CI: 1, 9] vs 1% [95% CI: 0, 5]; P = .009) than those with concordant organ-confined disease at MRI and pathologic analysis. At multivariable analyses, tumor extent at MRI (hazard ratio range, 4.1-5.2) and histopathologic evaluation (hazard ratio range, 3.6-6.7) was associated with the risk for BCR, metastases, and prostate cancer-specific mortality (P < .001 for all analyses). Conclusion The local extent of prostate cancer at MRI is associated with oncologic outcomes after prostatectomy, independent of pathologic tumor stage. This might inform a strategy on how to integrate MRI into a clinical staging algorithm. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Gottlieb in this issue.

The GPCR-ß-arrestin complex allosterically activates C-Raf by binding its amino terminus.

G protein-coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and ß-arrestins (ßarrs). In a ßarr-dependent signaling pathway, ßarrs link GPCRs to various downstream signaling partners, such as the Raf-mitogen-activated protein kinase extracellular signal-regulated kinase-extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR-ßarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, ßarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR-ßarr1 complexes in vitro. Furthermore, we show that ßarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates ßarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and ßarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between ßarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and ßarr1 bind to the same region on C-Raf. Furthermore, we found that ßarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR-ßarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how ßarrs regulate effector molecules to activate downstream signaling pathways.

Unique Positive Cooperativity Between the ß-Arrestin-Biased ß-Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the ß2-Adrenergic Receptor.

Among ß-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other ß-blockers in its ability to elicit ß-arrestin-biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased ß-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the ß2-adrenergic receptor (ß2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the ß2AR and enhances agonist-mediated transducer (G-protein and ß-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse ß-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the ß2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates ß-arrestin1- but not Gs-protein-mediated high-affinity binding of carvedilol at the ß2AR and ß-arrestin-mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phosphorylation, receptor endocytosis, and trafficking into lysosomes. Importantly, an analog of cmpd-6 that selectively retains positive cooperativity with carvedilol acts as a negative modulator of agonist-stimulated ß2AR signaling. These unprecedented cooperative properties of carvedilol and cmpd-6 have implications for fundamental understanding of G-protein-coupled receptor (GPCR) allosteric modulation, as well as for the development of more effective biased beta blockers and other GPCR therapeutics. SIGNIFICANCE STATEMENT: This study reports on the small molecule-mediated allosteric modulation of the ß-arrestin-biased ß-blocker, carvedilol. The small molecule, compound-6 (cmpd-6), displays an exclusive positive cooperativity with carvedilol among other ß-blockers and enhances the binding affinity of carvedilol for the ß2-adrenergic receptor. Cooperative effects of cmpd-6 augment the ß-blockade property of carvedilol while potentiating its ß-arrestin-mediated signaling functions. These findings have potential implications in advancing G-protein-coupled receptor allostery, developing biased therapeutics and remedying cardiovascular ailments.