β-Arrestins are multifunctional regulators of G-protein-coupled receptor (GPCR) signalling and orchestrate diverse downstream signalling events and physiological responses across the GPCR superfamily1-3. Although GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GPCR kinases, direct chemical tools to modulate β-arrestin activities have remained conspicuously absent. Here we report the identification of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical and structural analyses. These inhibitors disrupt β-arrestin engagement with agonist-activated GPCRs, impairing desensitization, internalization and β-arrestin-dependent physiological functions while sparing G protein-receptor coupling. Cryo-electron microscopy, molecular dynamics simulations and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a pocket within the central crest of β-arrestin1 formed by the middle, C and lariat loops, a critical receptor-binding interface, stabilizing a distinct conformation that is incompatible with full β-arrestin-receptor engagement. Together, these findings establish a mechanistic framework for β-arrestin modulation, reveal a novel allosteric site for structure-based drug design, and open new avenues for transducer-targeted, pathway-specific GPCR therapeutic agents.

Viral myocarditis is a major cause of sudden cardiac death and can lead to dilated cardiomyopathy in adults. However, effective treatments remain elusive due to an incomplete understanding of its molecular drivers. Here, we investigate the role of β-arrestins (βarrs), scaffolding proteins that regulate GPCR signaling, in acute viral myocarditis. Using global βarr1 and βarr2 knockout (KO) mice, we assessed immune cell infiltration and apoptosis as markers of cardiac inflammation under Coxsackievirus (CVB3) infection. CVB3-infected βarr1 and 2 KO mice exhibited suppressed recruitment of NK cells, monocytes, macrophages, dendritic cells, and T cells over a broad range of viral titers at 7 days postinfection along with reduced cardiac apoptosis. At 4 days postinfection, immune cell expansion in secondary lymphoid organs, including B cells, CD8+ T cells, CD64+ myeloid progenitors, and monocyte/macrophages was also impaired in βarr KO mice. Importantly, cardiomyocyte-specific βarr1 and 2 dual deletion mirrored the attenuated inflammatory response and apoptosis observed in global βarr KO mice. Mechanistically, cardiomyocytes lacking βarr1 or βarr2 displayed defective cGAS-STING pathway activation, with impaired STING, TBK1, and IRF3 phosphorylation and inhibited IFNβ production at 24 h post-CVB3 infection. These data highlight βarrs as critical mediators of the inflammatory response in the heart and secondary lymphoid organs during viral myocarditis and demonstrate that cardiomyocyte βarrs plays a fundamental role in the inflammatory response to CVB3 viral myocarditis.NEW & NOTEWORTHY Viral myocarditis can lead to dilated cardiomyopathy in adults, yet the molecular mechanisms that induce cardiac inflammation in viral myocarditis remain unclear. β-Arrestins are scaffolding proteins that mediate intracellular signaling during viral infection. We demonstrated that cardiomyocyte β-arrestins are necessary for immune cell recruitment, induction of cardiac apoptosis, and cGAS-STING pathway activation during acute coxsackievirus B3 infection. Targeting β-arrestins appears to decrease the inflammatory response in acute viral myocarditis.

Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we delineate structural mechanisms underlying βarr-mediated signal transduction. Using cryo-electron microscopy, we elucidate how βarr1 recruits and activates the non-receptor tyrosine kinase Src, a well-established signaling partner of βarrs. βarr1 engages Src SH3 through two distinct sites, each employing a different recognition mechanism: a polyproline motif in the N-domain and a non-proline-based interaction in the central crest region. At both sites βarr1 interacts with the aromatic surface of SH3, disrupting the autoinhibited conformation of Src and directly triggering its allosteric activation. This structural evidence establishes βarr1 as an active regulatory protein rather than a passive scaffold and suggests a potentially general mechanism for βarr-mediated signaling across diverse effectors.

Orthosteric beta blockers represent the leading pharmacological intervention for managing heart diseases owing to their ability to competitively antagonize β-adrenergic receptors (βARs). However, their use is often limited by adverse effects such as fatigue, hypotension, and reduced exercise capacity, due in part to nonselective inhibition of multiple βAR subtypes. These challenges are particularly problematic in treating catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease characterized by lethal tachyarrhythmias directly triggered by cardiac β1AR activation. To identify small-molecule allosteric modulators of the β1AR with enhanced subtype specificity and robust functional antagonism of β1AR-mediated signaling, we conducted a DNA-encoded small-molecule library screen and discovered Compound 11 (C11). C11 selectively potentiates the binding affinity of orthosteric agonists to the β1AR while potently inhibiting downstream signaling after β1AR activation. C11 prevents agonist-induced spontaneous contractile activity, Ca2+ release events, and exercise-induced ventricular tachycardia in the CSQ2-/- murine model of CPVT. Our studies demonstrate that C11 belongs to an emerging class of allosteric modulators termed positive allosteric modulator antagonists that positively modulate agonist binding but block downstream function. Its pharmacological properties and selective functional antagonism of β1AR-mediated signaling make C11 a promising therapeutic candidate for the treatment of CPVT and other forms of cardiac disease associated with excessive β1AR activation.

β-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR superfamily. While GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GRKs, comparable chemical tools to study β-arrestins remain lacking. Here, we report the discovery of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical, and structural analyses. These inhibitors disrupt β-arrestin-engagement with agonist-activated GPCRs, impairing desensitization, internalization, and β-arrestin-dependent functions while sparing G protein-receptor coupling. Cryo-EM, MD simulations, and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a cryptic pocket formed by the middle, C-, and lariat loops of β-arrestin1-a critical receptor-binding interface-stabilizing a distinct conformation incompatible with GPCR engagement. Together, these findings provide a mechanistic framework for β-arrestin modulation, introducing transducer-targeted strategies to fine-tune GPCR signaling and guide the development of pathway-specific therapeutics.

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 β2-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 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.

Abstract Purpose: Survival of patients with metastatic sarcoma remains poor, and there is a pressing need for new therapies. Most sarcoma subtypes are not responsive to immune checkpoint inhibition alone. Lenvatinib, a multireceptor tyrosine kinase inhibitor targeting tumor vasculature, has an immunomodulatory activity that contributes to its antitumor effects. Therefore, we hypothesized that a combination of lenvatinib and pembrolizumab would lead to improved clinical outcomes in patients with sarcoma. Patients and Methods: This was an open-label, single-arm study of lenvatinib and pembrolizumab in the following cohorts: (A) leiomyosarcoma, (B) undifferentiated pleomorphic sarcoma (UPS), (C) vascular sarcomas (angiosarcoma and epithelioid hemangioendothelioma), (D) synovial sarcoma or malignant peripheral nerve sheath tumor (MPNST), and (E) bone sarcomas (osteosarcoma and chondrosarcoma). The primary endpoint was the best overall response (BOR) rate documented by RECIST v1.1 by 27 weeks in each cohort, with a threshold of ≥2 responses among 10 patients. Secondary endpoints included progression-free survival, overall survival, duration of response, and safety. Results: Forty-six patients were evaluable for the primary endpoint, which was met in the UPS and MPNST/synovial cohorts (BOR rates by 27 weeks of 25% and 30%, respectively). There were seven partial responses overall with additional responses noted in angiosarcoma and osteosarcoma. Treatment-related adverse events of any grade and grade 3 or higher occurred in 50/51 (98%) and 29/51 (57%) of patients, respectively. Conclusions: We observed durable responses in MPNST, synovial sarcoma, and osteosarcoma. Patients with UPS and angiosarcoma also responded. Further exploration of this approach is warranted to confirm activity and determine optimal dosing schedules.

Supplementary Table 1: Representativeness of study participants

Supplementary Figure 1. Progression-free survival by histology.

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 β-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 β-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.

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

Supplementary Figure 2. Overall survival by (A) cohort and (B) histology.

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 β2-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.

While traditional β-blockers (i.e. competitive orthosteric antagonists of β-adrenergic receptors; βARs) are widely used as cardiovascular therapeutics, adverse effects such as fatigue and the nonselective inhibition of multiple receptor subtypes often limit maximal effectiveness. An emerging approach to enhance therapeutic targeting is to identify allosteric modulators that act cooperatively with orthosteric ligands. In contrast to orthosteric ligands which bind the endogenous ligand binding site, allosteric modulators bind to regions that are topographically distinct from the orthosteric pocket and can enhance (positive allosteric modulator; PAM) or reduce (negative allosteric modulator; NAM) the activities of orthosteric agonists/antagonists. Since allosteric regions exhibit greater sequence and structural diversity among receptor subtypes relative to the more highly conserved orthosteric pocket, allosteric modulators are more likely to be subtype specific and/or generate less adverse effects. We therefore embarked on a DNA-encoded small molecule library screen to identify novel allosteric modulators of the β 1 AR. Following multiple rounds of affinity selection using purified, functional, β 1 ARs reconstituted in lipid nanodiscs and HitGen’s OpenDEL® small molecule library containing more than 1 billion unique compounds, we identified Compound 11 (C11) as an allosteric modulator with unique pharmacological properties. Notably, C11 binds to the β 1 AR with micromolar affinity and enhances the binding affinity of orthosteric agonists and certain antagonists to the β 1 AR. In contrast to its positive cooperative effect on ligand binding, cell signaling assays showed C11 potently inhibits G protein and β-arrestin signaling downstream of the β 1 AR. Importantly, C11 showed high β 1 AR specificity with no effect on β 2 AR or AT 1 R signaling. These results suggest that C11 is a β 1 AR-specific PAM in terms of ligand binding but a NAM in terms of agonist efficacy, belonging to a largely under-characterized class of allosteric modulators termed PAM-antagonists. With an extremely unique pharmacological profile, C11 is a promising potential therapeutic and experiments evaluating its ability to modulate β 1 AR signaling in vivo are ongoing.

11517 Background: New treatment options are needed for sarcomas. Lenvatinib (L) is an oral, multi-tyrosine kinase inhibitor with significant inhibitory activity against VEGF receptor (VEFGR) 1-3, FGF receptor (FGFR) 1-3, KIT, platelet-derived growth factor receptor (PDGFR) alpha/beta, and RET. Experimental models suggest that L can favorably alter the tumor immune environment, and the combination of L and pembrolizumab (P) has proven to be synergistic and effective across multiple solid tumor types, providing rationale for this study. Methods: This is a pilot study evaluating the efficacy of L and P in select sarcomas. Patients who had at least 1 prior regimen but ≤ 3 were enrolled into one of five cohorts (n = 10 each) A: leiomyosarcoma (LMS); B: undifferentiated pleomorphic sarcoma (UPS); C: angiosarcoma and epithelioid hemangioendothelioma (EHE); D: synovial sarcoma (SS) and malignant peripheral nerve sheath tumor (MPNST); and E: osteosarcoma (OS) and chondrosarcoma (CS). Patients were treated with an initial 2-week run-in of L 20 mg orally daily. Subsequently, P was administered at 200 mg IV every 21 days. The primary endpoint for each cohort was the best objective response rate (ORR) documented by RECIST v1.1 by 27 weeks. The combination was considered worthy of further study if 2 or more responses were observed in a cohort. Secondary endpoints included progression-free survival (PFS), overall survival, duration of response, and safety of the combination. Archival tissue and on-treatment biopsies were collected. Results: As of January 31 st , 2023, cohorts A, D and E have completed accrual. The best response in the LMS cohort was stable disease (SD). In cohort D there were 3 partial responses (PR): 2 SS; 1 radiation associated high grade MPNST. In cohort E one of 6 patients with OS had a PR. To date, of the 6 patients with angiosarcoma, there was 1 PR in a patient with adrenal primary that occurred after the prespecified 27-week timepoint. One of 5 evaluable patients in Cohort B had a PR. Among the 44 patients evaluable for safety, most common AEs were hypertension (56.8%), diarrhea (45.5%), proteinuria (45.5%), fatigue (40.9%), headache (36.4%) and nausea (36.4%). Most common ≥ grade 3 AEs were hypertension (13.6%), dyspnea (6.8%), non-cardiac chest pain (6.8%), syncope (6.8%). Conclusions: A signal of activity was noted in this pilot study for patients with OS, MPNST, angiosarcoma and SS. In the LMS cohort, there were no responses, and PFS was poor. Enrollment to cohorts B and C is ongoing. Clinical trial information: NCT04784247 . [Table: see text]

Supplementary Data from A Phase 1 Dose-Escalation Study of Irinotecan in Combination with 17-Allylamino-17-Demethoxygeldanamycin in Patients with Solid Tumors

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

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

TPS11590 Background: Dedifferentiated liposarcoma (DDLPS) is characterized by near universal amplification of the cyclin-dependent kinase 4 ( CDK4) gene. Palbociclib (P) is a selective CDK4/6 inhibitor that has demonstrated promise in phase II studies of DDLPS. Anti-PD-1 therapy has also shown signals of efficacy, with an approximate overall response rate of 10% in DDLPS patients. CDK4/6 inhibitors upregulate antigen processing and presentation, suppress regulatory T cells, and increase inflammation within the tumor microenvironment. Combined with anti-PD-1 blockade, they can induce an inflamed T cell phenotype and tumor regression in pre-clinical models. We hypothesize that P combined with the anti-PD-1 inhibitor retifanlimab (R) will be safe and tolerable and have synergistic activity leading to activation of T cells and resultant clinical responses. Methods: NCT04438824 is a phase II study of P plus R with a safety lead-in phase. Patients with unresectable or metastatic DDLPS who have received any number of prior therapies are eligible to enroll. On the safety lead-in phase, 6 patients received P (125 mg once daily orally for 21 days followed by 7 days off) plus R (500 mg IV flat dose), repeated in 28-day cycles. P was initiated as monotherapy two weeks prior to initiation of R. The primary endpoint of the safety lead-in was to confirm the recommended phase two dose of the combination. Accrual to the safety lead-in has been completed and a pre-planned phase II expansion portion was opened to accrual. A study amendment was passed that revised the treatment schedule on the expansion cohort to start both P and R concomitantly on day 1 of each cycle. This revised schedule was based on recent data demonstrating the superior safety of a concurrent, rather than staggered, dosing schedule. The primary endpoint of the expansion phase is to estimate the best overall response rate (ORR) by RECIST 1.1. Secondary endpoints include describing the safety and estimating clinical benefit rate, duration of response, progression-free, and overall survival. A total of 30 patients treated with the revised dosing scheme will be enrolled onto the expansion phase. Twelve patients have been enrolled to date. An ORR of 5% will be considered not promising, while an ORR of 25% will be considered promising. The null hypothesis will be rejected if 4 or more patients have a confirmed objective response to treatment. This design has a type I error rate of 0.06 and a type II error rate of 0.04. Mandatory pre- and on-treatment biopsies will be performed for correlative analyses. Clinical trial information: NCT04438824 .

11518 Background: GD is an alternative to doxorubicin in the front line setting in advanced STS. In the Phase I portion of this study, the PD-1 antibody R, plus GD, was generally well tolerated. We hypothesized that R+GD would be more effective than historical controls treated with GD alone. Methods: This is an ongoing open-label single-center study of R+GD in pts with treatment-naïve unresectable or metastatic high-grade STS. G (900 mg/m 2 ) is administered on days 1 and 8 and D (75 mg/m 2 ) on day 8, in 21-day cycles. R (375 mg flat dose) is administered on day 1 starting in cycle 2 and continued as monotherapy ‘maintenance’ after completion of 6 cycles of GD. Up to fifty pts can accrue into five histology-specific cohorts of 10 pts each. The primary endpoint is to estimate the progression-free survival (PFS) rate at 24 weeks. Secondary endpoints included safety, best overall response rate (RR) by RECIST 1.1, disease control rate (DCR), and duration of response (DOR). Subgroup analyses to evaluate all endpoints within each histology-specific cohort are preplanned. An early stopping rule for excessive toxicity will halt accrual if a prespecified severe adverse event rate is surpassed. Results: As of January 11, 2023, 43 pts were enrolled and treated with R+GD. The leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS)/myxofibrosarcoma (MFS), dedifferentiated liposarcoma (DDLPS), and other STS cohorts (n = 10 pts each) were fully accrued. Three of 10 angiosarcoma (AS) pts were enrolled. Median age was 59.5 (range 21 – 81) and 27 (63%) were male. Two DDLPS patients were not yet evaluable for response. Of 41 evaluable pts, the best overall RR was 22% (95% confidence interval [CI]) 11 – 38). Confirmed responses were seen in UPS/MFS (n = 4), LMS (2), AS (2), and follicular dendritic cell sarcoma (1). Three pts (LMS, ossifying fibromyxoid tumor, AS) had an unconfirmed PR, for a best RR of 29% (95% CI 16 – 46). Median PFS was 32.7 weeks (95% CI 26.4 – not estimable [NE]) and median DOR was 24 weeks (95% CI 15 – NE). Safety was evaluated in all 43 pts and the early stopping rule was not triggered. Eighteen (42%) pts had at least one Grade (Gr) 3 or 4 treatment-related adverse event (TRAE). The most common ( > 5%) were anemia (16%), neutropenia (9%), febrile neutropenia (7%), lung infection (7%), and leukopenia (7%). Seven pts (16%) had pneumonitis: one Gr 1, four Gr 2, and two Gr 3. Six pts (14%) stopped treatment due to toxicity, including five with pneumonitis. Conclusions: The median PFS of R+GD appears promising compared to historical controls, although the primary endpoint analysis is pending completion of accrual. There was a higher incidence of pneumonitis with the combination compared to GD alone. Future studies of this combination will need to carefully consider the benefits and risks after the final efficacy and safety analyses are performed. Clinical trial information: NCT04577014 .