Allosteric modulators of the beta1-adrenergic receptor and methods of using same

Abstract

The present disclosure describes, in part, allosteric modulators of the β1- adrenergic receptor and methods of using same.

Description

ALLOSTERIC MODULATORS OF THE BETA1-ADRENERGIC RECEPTORAND METHODS OF USING SAMECross-Relation to Related Applications

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63 / 528,954, filed July 26, 2023, and U.S. Provisional Patent Application No.63 / 644,664, filed May 9, 2024, the contents of which are herein incorporated in their entirety by reference.Federal Funding Legend

[0002] This invention was made with Government support under Federal Grant no. HL056687 awarded by the National Institutes of Health (NIH). The Federal Government has certain rights to this invention.Background

[0003] While traditional P-blockers (i.e. competitive orthosteric antagonists of P- adrenergic receptors; pARs) are widely used as cardiovascular therapeutics, adverse effects such as fatigue and the nonselective inhibition of multiple receptor subtypes often limit maximal effectiveness. Hence, there is a need for the identification of allosteric modulators that act cooperatively with orthosteric ligands to enhance specificity and therapeutic efficacy.Summary

[0004] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0005] The present disclosure provides, in part, novel allosteric modulators of the Beta 1 (Pi)-adrenergic receptor (Pi AR) that have unique pharmacological properties and functional effects, and methods of making and using same.

[0006] Accordingly, one aspect of the present disclosure provides a compound comprising, consisting of, or consisting essentially of the general formula (I) (termed Compound 11; Cl l; Cmpd 11):or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.

[0007] In another aspect, the present disclosure provides a compound comprising, consisting of, or consisting essentially of the general formula (II) (termed Compound 11B; CUB; Cmpd 11B):or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.

[0008] Another aspect of the present disclosure provides a pharmaceutical composition comprising, consisting of, or consisting essentially of a compound as described herein and a pharmaceutically acceptable diluent, excipient, and / or carrier.

[0009] Another aspect of the present disclosure provides a method of enhancing the binding affinity of an agonist and / or antagonist to the Pi AR. comprising, consisting of, or consisting essentially of contacting a receptor with an effective amount of a compound as described herein and an agonist and / or antagonist such that the binding activity of said agonist and / or antagonist is enhanced.

[0010] In one embodiment, the agonist is selected from the group consisting of norepinephrine, epinephrine, isoproterenol, dobutamine and combinations thereof.

[0011] In another embodiment, the antagonist is selected from the group consisting of carvedilol, bucindolol, alprenolol, atenolol, and combinations thereof.

[0012] Another aspect of the present disclosure provides a method of suppressing PiAR-mediated G protein and P-arrestin signaling in a cell in response to an agonist, the method comprising, consisting of, or consisting essentially of contacting the cell that has been exposed to an agonist with an effective amount of a compound as provided herein such that the PiAR-mediated G protein and P-arrestin signaling are suppressed in the cell.

[0013] Another aspect of the present disclosure provides a method of suppressing an agonist-mediated response in a cardiomyocyte, the method comprising, consisting of, or consisting essentially of contacting the cardiomyocyte with an effective amount of a compound as provided herein such that the agonist-mediated response in the cardiomyocyte is suppressed.

[0014] Another aspect of the present disclosure provides a method of inhibiting basal contractility in a cardiomyocyte, the method comprising, consisting of, or consisting essentially of contacting the cardiomyocyte with an effective amount of a compound as provided herein such that the basal contractility in the cardiomyocyte is inhibited.

[0015] Another aspect of the present disclosure provides a method of suppressing PiAR-mediated G protein and P-arrestin signaling in a cell of a subject receiving an agonist, the method comprising, consisting of, or consisting essentially of administering to the subject that has received the agonist a therapeutically effective amount of a compound as provided herein, or a pharmaceutical composition thereof, such that the PiAR-mediated G protein and P-arrestin signaling are suppressed in the subject.

[0016] Another aspect of the present disclosure provides a method of suppressing an agonist-mediated response in a cardiomyocyte in a subject, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a compound as provided herein, or a pharmaceutical composition thereof, such that the agonist-mediated response in the cardiomyocyte is suppressed in the subject.

[0017] Another aspect of the present disclosure provides a method of suppressing sympathetic nervous system mediated PiAR triggered arrhythmic disorders such as Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), long QT syndrome, arrhythmogenic cardiomyopathy, atrial fibrillation, incessant ventricular tachycardia (known as VT storm).

[0018] Another aspect of the present disclosure provides all that is described and illustrated herein.Brief Description of the Drawings

[0019] The accompanying Figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG ”) relating to one or more embodiments, in which.

[0020] Figure 1 provides schematics showing the identification of novel PiAR allosteric modulators by DNA-encoded small molecule library (DEL) screening in accordance with one embodiment of the present disclosure. (A) Schematic of the five different screening conditions including the empty nanodisc and un-liganded (apo)- pi AR nanodisc controls, PiAR nanodiscs bound to the high-affinity orthosteric agonist, BI- 167107 (BI), and BLbound PiAR or P1V2RPP in complex with transducers (heterotrimeric Gsor P-arrestin 1, respectively). Transducer complexes were further reinforced using conformation-stabilizing nanobodies Nb35 (included in PiAR / G protein complex), Nb25, and Fab30 (included in PiV2Rpp / p-arrestin complex). (B) Purified PiARs reconstituted in biotinylated lipid nanodiscs were immobilized using neutravidin beads and incubated with HitGen’s OpenDEL® DNA- encoded small molecule library. Following several wash steps, bound compounds were eluted (Elution 1) and applied as input for a second round of affinity selection with fresh PiAR nanodiscs. Molecules from the final elution (Elution 2) were sent for high-throughput DNA sequencing for identification. (C-C’) qPCR was performed to measure the DNA copy number in eluted samples (El -2) following each round of affinity selection. Compared to library input (~1015molecules), copy number was reduced to ~107following two rounds of screening in each condition (C).Approximately 104'5molecules were lost in round 1 and ~102'3were lost in round 2 asindicated in the table (C’). (D-D’) 3-dimensional cubic plots of each screening condition (D’) and all five conditions merged (D) depict an enriched chemical line feature where Cl 1 was identified. This line feature was enriched in Apo-PiAR and BI-PiAR samples, minimally present in G protein and P-arrestin complex samples, and completely absent in the empty nanodisc condition. Axes enumerate the chemical building blocks utilized in each round of chemical synthesis (R1-R3). Data point size corresponds to the sequence count for a particular compound, and the copy number of Cl 1 in each condition is indicated within the respective cubic plot. (E) Schematic of the chemical structure of Cl 1 generated through three rounds (Rl-3) of chemical synthesis; Rl, R2, R3.

[0021] Figure 2 provides graphic data representations showing that Cl 1 potentiates the binding affinity of agonists and certain antagonists to the PiAR. (A-A”). PiAR nanodiscs were incubated with a fixed amount of radiolabeled orthosteric antagonist, I125-CYP, serial doses of unlabeled orthosteric ligand, and either DMSO (0.19%) or 30 pM Cl 1. The resulting competition binding curves (A-A’) and corresponding IC50 shift quantifications (A”) revealed that Cl 1 enhanced the binding affinity of agonists (norepinephrine, Nor; isoproterenol, Iso; dobutamine, Dob; and epinephrine, Epi) and certain antagonists (carvedilol, Carv; bucindolol, Buc; alprenolol, Alp; atenolol, Ate) to the PiAR with no effect on the binding affinity of the antagonists metoprolol (Met) or carazolol (Caraz). Dose response curves are presented as percent of maximum I125- CYP binding. IC50 values were calculated from the nonlinear fit (one-site binding; GraphPad prism) and plotted as the difference between ICsofDMSO) and ICso(Cl 1). T-tests were performed on paired IC50 values; *p<0.05, **p<0.01, ***p<0.001; data points represent mean ± SEM of at least 3 independent experiments performed in duplicate.

[0022] Figure 3 provides schematics and graphic data representations showing that Cl 1 suppresses G protein signaling and P-arrestin function downstream of agonist- activated PiAR in HEK293T cells. (A-A”) Schematic representation of the BRET- based Trupath G protein dissociation assay where Gas-RLuc8 dissociates from Gp / Gy-GFP upon PiAR activation, resulting in BRET signal decay (A). HEK293T cells transiently transfected with PiAR, Gas-RLuc8, Gp3 and G / 9-GFP were pretreated for 20 minutes with DMSO (0.19%) or 30 pM Cl 1 and then stimulated with serial doses of the agonist isoproterenol (Iso) for 10 minutes before measuring BRET.The resulting dose-response curve (A’) and corresponding Emax of the nonlinear fit (A”) revealed that Cl 1 treatment significantly reduced maximal G protein dissociation compared to vehicle. (B-B”) Schematic representation of the Giosensor luciferase-based biosensor that emits light in response to binding intracellular cAMP (B). HEK293T cells transiently transfected with PiAR and the Giosensor plasmid were pre-treated for 20 minutes with DMSO (0.19%) or 30 pM Cl 1 and then stimulated with serial doses of isoproterenol for 5 minutes before measuring luminescence. The resulting dose-response curve (B’) and corresponding Emax of the nonlinear fit (B”) demonstrated that Cl 1 treatment significantly reduced maximal cAMP accumulation compared to vehicle. (C-C”) Schematic representation of the BRET -based P-arrestin recruitment assay wherein P-arrestin-GFP is recruited to P1V2R-RLUCII upon receptor activation and generates a BRET signal (C). HEK293T cells transiently transfected with P-arrestin-GFP and P1V2R-RLUCII were pre-treated for 20 minutes with DMSO (0.19%) or 30 pM Cl 1 and then stimulated with serial doses of the isoproterenol for 20 minutes. The resulting dose-response curve (C’) and corresponding Emax of the nonlinear fit (C”) revealed that Cl 1 treatment significantly reduced maximal P-arrestin recruitment to P1V2R-RLUCII compared to vehicle. (D-D”) Schematic representation of the P-arrestin internalization assay (D). Upon P1V2R activation, the receptor / p-arrestin-RLucII complex is internalized into endosomes and a BRET signal is generated when internalized P-arrestin-RLucII and endosomal marker FYVE-GFP are in proximity. HEK293T cells transiently transfected with P1V2R, GFP-FYVE, and P-arrestin-RLucII were pre-treated for 20 minutes with DMSO (0.19%) or 30 pM Cl 1 and then stimulated with serial doses of isoproterenol for 25 minutes before measuring the BRET response. The resulting dose-response curve (D’) and corresponding Emax of the nonlinear fit (D”) demonstrated that Cl 1 treatment significantly reduced maximal P-arrestin internalization compared to vehicle; t-test, *p<0.05, **p<0.01, ***p<0.001; data points represent mean ± SEM of at least 3 independent experiments performed in duplicate or quadruplicate; curve fits were plotted using a log(agonist) three- parameter model in GraphPad Prism; net BRET ratios (emission of RLuc8 / GFP) are baseline-subtracted according to the non-linear fit of each treatment condition; luminescence values and Emax quantifications are presented as the percent of maximal signal in the vehicle condition.

[0023] Figure 4 provides immunoblots and graphic representations of data showing that Cl 1 suppresses phosphorylation of ERK mediated by isoproterenol or carvedilol downstream of agonist-activated PiAR in HEK293T cells. (A-A”) Representative immunoblots (A), relative densitometry quantifications (A’) and Emax values of the nonlinear curve fit (A”) demonstrated that Cl 1 significantly reduced maximal ERK phosphorylation (pERK) in response to isoproterenol (Iso) compared to vehicle control in HEK293T cells transiently expressing the PiAR. (B-B”) Representative immunoblots (B), relative densitometry quantifications (B’) and Emax values of the nonlinear curve fit (B”) demonstrated that Cl 1 significantly reduced maximal pERK in response to carvedilol (carv) compared to vehicle control in HEK293T cells transiently expressing PiAR; densitometric values of pERK were normalized to total ERK (tERK) and presented as percent of the maximal value in vehicle-treated cells; t- test, *p<0.05; data points represent mean ± SEM of at least 3 independent experiments; nonlinear curve fits and Emax values were calculated from a log(agonist) three-parameter model in GraphPad Prism.

[0024] Figure 5 provides graphic representations of data showing that Cl 1 does not suppress cellular signaling mediated by alternative receptors P2AR, AT1R, or M3R (A-A”) P2AR nanodiscs were incubated with a fixed amount of radiolabeled orthosteric antagonist, I125-CYP, serial doses of unlabeled orthosteric ligand, and either DMSO (0.19%) or 30 pM Cl 1. The resulting competition binding curves (A- A’) and corresponding IC50 shift quantifications (A”) revealed that Cl 1 enhanced the binding affinity of carvedilol to the P2AR, but only modestly increased isoproterenol binding. Dose response curves are presented as percent of maximum I125-CYP binding. IC50 values were calculated from the nonlinear fit (one-site binding; GraphPad prism) and plotted as the difference between ICsofDMSO) and ICso(Cl 1). T-tests were performed on paired IC50 values; *p<0.05. Data points represent mean ± SEM of at least 3 independent experiments performed in duplicate. (B-C’) HEK293T cells transiently overexpressing P2AR along with BRET constructs, Gas-Rluc8, GP, and Gy-GFP (B, Fig. 3 A) or P2V2R co-expressed with P-arrestin-RLucII and endosomal marker FYVE-GFP (C, Fig. 3D) were pre-treated with vehicle or 30 pM Cl 1 and stimulated with serial doses of isoproterenol. Cl 1 treatment did not have a significant effect on G-protein dissociation (B-B’) or P-arrestin internalization (C-C’) downstream of agonist-activated P2AR or P2V2R, respectively. (C-D’) HEK293T cellstransiently overexpressing AT1R along with BRET constructs, Gaq-Rluc8, GP, and Gy-GFP (D, Fig. 3 A) or AT1R co-expressed with P-arrestin-RLucII and endosomal marker FYVE-GFP (E, Fig. 3D) were pre-treated with vehicle or 30 pM Cl 1 and stimulated with serial doses of angiotensin II (Ang II). Cl 1 treatment did not have a significant effect on G-protein dissociation (D-D’) or P-arrestin internalization (E-E’) downstream of agonist-activated AT1R. (F-F’) HEK293T cells were pre-treated with vehicle or 30 pM Cl 1 and stimulated with serial doses of carbachol to activate endogenous Gq-coupled muscarinic M3 receptors (M3Rs). Cl 1 treatment did not have a significant effect on the resulting Ca2+response (F-F’). Representative time-course plots of the baseline-subtracted raw fluorescence at each carbachol dose depicted comparable Ca2+responses between vehicle- and Cl 1- treated cells (F”); data points represent mean ± SEM of at least 3 independent experiments performed in duplicate or triplicate; curve fits were plotted using a log(agonist) three-parameter model in GraphPad Prism; net BRET ratios (emission of RLuc8 / GFP) are baseline-subtracted according to the non-linear fit of each treatment condition. Ca2+responses are presented as the baseline-subtracted area under the curve (AUC) and normalized to the percent of DMSO maximum.

[0025] Figure 6 provides graphic data representations and immunoblots showing that Cl 1 reduces basal contractility and suppresses the isoproterenol response in isolated wild-type cardiomyocytes. (A-A”) Representative unloaded shortening contractions (A) from isolated wild-type cardiomyocytes treated with increasing doses of Cl 1 displayed a significant decrease in basal sarcomeric shortening in cardiomyocytes treated with 30 pM Cl 1 compared to vehicle, whereas treatment with Toss of function’ Cl 1 analog, Cl 1-G, had no effect (A’). (B) High doses of Cl 1 (30 pM) also elicited a significant reduction in basal sarcomeric shortening in [EAR ' cardiomyocytes, indicating that the Cl 1 -mediated reduction in contractility is partially non- selective. An intermediate dose of 10 pM was therefore selected for functional cardiomyocyte assays; one-way ANOVA, ***p<0.0002, ****p<0.0001; data points represent biological replicates (n = 4-7 hearts, 7-10 cells per treatment per heart). (C-D’) The dose-dependent enhancement of sarcomeric shortening (C) and contraction velocity (D) stimulated by serial doses of isoproterenol (Iso) was significantly blunted (C’, D’) in isolated wild-type cardiomyocytes treated with 10 pM Cl 1 compared to vehicle-treated cells. The magnitude of the isoproterenol-mediated increase is plotted as the difference between maximal isoproterenol dose (Logflso] = -6 M) and the non-stimulated (N.S.) condition (C’, D’); t-test, *p<0.05, **p<0.01; data points represent the mean ± SEM of n = 3 biological replicates (7-10 cells per treatment per heart). (E-E’) Representative immunoblots (E) and corresponding densitometric quantifications (E’) revealed significantly decreased levels of phosphorylated PLN (Seri 6), Tnl (Ser23 / 24), and PLN (Thrl7) in Cl 1- treated cardiomyocytes (10 pM) compared to vehicle control following isoproterenol stimulation. Phosphorylated proteins (pPLN and pTnl) are normalized to total levels (tPLN and tTnl). Statistical comparisons (one-way ANOVA) are shown between DMSO(vehicle) and DMSO(iso), Cl 1 (vehicle) and Cl l(iso), and between DMSO(iso) and Cl l(iso) groups only; *p<0.03, **p<0.002, ***p<0.0002, ****pO .0001; data points represent biological replicates (n = 5 hearts, each performed in duplicate); WT, wild-type; Pent, pentamer; Mon, monomer.

[0026] Figure 7 provides graphic data representations showing that Cl 1 restores regular contractile rhythm in cardiomyocytes isolated from CSQ2’ ’ mice. (A-A’”) Representative unloaded shortening contractions (A-A’) and corresponding quantifications (A” -A’”) revealed frequent and irregular beats in vehicle-treated CSQ2' ' cardiomyocytes stimulated with isoproterenol (Iso) during 1Hz pacing (i.e. 1 sec interval), reminiscent of ventricular tachycardia. Remarkably, isoproterenol- mediated spontaneous beating is completely attenuated in cells pre-treated with 10 pM Cl 1; data points represent biological replicates (n = 4 hearts, 7-10 cells per treatment per heart); one-way ANOVA, ****p<0.0001. (B) Poincare plots depict increased variability in the interval between consecutive cellular contractions in vehicle-treated CSQ2 ’ ’ cardiomyocytes stimulated with isoproterenol but not in cells pre-treated with 10 pM Cl 1; data points represent peak-peak intervals of all biological replicates combined.

[0027] Figure 8 provides graphic data representations showing that Cl 1 suppresses spontaneous Ca2+release events in isolated CSQ2 / _cardiomyocytes. (A-B) Spontaneous Ca2+release events (SREs) were measured in quiescent ventricular cardiomyocytes following pacing at 0.5Hz. Representative confocal line scans with associated fluorescent intensity profiles (A-A’”) and corresponding quantifications (B) revealed a robust increase in spontaneous Ca2+release event frequency in vehicle- treated CSQ2 ’ ’ cardiomyocytes after stimulation with isoproterenol (Iso, A’) that wassignificantly attenuated in cells pre-treated with 10 pM Cl 1 (A’”); data points represent biological replicates (n = 4 hearts, 11-13 cells per treatment per heart); oneway ANO V A, **p<0.0013, ****p<0.0001. (C-C”) Representative intracardiac electrocardiograms (C) immediately following isoproterenol injection (3 mg / kg, I.P.) depict episodes of sustained ventricular tachycardia (VT) that was detected in more than 50% of CSQ2 / _mice pre-treated with vehicle solution (n=3 / 5, C’) and was not observed in any of the mice pre-treated with 10 mg / kg Cl 1 (n=0 / 6, C’); ectopic beats are marked with asterisks. Both vehicle- and Cl 1 -treated CSQ2 / _mice exhibited a significant increase in heart rate (bpm) in response to isoproterenol (C”); two-way ANOVA, ***p<0.004, ****p<0.0001; data points represent biological replicates.

[0028] Figure 9 provides schematics and graphic data representations showing the generation and functional validation of PiAR and P1V2RPP nanodiscs. (A-A’) Snake diagrams of wild-type (A) and chimeric (A’) PiARs highlighting the cloning site of the sortase consensus sequence. The synthetic phospho-peptide corresponding to the V2R was ligated to PiAR truncated at G413 to generate P1V2RPP. (B-B’) Reconstituted nanodiscs containing PiAR (B) or P1V2RPP (B’) were functionally validated using radioligand competition binding experiments in the presence of increasing concentrations of heterotrimeric Gsor P-arrestinl, respectively, to confirm transducer cooperativity. Values are presented as percent of maximum I125-CYP binding. IC50 values (shown in parenthesis) were calculated from the nonlinear fit (one-site binding; GraphPad prism).

[0029] Figure 10 provides gel photographs and graphic data representations showing the optimization of DEL affinity selection protocol. (A-A’) The immobilization efficiency of biotinylated PiAR or P1V2RPP nanodiscs was evaluated by incubating a fixed quantity of pre-washed high-capacity neutravidin beads (10 uL) with increasing amounts of nanodisc (5-10 ug) at room temperature for 1 hour while rotating. After collecting flow through, the nanodisc-coated beads were washed three times and bound protein was eluted via boiling. Following SDS-PAGE and Coomassie Blue staining, a 1 : 1 ratio of receptor nanodisc (pg) to bead slurry (pL) was deemed optimal given minimal loss of nanodisc in the flow through. (B-B’) To evaluate the integrity of the G protein and P-arrestin transducer complexes, PiAR or P1V2RPP nanodiscs were immobilized to neutravidin beads along with 20 pM of the high-affinity agonist, BI- 167107 (BI), and a 1.2 molar excess of heterotrimeric Gsor P-arrestinl -me,respectively. To enhance complex stability, PiAR / Gscomplexes were supplemented with Nb35 (2.5 molar excess with respect to PiAR) and 0.05 U / mL apyrase, while the PiV2Rpp / p-arrestinl complex was further stabilized with Nb25 and Fab30 (2.5 or 1.7 molar excess relative to P1V2RPP, respectively). Following complex formation, the flow through was collected (FT #1) and the beads were washed three times with ice- cold binding buffer supplemented with 10 pM BI. To simulate incubation with DNA- encoded molecules, Img / mL salmon sperm DNA was applied to neutravi din- immobilized PiAR / Gs and PiV2Rpp / p-arrestinl complexes in binding buffer containing 20 pM BI for 1 hour while agitating. The secondary flow through (FT #2) was collected, bound protein was eluted via boiling at 95 °C, and samples were loaded onto 10% SDS-polyacrylamide gels for analysis by Coomassie Blue to confirm the stability of receptor-transducer complexes throughout the selection protocol. (C) To validate the retention of small molecule ligands to the PiAR or P1V2RPP nanodiscs during screening, 5 nM of the radiolabeled orthosteric antagonist,3H-DHA, was incubated with neutravidin-immobilized nanodiscs and washed three times with ice- cold binding buffer. To elute, beads were resuspended in 1.5% Fos-choline in water and incubated at 37 °C (15 min) then 95 °C (15 min) while agitating. The elution procedure was repeated a second time, combined, and applied to 5 mL scintillation fluid for overnight incubation.3H-DHA counts are presented as percent of input. Empty nanodiscs were utilized as a control to assess non-specific binding.

[0030] Figure 11 provides graphic data representations showing an evaluation of the affinity of Cl 1 for the PiAR. (A- A”) Isoproterenol competition binding curves testing serial doses of Cl 1 (A), and the corresponding IC50 shift quantifications (A’) plotted as a function of [Cl 1] (A”) demonstrated that the binding affinity of Cl 1 for the PiAR is in the sub-micromolar range (LogECso=-6.12 M); dose response curves are presented as percent of maximum I125-CYP binding. IC50 values were calculated from the nonlinear fit (one-site binding; GraphPad prism) and plotted as the difference between ICsofDMSO) and ICso(Cl 1). Data points represent mean ± SEM of at least 3 independent experiments performed in duplicate.

[0031] Figures 12A-12C: Figures 12A-12C show formulae of Cl l and variants Cl l- B through Cl 1-1. Fig. 12C shows how the R1 chemical group (solid rectangle) and R3 chemical group (dashed rectangle) were modified in analogs Cl 1-A through Cl 1- I.

[0032] Figure 13 provides graphic data representations showing the effect of Cl 1 analogs A-I on orthosteric ligand binding and PiAR-mediated signaling. (A-A”) PiAR nanodiscs were incubated with a fixed amount of radiolabeled orthosteric antagonist, I125-CYP, serial doses of unlabeled isoproterenol (A) or carvedilol (A’), and either DMSO (0.19%) or 30 pM of Cl 1 analogs. Quantification of IC50 log-shifts revealed that the ability of Cl 1 to potentiate isoproterenol (Iso) or carvedilol (Carv) to the PiAR is largely unaffected by modification of the R1 chemical group, whereas truncation of the R3 chemical group (Cl 1-G and H) resulted in complete loss of cooperativity. IC50 values were calculated from the nonlinear fit (one-site binding; GraphPad prism) and plotted as the difference between ICsofDMSO) and ICso(Cl 1). Data points represent mean ± SEM of at least 3 independent experiments performed in duplicate; one-way ANOVA, #p<0.05, ##p<0.01, ###p<0.001 (compared to Cl 1). (B-B”) Quantification of G protein dissociation (B) and P-arrestin recruitment (B’) via BRET revealed that the ability of Cl 1 to inhibit PiAR-mediated signaling is largely unaffected by modification of the R1 group, whereas truncation of the R3 group (Cl 1-G and H) resulted in complete loss of antagonistic function; data points represent mean ± SEM of at least 3 independent experiments performed in duplicate; curve fits were plotted using a log(agonist) three-parameter model in GraphPad Prism; net BRET ratios (emission of RLuc8 / GFP) are baseline-subtracted according to the non-linear fit of each treatment condition; one-way ANOVA, ##p<0.01, ###p<0.001 (compared to Cl 1), **p<0.01, ***p<0.001 (compared to vehicle).

[0033] Figure 14 provides graphic data representations showing the effect of Cl 1 on electrically stimulated Ca2+transients in isolated CSQ2 ’ ’ cardiomyocytes. (A-A’) Representative Ca2+transients obtained from CSQ2 ’ ’ ventricular cardiomyocytes pretreated with vehicle (A) or 10 pM Cl 1 (A’) during pacing at 0.5 Hz. (B-B”) No significant differences in Ca2+transient amplitude (F / Fo, B), time to peak (B’), or the decay constant tau (B”) were observed between groups; statistical significance was evaluated via t-test; data points represent biological replicates (n = 4 hearts, 11-13 cells per treatment per heart).

[0034] Figure 15 provides graphic data representations showing the effect of Cl 1 on caffeine-stimulated Ca2+release in isolated wild-type cardiomyocytes. (A-A’) Caffeine-induced Ca2+transients (A) were measured in quiescent wild-type cardiomyocytes pre-treated with vehicle or serial doses of Cl 1 following treatmentwith 10 mM caffeine. No significant differences in Ca2+transient amplitude (F / Fo, A’) were observed between groups; statistical significance was evaluated via one-way ANOVA, data points represent biological replicates (n = 3 hearts, 2-6 cells per treatment per heart).

[0035] Figure 16 provides graphic data representations showing the stability of Cl 1 in solution and in vivo. (A) Cl 1 (10 mM) prepared in a vehicle solution (50% DMSO, 50% PEG-400) was incubated at 37 °C for the indicated timepoints and applied to HPLC / MS to assess chemical stability. Quantification of peak area revealed that Cl 1 is stable for at least three days at 37 °C (n=l). (B) The pharmacokinetic profile of Cl 1 prepared in a vehicle solution (10%DMA, 40% PEG-300, 2% Tween 80 and 48% saline) was obtained following intraperitoneal injection (10 mg / kg) in wild-type mice (A’). Cl 1 is detectable at the highest concentration between 30 minutes to 1-hour post-injection in plasma (ng / mL) and in heart tissue (ng / g); data points represent the mean ± SD of all biological replicates (n=3-4 mice).

[0036] Figures 17A-17D provide schematics and graphic data representations which illustrate the experimental design, graded exercise protocol and observed arrhythmias in Casq2- / - Mice A. Experimental timeline of repeated vehicle / Cl 1 testing in mouse. B (left) A single-lane mouse treadmill with an adjustable incline ramp that can be increased from 0° to 25°. The treadmill uses negative reinforcement to encourage running, (right) Timeline for vehicle / Cl 1 administration, followed by exercise C. Illustrative graph of the 30-minute exercise protocol, showing equal intervals of increasing workload. D. Representative ECG snippets displaying various arrhythmia subtypes in Casq2- / - mice during the exercise protocol. Abnormal beats are indicated by dots.

[0037] Figure 18 provides graphic data representations showing quantification of arrhythmia subtypes in 30 minutes of graded treadmill exercise following vehicle / Cl 1 pretreatment in Casq2- / - mice. Preliminary data from 2 CSQ mice that had telemetry modules implanted and were exercised 4 times, each 4 days apart, after pretreatment with vehicle or Cl 1; A: Premature ventricular contractions; B: Total runs of bigeminy (defined as 3 or more altering regular and premature beats); C: Total duration of runs of ventricular tachycardia expressed in seconds.Detailed Description

[0038] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0039] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0040] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

[0041] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0042] As used herein, the transitional phrase "consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising."

[0043] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0044] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0045] As used herein, "treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. The term "effective amount" or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0046] As used herein, the term "administering" an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term "administering" is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0047] The term “biological sample” as used herein includes, but is not limited to, a sample containing tissues, cells, and / or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and tears. A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from athird party (e.g., received from an intermediary, such as a healthcare provider or lab technician).

[0048] "Contacting" as used herein, e.g., as in "contacting a sample" refers to contacting a sample directly or indirectly in vitro, ex vivo, or in vivo (i.e. within a subject as defined herein). Contacting a sample may include addition of a compound to a sample, or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture.

[0049] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient).

[0050] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.A. Compounds

[0051] Accordingly, one aspect of the present disclosure provides a compound comprising, consisting of, or consisting essentially of the general formula (I) (termed Compound 11; Cl l; Cmpd 11):or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.

[0052] Another aspect of the present disclosure provides a compound comprising, consisting of, or consisting essentially of the general formula (I) (termed Compound 11; Cl l; Cmpd 11):or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.B. Pharmaceutical Compositions

[0053] In another aspect, the present disclosure provides compositions comprising one or more of compounds as described herein and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.

[0054] When used to treat or prevent a disease, such as a bacterial infection, the compounds described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents (e.g., therapeutic agents) useful for treating such diseases and / or the symptoms associated with such diseases. Such agents may include, but are not limited to, steroids, membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgG isotype switching or IgG synthesis, b-agonists, tryptase inhibitors, asmlrin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to name a few.

[0055] The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.

[0056] Pharmaceutical compositions comprising the compound(s) may be manufactured by means of conventional mixing, dissolving, granulating, drageemaking levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.

[0057] The compounds may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

[0058] Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

[0059] For topical administration, the compound(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

[0060] Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and / or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0061] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0062] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.

[0063] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.

[0064] Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

[0065] For nasal administration or administration by inhalation or insufflation, the compound(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., di chi orodifluorom ethane, trichl orofluorom ethane, di chi orotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0066] For ocular administration, the compound(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.

[0067] For prolonged delivery, the compound(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The compound(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).

[0068] Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

[0069] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0070] The compound(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and / or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.

[0071] The amount of compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compound(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.

[0072] Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and / or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.

[0073] Dosage amounts will typically be in the range of from about 0.0001 mg / kg / day, 0.001 mg / kg / day or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and / or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and / or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.C. Methods

[0074] The compounds provided herein are novel and highly specific allosteric modulators of the piAR that act as a potent functional antagonist. These compounds show an extremely unique pharmacological profile and have been shown to be able to modulate cardiomyocyte function demonstrating that these compounds and their analog derivatives have many uses as therapeutics.

[0075] Hence, another aspect of the present disclosure provides a method of enhancing the binding affinity of an agonist and / or antagonist to the Pi AR. comprising, consisting of, or consisting essentially of contacting a cell with an effective amount of a compound as described herein and an agonist and / or antagonist such that the binding activity of said agonist and / or antagonist is enhanced.

[0076] In one embodiment, the agonist is selected from the group consisting of norepinephrine, epinephrine, isoproterenol, dobutamine and combinations thereof.

[0077] In another embodiment, the antagonist is selected from the group consisting of carvedilol, bucindolol, alprenolol, atenolol, and combinations thereof.

[0078] Another aspect of the present disclosure provides a method of suppressing PiAR-mediated G protein and P-arrestin signaling in a cell in response to an agonist, the method comprising, consisting of, or consisting essentially of contacting the cell that has been exposed to an agonist with an effective amount of a compound as provided herein such that the PiAR-mediated G protein and P-arrestin signaling are suppressed in the cell.

[0079] Another aspect of the present disclosure provides a method of suppressing an agonist-mediated response in a cardiomyocyte, the method comprising, consisting of, or consisting essentially of contacting the cardiomyocyte with an effective amount of a compound as provided herein such that the agonist-mediated response in the cardiomyocyte is suppressed.

[0080] Another aspect of the present disclosure provides a method of inhibiting basal contractility in a cardiomyocyte, the method comprising, consisting of, or consisting essentially of contacting the cardiomyocyte with an effective amount of a compound as provided herein such that the basal contractility in the cardiomyocyte is inhibited.

[0081] Another aspect of the present disclosure provides a method of suppressing PiAR-mediated G protein and P-arrestin signaling in a cell of a subject receiving anagonist, the method comprising, consisting of, or consisting essentially of administering to the subject that has received the agonist a therapeutically effective amount of a compound as provided herein, or a pharmaceutical composition thereof, such that the PiAR-mediated G protein and P-arrestin signaling are suppressed in the subject.

[0082] Another aspect of the present disclosure provides a method of suppressing an agonist-mediated response in a cardiomyocyte in a subject, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a compound as provided herein, or a pharmaceutical composition thereof, such that the agonist-mediated response in the cardiomyocyte is suppressed in the subject.D. Kits

[0083] The present disclosure further provides kits comprising the compounds and / or compositions provided herein and for carrying out the subject methods as provided herein. For example, in one embodiment, a subject kit may comprise, consist of, or consist essentially of a compound as provided herein and / or (v) pharmaceutical compositions as provided herein.

[0084] In other embodiments, a kit may further include other components. Such components may be provided individually or in combinations, and may provide in any suitable container such as a vial, a bottle, or a tube. Examples of such components include, but are not limited to, one or more additional reagents, such as one or more dilution buffers; one or more reconstitution solutions; one or more wash buffers; one or more storage buffers, one or more control reagents and the like. Components (e.g., reagents) may also be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.

[0085] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as apackage insert, in the labeling of the container of the kit or components thereof (z.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0086] Another aspect of the present disclosure provides all that is described and illustrated herein.

[0087] The following Examples are provided by way of illustration and not by way of limitation.ExamplesIntroduction

[0088] 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. Since PiARs are fundamental for the regulation of cardiovascular system and their dysregulation has been demonstrated in many cardiovascular diseases, the inventors therefore embarked on a DNA-encoded small molecule library screen to identify novel allosteric modulators of the PiAR. Following multiple rounds of affinity selection using purified, functional, PiARs reconstituted in lipid nanodiscs and HitGen’s OpenDEL® small molecule library containing more than 1 billion unique compounds, Compound 11 (Cl 1; Cmpd 11) was identified as an allosteric modulator with unique pharmacological properties.Notably, Cl 1 binds to the Pi AR with micromolar affinity and enhances the binding affinity of orthosteric agonists and certain antagonists to the PiAR. In contrast to its positive cooperative effect on ligand binding, cell signaling assays showed Cl 1 potently inhibits G protein and P-arrestin signaling downstream of the Pi AR. Importantly, Cl 1 showed high PiAR specificity with no effect on P2AR or AT1R signaling. Lastly, Cl 1 suppresses the isoproterenol response in isolated cardiomyocytes and completely blocks the development of spontaneous contractions, Ca2+release events, and stress-induced ventricular tachycardia in an established mouse model of CPVT. These results suggest that Cl 1 is a PiAR-specific PAM in terms of ligand binding but a NAM in terms of agonist efficacy, belonging to a largely undercharacterized class of allosteric modulators termed PAM-antagonists. Thus, Cl 1 represents a promising therapeutic molecule for the treatment of cardiac disease with a unique pharmacological profile.Example 1. A positive allosteric modulator of the PiAR with antagonist activity for catecholaminergic polymorphic ventricular tachycardia

[0089] G protein-coupled receptors (GPCRs) are integral regulators of cellular signaling in both health and disease and are readily modulated by an array of molecular modalities, including small molecules, peptides, and hormones (7, 2). Accordingly, GPCRs serve as exemplary therapeutic targets and currently comprise more than 30% of all biological entities targeted by FDA-approved drugs (3). As fundamental mediators of the chronotropic and inotropic response in the heart, the [3- adrenergic receptor (PAR) subfamily of GPCRs remains one of the most extensively pursued cardiovascular disease targets. pARs are activated via binding of the catecholamine hormones epinephrine and norepinephrine to the orthosteric (i.e., endogenous) ligand binding site on the extracellular surface. In turn, signaling cascades mediated by heterotrimeric Gsand / or P-arrestin transducer proteins are initiated to positively regulate heart rate and contractile dynamics (4). While acute stimulation of PARs, particularly via the more abundantly expressed cardiac PiAR subtype, is an essential physiological response to support increased cardiovascular demand, chronic catecholamine signaling is exceedingly damaging to the heart and is associated with maladaptive morphological remodeling, cardiomyocyte apoptosis, fibrosis, lethal arrhythmias, and heart failure (4).

[0090] For decades, traditional orthosteric P-blockers (i.e., competitive antagonists targeting the endogenous ligand binding site of pARs) have been widely used to combat pathological Pi AR over-activation and are highly efficacious in decreasing morbidity and mortality in heart failure (5). However, adverse side effects such as fatigue, reduced exercise capacity, and the nonselective inhibition of other PAR subtypes, such as the P2ARS expressed in vascular and respiratory tissues, often restrict maximal effectiveness. These limitations are particularly applicable to the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease characterized by extreme susceptibility to lethal ventricular tachyarrhythmia that develops in direct response to catecholamines (6). To date, nonselective P- blockers such as nadolol and propranolol are the most effective front-line therapy for CPVT (7). However, achieving a maximally tolerated dose without major side effects remains a major challenge (5, S). Thus, CPVT, among other cardiac disorders, could highly benefit from the discovery of novel, efficacious PAR ligands with improved subtype selectivity for the Pi AR.

[0091] While most GPCR-targeting drugs, including P-blockers, bind the orthosteric site, a rapidly expanding approach to identify ligands with enhanced specificity, efficacy, and modulatory function is to target allosteric sites of the receptor (9, 10). Allosteric modulators bind to regions that are topographically distinct from the endogenous ligand binding pocket and can increase (positive allosteric modulator, PAM) or suppress (negative allosteric modulator, NAM) the activity of receptors stimulated by an orthosteric ligand. Given that allosteric sites of receptors are more prone to evolutionary divergence and thereby more structurally diverse compared to the orthosteric region that is often highly conserved amongst closely related receptors, allosteric modulators are more likely to be subtype-selective with less potential for off-target effects. Moreover, allosteric modulators typically do not possess robust intrinsic activity of their own since they do not directly bind the orthosteric site and should exert a minimal effect on receptor function in the absence of an orthosteric ligand, potentially enabling increased tolerance at higher doses. Together, these properties of allosteric modulators expose their potential therapeutic advantages relative to their classical orthosteric counterparts (9, 10).

[0092] Therefore, a DNA-encoded small molecule library (DEL) screen was conducted to identify allosteric modulators of the Pi AR possessing functionalantagonism for use as a therapeutic molecule for cardiac disease. Following multiple rounds of affinity selection screening >1 billion unique small molecules comprising the OpenDEL® library (HitGen, Inc.) against purified, functional, PiARs reconstituted in lipid nanodiscs, a chemical feature enrichment analysis was performed, and Compound 11 (Cl 1) was discovered to be a highly selective allosteric modulator of the PiAR. Here, the unique pharmacological and functional properties of Cl 1 for modulating PiAR-mediated signaling are demonstrated and show its therapeutic potential for the treatment of CPVT.Materials and Methods:Materials

[0093] The following orthosteric ligands for pARs or AT1R were purchased commercially: isoproterenol hydrochloride, epinephrine hydrochloride, norepinephrine bitartrate, carvedilol, metoprolol tartrate, ICI 118,551 hydrochloride, atenolol, angiotensin-II (Sigma-Aldrich, St. Louis, MO), dobutamine hydrochloride, carazolol (Cayman Chemical, Ann Arbor, MI), bucindolol (Santa Cruz Biotechnology, Dallas, TX), and alprenolol hydrochloride (Tocris Bioscience, Minneapolis, MN). BI- 167107 was synthesized as previously described (77). Heterotrimeric Gs(72), minimal cysteine P-arrestinl truncated at amino acid 393 (P- arrestinl-mc) (13, 14), nanobody 25 (Nb25) (75, 16), nanobody 35 (Nb35) (72), and antibody fragment 30 (Fab30) (77) were expressed and purified as previously described. The DNA-encoded small molecule library OpenDEL® was synthesized by HitGen Inc. (Chengdu, China) through a ‘split-and-pool’ approach. Off-DNA synthesis of hit compounds was performed and validated with HPLC / MS (>90% purity) by HitGen Inc.Cell culture

[0094] Human embryonic kidney (HEK) 293T cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a humidified tissue culture incubator at 37 °C and 5% CO2. For bioluminescence resonance energy transfer (BRET) and GloSensor™ (Promega, Madison, WI) cellular signaling assays, HEK293T cells were transiently transfected using Lipofectamine™ 3000 (Thermo Fischer Scientific, Waltham, MA) according to the manufacturer’s standard protocol. Expi293T™ suspension cells stably expressing FLAG-PiAR (18), FLAG-P1AR G413, or FLAG-P2AR (19) werecultured in Expi293 Expression Medium (Invitrogen) with 10 pg / mL blasticidin and 10 pg / mL zeocin in a humidified tissue culture incubator maintained at 37 °C and 8% CO2 under constant shaking.Receptor purification and generation of receptor nanodiscs

[0095] Human FLAG-PiAR and FLAG-P2AR were expressed, purified, and reconstituted in high-density lipoprotein (HDL) particles to generate nanodiscs as previously described (14, 18). Biotinylation of the nanodisc membrane scaffold protein D1E3 (MSPD1E3) was utilized to facilitate immobilization to neutravidin beads. Chimeric PiAR containing the phosphorylated COOH-terminal tail of the vasopressin 2 receptor (V2R) was generated as previously described for the P2V2RPP (14). Briefly, the sortase recognition sequence (LPETGHH) was inserted into the COOH-terminus of human FLAG-PiAR after amino acid G413 to generate FLAG- P1AR G413 (Fig. 9A-A’). Following expression and detergent solubilization, FLAG- P1AR G413 was ligated to the synthetic phospho-peptide corresponding to the COOH-terminal tail of the V2R (GGG-V2Rpp) via incubation with sortase to generate FLAG-P1V2RPP (Fig. 9A') (14). Prior to screening, reconstituted FLAG-PiAR and FLAG-P1V2RPP nanodiscs were validated by radioligand binding to ensure nanodiscs contained functional receptors (Fig. 9B-B').Radioligand competition binding

[0096] Radioligand competition binding experiments were performed as previously described (18). Briefly, purified PiAR, P1V2RPP, or P2AR nanodiscs generated as described above were incubated with 60 pM of the radiolabeled orthosteric antagonist, [I125]-cyanopindolol (I125CYP; 2200 Ci / mmol, PerkinElmer, Waltham, MA), and serial concentrations of unlabeled orthosteric ligand in binding buffer (20 mM HEPES pH 7.4, 100 mM NaCl) supplemented with 0.1% bovine serum albumin (BSA) and 1 mM ascorbic acid at room temperature for 2 hours to reach equilibrium. Non-specific binding was evaluated in the presence of 20 uM propranolol. To validate nanodisc preparations, serial concentrations of purified heterotrimeric Gs(10-320 nM) or P-arrestinl-mc (0.1-1 pM) were included in the binding reaction with PiAR or P1V2RPP nanodiscs, respectively, to confirm transducer cooperativity (Fig. 9B-B'). To evaluate the effect of allosteric modulators on orthosteric ligand binding to pARs, reactions included the indicated concentration of allosteric compound (0.05-30 pM) or an equivalent volume of vehicle (0.19% DMSO). Equilibrated binding reactionswere harvested by rapid filtration onto 0.3% polyethyleneimine (PEI)-soaked GF / B glass fiber filter paper (Brandel) and washed extensively with ice-cold binding buffer. I-CYP was detected with the WIZARD22-detector Gamma Counter (PerkinElmer) and the raw counts per minute (cpm) were normalized to the percent of maximal I- CYP binding. The mean ± SEM of at least 3 independent experiments performed in duplicate were plotted in GraphPad Prism and fit to a one-site binding model to retrieve IC50. Cl 1 -mediated log shifts in the nonlinear fit are presented as AIC50 (Vehicle - Cl 1) and statistical analysis was performed using a paired two-tailed t-test of raw IC50 values. Statistical comparisons of the AIC50 between Cl 1 and its analogs (Cl 1 A-I) were conducted by one-way ANOVA with Dunnett’s post-hoc test (GraphPad Prism).Validation of the screening protocol

[0097] To measure the immobilization efficiency of purified PiAR or P1V2RPP biotinylated nanodiscs, a fixed quantity of pre-washed high capacity neutravidin beads (Pierce) were incubated with increasing concentrations of nanodisc in binding buffer (20 mM HEPES pH 7.4, 100 mM NaCl) at room temperature for 1 hour while rotating. After collecting flow through, the nanodisc-coated beads were washed three times with binding buffer and bound protein was eluted via boiling at 95 °C. Samples were loaded onto 10% SDS-polyacrylamide gels for analysis by Coomassie Blue and a 1 : 1 ratio of receptor nanodisc (pg) to bead slurry (pL) was deemed optimal given minimal loss of nanodisc in the flow through (Fig. 10A).

[0098] To confirm the integrity of the G protein and P-arrestin complexes throughout the screening procedure, PiAR or P1V2RPP nanodiscs were immobilized as described above to pre-washed high-capacity neutravidin beads along with 20 pM of the high- affinity agonist, BI- 167107 (BI), and a 1.2 molar excess of heterotrimeric Gs or P- arrestinl-mc, respectively. To enhance complex stability, PiAR / Gs complexes were supplemented with Nb35 (2.5 molar excess with respect to PiAR) and 0.05 U / mL apyrase, while the PiV2Rpp / p-arrestinl complex was further stabilized with Nb25 and Fab30 (2.5 or 1.7 molar excess relative to P1V2RPP, respectively). Following complex formation, the flow through was collected and the beads were washed three times with ice-cold binding buffer supplemented with 10 pM BI. To simulate incubation with DNA-encoded molecules, Img / mL salmon sperm DNA (Ambion) was applied to neutravidin-immobilized PiAR / Gs and PiV2Rpp / p-arrestinl complexes in bindingbuffer containing 20 pM BI for 1 hour while agitating (1150 rpm). The secondary flow through was collected, bound protein was eluted via boiling at 95 °C, and samples were loaded onto 10% SDS-polyacrylamide gels for analysis by Coomassie Blue to ensure the stability of receptor-transducer complexes throughout the selection protocol (Fig. 10B-B').

[0099] Finally, to validate the retention of small molecule ligands to the PiAR or P1V2RPP nanodiscs during screening, 5 nM of the radiolabeled orthosteric antagonist [H3]-dihydroalprenolol (H3-DHA, 105 Ci / mmol, PerkinElmer) was incubated with neutravidin-immobilized nanodiscs and washed three times with ice-cold binding buffer. To elute, beads were resuspended in 1.5% Fos-choline (Anatrace) in water and incubated at 37 °C (15 min) then 95 °C (15 min) while agitating (1150 rpm). The elution procedure was repeated a second time, combined, and applied to 5 mL scintillation fluid (LefkoFluor) for overnight incubation. H3-DHA counts were obtained with a TriCarb 2800TR liquid scintillation counter (PerkinElmer). Empty nanodiscs were utilized as a control to assess non-specific binding (Fig. 10C).DNA-encoded small molecule library screening

[0100] Dried aliquots of OpenDEL® were resuspended in 50 pL water and incubated overnight at 4 °C to dissolve fully. Approximately 60 pg (~0.5 nmol) of PiAR or P1V2RPP nanodiscs were immobilized to pre-washed high capacity neutravidin beads along with transducer proteins (if applicable), 20 pM BI (if applicable) and conformation stabilizing reagents as described above in 500 pL of binding buffer. Complexes were incubated for one hour at room temperature while rotating and washed three times with 1 mL of ice-cold binding buffer containing 10 pM BI. Prior to library incubation, 1 pL (2%) of dissolved OpenDEL® was set aside for qPCR as input. The washed nanodisc-coated beads were resuspended in OpenDEL®, further diluted to 100 pL in ice-cold binding buffer supplemented with 1 mg / mL salmon sperm DNA and 20 pM BI, and incubated for 1 hour at room temperature while agitating (1150 rpm). To remove unbound molecules, beads were washed three times with 500 pL ice-cold binding buffer containing 1 mg / mL salmon sperm DNA and 20 pM BI. Samples were eluted twice in 53 pL water containing 1.5% Fos-choline while agitating at 37 °C (15 min) then 95 °C (15 min). The combined elution was applied to the QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany) and eluted in 70 pL water to isolate DNA-encoded molecules. Following DNA purification, 1.4 pL(2%) was set aside for qPCR analysis and the remaining elution was diluted to 100 pL in binding buffer containing 1 mg / mL salmon sperm DNA and 20 pM BI to apply as library input for a second round of affinity selection with freshly immobilized protein complexes. Following a second round of selection and purification of DNA-encoded molecules, samples were PCR amplified and subjected to next-generation DNA sequencing (HitGen Inc.) to decode binders. Of note, BI was omitted from wash buffers in the apo-PiAR and empty nanodisc conditions. All solutions were prepared in DNase / RNase free UltraPure distilled water (Invitrogen, Waltham, MA) and all centrifugation steps were performed at 3000 xg for 1 min to pellet beads.Determining library decay by qPCR

[0101] Aliquots of library input and elution collected as described above were diluted in qPCR sample buffer (10 mM Tris pH 8, 0.05% tween-20) and amplified along a standard curve of OpenDEL® reference library samples using 2X SYBR Green qPCR Mix (Thermo Fischer Scientific) according to the manufacturer’s protocol. Universal forward and reverse primers (OpenDEL®) were used to target the 5’ and 3’ ends of the DEL sequence which are identical across all molecules. Thermocycling was conducted as follows on a QuantStudio5™ (Applied Biosystems): 95 °C 10 min, ([95 °C 10s, 55 °C 10s, 72 °C 13s *collect signal] x 40 cycles). All solutions were prepared in DNase / RNase free UltraPure distilled water (Invitrogen) and each sample was performed in duplicate.Next-generation sequencing

[0102] The selection output was amplified by PCR using Q5 Hot Start High-Fidelity 2X Master Mix (NEB, M0494L). Amplicons were purified by QIAGEN-MinElute PCR Purification Kit (QIAGEN, 28006) and quantified using Qubit DNA High- Sensitivity kit (Invitrogen, 32854) before library construction and sequencing. The library preparation was performed with Nextflex Rapid DNA-Seq Kit (BI00 Scientific, 5144-08) following the manufacturer's manual and libraries were sequenced on the Illumina NovaSeq platform (Illumina, USA) by HitGen. After sequencing, samples were decoded and analyzed as previously reported (20), and the results were visualized in DataWarrior (OpenMolecules) with each dimension representing one cycle of DEL construction.Chemical feature enrichment analysis

[0103] The enrichment of a particular DEL chemical group (i.e., chemical feature) was assessed as previously described (20). Feature intensity enrichment scores were calculated as follows: sum of sequence counts for one feature divided by the average of the sum of sequence counts for all possible parallel features in the library. Chemical features that were highly enriched in the BLbound PiAR condition, potentially enriched in the apo-PiAR sample, and minimally present in the G protein or P-arrestin transducer complex conditions were selected as potential hits. Signals that were present in the empty nanodisc control were excluded, as well as imidazole- like binders. The structures of enriched features were further examined, and promiscuous features were cross-checked with the HitGen selection database (20).G protein dissociation assay (TRUPATH)

[0104] G protein dissociation was evaluated by the BRET-based TRUPATH assay as originally described in (21) with minor modifications. Briefly, 2.25 x 106HEK293T cells maintained in growth media were seeded in a 10 cm dish and incubated overnight. On the next day, cells were transfected with 0.75 pg of human FLAG- PiAR (or FLAG-p2AR), with GasS-RLuc8, Gp3, and G / 9-GFP at a 1 : 1 : 1 : 1 DNA ratio. For AT1R counter assays, 0.75 pg of human FLAG-AT1R (22) was transfected along with 0.75 pg of Gaq-RLuc8, GP3, and G / 9-GFP. After 24 hours, cells were trypsinized, resuspended in low-serum medium (IX MEM without phenol red, supplemented with 2% FBS, 1% HEPES, 1% anti-anti, 1% glutamine, and 1% P / S), and re-plated in a white, clear bottom 96-well assay plate at a density of 100,000 cells / well for overnight incubation. Prior to experimentation, low-serum media was aspirated and replaced with IX Assay Buffer (Hank’s Balanced Salt Solution, HBSS, supplemented with 20 mM HEPES) and incubated for 10 minutes at 37 °C. Cells were pre-treated with vehicle (0.19% DMSO) or 30 pM Cl 1 prepared in IX Assay Buffer for 20 minutes at 37 °C. To assess PiAR-mediated G protein dissociation, 100 nM ICL118,551 was included during pre-treatment to block activation of endogenous P2ARS. Cells were then stimulated with serial doses of isoproterenol for 10 minutes at 37 °C. BRET emission ratios (GFP / RLucII) were measured immediately following the addition of 5 pM coelenterazine 400a (Nanolight Technology, Norman, OK) on a Biotek Neo2 microplate reader (Agilent Technologies, Santa Clara, CA) using the 410nm (donor) and 515 nm (acceptor) filter pair. At least three independent experiments performed in duplicate were fitted to a log(agonist) vs response (three parameter) model in GraphPad Prism and baseline subtracted to assess net BRET ratio. Statistical analysis of the nonlinear curve fit (Emax) was evaluated by two-tailed t-test.GloSensor™ assay

[0105] The Giosensor™ assay (Promega) was performed as previously described with minor modifications (7S). Briefly, 2.25 x 106HEK293T cells maintained in growth media were seeded in a 10 cm plate and transfected 24 hours later with 25 ng of human FLAG-PiAR and 6 pg of the GloSensor cAMP biosensor plasmid (Promega). On the following day, cells were re-plated into a white, clear bottom 96- well plate at a density of 50,000 cells / well in low-serum medium (IX MEM without phenol red, supplemented with 2% FBS, 1% HEPES, 1% anti-anti, 1% glutamine, and 1% P / S) and incubated overnight. Prior to experimentation, low-serum media was aspirated and replaced with the Giosensor™ reagent (2 mM; Promega) prepared in IX Assay Buffer (HBSS with 20 mM HEPES) and incubated at room temperature for 1.5 hours. Cells were pre-treated with vehicle (0.19% DMSO) or 30 pM Cl 1 prepared in IX Assay Buffer for 20 minutes at room temperature, along with 100 nM ICI- 118,551 to block activation of endogenous P2ARS. Cells were then stimulated with serial doses of isoproterenol for 5 minutes and luminescent values were recorded using the Biotek Neo2 microplate reader (Agilent Technologies). The mean ± SEM of at least three experimental replicates performed in duplicate were fit to a log(agonist) vs response (three parameter) model in GraphPad Prism and normalized to the percent of vehicle maximum. Statistical analysis of the nonlinear curve fit (Emax) was evaluated by two-tailed t-test.BRET-based -arreslin recruitment and internalization assays

[0106] Evaluation of P-arrestin recruitment and internalization was performed using BRET-based biosensors as described below. FLAG-P1V2R and FLAG-P1V2R-RLUCII constructs were generated using HiFi DNA assembly (New England Biolabs, Ipswich, MA) according to the manufacturer’s guidelines. Fragment inserts of human Pi AR truncated at residue G413 (P1AR G413), the COOH-terminal tail of human vasopressin 2 receptor (V2R) encompassing amino acids 343-371 (V2R_343-371), and V2R 343-371 conjugated to RLucII were amplified by polymerase chain reaction (PCR) from pcDNA3 FLAG-PiAR (Addgene plasmid 14698; (23)) or V2R-RLUCII (agift from Dr. Sudar Rajagopal) using primers listed in Table 1, below.Table 1. Primer sequences utilized to clone BRET biosensors0107] Fragments consisting of pi AR truncated at residue G413 (P1AR G413) and V2R were amplified and inserted into a linearized pcDNA3 empty vector to generate P1V2R via HiFi DNA assembly. Fragments consisting of P1AR G413 and V2R- RLucII were amplified and inserted into a linearized pcDNA3 empty vector to generate pi V2R-RLucII via HiFi DNA assembly.

[0108] Gel-purified fragments were incubated with HiFi Master Mix (New England Biolabs) and inserted into a linearized pcDNA3 empty vector (Addgene plasmid 10792). Plasmids were transformed into TOP 10 E. coli (Thermo Fisher Scientific, Waltham, MA) and validated by DNA sequencing.

[0109] One day prior to transfection, 2.25 x 106HEK293T cells maintained in growth media were seeded in a 10 cm dish. For P-arrestin recruitment assays, cells were cotransfected with 2 pg FLAG-P1V2R-RLUCII and 1 pg P-arrestin2-eGFP (Addgene plasmid 35411; (24)), while P-arrestin internalization was evaluated via cotransfection of 2 pg FLAG-P1V2R (or FLAG-P2V2R or FLAG-AT1R for counter assays), with 1.5 pg P-arrestin2-RLucII, and 2.5 pg rGFP-FYVE (25). After 24 hours, cells were trypsinized, resuspended in low-serum medium (IX MEM without phenol red, supplemented with 2% FBS, 1% HEPES, 1% anti-anti, 1% glutamine, and 1% P / S), and re-plated at a density of 100,000 cells / well in a white, clear bottom 96-well plate for overnight incubation. Prior to experimentation, low-serum media wasaspirated and replaced with IX Assay Buffer (HBSS with 20 mM HEPES) and incubated for 10 minutes at 37 °C. Cells were pre-treated with vehicle (0.19% DMSO) or 30 pM Cl 1 prepared in IX Assay Buffer for 20 minutes at 37 °C. To evaluate BRET responses mediated by Pi AR only, 100 nM ICI-118,551 was included during pre-treatment to block activation of endogenous P2ARS. Cells were then stimulated with serial doses of isoproterenol for 20 minutes (recruitment) or 25 minutes (internalization) at 37 °C. BRET emission ratios (GFP / RLucII) were measured immediately following the addition of 5 pM coelenterazine 400a (Nanolight Technology) on a Biotek Neo2 microplate reader (Agilent Technologies) using the 410 nm (donor) and 515 nm (acceptor) filter pair. At least three independent experiments performed in duplicate were fitted to a log(agonist) vs response (three parameter) model in GraphPad Prism and baseline subtracted to assess net BRET ratio. Statistical analysis of the nonlinear curve fit (Emax) was evaluated by two-tailed t-test.FLIPR Ca2+Assay (HEK293T)

[0110] HEK293T cells were plated at a density of 50,000 cells / well in poly-D-lysine- coated 96-well black well plates and incubated overnight at 37 °C and 5% CO2. Media was removed from plates and the cells were incubated with Ca2+-sensitive fluorescent dye from the FLIPR Calcium 6 assay kit (Molecular Devices, San Jose, CA) for 2 hours according to the manufacturer’s instructions. To study the effect of C l 1 on the muscarinic receptor, cells were pre-treated with vehicle (0.19% DMSO) or 30 pM Cl 1 for 20 minutes. Fluorescence was measured with the FlexStation 3 microplate reader (Molecular Devices) for 2 minutes. Cells were stimulated with carbachol 20 seconds after starting fluorescence measurements. Baseline adjustments for each well were done by subtracting the average signal from the first 10 seconds of measurement. All Ca2+responses were quantified as baseline-adjusted area under the curve of the fluorescent signal and presented as percent DMSO maximum.Phospho-ERK assay

[0111] Evaluation of PiAR-mediated activation of extracellular signal-regulated kinase (ERK) was performed as previously described with minor modifications (7S). Briefly, 2.25 x 106HEK293T cells maintained in growth media were seeded in a 10 cm dish, incubated overnight, and transfected with 2 ug human FLAG-PiAR. After 24 hours, cells were re-plated in 6-well assay plates at a density of 7.5 x 105cells / well ingrowth media. On the final day, cells were starved for 3 hours in serum-free media (MEM supplemented with 0.1% BSA, 10 mM HEPES, and 1% P / S), pre-treated with vehicle (0.19% DMSO) or 30 pM Cl 1 for 20 minutes at 37 °C along with 100 nM ICI-118,551 to block endogenous P2ARS, and stimulated with serial doses of isoproterenol or carvedilol for 5 minutes. Cells were subsequently harvested in ice- cold lysis buffer (20 mM Tris pH 7.4, 137 nM NaCl, 20% glycerol, 1% Nonidet P-40, 2 mM sodium orthovanadate, 1 mM phenylmethyl sulphonyl fluoride, 10 mM sodium fluoride, 10 pg / mL aprotinin, 5 pg / mL leupeptin, and phosphatase inhibitors) and rotated for 30 minutes at 4 °C. Cell lysates were separated on a 10% SDS- polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Blocked membranes were probed with anti-p44 / 42 MAPK (Cell Signaling Technology, Danvers, MA) or anti-MAPK 1 / 2 (Millipore, Burlington, MA) primary antibodies, and horseradish peroxidase (HRP)-conjugated secondary antibodies (1 :3000; donkey anti-rabbit IgG, NA934V, and sheep anti-mouse IgG, NA931VS, Cytiva, Marlborough, MA). Following incubation with ECL chemiluminescent substrate (SuperSignal™, Thermo Fischer Scientific), immunoreactive bands were visualized with a ChemiDoc XRS+ imager (Bio-Rad, Hercules, CA). Densitometry of phospho-ERK was performed with ImageJ and normalized to total-ERK. The mean ± SEM of at least 3 technical replicates were plotted in GraphPad Prism and fit to a log(agonist) vs response (three parameter) model. Statistical analysis of the nonlinear curve fit (Emax) was evaluated by two-tailed t-test.Animal studies

[0112] All animal procedures were performed in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) and adhered to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University Medical Center. Cardiomyocytes were isolated from homozygous 12-16 week old C57BL / 6J wild-type, PiAR knock-out (PiAR' / _) (26, 27), or calsequestrin 2 (CSQ2A) (27, 28) mice, while in vivo intracardiac electrophysiology was performed with 8-12 week old CSQ2 / _mice. Genotypes were confirmed via PCR using previously described protocols (26-29).Cardiomyocyte isolation

[0113] Ventricular cardiomyocytes were isolated using standard Langendorff perfusion procedures (30). Mice were injected intraperitoneally with 200U of heparinand anesthetized under 3% isoflurane. Dissected whole hearts were placed immediately into perfusion buffer (120 mM NaCl, 14.8 mM KC1, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4-7H20, 10 mM HEPES, 4.6 mM NaHCO3, 30 mM taurine, and 5.6 mM glucose, pH 7.3) and cannulated through the aorta. Hearts were perfused in retrograde for 3 minutes with oxygenated perfusion buffer and then for ~8 minutes with digestion buffer containing 2.4 mg / mL collagenase (Worthington) at 37 °C. To terminate enzymatic digestion, ventricular tissues were transferred to perfusion buffer containing 10% calf serum with 12.5 pM CaCl2. Myocytes were dissociated by trituration and gradually brought to physiological Ca2+(1.2 mM).Contractility measurements

[0114] Cardiomyocytes isolated as described above were pre-treated with DMSO (0.3%) or Cl 1 (3-30 pM) for 20 minutes. Following pre-treatment, cells were stimulated with isoproterenol (0.01-1 pM) or left un-treated (basal condition) and plated immediately in a FHD rotational cell chamber (lonoptix, Westwood, MA) mounted on a Nikon Eclipse TE300 inverted microscope (40X 0.9 NA objective, MRF00400, Nikon). Myocytes were paced at 1Hz (20V, MyoPacer, lonoptix) and sarcomere length was recorded with lonWizard 7.2 using the MyoCam-S camera (lonoptix). Ten consecutive contractions per cell (7-10 cells per condition) were averaged for quantification of contractile magnitude and kinetics (lonWizard 7.2). Time intervals between consecutive contractions (i.e., peak-peak intervals) were measured during a representative five second recording in each cell. Interval variability plots were generated by plotting the peak-peak interval (n) against the peak-peak interval of the subsequent cellular contraction (n+1) for all cells in a particular treatment group. Only the myocytes that exhibited proper morphology (i.e., rod-shaped and striated) and were responsive to electrical stimulation were utilized for experimentation. Statistical comparisons between conditions were evaluated by one-way ANOVA with Tukey’s post-hoc test.Phospho-PLN and phospho-Tnl assay

[0115] Cardiomyocytes isolated as described above were pretreated with DMSO (0.3%) or 10 pM Cl 1 for 20 minutes at room temperature prior to stimulation with 10 pM isoproterenol for 15 minutes while rotating. Cells were pelleted via centrifugation and homogenized in a 1 : 1 mixture of urea-thiourea lysis buffer (8 M urea, 2M thiourea, 3% SDS, 0.05 M tris-HCl, 0.03% bromophenol blue, 0.075 M DTT, pH 6.8)supplemented with protease and phosphatase inhibitors (Halt, Protease and Phosphatase Inhibitor Cocktail #78440, Thermo Fisher Scientific, Rockford IL) and 50% glycerol at 60 °C as described in (37). Lysates were separated on a 15% SDS- polyacrylamide gel and immunoblotting procedures were conducted as indicated above. Membranes were probed the following primary antibodies: rabbit polyclonal antibodies targeting pPLN Serl6 (1 : 1000, 07-052, Millipore, Temecula, CA), pTnl Ser23 / 24 (1 : 1000, 4004S, Cell Signaling Technology, Danvers, MA), pPLN Thrl7 (1 : 1000, Arigo Biolaboratories, Taiwan) and Tnl (1 : 1000, 4002S, Cell Signaling Technology), or mouse monoclonal antibodies to PLN (ab2865 Abeam, Cambridge, MA). Densitometric analysis of phospho-PLN and phospho-Tnl was performed with Imaged and normalized to total PLN or Tnl, respectively. Each experimental condition was performed in duplicate and averaged per biological replicate. Statistical analysis was performed with one-way ANOVA and Tukey’s post-hoc test.Cardiomyocyte Ca2+imaging

[0116] To conduct live cell Ca2+imaging, ventricular myocytes isolated as described above were loaded with 10 pM CAL-520 (ab 171868, Abeam) for 1 hour in IX Ca2+- free Tyrode solution. Cells were then incubated with a 1 : 1 solution of RPMI / B27+ [RPMI 1640 (11875199, Thermofisher Scientific) with 2% B27 with insulin (17504044, Life Technologies)] and IX Tyrode with 1.8mM CaCh for 20 minutes prior to imaging. Cells were pretreated with DMSO (0.3%) or 10 pM Cl 1 for 20 minutes and plated onto 200pg / ml laminin (L2020, Sigma-Aldrich) coated coverslips. Line scans were acquired on a Zeiss Laser Scanning Confocal 510 Meta Microscope (Carl Zeiss AG) at 0.1pm per pixel along the longitudinal axis of cardiomyocytes. Cells were paced at 0.5Hz with an lonOptix MyoPacer field stimulator (lonOptix) for ten seconds with a total imaging time of 34 seconds per cell. Fiji Imaged v.1 ,53c (National Institutes of Health) was used to analyze Ca2+transients. Cells were scored as having sustained spontaneous calcium release events (SREs) if there were greater than 10 SREs less than 500ms apart. For caffeine-induced Ca2+transients, cells were paced at 0.5 Hz for 10 seconds and then treated with 10 mM caffeine (10 seconds after termination of pacing) on a Zeiss spinning disk Axio Observer.Zl (40X, 215ms intervals). All Ca2+imaging experiments were performed at room temperature.High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) Analysis

[0117] Stability analysis of Cl 1 was performed on a 6224 TOF LC / MS system (Agilent Technologies), consisting of a 1200 HPLC (degasser, binary pump, thermostated column compartment, diode array detector (DAD)) coupled to a 6224 accurate-mass time-of-flight mass spectrometer. The mass spectrometer was equipped with a Dual ESI source, and accurate mass data was obtained by internal calibration (reference ion 922.009798 m / z) using a secondary nebulizer to deliver the reference solution continuously. Positive-ion mass spectral data were acquired in full-scan mode over the range of 75-3200 m / z using the following source parameters: gas temperature 325 °C, gas flow 11 L / min, nebulizer pressure 33 psig, VCap 3500 V, and fragmented voltage 150 V. Aliquots of 10 mM Cl 1 formulated in a mixture of DMSO / PEG400 (50% / 50%, v / v) were incubated at 37 °C and withdrawn at different time points (0, 1, 6, 12, 24, 48, and 72 hours), flash-frozen in liquid nitrogen, and afterward transferred into a -80 °C freezer until HPLC-MS analysis. All Cl 1 samples were first diluted with 50% acetonitrile in ddH2O to obtain 250 pM solutions. Subsequently, HPLC separations were achieved on an Agilent Zorbax SB-C18 column (2.1 x 150 mm I.D., particle size 3.5 pm) using a linear gradient of mobile phase B in A, a flow rate of 0.5 mL / min. Mobile phase A was prepared by combining 400 mL ultrapure water with 12 mL methanol and 1.2 mL formic acid. Mobile phase B was prepared by mixing 400 mL acetonitrile with 12 mL ultrapure water and 1.2 mL formic acid. The gradient program included an initial hold at 0% solvent B for 0.5 min, followed by a linear increase to 100% solvent B from 0.5-8 min, hold at 100% solvent B from 8.1-9 min, and re-equilibration back to 0% B for a total run time of 15 min. Samples were analyzed using a 1 pL injection volume. Target compound (Cl 1) was confirmed by mass spectral data in positive ion mode, and U.V. spectra peaks (254 nm) were integrated to determine values for relative content and %-area purity (relative to t = 0 as 100% and then plotted as a function of time). Little to no impurities were detectable in Cl 1. Cl 1 had a purity of over 95% with a molecular ion peak, [M+H] at 561.34323 m / z.Pharmacokinetics of Cll in mice

[0118] Male CD-I mice (n=4; average body weight 28 g) were injected intraperitoneally with 10 mg / kg of Cl 1 as 100 pL formulation containing 10% DMA, 40% PEG-300, 2% Tween 80, and 48% saline. For plasma, whole blood (~30 pL) was collected serially (“tail snip”) at 5, 15, 30 minutes, 1, 3, 8, and 24 hours into vialscontaining 1 pL of 75 mg / mL K2EDTA in water, and immediately frozen until the day of analysis. Heart tissue was harvested from 3 mice per time-point (30 minutes, 1 hour, and 3 hours) without saline perfusion. For liquid chromatography tandem-mass spectrometry (LC / MS / MS) analysis, 10 pL of each plasma sample was mixed with 20 pL of methanol / chloroform (1 : 1) fortified with 20 ng / mL Cl 1-A (internal standard) and vigorously agitated in FastPrep FP120 apparatus (Thermo-Savant) at speed 4 for 45 seconds. After precipitation at -20 °C for 15 min and centrifugation at 14,000 xg for 5 min at room temperature, 20 pL of supernatant was mixed with 20 pL of mobile phase A (see below), and 5 pL injected into LC / MS / MS system. For heart tissue analysis, the sample was homogenized with 3 parts water and 100 pL homogenate mixed with 10 pL of 20 ng / mL Cl 1-A and 200 pL chloroform. After agitation and centrifugation, 150 pL of organic (lower) layer was evaporated to dryness (nitrogen stream), reconstituted with mobile phase A / mobile phase B (1 : 1) and 10 pL injected into LC / MS / MS system. LC / MS / MS (Agilent 1200 series HPLC and Sciex / Applied Biosystems API 5500 QTrap) was utilized to quantify Cl 1. Analytical column: Agilent Eclipse Plus (Cis, 1.8 pm, 50 x 4.6 mm), at 40 °C. Mobile phase: (A) 0.1% formic acid, 2% acetonitrile in water, (B) acetonitrile. Isocratic elution: 30% A, 70% B. Run time: 2 min. Mass spectrometer parameters (voltages, gas flow, and temperature) were optimized by infusion of 100 ng / mL of analytes in mobile phase at 10 pL / min using Analyst 1.6.2 software tuning module. The MS / MS (m / z) transitions used for quantification: 561.2 / 353.1 (Cl 1), 575.4 / 353.1 (Cl l-A; internal standard). A set of calibrator samples in drug-free matrix was prepared by adding appropriate amounts of pure analyte (Cl 1) in 0.243 - 100 ng / mL range. The calibration samples were analyzed alongside the experimental samples. Accuracy acceptance criteria was 85% for each but the lowest level (80%, LLOQ = 0.243 ng / mL). Non-compartmental approach within WinNonlin (2.1) software was used for modeling of concentration / time data to calculate relevant pharmacokinetic parameters.Intracardiac electrophysiology

[0119] CSQ2 'Amice were pretreated with an intraperitoneal injection of vehicle solution (10% dimethylacetamide, 40% PEG300, and 2% Tween80 in 48% saline) or 10 mg / kg Cl 1 (blinded) for 45 minutes. Mice were anesthetized using a volatile anesthetic system with induction chamber (R5835, RWD Life Science, Dover, Delaware) with 2% isofluorane mixed with 2L / min 100% 02. Subdermal leads wereplaced for surface electrocardiographic (ECG) analysis which includes two total leads: I and II. ECGs were recorded at 1000 samples / second. When mice reached internal temperature of 37 °C, jugular venous cutdown was performed using a dissection microscope (A60, Leica, Buffalo Grove, Illinois) and an 8-lead 1.1F, 8E, 1.0mm octapolar electrophysiology catheter (iWire-BIO8, ADInstruments, Colorado Springs, Colorado) was placed in the internal jugular vein and positioned in the right ventricle and right atrium (32). After the cutdown, isofluorane was reduced to 1.5%. Electrical pacing was performed utilizing customized electrical stimulator to elicit ectopy and arrhythmia generation with progressive electrical challenge (33). Induced ventricular arrhythmias were tested using this electrical stimulation. Following ventricular pacing, mice were injected intraperitoneally with 3mg / kg isoproterenol and observed for spontaneous ventricular arrhythmias. Ventricular tachycardia was scored as greater than 2 ventricular ectopic beats in a row. Ventricular pacing was then repeated. Rhythm detection was captured by an iWorx-RA-834 Eight Channel 16bit Data Acquisition System (iWorx, Dover New Hampshire, United States). Data was viewed using a custom-built ECG Analysis Module software program for LabScribe v4. Only the mice with a baseline heart rate between 350 - 500 bpm were included in analysis. Statistical comparisons of the proportion of mice in each treatment group exhibiting sustained ventricular tachycardia was assessed by Fisher’s Exact test and heart rate analysis by two-way ANOVA (GraphPad Prism).Statistical analysis

[0120] Statistical significance was determined by two-tailed t-test or one-way ANOVA with Tukey’s post-hoc test for multiple comparisons unless indicated otherwise. Error bars represent mean ± SEM. Sample sizes are indicated in the corresponding figure legends.ResultsDiscovery of Compound 11 (Cll) through DNA-encoded small molecule library screening

[0121] To discover allosteric modulators of the PiAR with pharmacological and functional properties suitable for potential use as a therapeutic, a DEL screen was conducted using purified PiARs reconstituted in lipid nanodiscs mimicking the native membrane environment. To enable the discovery of molecules that target unique conformational states of the PiAR, five different conditions were screened: PiARbound to the high-affinity agonist BI-167107 (BI), Bl-bound PiAR in complex with either heterotrimeric Gsor P-arrestinl, and the empty nanodisc and un-liganded PiAR (apo-PiAR) controls (Fig 1 A). Chimeric PiARs harboring the phosphorylated COOH- terminal tail of the V2R (B1V2RPP) were engineered to convert PiAR to a Class B GPCR, thereby strengthening P-arrestinl complex stability (34) (Fig. 1A). Transducer complexes were further reinforced via conformation stabilizing antibody fragments (i.e., Nb35, Nb25, Fab30; Fig. 1 A). Prior to the screen, nanodiscs were functionally validated via radioligand binding (Fig. 9B-B') and the affinity selection protocol was optimized as described in Materials and Methods and Fig. 10.

[0122] To facilitate the purification of small molecule PiAR binders from the >1 billion unique compounds comprising the OpenDEL® library, nanodisc PiAR complexes were immobilized to neutravidin beads via biotinylation of the nanodisc membrane scaffold protein, MSPD1E3 (Fig. IB). Following two consecutive rounds of affinity selection, eluted molecules were purified, PCR amplified, and subjected to high-throughput next-generation sequencing to decode binders (Fig. IB). The decay of library molecules throughout each round of selection was monitored via qPCR using a universal primer set that amplifies all molecules in the library (Fig. 1C-C’). In each experimental condition, approximately IxlO7molecules were collected in the final elution from the IxlO15molecules applied as input (Fig. 1C-C’).

[0123] Putative hit molecules were identified via comprehensive bioinformatics analysis of the chemical structure similarities among molecules present in the decoded dataset. Enriched chemical features that aligned with the scenario of interest (i.e., an unbiased negative allosteric modulator) were evaluated based on their abundance (or absence) in a particular experimental condition. Using this filtering criteria, a family of compounds sharing a common partial structure was discovered (i.e., R2 and R3) that was significantly enriched in the Bl-bound PiAR and apo-PiAR conditions, minimally present in the Gsand P-arrestinl samples, and completely absent in the empty nanodisc control (Fig. 1D-D’). From this family, Compound Cl 1 (Cl 1) was selected for further characterization off-DNA given its potential as a PiAR negative allosteric modulator (Fig. IE).Cl 1 potentiates the binding affinity of agonists and certain antagonists to the ffiAR

[0124] To interrogate the pharmacological properties of Cl 1 on the PiAR, the ability of Cl 1 to modulate orthosteric ligand binding affinity via radioligand competition binding experiments was first investigated. Purified PiAR nanodiscs were incubated with the radiolabeled PiAR antagonist,125I-CYP, and serial doses of un-labeled orthosteric PiAR agonists or antagonists. In the presence of Cl 1, the binding affinity of norepinephrine, isoproterenol, dobutamine, and epinephrine was significantly enhanced to the PiAR evidenced by a ~0.4-log leftward shift and corresponding -2-3- fold decrease in the IC50 of the competition binding curve compared to vehicle control (Fig. 2A-A”), demonstrating positive cooperativity between Cl 1 and PiAR agonists. Interestingly, Cl 1 exhibited a probe-dependent effect with respect to orthosteric antagonist binding to the PiAR since it potentiated the binding of certain antagonists or biased ligands (i.e., carvedilol, bucindolol, alprenolol, and atenolol; up to 0.5-log leftward shift and ~3-fold IC50 decrease) without affecting metoprolol or carazolol binding affinity (Fig. 2A’-A”). This indicates that the effect of Cl 1 on orthosteric ligand binding to the PiAR is unique to the ligand bound.

[0125] To estimate the affinity of Cl 1 for the PiAR, the Cl 1-mediated dosedependent increase in isoproterenol binding to the receptor (quantified as AIC50 of the competition binding curve) was plotted as a function of increasing doses of Cl 1 (Fig. 11 A-A"). The resulting LogECso (-6.12, -0.76 uM) derived from the non-linear fit revealed low micromolar affinity between Cl 1 and the PiAR (Fig. 11 A-A").Cl 1 suppresses G protein and f-arrestin signaling downstream of agonist- activated fiiAR

[0126] Given the positive cooperativity between Cl 1 and orthosteric agonist binding to the PiAR, it was anticipated that Cl 1 would enhance PiAR downstream signaling functioning as a PAM. The functional impact of Cl 1 on G protein and P-arrestin signaling in response to PiAR activation was assessed via BRET-based and / or luciferase-based cellular signaling assays. To measure dissociation of GasPy upon PiAR stimulation, HEK293T cells transiently overexpressing PiAR with TRUPATH biosensor proteins Gas-RLuc8, Gp3, and G / 9-GFP (27) were pre-treated with vehicle (DMSO) or 30 pM Cl l and stimulated with serial doses of isoproterenol (Fig. 3 A). Surprisingly, despite its positive cooperativity on agonist binding, Cl l substantially reduced maximal G protein dissociation (Fig. 3A’-A”) evidenced by an attenuation of the BRET signal decay. To measure Gas-mediated signaling downstream of agonist-activated PiARs, intracellular cAMP generation was quantified utilizing the luciferase-based GloSensor™ cAMP biosensor (Fig. 3B). Consistent with TRUPATH results, pre-treatm ent with Cl l significantly diminished maximal cAMP generation compared to vehicle-treated cells (Fig. 3B’-B”).

[0127] Utilizing similar BRET-based approaches, the effect of Cl 1 on the various functions of P-arrestin was evaluated. To measure P-arrestin recruitment to agonist- activated PiAR, a BRET sensor pair was developed, consisting of P-arrestin2-GFP and a chimeric PiAR containing the COOH-terminal tail of the V2R (P1V2R) conjugated to RLucII (Fig. 3C) that was utilized to enhance P-arrestin affinity to agonist occupied PiAR. To measure P-arrestin-mediated receptor internalization, P1V2R was co-expressed with P-arrestin2 -RLucII and the early endosomal marker FYVE-rGFP (25) (Fig. 3D). Strikingly, pre-treatment with Cl l induced a robust decrease in both P-arrestin recruitment to P1V2R (Fig. 3C-C”), and P-arrestin- mediated receptor internalization into endosomes (Fig. 3D’-D”). These findings indicate that Cl l acts as an unbiased functional NAM of agonist-activated PiAR since it potently inhibits both Gs and P-arrestin signaling.

[0128] Given that ERK is one of the major cellular effectors of both Gs and P-arrestin signaling cascades, the effect of Cl 1 on PiAR-mediated ERK phosphorylation was next evaluated via immunoblotting. Consistent with BRET-based assays, HEK293T cells transiently overexpressing PiAR displayed a dose-dependent increase in ERK phosphorylation in response to serial doses of isoproterenol that is suppressed in cells pre-treated with Cl l (Fig. 4A-A”). Notably, ERK phosphorylation stimulated by carvedilol, a P-arrestin biased PAR ligand that has been previously shown to promote ERK phosphorylation in a P-arrestin dependent manner (18, 35-37), was also attenuated in the presence of Cl 1 (Fig. 4B-B”). This indicates that Cl l serves as a functional NAM of the PiAR irrespective of the nature of the orthosteric ligand (i.e., full agonists versus biased ligands).

[0129] Taken together, in striking opposition to the positive cooperativity of agonist binding, cellular signaling assays revealed that Cl l is a potent inhibitor of PiAR- mediated Gasand P-arrestin signaling. With such a unique pharmacological and functional profile, studies herein indicate that Cl l likely belongs to a recently established class of allosteric modulators termed PAM-antagonists that potentiate agonist affinity to receptors while antagonizing downstream signaling (38). Whilelargely under-characterized to date, PAM antagonists are predicted to be especially favorable therapeutically due to their positive cooperativity with agonists (38).The antagonist function of Cl 1 is highly selective for the fiiAR subtype

[0130] The pharmacological selectivity of Cl 1 for the Pi AR was next evaluated by interrogating its effect on orthosteric ligand binding to the P2AR subtype. Radioligand binding experiments utilizing P2AR nanodiscs demonstrated that Cl 1 induced a statistically significant, albeit modest, leftward shift (<0.08-log shift; 1.2-fold IC50 decrease) in the isoproterenol binding curve compared to vehicle (Fig. 5A-A”), whereas carvedilol binding to the P2AR is potentiated by ~2.6-fold (~0.4-log leftward shift) in the presence of Cl 1 (Fig. 5A’-A”). These data indicate that Cl 1 also binds and modulates ligand binding to the P2AR, given that it potentiates carvedilol binding to both subtypes, but does not elicit a considerable increase in affinity between P2AR and the agonist isoproterenol. Of note, this PAR subtype-dependent activity is reminiscent of the recently discovered P2AR positive allosteric modulator, Compound 6, that potentiates carvedilol binding to both PiAR and P2AR while selectively increasing agonist binding to the P2AR only (18, 39, 40).

[0131] To determine the functional selectivity of Cl 1 for the Pi AR, various BRET- based counter assays were performed, interrogating G protein and P-arrestin signaling stimulated by alternative GPCRs of the cardiovascular system including the P2AR, AT1R, and M3R. Importantly, Cl 1 did not significantly impact G protein dissociation or P-arrestin internalization downstream of either agonist-activated P2AR (Fig. 5B-C’) or AT1R (Fig. 5D-E’). Moreover, Cl 1 had no effect on intracellular Ca2+release downstream of carbachol-activated endogenous M3Rs (Fig. 5F-F”). Together, these experiments indicate that the functional effects of Cl 1 are highly selective for the PiAR subtype.Cl 1 is a superior PAM antagonist relative to structurally related OpenDEL® analogs.

[0132] In order to gain mechanistic insights on the chemical features that support the pharmacological and functional effects of Cl 1 on the PiAR and to evaluate whether small modifications in its structure would confer altered efficacy as a PAM antagonist, a panel of Cl 1 analogs (Cl 1-A through Cl 1-1; Figs. 12A-12C) comprising additional members of the enriched family of structurally related OpenDEL® molecules detected via bioinformatics analysis (Fig. 1D-D’; Cl 1-A through F and H)as well as several truncated versions (Cl 1-G and I) was characterized and their effects on ligand binding and PiAR-mediated signaling was evaluated. The ability of Cl 1 to potentiate agonist and antagonist binding to the PiAR was largely unaffected by modifying the R1 chemical group, given the lack of substantial differences in the IC50 shifts between Cl 1 and analogs A-F and H (Fig. 14A-A"). Remarkably, truncation of the extended carbon chain comprising the R3 chemical group in Cl 1 and Cl 1-H (generating Cl 1-G and Cl 1 -I, respectively) resulted in complete loss of the positive cooperativity of isoproterenol and carvedilol binding to the PiAR (Fig. 13A-A'"). These results are corroborated by evaluation of Gasdissociation and P-arrestin recruitment to the PiAR via BRET, wherein the R3-truncated Cl 1-G and Cl 1-1 analogs completely lose the antagonistic functions of Cl 1 (Fig. 13B-B") while the remaining analogs that vary in the R1 group remain functional antagonists to a similar or lesser extent than Cl 1. Together, these data demonstrate that the identity of the R1 chemical group is largely exchangeable, while the R3 extended carbon chain is necessary for its function. These findings provide several mechanistic insights into the precise chemical features that mediate the activity of Cl 1 and indicate that Cl 1 is a superior PAM antagonist relative to a panel of structurally related molecules selected from the OpenDEL® library. Lastly, Cl 1-G, the R3 -truncated form of Cl 1, is revealed as a complete Toss-of-function’ analog.Cl 1 reduces basal contractility and suppresses the isoproterenol response in isolated wild-type cardiomyocytes

[0133] Following comprehensive pharmacological and cellular characterizations, the impact of Cl 1 on cardiac signaling and function in primary cardiomyocytes expressing endogenous levels of the PiAR was investigated. Ventricular cardiomyocytes isolated from wild-type mice were pre-treated with DMSO or serial doses of Cl 1 and paced at 1 Hz. Pre-treatment with 30 pM Cl 1 induced a significant reduction in basal fractional shortening compared to cells pre-treated with DMSO (Fig. 6 A- A’; Table 2, below).Table 2. Effect of serial doses of Cl 1 on contractility in isolated cardiomyocytes.0134] Contractility parameters were measured from isolated wild-type or B1AR- / - cardiomyocytes during 1 Hz pacing (n= 4-7 hearts; 7-10 cells per treatment per heart). Values are represented ± SEM. SL, sarcomere length.

[0135] To evaluate whether this inhibitory effect was specific to Cl 1 and not a non- selective consequence of its high-dosage (up to 30 pM), the loss-of-function analog, Cl 1-G, was employed that does not modulate PiAR-mediated signaling (Fig. 13A- B"). Here, pre-treatment with 30 pM Cl 1-G did not affect basal contractility in wildtype cardiomyocytes compared to vehicle control indicating a direct effect of Cl 1 on cardiomyocyte contractility (Fig. 6A-A’; Table 2). To assess whether the Cl 1- mediated reduction in basal contractility results from direct modulation of the PiAR, ventricular cardiomyocytes isolated from i AR" mice were pre-treated with DMSO or serial doses of Cl 1. Importantly, 10 pM Cl 1 showed little effect on basal contractility while 30 pM Cl 1 also elicited a significant reduction in basal contractility in Pi AR" cardiomyocytes (Fig. 6B), albeit to a lesser extent than in wildtype (Fig. 6A-A’). These data suggest that the Cl 1 -mediated suppression of basal contractility at high dose (i.e., 30 pM Cl 1) has partial off-target effects, while 10 pMCl 1 shows little off-target effect. Therefore, 10 pM Cl 1 was chosen for subsequent functional and signaling assays in isolated cardiomyocytes.

[0136] To determine the effect of Cl 1 on the isoproterenol-induced contractile response, isolated wild-type cardiomyocytes were stimulated with serial doses of isoproterenol in the presence or absence of 10 pM Cl 1. In line with its ability to suppress Gasand P-arrestin signaling downstream of agonist-activated Pi AR, Cl 1 substantially suppressed the dose-dependent increase in fractional shortening and contractile kinetics mediated by isoproterenol (Fig. 6C-D’). To corroborate this finding biochemically, representative Ca2+cycling or sarcomeric cellular effectors of PiAR activation were selected, namely phospholamban (PLN) and troponin I (Tnl), and assessed their phosphorylation status in isolated cardiomyocytes in the presence of isoproterenol via immunoblotting. Pre-treating wild-type cardiomyocytes with 10 pM Cl 1 robustly suppressed the isoproterenol-induced phosphorylation of PLN and Tnl at the canonical protein kinase A (PKA) phosphorylation sites (pPLN Seri 6 and pTnl Ser23 / 24) as well as Thrl7 mediated by Ca2+ / calmodulin protein kinase II (CaMKII) on PLN (Fig. 6E-E’), providing further evidence of Cl l’s ability to block PiAR-mediated signaling in the heart.Cl 1 restores regular contractile rhythm and suppresses spontaneous Ca2+release in CSQ2~ / ~ cardiomyocytes

[0137] Given the robust inhibition of the isoproterenol response in cardiomyocytes pre-treated with Cl 1, its potential as a therapeutic molecule for CPVT was next evaluated utilizing cardiomyocytes from mice that are constitutively null for CSQ2 (28). Previous studies have demonstrated that CSQ2'Amice are highly susceptible to catecholamine-induced arrhythmia in the form of frequent premature ventricular contractions (PVCs), increased heart rate variability, and increased diastolic Ca2+leak due to spontaneous Ca2+release events (28). Consistent with this, isolated ventricular cardiomyocytes from CSQ2 ’ ’ mice developed an arrhythmic-like phenotype when stimulated with isoproterenol during 1 Hz pacing, evidenced by a robust increase in both the frequency of contractions and the variability in the time interval between consecutive beats that is reminiscent of the irregular contractile rhythms observed in vivo (Fig. 7A-B) (28). Remarkably, application of 10 pM Cl l completely attenuated isoproterenol-mediated spontaneous cellular beating and restored regular contractile frequency in CSQ2 ’ ’ cardiomyocytes (Fig. 7A-B). To further corroborate theprotective effect of Cl 1 in CSQ2 / _cardiomyocytes, spontaneous Ca2+release events elicited by isoproterenol immediately following termination of 0.5 Hz pacing were measured. In line with the blockade of isoproterenol-induced spontaneous contractile activity in CSQ2’ ’ cardiomyocytes (Fig. 7A-B), pre-treatment with 10 pM Cl l also prevented the development of spontaneous Ca2+release events following isoproterenol stimulation (Fig. 8A-B). Of note, 10 pM Cl l had no significant effect on the amplitude or kinetics of paced Ca2+transients in CSQ2 / _cardiomyocytes (Fig. 14A-B"), nor did it affect caffeine-induced Ca2+release in wild-type cells (Fig. 15A- A'), indicating that Cl l has no direct impact on baseline Ca2+signaling.Cll blocks the development of isoproterenol-induced ventricular tachycardia in vivo

[0138] To evaluate the therapeutic potential of Cl 1 in vivo, the cardiac electrical activity of vehicle- or Cl 1 -treated CSQ2 ’ ’ mice was monitored via intracardiac ECG. It was first confirmed that Cl 1 is stable long-term at 37 °C in vehicle solution via HPLC / MS (Fig. 16A). The pharmacokinetic profile of Cl 1 following intraperitoneal injection (10 mg / kg) was also evaluated and it was determined that Cl l is detectable in plasma and heart at the highest level between 45 minutes to 1-hour post-injection (Fig. 15B). Guided by pharmacokinetic findings, CSQ2 / _mice were pre-treated for 45 minutes with either vehicle solution or 10 mg / kg Cl l and the incidence of spontaneous arrhythmic events induced by isoproterenol (3 mg / kg) was compared. Remarkably, while -50% of the vehicle-treated CSQ2 ’ ’ mice developed episodes of sustained ventricular tachycardia, evidenced by consecutive ectopic beats and bidirectional QRS waveforms, CSQ2 / _mice pre-treated with 10 mg / kg Cl l retained normal electrical rhythm after isoproterenol stress (Fig. 8C-C’). Notably, while completely suppressing isoproterenol-induced spontaneous ventricular arrhythmia, Cl l treatment had no effect on the isoproterenol -mediated increase in heart rate (Fig. 8C”). This important observation may suggest that Cl l would not significantly suppress the physiological heart rate response to catecholamines, which is a major limitation of classical orthosteric P-blockers. Taken together, these results demonstrate the therapeutic capability of Cl 1 in blocking pathological Pi AR overactivation in the heart and underscore its potential as a new class of drug to block thePiAR in disease states.Discussion

[0139] Herein, the discovery of the PiAR-selective allosteric modulator, Cl l (and its analog molecules), via DEL screening and comprehensively interrogate its unique pharmacological properties, functional effects, and therapeutic applications in the heart is reported. Cl l was revealed to bind to the Pi AR with sub-micromolar affinity and potentiates the binding of agonists and certain antagonists to the PiAR in a subtype-specific fashion. Remarkably, in contrast to its positive cooperativity with orthosteric agonists, Cl l potently decreases agonist-activated PiAR signaling, suppresses the isoproterenol response in isolated cardiomyocytes, and completely blocks the development of spontaneous contractile and Ca2+release events in a model of CPVT. Most importantly, treatment with Cl l completely prevents isoproterenol- induced ventricular tachycardia in CPVT mice. Cl l therefore represents a promising therapeutic molecule for the treatment of cardiac disease, illustrated herein in the context of CPVT.

[0140] CPVT is an inherited arrhythmic disorder characterized by the heightened susceptibility to catecholamine-induced polymorphic or bidirectional ventricular tachycardia initiated by exercise or emotional stress in the absence of structural heart disease (6). The genetic basis is commonly associated with mutations in proteins of the sarcoplasmic reticulum (SR) Ca2+release complex (i.e., RyR2 or CSQ2), leading to SR Ca2+overload, spontaneous Ca2+release due to hyperactive RyR2, and arrhythmogenic Ca2+-induced delayed after depolarizations potentially culminating in sudden cardiac death (6, 47). CPVT is a severe life-threatening disorder with mortality rates ranging from 30-50% in untreated patients by the age of 40 (42). [3- blockers such as nadolol are currently the most efficacious therapy for CPVT and have been successful in decreasing arrhythmic risk and severity (7, 43). However, [3- blockers are not completely effective as recurrent arrhythmic events occur in up to 37.2% of treated patients, with 15.3% being near-fatal and 6.4% fatal during an 8-year follow up period after beginning treatment (44). A major obstacle that remains is achieving a maximally tolerated dose due to adverse effects in vascular tissues (i.e., hypotension) where the P2AR is highly expressed (5, S). Moreover, cardio- selective PAR antagonism would be especially optimal in the presence of respiratory disorders such as asthma or COPD. As a PiAR-selective allosteric modulator, Cl l has the potential to be used at a therapeutically effective dose with minimal off-target concerns.

[0141] While the therapeutic potential of Cl 1 utilizing a model of CPVT where the primary pathological mechanism is directly related to PiAR activation is demonstrated herein, Cl 1 could be applied as a treatment for a wide range of cardiac diseases (i.e., hypertrophic and dilated cardiomyopathy) or arrhythmic disorders (i.e., long QT syndrome, arrhythmogenic cardiomyopathy, atrial fibrillation) that are aggravated by excessive sympathetic stimulation and where P-blockers have been previously shown to be beneficial (7). It is also interesting to speculate that Cl 1 could possibly modulate the activity of PiAR-activating autoantibodies that develop during chronic heart failure (45-47), and in conditions where enhanced sympathetic activity results in high synaptic catecholamine release such as exertional angina and postural orthostatic tachycardia syndrome. An important consideration in the broad clinical use of Cl 1 would be the probe dependence of its modulatory effects on distinct orthosteric P-blockers. Studies herein have shown that Cl 1 potentiates the affinity of certain antagonists to the PiAR such as the P-arrestin-biased ligand carvedilol and suppresses carvedilol-mediated ERK activation. It remains to be determined whether Cl 1 also diminishes the antagonist function of carvedilol on G protein signaling or whether Cl 1 potentiates the inhibition. This would influence whether a patient could be treated simultaneously with Cl 1 and orthosteric P-blockers such as carvedilol or whether an antagonist like metoprolol that is unaffected by Cl 1 could be used in combination.

[0142] The unique pharmacological and functional profile exhibited by Cl 1 shows features similar to a recently established entity of allosteric modulators termed PAM- antagonists that increase the affinity but decrease the efficacy of the agonist (38). There are several major advantages to this mechanism of signaling blockade relative to traditional orthosteric antagonists like P-blockers. Specifically, in contrast to orthosteric antagonists and even canonical NAMs that act competitively with the agonist by favoring an inactive receptor conformation, the affinity and efficacy of a PAM-antagonist is potentiated in presence of increasing agonist due to cooperativity. Moreover, PAM-antagonists such as Cl 1 are further advantageous in that they have a higher propensity to bind agonist-bound receptors, thereby preferentially targeting pre-existing pathological overactivation (38). Along with its selective effects on the PiAR, the pharmacological properties conferred by PAM-antagonism underscore thepotential therapeutic superiority of Cl 1 over canonical non-selective orthosteric P- blockers.

[0143] While Cl 1 has shown reciprocal effects on agonist binding affinity and efficacy, the precise inhibitory mechanism, binding site, and receptor conformation it promotes remain outstanding questions. To date, only several PAM-antagonists are currently described, and their mechanism of action is largely elusive. The first reported PAM-antagonist, ifenprodil, was discovered in 1996 as a neuroprotective modulator of the N-methyl-D-aspartate (NMD A) glutamate receptor ion channel (48). In the following decades, PAM-antagonists targeting the free fatty acid 3 (FFA-3) receptor (49) and the cannabinoid 1 (CB1) receptor (50, 51) were identified. Previous studies utilizing site-directed fluorescent labeling characterizing the CB1 PAM- antagonist, Org27569, have reported that Org27569 blocks conformational changes associated with G protein binding suggesting that it locks the CB 1 receptor in an agonist-bound, non-signaling, early-activation intermediate state (52). Alternative hypotheses regarding the mechanism of PAM-antagonists include stabilizing a quaternary complex comprised of receptor, agonist, modulator, and transducer, or perhaps steric blockade of transducer binding. While some structure-activity insights of Cl 1 via characterizing a panel of analogs have been generated, future biophysical and structural studies will be required to precisely determine the binding location and details of the mechanism of action of Cl 1.

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[0145] Intense exercise stress is the primary cause of ventricular tachycardia and sudden cardiac death in CPVT. To investigate the therapeutic potential of Cl 1 in a physiologically relevant setting, we recorded cardiac electrical activity in conscious, non-anesthetized, CSQ2 ’ ’ mice via telemetry during graded treadmill exercise (Fig. 17A-17C) and quantified the presence of premature ventricular contractions (PVCs) and episodes of ventricular tachycardia (Fig. 17D). Mice were implanted with telemetry devices and pre-treated with either vehicle solution or 10 mg / kg Cl 1 (Fig. 17B). Following 45 minutes of pre-treatment, mice were subjected to forced treadmill running where workload (i.e. speed and incline) was periodically increased during each 30-minute run and repeated every four days (Fig. 17C). Individual mice were delivered alternating treatments of vehicle or Cl 1 on every fourth day and thus each mouse served as its own internal control (Fig. 17A). Our initial pilot experiment demonstrated that exercise induced fewer arrhythmic events following pre-treatment with Cl 1 compared to when these same mice were pre-treated with vehicle (Fig. 18A- 18C; n=2 mice). Specifically, Cl 1 -treatment appeared to reduce the overall number of PVCs (Fig. 18 A) and episodes of bigeminy (Fig. 18B) defined as 3 or more altering regular and premature beats. Moreover, the overall duration of ventricular tachycardia was decreased following Cl 1 treatment compared to vehicle (Fig. 18C). This experiment highlights the potential therapeutic capability of Cl 1 in suppressing the development of arrhythmic events in CPVT following physiological exercise stress.

[0146] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[0147] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

Claims

What is claimed is:

1. A compound comprising the general formula (I) (termed Compound 11; Cl 1;Cmpd 11):or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.

3. A compound comprising the general formula (II) (termed Compound 1 IB;CUB; Cmpd 11B):or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.

3. A pharmaceutical composition comprising a compound according to claims 1 or 2 and a pharmaceutically acceptable diluent, excipient, and / or carrier.

4. A method of enhancing the binding affinity of an agonist and / or antagonist to the PiAR comprising contacting a cell with an effective amount of a compound according to claims 1 or 2 and an agonist and / or antagonist such that the binding activity of said agonist and / or antagonist is enhanced.

5. The method according to claim 3 in which the agonist is selected from the group consisting of norepinephrine, epinephrine, isoproterenol, dobutamine and combinations thereof.

6. The method according to claim 3 in which the antagonist is selected from the group consisting of carvedilol, bucindolol, alprenolol, atenolol, and combinations thereof.

7. A method of suppressing PiAR-mediated G protein and P-arrestin signaling in a cell in response to an agonist, the method comprising contacting the cell that has been exposed to an agonist with an effective amount of a compound according to claims 1 or 2 such that the PiAR-mediated G protein and P-arrestin signaling are suppressed in the cell.

8. A method of suppressing an agonist-mediated response in a cardiomyocyte, the method comprising contacting the cardiomyocyte with an effective amount of a compound according to claim 1 or 2 such that the agonist-mediated response in the cardiomyocyte is suppressed.

9. A method of inhibiting basal contractility in a cardiomyocyte, the method comprising contacting the cardiomyocyte with an effective amount of a compound according to claim 1 or 2 such that the basal contractility in the cardiomyocyte is inhibited.

10. All that is described and illustrated herein.

10. Any and all methods, processes, devices, systems, devices, kits, products, materials, compositions and / or uses shown and / or described expressly or byimplication in the information provided herewith, including but not limited to features that may be apparent and / or understood by those of skill in the art.

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