Discovery of SYD5115, a novel orally active small molecule TSH-R antagonist.

The thyrotropin receptor (TSH-R) regulates the thyroid gland and is normally activated by thyrotropin. In patients with Graves' disease, TSH-R is also stimulated by stimulatory TSH-R autoantibodies leading to hyperthyroidism. In this paper, we describe the discovery of SYD5115 (67), a novel small molecule TSH-R antagonist with nanomolar potency. SYD5115 also blocks stimulating antibody induced synthesis of the thyroid hormone thyroxine (T4) in vivo, after a single oral dose. During optimization, several issues had to be addressed such as the low metabolic stability and the potential mutagenicity of our first series of compounds.

A Platform for the Generation of Site-Specific Antibody-Drug Conjugates That Allows for Selective Reduction of Engineered Cysteines.

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an in silico screening procedure to select for optimal sites in an antibody to which a hydrophobic linker-drug can be conjugated. Sites were identified inside the cavity that is naturally present in the Fab part of the antibody. Conjugating a linker-drug to these sites demonstrated the ability of the antibody to shield the hydrophobic character of the linker-drug while resulting ADCs maintained their cytotoxic potency in vitro. Comparison of site-specific ADCs versus randomly conjugated ADCs in an in vivo xenograft model revealed improved efficacy and exposure. We also report a selective reducing agent that is able to reduce the engineered cysteines while leaving the interchain disulfides in the oxidized state. This enables us to manufacture site-specific ADCs without introducing impurities associated with the conventional reduction/oxidation procedure for site-specific conjugation.

Chemokine cooperativity is caused by competitive glycosaminoglycan binding.

Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

ß-Arrestin recruitment and G protein signaling by the atypical human chemokine decoy receptor CCX-CKR.

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with ß-arrestin2-GFP translocation. Moreover, recruitment of ß-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold ß-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.

Thyrotropin Receptor Antagonism by a Novel Small Molecule: Preclinical In Vitro Observations.

Background: Treatment of Graves' hyperthyroidism (GH) and Graves' orbitopathy (GO) is far from adequate, and hence, new substances that specifically target the autoantigens in GH/GO are warranted. This study determined the preclinical in vitro efficacy of SYD5115, a novel low-molecular-weight compound that inhibits the thyrotropin receptor (TSH-R). Methods: The TSH-R inhibiting capability of SYD5115 was tested through stimulation of wild-type and chimeric TSH-R expressed in Chinese hamster ovary (CHO) cells using two functional (stimulatory and blocking) cell-based TSH-R-Ab bioassays. TSH-R expressing human orbital fibroblasts, collected from GH+GO patients (GOF), were stimulated with the monoclonal antibody M22 or with stimulatory TSH-R-Ab (TSAb)-positive sera with cyclic adenosine monophosphate (cAMP) or hyaluronic acid (HA) release as readouts. The effect of SYD5115 on the viability of GOF was tested in 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and scratch cell growth assays. Results: SYD5115 significantly and dose dependently inhibited the TSH-R activation through M22 or TSAb-positive sera in all performed bioassays. Inhibition showed similar levels in the TSAb reporter bioassay and in the cAMP assay with GOF. The % inhibition and compound concentration showed a sigmoidal relationship, with all seven TSAb-positive sera markedly inhibited by SYD5115. An SYD5115 dose-dependent inhibition of M22 (10 ng/mL, 6 hours)-stimulated HA and/or cAMP-release from GOF was observed. Strong SYD5115-induced inhibitions of M22-stimulated cAMP production in GOF were registered with SYD5115 concentrations of 1 (p = 0.0029), 10 (p < 0.0001), 100 (p < 0.0001), 1,000 (p < 0.0001), and 10,000 (p < 0.0001) nM, respectively. SYD5115-induced inhibition of M22-stimulated HA production was noted with SYD5115 concentrations of 100 (p = 0.0392), 1000 (p = 0.0431), and 10,000 (p = 0.0245) nM, respectively. The inhibitory activity of SYD5115 was confirmed in a human osteosarcoma U2OS cell line stably expressing human TSH-R with cAMP as readout. SYD5115 induced 100% inhibition of the M22-induced cAMP levels with a potency of 193 nM. Compared with control, SYD5115 did neither impact the growth nor the migration of cultivated GOF. In addition, SYD5115 did not alter the viability of GOF. Conclusions: SYD5115 blocked M22- and TSAb-induced TSH-R activity with a nanomolar potency in TSH-R-overexpressed CHO cells as well as primary GOF, which demonstrates the ability of this small molecule to block TSH-R overactivity.

Preclinical Profile of BYON3521 Predicts an Effective and Safe MET Antibody-Drug Conjugate.

MET, the cell-surface receptor for the hepatocyte growth factor/scatter factor, which is widely overexpressed in various solid cancer types, is an attractive target for the development of antibody-based therapeutics. BYON3521 is a novel site-specifically conjugated duocarmycin-based antibody-drug conjugate (ADC), comprising a humanized cysteine-engineered IgG1 monoclonal antibody with low pmol/L binding affinity towards both human and cynomolgus MET. In vitro studies showed that BYON3521 internalizes efficiently upon MET binding and induces both target- and bystander-mediated cell killing. BYON3521 showed good potency and full efficacy in MET-amplified and high MET-expressing cancer cell lines; in moderate and low MET-expressing cancer cell lines good potencies and partial efficacy were observed. In mouse xenograft models, BYON3521 showed significant antitumor activity upon single-dose administration in multiple non-MET-amplified tumor types with low, moderate, and high MET expression, including complete tumor remissions in models with moderate MET expression. In the repeat-dose Good Laboratory Practice (GLP) safety assessment in cynomolgus monkeys, BYON3521 was well tolerated and based on the observed toxicities and their reversibility, the highest non-severely toxic dose was set at 15 mg/kg. A human pharmacokinetics (PK) model was derived from the PK data from the cynomolgus safety assessments, and the minimal efficacious dose in humans is estimated to be in the range of 3 to 4 mg/kg. In all, our nonclinical data suggests that BYON3521 is a safe ADC with potential for clinical benefit in patients. A first-in-human dose-escalation study is currently ongoing to determine the maximum tolerated dose and recommended dose for expansion (NCT05323045).

BYON4228 is a pan-allelic antagonistic SIRPα antibody that potentiates destruction of antibody-opsonized tumor cells and lacks binding to SIRPγ on T cells.

BACKGROUND: Preclinical studies have firmly established the CD47-signal-regulatory protein (SIRP)α axis as a myeloid immune checkpoint in cancer, and this is corroborated by available evidence from the first clinical studies with CD47 blockers. However, CD47 is ubiquitously expressed and mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy. METHOD: We generated BYON4228, a novel SIRPα-directed antibody. An extensive preclinical characterization was performed, including direct comparisons to previously reported anti-SIRPα antibodies. RESULTS: BYON4228 is an antibody directed against SIRPα that recognizes both allelic variants of SIRPα in the human population, thereby maximizing its potential clinical applicability. Notably, BYON4228 does not recognize the closely related T-cell expressed SIRPγ that mediates interactions with CD47 as well, which are known to be instrumental in T-cell extravasation and activation. BYON4228 binds to the N-terminal Ig-like domain of SIRPα and its epitope largely overlaps with the CD47-binding site. BYON4228 blocks binding of CD47 to SIRPα and inhibits signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates macrophage-mediated and neutrophil-mediated killing of hematologic and solid cancer cells in vitro in the presence of a variety of tumor-targeting antibodies, including trastuzumab, rituximab, daratumumab and cetuximab. The silenced Fc region of BYON4228 precludes immune cell-mediated elimination of SIRPα-positive myeloid cells, implying anticipated preservation of myeloid immune effector cells in patients. The unique profile of BYON4228 clearly distinguishes it from previously reported antibodies representative of agents in clinical development, which either lack recognition of one of the two SIRPα polymorphic variants (HEFLB), or cross-react with SIRPγ and inhibit CD47-SIRPγ interactions (SIRPAB-11-K322A, 1H9), and/or have functional Fc regions thereby displaying myeloid cell depletion activity (SIRPAB-11-K322A). In vivo, BYON4228 increases the antitumor activity of rituximab in a B-cell Raji xenograft model in human SIRPαBIT transgenic mice. Finally, BYON4228 shows a favorable safety profile in cynomolgus monkeys. CONCLUSIONS: Collectively, this defines BYON4228 as a preclinically highly differentiating pan-allelic SIRPα antibody without T-cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2023.

Nonpeptidergic allosteric antagonists differentially bind to the CXCR2 chemokine receptor.

The chemokine receptor CXCR2 is involved in different inflammatory diseases, like chronic obstructive pulmonary disease, psoriasis, rheumatoid arthritis, and ulcerative colitis; therefore, it is considered an attractive drug target. Different classes of small CXCR2 antagonists have been developed. In this study, we selected seven CXCR2 antagonists from the diarylurea, imidazolylpyrimide, and thiazolopyrimidine class and studied their mechanisms of action at human CXCR2. All compounds are able to displace (125)I-CXCL8 and inhibit CXCL8-induced beta-arrestin2 recruitment. Detailed studies with representatives of each class showed that these compounds displace and antagonize CXCL8, most probably via a noncompetitive, allosteric mechanism. In addition, we radiolabeled the high-affinity CXCR2 antagonist SB265610 [1-(2-bromophenyl)-3-(4-cyano-1H-benzo[d] [1,2,3]-triazol-7-yl)urea] and subjected [(3)H]SB265610 to a detailed analysis. The binding of this radioligand was saturable and reversible. Using [(3)H]SB265610, we found that compounds of the different chemical classes bind to distinct binding sites. Hence, the use of a radiolabeled low-molecular weight CXCR2 antagonist serves as a tool to investigate the different binding sites of CXCR2 antagonists in more detail.

beta-Arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1.

beta-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of beta-galactosidase to measure receptor-beta-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter beta-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed beta-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter cell line at the level of beta-arrestin recruitment, G(i/o) protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of beta-arrestin recruitment was largely unaffected upon blockade of G(i/o) protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of beta-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully G(i/o) protein regulated. The data indicate that the beta-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of "biased ligands" (i.e., compounds that differ in G-protein coupling and beta-arrestin-mediated cellular effects).

Unraveling the Interaction between Carboxylesterase 1c and the Antibody-Drug Conjugate SYD985: Improved Translational PK/PD by Using Ces1c Knockout Mice.

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody-drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c-/- SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c-/- SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389-98. ©2018 AACR.

Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitors.

The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.

A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA.

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

SYD985, a Novel Duocarmycin-Based HER2-Targeting Antibody-Drug Conjugate, Shows Antitumor Activity in Uterine Serous Carcinoma with HER2/Neu Expression.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels by IHC while an additional 40% to 50% express HER2/neu at moderate (2+) or low (1+) levels. We investigated the efficacy of SYD985, (Synthon Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in USC. We also compared the antitumor activity of SYD985 in head-to-head experiments to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary USC cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Using antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing assays as well as propidium iodide-based flow cytometry assays and multiple in vivo USC mouse xenograft models, we demonstrate for the first time that SYD985 is a novel ADC with activity against USC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is 10- to 70-fold more potent than T-DM1 in comparative experiments and, unlike T-DM1, it is active against USC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in patients harboring chemotherapy-resistant USC with low, moderate, and high HER2 expression are warranted. Mol Cancer Ther; 15(8); 1900-9. ©2016 AACR.

The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers.

SYD985 is a HER2-targeting antibody-drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need.

ß-Arrestin-biased signaling of PTH analogs of the type 1 parathyroid hormone receptor.

Parathyroid hormone (PTH) is an anabolic agent that mediates bone formation through activation of the Gα(s)-, Gα(q)- and ß-arrestin-coupled parathyroid hormone receptor type 1 (PTH1R). Pharmacological evidence based on the effect of PTH(7-34), a PTH derivative that is said to preferentially activate ß-arrestin signaling through PTH1R, suggests that PTH1R-activated ß-arrestin signaling mediates anabolic effects on bone. Here, we performed a thorough evaluation of PTH(7-34) signaling behaviour using quantitative assays for ß-arrestin recruitment, Gα(s)- and Gα(q)-signaling. We found that PTH(7-34) inhibited PTH-induced cAMP accumulation, but was unable to induce ß-arrestin recruitment, PTH1R internalization and ERK1/2 phosphorylation in HEK293, CHO and U2OS cells. Thus, the ß-arrestin bias of PTH(7-34) is not apparent in every cell type examined, suggesting that correlating in vivo effects of PTH(7-34) to in vitro pharmacology should be done with caution.

Pharmacological characterization of receptor redistribution and beta-arrestin recruitment assays for the cannabinoid receptor 1.

Receptor redistribution and beta-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular beta-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution assay and 2 nonimaging-based beta-arrestin recruitment assays, Tango and PathHunter, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Galpha(i) coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via beta-arrestin recruitment and Galpha(i)-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed.