Biased Ligands of G Protein-Coupled Receptors (GPCRs): Structure

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Perspective

Biased Ligands of G Protein-Coupled Receptors (GPCRs): StructureFunctional Selectivity Relationships (SFSRs) and Therapeutic Potential Liang Tan, Wenzhong Yan, John D. McCorvy, and Jianjun Cheng J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00435 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Journal of Medicinal Chemistry

Biased Ligands of G Protein-Coupled Receptors (GPCRs): Structure-Functional Selectivity Relationships (SFSRs) and Therapeutic Potential Liang Tan,† Wenzhong Yan,† John D. McCorvy*,‡ and Jianjun Cheng*,† †

iHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Pudong

District, Shanghai 201210, China ‡

Department of Cell Biology, Neurobiology and Anatomy, Medical College of

Wisconsin, 8701 W Watertown Plank Road, Milwaukee, WI 53226, USA ABSTRACT G protein-coupled receptors (GPCRs) signal through both G protein-dependent and G protein-independent pathways, and β-arrestin recruitment is the most recognized one of the latter. Biased ligands selective for either pathway are expected to regulate biological functions of GPCRs in a more precise way, therefore providing new drug molecules with superior efficacy and/or reduced side effects. During the past decade, biased ligands have been discovered and developed for many GPCRs, such as the µ opioid receptor, the angiotensin II receptor type 1, the dopamine D2 receptor, and many others. In this Perspective, recent advances in this field are reviewed by discussing the structure-functional selectivity relationships (SFSRs) of GPCR biased ligands and the therapeutic potential of these molecules. Further understanding into the biological functions associated with each signaling pathway and structural basis 1

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for biased signaling, will facilitate future drug design in this field. INTRODUCTION G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, with more than 800 members.1 According to the “GRAFS” classification system, GPCRs can be grouped in five families: Glutamate, Rhodopsin-like, Adhesion, Frizzled/Taste2 and Secretin, while the rhodopsin-like receptors are also known as class A, secretin as class B and glutamate as class C.2 All GPCRs share a common architecture characterized by the seven transmembrane (7TM) domains, also known as seven transmembrane receptors (7TMRs), but they play diverse physiological roles and can be activated by various stimuli, such as photons, ions, neurotransmitters, and hormones.3 GPCRs are the targets for more than 30% of all approved drugs, and they remain the most successful and promising class of target proteins for drug discovery.4, 5 As membrane proteins, GPCRs are responsible for transferring extracellular signals to the cytosol. As indicated by their name, GPCRs couple to G proteins that bind and hydrolyze guanosine-5'-triphosphate (GTP) to mediate downstream signaling. G proteins associated with GPCRs are heterotrimeric and composed of three subunits: Gα, Gβ, and Gγ. Gα proteins are further divided into four main sub-groups: Gαs, Gαi/o, Gαq/11, and Gα12/13. Depending on the type of G protein that a specific GPCR couples to, receptor activation can lead to secondary messengers, such as inositol triphosphates (Gq) or cyclic adenosine monophosphate (cAMP) (Gs/Gi).

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Recently, other G protein-independent pathways have been identified, and the most recognized one is the β-arrestin-dependent signaling pathway.6 Upon the activation of GPCRs, a family of protein kinases called G protein-coupled receptor kinases (GRKs) will phosphorylate the intracellular domains of GPCRs after their associated G proteins have been released and activated. Phosphorylated GPCRs will recruit β-arrestins, which mediate the desensitization of GPCR signaling, internalization of GPCRs and therefore serve to “turn-off” signaling, resulting in negative feedback of G protein-dependent GPCR signaling. β-Arrestins have also been associated with the activation of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinases 1 and 2 (ERK1/2).7 However, ERK1/2 signaling can be either G protein-dependent or β-arrestin-mediated or both, depending on the cellular context in which the response is measured. Two types of β-arrestins (non-visual arrestins) have been identified, namely β-arrestin1 (also referred to as arrestin2) and β-arrestin2 (also referred to as arrestin3), along with two visual arrestins, arrestin1 and arrestin4. Although visual arrestins are only expressed in the retina where they regulate the functions of rhodopsin, β-arrestins are widely expressed and they modulate the signaling of other GPCRs. It remains unclear if all GPCRs that recruit β-arrestins cause β-arrestin-dependent signaling, and to what extent β-arrestin-dependent signaling occurs. Historically, GPCR signaling resulting from the activation by a specific ligand was assumed to show equal efficacy across systems, a property of the ligand termed “intrinsic efficacy.” In fact, current models of GPCR activation states, including the 3



ternary and extended ternary complex models of receptor activation, propose that receptors exist in either “active” or “inactive” states, and specific states of activation have also been discovered for rhodopsin receptors.8 The concept of biased signaling of GPCRs was first described by Kenakin more than two decades ago in 1995.9 It wasn’t until accumulating evidence demonstrated that ligands could show efficacy for a variety of cellular responses that the notion of functional selectivity or ligand bias began to take hold.10 “Functional selectivity” is an older term and simply denotes difference in efficacy for one or more functional readouts, while “ligand bias” usually involves a formal analysis of opposing pathways (e.g. G protein versus arrestin) and can be quantitative. With the identification of G protein-independent signaling (e.g. β-arrestins), which can directly bind to GPCRs to stabilize separate receptor active states, GPCRs have now been posited to exist in multiple, distinct conformational states, which is a theoretical notion that GPCRs form ensembles of several active and inactive conformational populations.11 Although the detailed molecular mechanisms of biased signaling is not yet well understood, it has been reported that biased GPCR ligands induce a unique receptor conformation (Figure 1), which in turn activates a particular signaling pathway via an effector (e.g. G proteins or β-arrestins). Although theoretically it has been assumed that GPCR conformations stabilized by a G protein-biased ligand are distinct from the conformations stabilized by a β-arrestin-biased ligand, further study is required to determine how GPCRs adopt specific conformations that lead to preference for either effector.

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Our increased understanding of this phenomenon has opened a new era for the study of GPCR signaling as well as GPCR-targeted drug discovery. Biased ligands have been discovered through evaluating ligand efficacy in various pharmacological assays rather than solely from canonical G protein-dependent assays. Subsequently, methods of detecting and quantifying bias properties of a given GPCR ligands have been substantially developed and evolved.12 During the past decade, both G protein-biased and β-arrestin-biased ligands have been discovered and developed for many GPCRs. Although a few previous reviews have summarized the progress in this field,13-17 the rapid discovery of new biased ligands in this field necessitates an up-to-date and comprehensive review. In this Perspective, we summarize recent advances in biased ligand discovery for around 30 different GPCRs in classes A, B and C (Figure 2, Table 1), with an emphasis on structure-functional selectivity relationships (SFSRs) analyzed from a medicinal chemistry perspective. In a number of cases, therapeutic advantages of biased ligands have been studied in preclinical animal models or clinical trials (Table 1). Challenges in the discovery of biased ligands and medicinal chemistry strategies that can be used are also discussed.

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Figure 1. Biased signaling at a glance. GPCR represented is from the crystal structure of β2 adrenergic receptor (PDB: 2RH1); the GPCR-G protein complex represented is from

the β2 adrenergic

receptor-G

protein

complex (PDB: 3SN6);

the

GPCR-β-arrestin complex represented is from the structure of rhodopsin-β-arrestin complex (PDB: 4ZWJ). Throughout this review, G protein-dependent activity of ligands is represented in red, and G protein-independent signaling in blue.

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Figure 2. Biased ligands have been reported for 30 GPCR targets. ADORA1, adenosine A1 receptor (A1AR); ADORA3, adenosine A3 receptor (A3AR); ADRB1, adrenergic β1 receptor (β1AR); ADRB2, adrenergic β2 receptor (β2AR); AGTR1, angiotensin II receptor type 1 (AT1R); AGTRL1, apelin receptor (APJ); CASR, calcium-sensing receptor (CaSR); CNR1, cannabinoid receptor 1 (CB1R); CNR2, cannabinoid receptor 2 (CB2R); CXCR3, chemokine receptor CXCR3; DRD1, dopamine D1 receptor (D1R); DRD2, dopamine D2 receptor (D2R); EDG1, sphingosine-1-phosphate receptor 1 (S1P1R); EDNRA, endothelin A receptor (ETA);

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FPR2, formyl peptide receptor 2; GLP1R, glucagon-like peptide 1 receptor; GRM1, metabotropic glutamate receptor 1 (mGlu1R); GRM5, metabotropic glutamate receptor 5 (mGlu5R); HM74, niacin receptor (HM74A for GPR109A); HRH2, histamine H2 receptor (H2R); HRH4, histamine H4 receptor (H4R); HTR1A, serotonin 1A receptor (5-HT1AR); HTR2B, serotonin 2B receptor (5-HT2BR); HTR2C, serotonin 2C receptor (5-HT2CR); NTSR1, neurotensin receptor type 1 (NT1R); OPRK1, κ opioid receptor (KOR); OPRL1, nociception receptor (NOPR); OPRM1, µ opioid receptor (MOR); PTGER2, prostaglandin E2 receptor 2 (EP2R); PTHR1, parathyroid hormone 1 receptor. The graph was produced based on the dendrogram of the human GPCR superfamily (“Figure 1” in Katritch et al., “Structure-Function of the G Protein-Coupled Receptor Superfamily”.18), with permission from the authors. Table 1. Biased ligands at class A, class B and class C GPCRs. GPCR

Biased Ligand

Type of Ligand Bias

Therapeutic Area

β1AR

metoprolol;23 alprenolol and carvedilol24

β-arrestin-biased agonists

Heart diseases

β2AR

ethylnorepinephrine, isoetharine, β-arrestin-biased agonists N-cyclopentylbutanepherine;22 carvedilol;25 propranolol and ICI-118,55126

AT1R

TRV120023 and TRV12002728


Acute heart failure

MOR

TRV130;35,37 PZM21;38

G protein-biased agonists

Pain

Class A

mitragynine and analogs;41 SR-17018, SR-15099 and SR-1509842 8


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KOR

33;49 35;48 RB-64;52 6’-GNTI;53 nalfurafine55


Pain

NOPR

MCOPPB;58 NNC 63-0532;58,59


Pain

UNC9975, UNC9994 and UNC9995;67 55.74


Schizophrenia

49;71 50;72 52;73 cariprazine;77 60;78 6380


BRD581483

β-arrestin-biased antagonist /G protein agonist

D1 R

SKF38393, SKF75670, SKF-77434, SKF83959 and SKF82957;86 PF-8294 and PF-614288


Cognition; Parkinson’s disease

5-HT2B

ergotamine93

β-arrestin-biased agonist

-a

5-HT2C

80;97 8198


Obesity; schizphrenia

5-HT1A

83, 84 and 8599


Anxiety

H4 R

JNJ-7777120;104 VUF5223 and VUF10214106

β-arrestin biased agonists

Inflammation and pruritus

VUF5222, VUF10778 and VUF10185106


2-AG and AEA116


CP55,940 and THC116

β-arrestin1-biased agonists

WIN-55212, THC, JWH-133, HU-308 and JWH-015119

G protein-biased partial agonists

AM-1248, STS-135 and UR-144119


Pain, sclerosis and Huntington’s disease

MM07;124 110;125 CMF-019128


Heart failure

RTI-4229-819 and RTI-4229-85658 D2 R

CB1R

CB2R

APJ

9


Huntington’s disease; pain; nausea; obesity


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113135

β-arrestin-biased NAM

114136

G protein-biased NAM

FAUC1036133

β-arrestin-biased allosteric agonist

FAUC1104133

G protein-biased allosteric agonist

EP2

119, 120, 121 and 123140


Lowing intraocular pressure

ETA

ET-2 and ET-3144


bosentan144,145

β-arrestin-biased antagonist

Pulmonary arterial hypertension (PAH); cancer

GPR109A

MK-0354150

G protein-biased agonist

Lowering triglycerides

S1P1R

BMS-986104154


Multiple sclerosis

NTR1

ML314162


Schizophrenia

FPR2

F2Pal10159


Inflammation

A1AR

VCP746;165, 166 139167

agonists; cAMP inhibition over pERK1/2 or calcium mobilization

Ischemia

141167

agonist; calcium mobilization over cAMP inhibition

143-148169

agonist; cell survival over cAMP inhibition

P5175


oxyntomodulin;173 exendin-4;173 154-156177


CXCR3

A3AR

Autoimmune diseases and cancer

Class B GLP-1R

10


Type 2 diabetes

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PTH-barr171


Osteoporosis

quisqualate;182 DHPG183


Neuroprotection

glutaric acid and succinic acid183


mGlu5R

VU0409551;189 NCFP190

PAMs that bias toward calcium mobilization over pERK1/2

CNS disorders

CaSR

cinacalcet194

Bias for Ca2+ mobilization and IP1 accumulation

Hyperparathyroi dism; hypercalcemia

AC-265347 and R,R-calcimimetic B194

Bias for pERK1/2 and IP1 accumulation

S,R-calcimimetic B194

Bias for IP1 accumulation

PTHR1 Class C mGlu1R

a

Ergotamine is an approved drug for the treatment of migraine but not based on its

action at 5-HT2B. Biased Ligands of Class A GPCRs The rhodopsin-like or class A is the largest family which accounts for 85% of all GPCRs, including many drug targets as well as olfactory and orphan receptors. Numerous class A GPCRs such as the aminergic receptors and purinergic receptors are well-validated drug targets, and the druggability of many emerging targets are being explored with drug candidates in preclinical studies and clinical trials.5 Biased ligands of class A GPCRs are discussed in the following, outlined by the specific targets they interact with. The β Adrenergic Receptors (βARs). βARs can be genetically subdivided into three subtypes: β1AR, β2AR and β3AR,19 all of which have been long known as drug

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targets. Among them, the β2AR has been the first non-rhodopsin human GPCR for which crystal structures have been solved and remains as a prototype receptor for the structural and functional investigations of GPCRs.20,

21

Previously identified βAR

ligands have a common structural feature in that they all contain a catecholamine framework, as seen in norepinephrine (1) and isoproterenol (2) (Figure 3). The study by Drake et al. demonstrated that three catecholamine derivatives ethylnorepinephrine (3), isoetharine (4) and N-cyclopentylbutanepherine (CPB, 5) show marked bias toward β-arrestin signaling pathway at the β2AR.22 These ligands appeared to stimulate β-arrestin-dependent receptor activities (in β-arrestin translocation FRET assays) to a much greater extent than their efficacy in G protein-dependent signaling (in cAMP FRET assays). Structural comparison between these compounds and compounds 1 and 2 clearly shows that the biased ligands share a common structural feature critical for their β-arrestin recruitment activity, which is the ethyl substituent on the catecholamine α-carbon (Figure 3). Compound 5 was characterized as the most biased agonist among the three (bias factor > 2), and its stimulation of the β2AR resulted in rapid β-arrestin-mediated receptor internalization.22

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Figure 3. Catecholamine derivatives that are β-arrrestin-biased agonists of βARs. The “bias factor” was calculated as a ratio of β-arrestin efficacy (in β-arrestin translocation FRET assay) to G protein efficacy (in cAMP assay).22 Inserting an “–OCH2–” unit between the aryl and the α-carbon of aryl ethanolamine provided a class of βAR antagonists (also known as β blockers) such as metoprolol (6), carazolol (7) and propranolol (8) (Figure 4). Their antagonist activity was defined by the inhibition of G protein-mediated signaling at the receptors, and Nakaya et al. reported that metoprolol shows agonist activity at β1AR when tested for β-arrestin2-dependent signaling.23 They found that metoprolol could induce cardiac fibrosis and impaired diastolic function through the β-arrestin2-dependent pathway although metoprolol is used for the treatment of various heart diseases.

Figure 4. Some β blockers are β-arrestin-biased agonists of βARs. A study conducted by Kim et al. showed that alprenolol (9) and carvedilol (10) (Figure 4) could stimulate β-arrestin-mediated EGFR transactivation and downstream 13



ERK signaling at the β1AR.24 Wisler et al. also reported that carvedilol is an inverse agonist of G protein-mediated signaling at β2AR but an agonist of β-arrestin signaling, the latter of which was measured by an increase in the phosphorylation of β2AR, ERK activation and receptor internalization.25 Furthermore, aryl ethanolamine β-blockers propranolol (8) and ICI-118,551 (11) were also demonstrated by Azzi et al. to induce ERK1/2 phosphorylation via a β-arrestin-mediated mechanism at β2AR.26 Together, these results suggest that the β-arrestin signaling may play important roles in the therapeutic or adverse effects of β blockers, which do not simply work by “blocking” all the effects mediated by βARs. For these βAR ligands, Liu et al. studied the structural basis for their biased properties using the

19

F-NMR methodology.27 Their results demonstrated that

compared to the balanced prototypes isoproterenol and carazolol respectively, isoetharine and carvedilol induced a larger shift of helix VII (TM7) of β2AR, which may form the structural basis for the signaling bias toward β-arrestins at the β2AR. Comparing the structures of isoetharine versus isoproterenol, and carvedilol versus carazolol, the additional ethyl or phenyl substitutions probably directly interacts with TM7 of the receptor or through changing the overall conformation of the ligand itself. The Angiotensin II Type 1 Receptor (AT1R). AT1R is a primary blood pressure modulator, and angiotensin receptor blockers (ARBs), which are AT1R antagonists and are widely prescribed drugs indicated for hypertension. Unlike the β blockers, most of these ARBs have been demonstrated as balanced antagonists for both the G protein and β-arrestin signaling in subsequent studies.28 It has been anticipated that 14


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β-arrestin-biased AT1R agonists, which would engage the β-arrestin-mediated receptor desensitization, internalization, as well as associated signaling pathways, could provide novel therapeutics for congestive heart failure.

Figure 5. β-Arrestin-biased AT1R agonists. The amino acid sequences are shown from the N-terminus (left) to the C-terminus (right). Through sequence modifications of angiotensin II (Ang II, 12, Figure 5), Trevena scientists discovered peptide SII (13) as the first β-arrestin-biased agonist of AT1R, which was designed by the substitution of Asp1 with sarcosine, and Tyr4 and Phe8 with isoleucines.29 SII binds to AT1R in an AngII-competitive manner with moderate affinity (KD = 300 nM), which was 187 times lower than that of Ang II (KD = 1.6 nM). The binding of SII did not induce the coupling of AT1R to G protein, but caused recruitment of β-arrestins and internalization of the receptor and connected the receptor to the β-arrestin-dependent activation of the MAPK pathway. To enhance the binding affinity of SII, a series of new Ang II analogs were designed, and peptides TRV120023 (14) and TRV120027 (15) displayed improved affinity for AT1R.28 15



TRV120023 and TRV120027 selectively induced β-arrestin2 recruitment (EC50 = 44 nM and 17 nM respectively at the human AT1R) while antagonizing Ang II-stimulated G protein signaling. In vivo studies in rats showed that, unlike the balanced AT1R antagonists, which decreased cardiac performance while reducing blood pressure, TRV120027 increased cardiac performance and preserved cardiac stroke volume.28 These results support the development of β-arrestin-biased AT1R agonists for the treatment of hypertension and heart failure, which would provide cardiac benefits over the existing ARBs. TRV120027 has been advanced into clinical evaluations for the treatment of acute heart failure (AHF), however, it did not confer any benefits over placebo in patients and failed to meet its primary or secondary endpoints in a phase IIb trial (BLAST-AHF).30 A post-hoc analysis revealed that TRV120027 exhibits differential effects depending on systolic blood pressure, and positive data may be extracted for a narrowly defined subgroup.31, 32 The Opioid Receptors. Opioid receptors play critical roles in pain management, drug abuse/addiction, and mood disorders. There are four major subtypes: the δ opioid receptor (DOR), the κ opioid receptor (KOR), the µ opioid receptor (MOR) and the nociceptin receptor (NOPR). The MOR primarily mediates the most pronounced analgesic effects via opioid drugs. Recently, MOR has become another example of GPCRs that the ‘on-target’ benefits can be separated from adverse effects through selective activation of downstream pathways that couple to the receptor. Studies have suggested that the opioid-induced analgesia results from the Gi/o signaling through MOR, while the on-target side effects, such as respiratory depression and constipation, 16


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may be conferred via the β-arrestin-dependent signaling.14, 33, 34 Therefore, Gi/o-biased agonists of MOR have been investigated to explore their therapeutic potential. A lead discovery was made by Trevena who screened its internal compound collection and identified compound 16 (Figure 6) as a novel MOR agonist. Compound 16 showed submicromolar activation of G protein signaling (pEC50 = 6.3) and lower MOR β-arrestin recruitment activity (Emax = 32% compared to 100% for morphine).35 An SAR investigation with the aim of improving its MOR G protein potency while reducing β-arrestin recruitment activity led to the identification of G protein-biased compound 17. Compound 17 showed much improved bias properties compared to the hit compound, however, it poses a potential risk for arrhythmia (hERG IC50 = 2.3 µM). Further SAR studies focusing on the thiophene moiety led to the discovery of compound TRV130 (18), which displayed similar bias for G protein-dependent signaling but with somewhat reduced hERG inhibition (IC50 = 6.2 µM).35 Thiophene is, however, a structural alert in medicinal chemistry that needs special attention due to metabolic liabilities.36 In animal models, compound TRV130 showed potent analgesic effects with reduced respiratory depression and constipation compared to morphine.37 These results showed that TRV130 or other G protein-biased MOR agonists might function as effective analgesics with fewer side effects than existing nonbiased MOR agonists like morphine. TRV130 (also known as oliceridine, OLINVO) has finished phase 3 clinical trials for the treatment of moderate to severe acute pain and is awaiting the approval from US Food and Drug Administration (FDA). If successful, TRV130 will be the first of its kind in the market. 17



Using a structure-based approach, Manglik et al. recently reported another G protein-biased MOR agonist.38 Based on the crystal structure of MOR,39 three million molecules were computationally docked to the receptor, with the goal of finding novel chemotypes that can stabilize the receptor in unexplored conformations. Compound 19 (Figure 6) was identified as a MOR agonist with moderate binding to the receptor (Ki = 2.5 µM) but displayed a strong bias for Gi/o signaling.38 Structural modifications of 19 led to the discovery of compound 20 (Ki = 42 nM), which strongly activated Gi/o (EC50 = 180 nM, Emax = 88%) with low levels of β-arrestin2 recruitment (EC50 = 940 nM, Emax = 9.4%). Finally, the structure-guided optimization of 20 by synthesizing its stereochemical isomers and introducing a phenolic hydroxyl provided compound PZM21 (21), which had an EC50 of 4.6 nM in a Gi/o activation assay (Emax = 77%), and a Ki of 1.1 nM in a radioligand binding assay.38 As a potent Gi/o activator with exceptional selectivity for MOR and minimal β-arrestin2 recruitment, PZM21 decreases affective pain perception and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses compared to that of morphine.38 However, in later studies, PZM21 was demonstrated to be a low efficacy MOR agonist in both G protein and β-arrestin2 assays, and it depresses respiration in a manner similar to morphine.40

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Figure 6. G protein-biased ligands at MOR. Another family of natural products that were found to be biased MOR agonists are mitragynine and its analogs. Mitragynine (22, Figure 6) has an indole-based scaffold distinct from that of morphine. Mitragynine and an oxidative derivative of this compound, 7-OH mitragynine (23), as well as a further rearrangement product mitragynine pseudoindoxyl (24), are all MOR agonists that showed activity as antinociceptive agents. As shown in Figure 6, both potency and efficacy at the MOR were gradually increased from mitragynine to 7-OH mitragynine, and then to mitragynine pseudoindoxyl. Interestingly, all three compounds were inactive for β-arrestin2 recruitment, suggesting that they are G protein-biased MOR agonists.41

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Although all three compounds showed significant binding to the KOR and DOR as well, they function only as weak antagonists rather than as agonists.41 In animal tests, mitragynine pseudoindoxyl exhibited better antinociceptive effects than mitragynine and 7-OH mitragynine, which was equal to or more potent than those of morphine.41 However, mitragynine pseudoindoxyl developed tolerance more slowly than morphine, and it showed fewer side effects such as physical dependence, constipation and respiratory depression, which provided further evidence for the potential of G protein-biased MOR agonists as a new generation of superior analgesics. More recently, a series of new but structurally related compounds were studied by Schmid et al. for their bias properties at MORs and therapeutic potential as safer opioid analgesics.42 Fentanyl (25) and its analog sufentanil (26) are clinically relevant analgesics, and they show comparable G protein-activity (in GTPγS binding assay) and β-arrestin2 recruitment activity (Figure 7). However, when their activity was compared to DAMGO (GTPγS binding, EC50 = 33 nM (Emax = 100%); β-arrestin2 recruitment, EC50 = 229 nM (Emax = 100%)), both compounds are β-arrestin-biased agonists of MORs. Multiple systems were used to study fentanyl, sufentanil, morphine, and piperidine compounds such as SR-17018 (27), SR-15099 (28), SR-15098 (29) and SR-11501 (30), to demonstrate their signaling bias properties. As a result, compounds 27, 28 and 29 are G protein-biased agonists of MOR in both GTPγS binding and cAMP inhibition assays, while compound 30 is a β-arrestin-biased agonist (Figure 7).42 Morphine, on the other hand, was shown as a balanced agonist of MOR. In vivo efficacy of these compounds were tested in the hot 20


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plate and tail flick models, and their antinociceptive effects were compared to respiratory suppression effects to calculate the therapeutic window of in vivo efficacy. Results from this study showed that the GTPγS-binding assay-derived bias factors were more predictive of the calculated therapeutic windows than the cAMP assay-derived bias factors, and MOR agonists with higher G protein-bias factors showed better therapeutic effects and less respiratory suppression.42 A bias factor of 85 was calculated for compound 27 at human MOR using GTPγS-binding assay-derived method, and this compound showed much broader therapeutic windows compared to either fentanyl or morphine. Although structurally different from fentanyl and sufentanil, compounds 27-30 share the overall scaffold with them as highlighted in Figure 7, and it seems the dichloro-substitutions of the right-hand phenyl ring contributed to the G protein-bias properties of compounds 27, 28 and 29.42 Further characterization of these compounds as potential drug candidates is to be expected.

21



Figure 7. Piperidine compounds as G protein-biased agonists of MOR. DAMGO was the reference compound for the calculation of bias factors. Unlike the MOR, KOR agonists have long been recognized to be analgesics with no addiction and tolerance liability.43 However, most KOR agonists cause other CNS side effects such as dysphoria.44 Similarly, it has been hypothesized that the analgesia effect results from G protein-mediated signaling pathway and the side effects are mediated through β-arrestin2 recruitment.45 Therefore, developing KOR agonists that are biased toward G protein signaling but devoid of β-arrestin2 recruitment will be therapeutically beneficial and avoid dysphoric side effects.46 22


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Recently, a number of selective KOR agonists displaying G protein-biased properties have been reported. The triazole compound 31 (Figure 8) was identified from high-throughput screening (HTS) campaign, which was a KOR agonist showing moderate functional potency in the DiscoveRx β-arrestin PathHunter assay (EC50 = 0.87 µM, Emax ~140% compared to the reference compound U69,593), with high selectivity over MOR and DOR.47 Further structural modifications and SAR studies focused on the benzene ring led to the discovery of a series of triazole analogues that showed G protein-biased activity. Among them, compound 32 showed high potency (EC50 = 31 nM, Emax = 94%) in the [35S]GTPγS binding assay, slightly more potent than U69,593 (EC50 = 52 nM, Emax = 100%).47 However, compound 32 showed much lower potency in two different β-arrestin2 assays (EC50 = 4129, 3138 nM for 32, versus 131 and 205 nM for U69,593).48 In further characterization of the compounds at a downstream signaling pathway, compound 32 showed lower potency in ERK1/2 phosphorylation (EC50 = 329 nM) than the unbiased agonist U69,593 (EC50 = 5.4 nM).48 When the furan substituent was replaced for a pyridine ring, compound 33 maintained the bias for G protein signaling (EC50 = 76 nM, Emax = 98%) over β-arrestin2 recruitment (EC50 = 10,090 nM, Emax = 101%).49 In these studies, ERK1/2 activation was tested in parallel with G protein and β-arrestin signaling, which led to the conclusion that bias for ERK1/2 activation can be altered while preserving G protein-biased properties with respect to β-arrestin recruitment, which supported that KOR ERK activation can occur via G protein-dependent or β-arrestin-dependent signaling pathways.49 23



Another screening program by Frankowski et al. led to the discovery of the isoquinolinone compound 34 (Figure 8) as a selective KOR agonist in the cAMP inhibition assay (EC50 = 63 nM), showing high selectivity in binding assays for KOR over MOR, DOR, and other GPCRs.50 Interestingly, replacement of the 2-benzyl with a 2-fluorobenzyl group together with the substitution of the chlorine with a methyl provided a G protein-biased ligand 35. It demonstrated comparable potency (EC50 = 85 nM, Emax = 89%) in [35S]GTPγS binding assay using U69,593 as the reference compound, yet showing weak β-arrestin2 recruitment (EC50 > 10 µM).48 Both compounds 32 and 34 were proved to be brain-penetrant and produced antinociception in the mouse tail flick test, which correlates with the ability of these compounds to potently activate G protein-dependent signaling in cell-based assays.48 Salvinorin A (36, Figure 8) is a terpenoid compound which has recently been characterized by White et al. as a balanced KOR agonist at both the human and the mouse KORs.51,

52

However, replacement of the 2-acetoxy group with a

2-thiocyanatoacetoxy group gave compound RB-64 (37), which showed potent activity stimulating G protein signaling (cAMP assay, EC50 = 5.2 nM, Emax = 99%) but much less potent in β-arrestin translocation (EC50 = 1,130 nM) and mouse KOR internalization assays, suggesting that RB-64 was G protein-biased (bias factor = 96).52 In vivo studies using both wild-type and β-arrestin2 knockout mice showed that RB-64 had no effect on motor coordination, sedation, or anhedonia, suggesting that these KOR-related adverse effects are probably mediated by the β-arrestin2 signaling pathway. 24


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Figure 8. G protein-biased ligands at KOR. Rives et al. reported that the naltrindole-derived ligand 6’-GNTI (38, Figure 8) was a potent partial agonist in both G protein activation (BRET-based) and cAMP inhibition assays compared to the reference agonist ethylketocyclazocine, yet an 25



antagonist for the recruitment of β-arrestin2 (BRET-based) in HEK-293 cells.53 Subsequently, Schmid et al. confirmed the delineation of this bias in vitro and further explored it in cultured striatal neurons.54 6’-GNTI was reported to activate Akt but not ERK1/2 in striatal neurons, while U69,593 activates both kinases, suggesting that KOR utilizes β-arrestin2 to activate ERK in the endogenous setting.54 Taken into consideration the conclusion from Lovell et al.,49 which suggested ERK activation can occur via G protein-dependent or β-arrestin-dependent signaling pathways, understanding of the correlation of ERK activation with G protein and β-arrestin signaling pathways requires further studies. Meanwhile, some recent studies have questioned the therapeutic benefits of G protein-biased KOR agonists for pain management. For example, nalfurafine (39, Figure 8) has been demonstrated as a G protein-biased agonist of both human and rat KORs (hKOR and rKOR respectively), for which the G protein activity was measured as the early phase ERK phosphorylation and β-arrestin-mediated activity as the late phase of p38 MAPK signaling (Figure 8).55 Nalfurafine has been approved in Japan for the treatment of uremic pruritus, but not nalfurafine or any other KOR agonists has been approved for the treatment of pain. Lazenka et al. reported that in animal models, nalfurafine-induced decreases in pain/itch-stimulated behaviors may reflect nonselective decreases in motivated behavior rather than analgesic effects.56 Further study is needed to demonstrate whether KOR agonists would serve as effective analgesics and whether G protein-biased KOR agonists would be clinically safer in the treatment of other KOR-related diseases. 26


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The most recently identified member of the opioid receptor family is NOPR, also known as the nociceptin/orphanin FQ (N/OFQ) receptor. The progress in understanding NOPR pharmacology supports it as a potential drug target for pain treatment and substance abuse.57 Recently, Chang et al. conducted the first functional selectivity analysis of the NOPR by a quantitative analysis of novel NOPR-selective ligands along with commercially available small molecule- and peptide-based NOPR ligands.58 As a result, compound MCOPPB (40, Figure 9) was characterized as a G protein-biased NOPR agonist, being approximately 105-fold more potent in the G protein-mediated cAMP inhibition assay than for β-arrestin1 and β-arrestin2 recruitment. In addition, the spiroxatrine scaffold compound NNC 63-0532 (41),59 a partial agonist at NOPR, also exhibited marked bias towards the G protein pathway and showed lower efficacies in β-arrestin recruitment assays.58 Interestingly, the balanced antagonism of NOPR by J113,397 (42) was converted to G protein-biased agonism with minor structural modifications to the “message” moiety of the ligand by replacing the cyclo-octymethyl with a decahydronaphthalen-1-methyl as in RTI-4229-819 (43), or an isopropylcyclohexane as in RTI-4229-856 (44). Both compounds exhibited moderate to high potency at NOPR (pEC50 = 7.14 and 8.14, respectively) and good functional selectivity against the β-arrestin pathway.58

27



Figure 9. G protein-biased ligands at NOPR (ND, no determinable EC50). The opioid receptors are very important drug targets for pain management and extensive efforts have been put into the discovery of novel biased agonists of these receptors. While the roles of β-arrestin-mediated signaling in the therapeutic/adverse effects associated with these receptors are not yet fully understood, future explorations will benefit from the availability of biased ligands as tool compounds. A milestone will be achieved if the most advanced compound TRV130 gains FDA approval, which will definitely boost GPCR biased ligand discovery. The Dopamine Receptors. Dopamine receptors are comprised of five subtypes that can be classified into two subfamilies, the D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptors.60 The D2 receptor (D2R) is one of the most important drug 28


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targets in neurological disease being one of the primary targets for drugs that treat Parkinson’s disease, schizophrenia, and other affective disorders.61 Treatments for schizophrenia have traditionally involved typical antipsychotics, which block the function of D2R as antagonists or inverse agonists;62 however newer generation antipsychotics target D2R as partial agonists, represented by aripiprazole (45, Figure 10).63 Although these drugs have formed the basis for schizophrenia treatment, most of them are either less efficacious than desired or possess adverse side effects, including tardive dyskinesia, an irreversible movement disorder resulting from prolonged use of typical antipsychotics.64 And aripiprazole has been reported to induce movement disorders as well.65 Based on the scaffold of aripiprazole, a number of biased D2R agonists have been discovered and their effects as potential antipsychotics in animal models described.66-69 For example, Chen et al. studied the SFSRs of aripiprazole analogues through a diversity-oriented medicinal chemistry strategy.68 As summarized in Figure 10,

the structural

modifications

of

the piperazine,

the

linker, and the

dihydroquinolinone moieties led to the discovery of compounds UNC9975 (46), UNC9994 (47) and UNC9995 (48), which were demonstrated to be high-affinity β-arrestin-biased ligands at the D2R. These compounds were potent for D2R β-arrestin recruitment, while showing no significant activity in the G protein-dependent cAMP assays. In animal models of psychosis, compounds UNC9975 and UNC9994 displayed

potent

inhibition

of

amphetamine

(AMPH)-

and

phencyclidine

(PCP)-stimulated hyperlocomotion, which were attenuated or completely abolished in 29



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β-arrestin2 knockout mice.67, 70 These results showed that β-arrestin signaling at D2R is a significant contributor to the antipsychotic efficacy of these compounds. These β-arrestin-biased

D2R

ligands

UNC9975

and

UNC9994

also

reduced

schizophrenia-like behaviors in PCP-treated or NR1-knockdown hypoglutamatergic mice, but induced a lower level of catalepsy than the typical antipsychotic and D2R antagonist, haloperidol.70 The SFSRs results from this series of compounds show that slight structural modifications to a GPCR ligand may provide ligands with distinct signaling bias properties, and known drug molecules can be used as lead compounds for discovering novel biased GPCR ligands. Biased ligands with the opposing pharmacology for the D2R, that is, biased toward stimulation of G protein signaling pathways, have also been identified based on the same scaffold of aripiprazole. Möller et al. reported that the bioisosteric replacement of the heterocyclic appendage of aripiprazole with a pyrazolo[1,5-a]pyridine moiety led to compound 49, which behaved as a potent partial agonist in G protein-signaling but as an antagonist for β-arrestin2 recruitment.71 Also shown in Figure 10, by introducing a 2-methyl group to the benzothiazole of UNC9995 and changing the middle linker from propoxy back to butoxy, Chen et al. unexpectedly converted the β-arrestin-biased UNC9995 to a G protein-biased D2R agonist 50.72 Further SFSR studies that focus on the two aromatic moieties of compound 50 led to compound 51, which showed no activity for β-arrestin recruitment and therefore a G protein-biased D2R partial agonist. Finally, a structural hybridization of compounds 49 and 51 afforded compound 52, which was a highly potent G protein-biased partial agonist 30


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devoid of β-arrestin recruitment activity at D2R.73 In vivo antipsychotic activity of compound 52 was also studied in rat behavioral models,73 and it seemed more effective than aripiprazole treatment in inhibiting AMPH-induced hyperlocomotor activity. Compared to the results of compounds 46-48, since both G protein-biased and β-arrestin-biased agonists of D2R have been shown to be effective as antipsychotic agents, further studies are required for a complete understanding of the roles each signaling pathway plays in the physiology and treatment of schizophrenia.

31



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Figure 10. Aripiprazole-based biased agonists at D2R. More recently, novel aripiprazole analogs with biased properties have also been discovered through a structure-based approach.74 As shown in Figure 11, the dichlorophenyl-piperazine

portion

of

aripiprazole

was

replaced

with

an

indole-piperazine as in compound 53. This indole-piperazine compound displayed comparable activity in both Gi/o-mediated cAMP inhibition and β-arrestin2 recruitment assays with a weak bias factor of 2.5 for β-arrestin signaling. Based on molecular docking with a D2R homology model, the indole NH of compound 53 was predicted to form a hydrogen bond with Ser5.42 in transmembrane 5 (TM5), which has been shown to contribute to G protein-dependent signaling for β2 adrenergic receptors (Figure 11). Inspired by structural information from the serotonin 5-HT2B receptor,75 where ligand interactions with hydrophobic residues on EL2 appear to promote β-arrestin recruitment, a methyl group was added to preclude G protein-dependent signaling via Ser5.42 interactions and instead promote EL2 hydrophobic interactions. This strategy led to the N-methyl compound 54, which shows β-arrestin recruitment and no G protein-dependent signaling activity at D2R. To strengthen the hydrophobic interactions with the isoleucine residue on EL2, a second methyl was introduced to position 2 of the indole ring, giving compound 55. Both potency and efficacy of compound 55 with respect to β-arrestin2 recruitment were significantly enhanced compared to those of compound 54, leading to a β-arrestin bias factor of 20 (Figure 11).74 This is the first example of using GPCR 32


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structural information for the rational design of biased ligands of GPCRs.

EL2 EL2.52 β-arrestin recruitment activity

TM5 Ser5.42 Ser5.43

G protein-dependent activity

Ser5.46

Figure 11. Structure-based design of β-arrestin-biased D2R agonists. Bias factors were calculated based on transduction coefficients using quinpirole as the reference compound.74 Cariprazine (56, Figure 12) is an analogue of aripiprazole and has been approved by the FDA in 2015 for the treatment of schizophrenia. It shares the 1,4-disubstituted aromatic piperazine (1,4-DAP) scaffold of aripiprazole and is a D2R and D3R partial agonist with higher affinity towards D3R.76 Shonberg et al. reported that using dopamine as the reference compound, cariprazine demonstrated a 230-fold bias for the stimulation of D2R-mediated inhibition of cAMP production, versus the 33



phosphorylation of ERK1/2.77 In their studies, transduction coefficients (τ/KA) of both cAMP inhibition and ERK1/2 phosphorylation were calculated as a measure of the activity of an agonist. Values of log(τ/KA) were normalized to dopamine to give values of ∆log(τ/KA), which were compared across pathways for each ligand to give values of ∆∆log(τ/KA), a quantitative measure of bias between two different pathways. Substitution of the N, N-dimethylurea tail group of cariprazine with a tert-butyl carbamate gave compound 57 (Figure 12), which showed decreased bias toward cAMP inhibition versus ERK1/2 phosphorylation.77 Molecular modeling studies suggested that these subtle changes in the spacer and tail regions can influence the orientation of the head group within the orthosteric pocket, and these distinct orientations may underlie patterns of biased agonism.77 In later studies, Szabo et al. found that the bias for G protein signaling was improved when the cyclohexane was opened to a five-carbon spacer (compound 58).78 Keeping this five-carbon spacer, when the tail group was changed to a thienopyridine, the substitution pattern had a significant effect on ligand bias. As shown

in Figure 12, while the

5-chloro-6-methoxy-4-methyl analogue 59 is not biased, the 4,6-dimethyl analogue 60 shows significant bias toward the G protein signaling.78 Interestingly, when the 2,3-dichloro substitution pattern of 59 was changed to 2-methoxyl as in compound 61, the compound’s efficacy in cAMP assay was decreased while ERK activity was enhanced, therefore leading to a reversal of bias (a negative ∆∆log(τ/KA) value). It is worth mentioning that although compounds 56-61 have been studied for their biased agonism of D2R in two distinct assays, it is not clear yet whether ERK 34


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phosphorylation of D2R is G protein-dependent or β-arrestin-dependent. As a matter of fact, it has been reported that G protein is responsible for the regulation of ERK phosphorylation at D2R, and β-arrestin may play an inhibitory role.79 Therefore, further study of these compounds (i.e. in β-arrestin recruitment assays) is needed to fully demonstrate their biased properties.

Figure 12. Other 1,4-DAPs scaffold-based G protein-biased agonists of D2R. The 35



Page 36 of 124

values of pEC50 and Emax can be found in the supporting information of the references.77, 78 Dopamine was used as the reference compound for the calculation of transduction coefficients. Very recently, Bonifazi et al. reported novel bivalent derivatives of sumanirole (62, Figure 13), which showed biased properties at the D2R.80 Sumanirole is a selective D2R agonist that had been studied in clinical trials for the treatment of Parkinson’s disease and restless leg syndrome but has never been approved.81,

82

Presumably,

sumanirole binds to the orthosteric binding pocket of the D2R, in the same way as dopamine. A secondary pharmacophore was attached to the primary pharmacophore of sumanirole through a linker, which gave bivalent ligands that presumably bind to both the orthosteric binding pocket and a secondary binding pocket. Three series of compounds were synthesized and compound 63 was identified as a biased ligand with a preference for G protein signaling compared to sumanirole, with the EC50 ratio of β-arrestin/cAMP of 517 indicating preference for G protein-dependent signaling.

Figure 13. A G protein-biased D2R agonist derived from sumanirole. 36


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More recently, Weïwer et al. characterized β-arrestin biased D2R antagonists with novel scaffold.83 As shown in Figure 14, the hit compound BRD7640 (64), which came from a high-throughput screen, is a micromolar antagonist of the D2R/β-arrestin pathway (EC50 = 2.14 μM, Emax = 80%) and a partial agonist of the Gi/cAMP pathway (EC50 = 0.12 μM, Emax = 75%). In their initial SAR exploration, the pyrrolidine core of BRD7640 was replaced by an achiral 4-piperidine core, which converted it to an agonist in both assays (65). Introduction of an ortho CF3 led to the discovery of BRD5814 (66, Figure 14), which showed antagonist activity in the β-arrestin assay (EC50 = 0.061 µM, 80%) but agonist activity in the G protein assay (EC50 = 0.54 µM, 85%). It was suggested based on docking results that the ortho CF3 group of BRD5814 interacts with H3936.55, a major determinant of biased signaling in D2R,84 and prevents its rotation, resulting in a G protein-preferred active conformation. BRD5814 exhibited efficacy in an amphetamine-induced hyperlocomotion mouse model with reduced motoric side effects in a rotarod performance test.83

Figure 14. BRD5814 is a G protein pathway agonist and β-arrestin pathway antagonist at D2R (red: inhibition of cAMP accumulation; blue: β-arrestin2 37



recruitment). The D2R has been an extensively studied GPCR with identified biased ligands towards both G protein and β-arrestin pathways, displaying agonist, partial agonist or antagonist profiles. Therapeutic potential has been demonstrated in both efficacy and side effect models in animals for a number of these ligands. However, at this stage, it is not yet clear which signaling pathway is more relevant to therapeutic or adverse effects at this receptor and what profile an ideal ligand should have in order to show the best therapeutic effects in the clinic. Further studies in this field are warranted. Meanwhile, although D2R has been a validated drug target for many years, its crystal structure has only recently been solved in an inactive conformation,85 and more structure-based drug design is expected in the future. The D1R is also an important drug target that shows great potential for the treatment of various neuropsychiatric disorders, including Parkinson’s disease and the cognitive impairment associated with schizophrenia. However, significant side effects such as hypotension and tolerance have precluded the approval of a number of candidate compounds. It was thus postulated that biased agonists at the D1R might provide novel therapeutics devoid of the aforementioned side effects.86, 87 Conroy et al. studied the bias properties of a set of substituted benzazepine compounds which are summarized in Figure 15.86 Compounds SKF38393 (67), SKF75670 (68) and SKF-77434 (69), which have hydrogen, methyl and allyl substituents attached to their nitrogen atoms respectively, behaved as very potent agonists in G protein-mediated cAMP accumulation assays, while showing no discernible β-arrestin recruitment. 38


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When tested as antagonists, these compounds showed potent inhibition of dopamine-induced β-arrestin recruitment and D1R internalization. The introduction of a chlorine atom to the catechol benzene ring led to the loss of bias in N-H and N-allyl compounds (70, 72, 73 and 75) but not N-methyl compounds SKF83959 (71) and SKF82957 (74). In terms of β-arrestin to cAMP EC50 ratios, compounds 67, 68, 69, 71 and 74 represent the most biased GPCR ligands reported so far, and further characterization in other assays and animal models are expected which should show their therapeutic advantages compared to balanced D1R ligands.

Figure 15. G protein-biased agonists at D1R (red: stimulation of cAMP accumulation; 39



blue: β-arrestin2 recruitment). However, catechols suffer from poor pharmacokinetic properties such as negligible bioavailability and low central nervous system (CNS) penetration, which has prevented the development of such compounds as clinical drugs. More recently, Gray et al. have reported non-catechol agonists of the D1R for which G protein-bias properties have been observed.88 As shown in Figure 16, compound PF-4211 (76) was identified from an HTS of about 3 million compounds and displayed modest potency and efficacy as a non-catechol D1R agonist (EC50 = 2,519 nM, Emax = 42% in cAMP assay). Extensive SAR study and iterative medicinal chemistry optimizations led to the discovery of compounds PF-8294 (77) and PF-6142 (78), which are potent and selective D1R agonists with good pharmacokinetic profiles. More importantly, these compounds showed no significant desensitization of the receptor after 2 h treatment of the cells with the compounds, whereas significant desensitization was measured for catechol D1R ligands such as dopamine and dihydrexidine. In this study, live cell total internal reflection fluorescence microscopy imaging assay measuring β-arrestin recruitment revealed that compounds 77 and 78 showed impaired β-arrestin recruitment. In an animal efficacy study, orally-administered of another compound in the series led to robust and more sustained effects in both non-human primate eye blink response89 and the unilateral 6-OHDA lesion rodent model of parkinsonism.88

40


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Figure 16. Non-catechol, G protein-biased agonists of D1R. The Serotonin Receptors. The serotonin receptors (also known as 5-HT receptors) are the targets of a variety of pharmaceutical drugs, including many antidepressants, antipsychotics, and anorectics.90 All serotonin receptors are GPCRs except the 5-HT3 receptor, which is a ligand-gated ion channel. In terms of signaling bias, the 5-HT2A/2C receptors are among the very first GPCRs for which functionally selective ligands have been reported91; however, interrogation of specific downstream signaling pathways (e.g. G protein versus β-arrestin) have only recently been explored. Ergotamine (79, Figure 17), a natural ergoline alkaloid that shows anti-migraine properties, is an agonist at many serotonin receptors.92 Like most ergolines, such as the hallucinogen lysergic acid diethylamide (LSD), ergotamine has been reported to be β-arrestin-biased at the 5-HT2B receptor.75, 93 Structures of the 5-HT2B receptor and 5-HT1B receptor co-crystallized with ergotamine revealed a β-arrestin-biased and non-biased ‘active’ states of these receptors, respectively.93, 94 These studies provided the first structural features for biased signaling, which will in turn guide the rational design of biased ligands of other GPCRs. With the discovery of more biased GPCR ligands, more crystal structures with biased ligands bound to the receptor are to be 41



Page 42 of 124

expected in the future. Another member of the 5-HT2 subfamily, the 5-HT2C receptor, has been demonstrated as a potential drug target for a variety of CNS diseases, such as obesity, schizophrenia

and

drug

addiction.95,

96

We

have

reported

that

2-arylcyclopropylmethylamines such as compound 80 (Figure 17) shows functional selectivity for the G protein signaling over β-arrestin2 recruitment.97 Compared to serotonin, compound 80 shows less receptor desensitization in both calcium flux and phosphoinositide hydrolysis assays, which is a result expected with weaker efficacy for β-arrestin2 recruitment. More recently, we discovered that introducing a methyl substitution to the primary amine led to a markedly G protein-biased compound 81, although a 20-fold decrease of potency (EC50 = 23 nM versus 1.2 nM) in the calcium flux assay was observed.98 When the substituent was enlarged to a 2-methoxybenzyl group as in compound 82, the potency in G protein signaling was unchanged but β-arrestin2 recruitment recovered, therefore functional selectivity was reduced compared to 81. Future study is needed to shed light on the role of functional selectivity at this receptor with respect to in vivo effects. For another 5-HT1 subtype, the 5-HT1A receptors, a series of long-chain arylpiperazines have been characterized as functionally selective agonists for G protein-dependent cAMP inhibition versus β-arrestin2 recruitment.99 As shown in Figure 17, all the arylpiperazines (83-85) displayed a sizable preference for G protein-mediated cAMP inhibition over β-arrestin2 recruitment with respect to both potency and efficacy. The strong pathway preference shown by the naphthalenyl 42


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derivative 84, compared to those of the tetrahydronaphthalenyl analogs 83 and 85, suggests that modification of the terminal fragment of these arylpiperazine derivatives might provide pathway selective compounds that favor G protein-dependent signaling over β-arrestin-2 recruitment. In vivo effects of these compounds are not reported.

Figure 17. Biased agonists at serotonin receptors. The Histamine Receptors. Histamine is a neurotransmitter that plays physiological roles in cellular events through binding to four types of G protein-coupled histamine receptors (H1R-H4R) that are differentially expressed in 43



various cell types. For example, the H2R is mainly involved in gastric acid production and has been a classic pharmacological target to treat gastric acid-related conditions.100 Regarding ligand bias properties, a retrospective study of the commonly used H2R blocker famotidine has proved that although an inverse agonist in cAMP assays, famotidine mimics the effects of histamine concerning receptor desensitization and internalization.101 This adds to the conclusion that for many known GPCR ligands that have been characterized as antagonists or inverse agonists in the traditional G protein assays, they could have positive efficacy concerning receptor desensitization and internalization which are mediated by β-arrestins. Another subtype of the histamine receptor, H4R, represents a promising therapeutic target for pathologies such as airway inflammation, inflammatory bowel disease, and atopic dermatitis.102 Recently, a comparative study of biased signaling for a number of H4R antagonists has been reported. Compound JNJ-7777120 (86, Figure 18), which had been initially identified as a selective antagonist of the H4R,103 was characterized as a β-arrestin biased agonist. It showed no effects in the [35S]GTPγS binding assay, but demonstrated potent partial agonist activity in the β-arrestin2 recruitment assay (pEC50 = 7.9, Emax = 64%).104 Based on its indolecarboxamide scaffold, various JNJ-7777120 analogues have been recently developed with similar biased properties toward β-arrestin2, but interestingly the nitrated compound 87 was found to be a balanced hH4R agonist.105 Unlike these indolecarboxamides, the isothiourea compounds gives a broader spectrum of efficacies upon relatively modest structural changes. For example, introducing a 2-Cl substitution to the phenyl group of the 44


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unbiased H4R antagonist VUF9107 (88) converts it into a G protein-biased agonist VUF5222 (89), and a 3-Cl substitution converts it into a β-arrestin2-biased compound VUF5223 (90).106 Other chemical classes of G protein-biased ligands including VUF10778 (91) and VUF10185 (92), and β-arrestin2-biased ligand VUF10214 (93) were also reported.106 Only a few H4R antagonists have entered clinical trials and JNJ-7777120 has never been advanced into clinical trials due to a short half-life and preclinical toxicity, but it serves as a tool compound for showing the role of the H4R in inflammation and pruritus.107

Figure 18. Biased ligands at H4R. The Cannabinoid Receptors. The cannabinoid receptors are a family of GPCRs that play important roles in physiology,108 which have mainly two subtypes CB1R and CB2R (other putative cannabinoid receptors include GPR18 and GPR55). CB1R is 45



mainly expressed in the CNS whereas CB2R is expressed in the immune system, and both of them are important drug targets that have drawn a lot of attention.109-111 Two recent review articles have summarized the ligand bias phenomenon at these receptors,112, 113 and some of the studies are highlighted in the following. The CB1R couples primarily to Gi/o, but has also been observed to couple with Gs and Gq when activated by specific agonists.112 A line of evidence has shown that most CB1 agonists do not activate associated G proteins equally, therefore showing functional selectivity towards specific downstream signaling pathways. For example, the synthetic cannabinoid WIN-55212 (94, Figure 19) is equally efficacious for inhibiting (Gi) and stimulating (Gs) cAMP accumulation (Emax both normalized to 100%), while the endogenous cannabinoid anandamide (AEA, 95) favor Gi over Gs (Emax = 81% versus 27%).114 Biased properties of CB1R ligands between the G protein-dependent signaling versus β-arrestin signaling have also been studied. For example, using a cell culture model of striatal medium spiny projection neurons, the endogenous cannabinoids 2-arachidonoylglycerol (2-AG, 96, Figure 19) and AEA, the phytocannabinoids ∆9-tetrahydrocannabinol (THC, 97) and canabidiol (CBD, 98) and the synthetic cannabinoids CP-55940 (99) and WIN-55212 have been evaluated for their signaling bias properties at CB1R, in various signaling activities associated with Gi/o, Gs, Gq and β-arretin1.115 Signaling bias was more recently quantified using the Black and Leff operational model by the same group, and the effects were compared between wild-type and mutant huntingtin protein.116 In these studies, Gi-mediated activity was 46


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evaluated in pERK1/2 assay and the interactions between CB1R and β-arrestin1 were quantified via BRET2.116 Using WIN-55212 as the reference compound, 2-AG and AEA were shown to be Gi-biased and normalized CB1R protein levels and improved cell viability, whereas CP55,940 and THC were β-arrestin1-biased and reduced CB1R protein levels and cell viability in Huntington’s disease (HD) cells.116 Consistent with the literature, CBD was not a CB1R agonist, but it inhibited THC-dependent signaling. The correlation between ligand bias and cell viability suggests that enhancing Gi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD, while β-arrestin-biased cannabinoids, such as THC may be detrimental to CB1 signaling in HD where CB1 levels are already reduced.116

Figure 19. Biased agonists at CB1R. Due to the sequence similarity between CB1R and CB2R (44% overall identity, 68% in the TM region),117 some of the aforementioned CB1 agonists activate CB2R as well due to their weak selectivity. Such compounds WIN55212 and CP-55940 have 47



been profiled for their functional selectivity properties at CB2R by Atwood et al.118 It was found that compound WIN-55212 induced β-arrestin recruitment to a much less degree than CP-55940 at CB2R, and WIN-55212 not only did not lead to receptor internalization but also antagonized the effects induced by CP-55940.118 These compounds were later studied together with more CB2R ligands by the same group in more functional assays, for which their biased activities were profiled and bias factors calculated.119 As shown in Figure 20, using CP-55940 as the reference compound, WIN-55212, THC, JWH-133 (100), HU-308 (101) and JWH-015 (102) behaved as G protein-biased partial agonists, whereas AM-1248 (103), STS-135 (104) and UR-144 (105) show β-arrestin-biased properties. It is also worth noting that within each chemical class, that is the phytocannabinoids and analogs (CP-55940, THC, JWH-133 and HU-308) or the synthetic indoles (AM-1248, STS-135, UR-144 and JWH-015), opposite biased properties have been observed despite the light structural differences (Figure 20). Meanwhile, it has also been reported that while rimonabant (106) behaved as an inverse agonist at CB2R in both Gi-mediated cAMP inhibition and β-arrestin recruitment assays, the indole compound AM-630 (107) displayed inverse agonist activity in Gi signaling but partial agonist activity in arrestin recruitment (Figure 20).119

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Figure 20. Biased ligands at CB2R. “NA”, not applicable (activation < 20%

49



threshold); negative Emax values (for 106 and 107) represent inverse agonist activity. Targeting the CB1R as an inverse agonist, rimonabant had been approved for the treatment of obesity but was quickly withdrawn due to CNS side effects.120 The phytocannabinoid THC and its analog nabilone have been approved for the treatment of pain and nausea in cancer and HIV patients, but the hallucinogenic effects of THC have been well known. Whether biased ligands at either CB1R or CB2R could provide safer therapeutics remains to be seen, as how the abovementioned biased properties correlate to therapeutic or unwanted side effects are not yet understood. Further research in this area is warranted. The Apelin Receptor (APJ). APJ is a class A GPCR that has been discovered in 1993,121 and it has been suggested as a potential target for cardiovascular diseases.122 It has been demonstrated that the activation of APJ by apelin triggers intracellular G protein-dependent signaling pathways which increase both myocardial contractility and vasodilatation; but mechanical stretch activates APJ-mediated β-arrestin signaling, which leads to cardiac hypertrophy.123 Therefore, from a drug discovery point of view, G protein-biased APJ agonists would be more effective for the treatment of heart failure. Based on the peptide sequence of [Pyr1]apelin-13 (108, Figure 21), which is the main active form of apelinergic peptides, Brame et al. designed a series of cyclic analogues with the aim of discovering biased agonists of APJ.124 Of more than 100 peptides that were designed, MM07 (109) was identified as a G protein-biased agonist, which has an intramolecular macrocycle formed by a disulfide bridge 50


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between two cysteine residues (Figure 21). In the β-arrestin recruitment assay, MM07 was ~790-fold less potent than [Pyr1]apelin-13; and in a receptor internalization assay, MM07 was ~215-fold less potent.124 Given that MM07 has comparable potency in a G protein-dependent saphenous vein contraction assay to [Pyr1]apelin-13 (pEC50 = 9.54 for MM07 versus 9.93 for [Pyr1]apelin-13), these translated into bias factors of ~350-fold (against β-arrestin recruitment) and ~1300-fold (against receptor internalization) respectively for MM07. The therapeutic advantages of MM07 over [Pyr1]apelin-13 were demonstrated in a rat cardiac output experiment and a forearm blood flow test in human volunteers, which supports that biased agonism of the APJ receptor could translate to improved efficacy in vivo.124 Murza et al. also reported a series of macrocylic analogues of apelin such as 110 that showed biased properties as APJ agonists.125 Peptide 110 incorporates a 17-membered ring in its scaffold and showed nanomolar potency in G protein assays, with excellent functional selectivity against β-arrestin recruitment. Although less potent than [Pyr1]apelin-13, significantly improved signaling bias has been demonstrated for 110. The authors concluded from the SFSR results that the position and the nature of the C-terminal aromatic residue is a determinant for APJ interaction and β-arrestin recruitment.125 This is in agreement with a previous report that the deletion of the C-terminal phenylalanine provides an analogue with reduced β-arrestin activity and therefore becomes biased toward G protein signaling.126 However, compound 110 failed to induce hypotensive effect in vivo, whereas a less biased analogue induced a significant drop in mean arterial blood pressure. The reason for 51



this is probably that hypotensive effects of APJ elicited by apelin analogues depends on the ability of these ligands to recruit β-arrestins and induce receptor internalization.126 Nevertheless, these results support the idea that the distinct signaling pathways coupled to a specific GPCR could be the mediators of distinct physiological functions of the same receptor, and biased ligands could be useful probes for the study of such effects. For small molecule APJ agonists, Read et al. reported that compound CMF-019 (111, Figure 21), the carboxylic potassium salt of a benzoimidazole compound patented by Sanofi,127 was a G protein-biased small molecule agonist of APJ.128 Compared to apelin-13, CMF-019 exhibited similar potency in the cAMP assay (pEC50 = 10.00 versus 9.34 for apelin-13), whereas it was much less potent in β-arrestin recruitment and receptor internalization assays (pEC50 = 6.65 versus 8.65 and pEC50 = 6.16 versus 9.28 respectively). These translated into a bias factor of ~400 for the Gi signaling over β-arrestin recruitment and ~6000 over receptor internalization.128 Furthermore, CMF-019 induced a significant increase in cardiac contractility in a rat model.128 These results demonstrated that the ligand bias properties at APJ can be retained in a non-peptide compounds and such bias could translate into therapeutic benefits.

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Figure 21. G protein-biased APJ agonists. The amino acid sequences of the peptides are shown from the N-terminus (left) to the C-terminus (right). The Chemokine Receptors. The chemokine receptors also belong to class A GPCRs, and they bind to chemokines (8-12 kDa peptides). The chemokine receptor family contains 24 members in humans and can be subdivided, based on the class of chemokines they bind, into four subfamilies: CCR1-10, CXCR1-7, CX3CR1, and XCR1. Chemokine receptors mediate the trafficking of leukocytes and dysregulation of chemokine receptors are involved in autoimmune conditions, inflammatory diseases, viral infections and cancer.129 Two drugs targeting this family of receptors have so far made it to the market: the CCR5 antagonist maraviroc for the treatment of HIV infections, and the CXCR4 antagonist plerixafor for the mobilization of hematopoietic stem cells into the bloodstream in cancer patients. Currently, there are

53



more than 50 chemokines that have been identified, and a complex network between chemokines and chemokine receptors has been depicted.130 Bias properties of these chemokines at chemokine receptors have been studied, which could in part explain the ligand redundancy for this family of receptors.131, 132 The chemokine receptor CXCR3 is a potential drug target for the treatment of autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis) and cancer.133 Bernat et al. recently reported that for CXCR3, light structural modifications of a negative allosteric modulator (NAM) 112 (Figure 22) led to the discovery of compounds biased for either the β-arrestin or G protein pathway. Based on the 8-azaquinazolinone scaffold of compound 112, a boronic acid which is known for its unique ability to interact with proteins in a reversible covalent way,134 was used to investigate the allosteric modulation of CXCR3.135 This led to the discovery of compound 113, which was reported as the first biased NAM of CXCR3 that preferentially inhibited the CXCL11-mediated recruitment of β-arrestin2 to CXCR3, but not the activation of G proteins. In later work, in order to reduce the scaffold rigidity and therefore improve the physicochemical properties of the compounds, the benzene moiety on the right-hand was changed to a flexible fluorooxoalkyl chain as in compound 114.136 This compound showed 187-fold preference for the inhibition of G protein signaling versus β-arrestin recruitment. The authors reasoned that structurally similar allosteric modulators can bind to CXCR3 in different orientations, and these orientations elicit different functional responses, leading to biased signal transduction. The same group also reported the discovery and characterization of two strongly 54


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biased allosteric agonists of CXCR3.133 Based on the tetrahydroisoquinoline carboxamide core of compound VUF10661 (115, Figure 22), the positive charge at the amine on the side chain of the lysine moiety was shown to be dispensable. The elongation of the linker connecting the dibenzyl moiety led to compound FAUC1036 (116), which was characterized as a fully β-arrestin-biased allosteric agonist of CXCR3. Compound FAUC1036 showed improved potency in the β-arrestin2 assay (pEC50 = 6.64 compared to 5.89 of VUF10661) while displaying no effect in the [35S]GTPγS binding assay. Interestingly, an introduction of a para-methoxy group on the upper benzene ring afforded compound FAUC1104 (117), which showed a bias for the G protein pathway.133 Although slightly weaker in the G protein assay (pEC50 = 5.22, Emax = 50%), this compound shows no measureable β-arrestin2 recruitment. In CXCR3

internalization

assays,

compound

FAUC1036

induced

receptor

internalization at concentrations of 10 µM and 1 µM, similar to the effect observed with 100 nM of CXCL11, the endogenous ligand; but for compound FAUC1104, no internalization was observed. Interestingly, both compounds FAUC1036 and FAUC1104 induced cell migration in a standard Transwell assay, suggesting that both G protein activation and β-arrestin recruitment account for the functional chemotaxis response.133 Therapeutic potential of these compounds in a disease context has not been reported yet.

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Figure 22. Biased allosteric modulators of CXCR3. The Prostaglandin EP2 Receptor. Prostaglandin E2 (PGE2) exerts a wide variety of biological actions through four receptor subtypes, EP1-EP4, in various tissues. The EP2 receptor plays important roles in inflammation and is a potential target for the treatment of various CNS and peripheral diseases.137 The EP2 receptor modulates beneficial neuroprotective effects in the brain via G protein-mediated cAMP signaling,138 however, the activation of β-arrestin-mediated EP2 receptor signaling led to deleterious effects, such as tumorigenesis and angiogenesis.139 Ogawa et al. 56


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recently reported the results of their studies of biased ligands at prostanoid receptors. As shown in Figure 23, the prostacyclin analogue 118 showed very weak EP2 agonist activity (EC50 = 8,900 nM in the cAMP assay). Substitution of the upper flexible alkyl chain with a thiazole, along with the removal of the 15-hydroxyl group from the ω-chain afforded a potent EP2 agonist 119, which was biased toward the G protein pathway (cAMP assay EC50 = 3.9 nM, Emax = 98%) and away from the β-arrestin pathway (EC50 > 10,000 nM, Emax = 38%).140 Adjustment of the linker length between the cyclopentane scaffold and the phenoxy moiety provided a short ω chain derivative 120, with a similar biased profile.140 SFSR studies around the benzene ring led to the discovery of compound 121, with a 3-methyl-4-chloro substitution pattern, which showed enhanced potency in the G protein assay maintaining excellent functional selectivity against β-arrestin recruitment.141 Notably, adding another methyl substitution to the benzene ring, as shown with compound 122, led to a sharp increase of β-arrestin activity (EC50 = 2.3 nM, Emax = 98%), thus a significant loss of functional selectivity. The inversion of the hydroxyl group on the cyclopentane moiety of 121 gave compound 123, which showed a 5-fold increase in its G protein activity (EC50 = 0.14 nM versus 0.69 nM of 121) without any increase in β-arrestin activity.140 These results add to the conclusion that subtle modifications of molecular structure could lead to significant changes of bias profiles of the ligands. G protein-biased EP2 agonists have been reported to show potent intraocular lowering effects in rabbit and monkey.142

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COOH

COOH

COOH

N

S

S

N

O O

O O

HO

O

OH

O

HO

118, hEP2 agonist cAMP assay: EC50 = 8900 nM

119, G protein-biased cAMP assay: EC50 = 3.9 nM (98%) -arrestin recruitment: EC50 > 10,000 nM (38%)

COOH S

HO 120, G protein-biased EC50 = 13 nM (118%) EC50 > 10,000 nM (28%)

COOH

N

S

O

COOH

N

S

O

O

Me

HO

O

O

Me

HO

O

Me

HO

Cl 121, G protein-biased EC50 = 0.69 nM (88%) EC50 > 10,000 nM (23%)

N

Cl

Cl

122 EC50 = 0.17 nM (102%) EC50 = 2.3 nM (98%)

123, G protein-biased EC50 = 0.14 nM (97%) EC50 > 10,000 nM (21%)

Me

Figure 23. Biased prostaglandin EP2 agonists. The Endothelin A (ETA) Receptor. Endothelin peptides mediate their cellular actions via interactions with two GPCRs, ETA and ETB. Endothelin antagonists are approved drugs for the treatment of pulmonary arterial hypertension (PAH), and hold promise for cancer and fibrosis therapy.143 Like other GPCRs, there have been emerging interests in the study of ligand bias properties at these receptors, which were recently reviewed.144 For example, it has been reported that the potency and efficacy of the endogenous ligands ET-1, 2, and 3 vary in the G protein-dependent saphenous vein constrictor assays and the β-arrestin recruitment assay.145 ET-1 (124) was taken as the reference compound, and transduction efficiency (τ/KA) was compared between these peptides which showed that both ET-2 (125) and ET-3 (126) are G protein-biased agonists of ETA although ET-3 is much less potent (Figure 24).144 Of 58


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more importance is the biased profile of bosentan (127), a non-selective ETA/ETB antagonist that is used in the clinic for the treatment of PAH. It was found that bosentan is a β-arrestin-biased antagonist of ETA, while it is balanced at ETB (Figure 24).144,

145

It is not yet clear whether the β-arrestin-biased antagonism of ETA is

beneficial or detrimental for its effects in treating PAH, which necessitate further studies. But as pointed out,144 ET-1 stimulated ETA-mediated β-arrestin signaling leads to activation of the NF-κB in epithelial ovarian cancer,146 and that β-arrestin1 epigenetically regulates ET-1-induced β-catenin signaling,147, 148 thus the β-arrestin pathway plays important roles in cancer. Better understanding of the signaling pathways will definitely help with further drug discovery efforts targeting the ETA receptor.

Figure 24. Biased ligands at the ETA receptor. The GPR109A Receptor. GPR109A (also known as the hydroxy-carboxylic acid receptor HCAR2) is an orphan GPCR which was thought to be the receptor responsible for niacin’s biological effects.149 Niacin (128, Figure 25), both as a 59



medication and diet supplement, has been primarily used for lowering triglycerides and raising plasma high-density lipoprotein (HDL). However, niacin causes side effects, among which the most common is facial flushing. It was reported by Walters et al. that niacin was active in a series of assays testing β-arrestin1/2 membrane recruitment, as well as conformational changes in β-arrestin2 upon activation of GPR109A, phosphorylation of ERK and others, demonstrating that β-arrestin1 mediates niacin-induced flushing, but not its antilipolytic effect.150 A G protein-biased GPR109A agonist MK-0354 (129), which is a synthetic tetrazole bioisostere of niacin, was discovered by Merck.151 Compared to niacin, stimulation with MK-0354 failed to induce the conformational change of β-arrestin2 upon GPR109A activation and MK-0354 did not induce the recruitment of β-arrestin1.150 As a biased agonist of GPR109A, MK-0354 had been evaluated in clinical trials but failed to elevate HDL levels. This led to the conclusion that the mechanism by which niacin alters HDL levels is GPR109A independent.152 Nevertheless, this example provided further evidence that biasing a ligand toward GPCR (in this case GPR109A) agonism could separate the receptor mediated G-protein activation from the known side effect (in this case, flushing) which has been linked to the β-arrestin pathway.

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Figure 25. MK-0354 is a G protein-biased GPR109A agonist. The Sphingosine 1-Phosphate Receptor 1 (S1P1R). S1P1R is a validated drug target for the treatment of multiple sclerosis. Fingolimod, a modulator of S1P1R, is the first oral therapy for relapsing remitting multiple sclerosis, however, it causes a dose-dependent reduction in heart rate (bradycardia). Clinical studies with selective S1P1R agonists suggested that the bradycardia in human is at least in part mediated by the agonism of S1P1R,153 therefore an “on-target” side effect that could only be avoided by biased agonists. Dhar et al. reported the discovery of BMS-986104, a differentiated S1P1R modulator with improved side effect profile.154 Due to the fact that both fingolimod and BMS-986104 are prodrugs, the phosphate forms were compared to determine their bias profiles. As shown in Figure 26, both compounds 130 (fingolimod phosphate) and 131 (BMS-986104 phosphate) were highly potent in cAMP inhibition, GTPγS binding, ERK phosphorylation and receptor internalization assays. Compared to 130, compound 131 showed lower efficacy in internalization assays (Emax = 68% versus 100% for 130), and an over 1400-fold potency difference 61



between them in ERK phosphorylation was observed (8.16 nM versus 0.0056 nM). In addition, compound 131 was demonstrated to be cAMP-biased (~1000-fold) relative to ERK phosphorylation.154 In cultured human cardiomyocytes, compound 131 showed less reduction in heart rate compared to compound 130, and BMS-986104 has been advanced to human clinical trials. It is not clear at this stage whether ERK if phosphorylation is G protein-dependent or β-arrestin-dependent at S1P1R, but the overall differential functional profile of 131 might contribute to its superior safety profile.

Figure 26. BMS-986104 phosphate is a G protein-biased agonist at S1P1R. The Neurotensin Receptor 1 (NTR1). The physiological roles of neurotensin are mainly mediated by NTR1, which has been demonstrated as a drug target for both CNS disorders such as schizophrenia and drug addiction and peripheral diseases such as cancer.155,156 While most known NTR1 agonists are peptide analogues of neurotensin, small molecule agonists have also been reported. For example, to identify nonpeptidic, β-arrestin-biased NTR1 agonists, Peddibhotla et al. carried out a high-content screening (HCS) of receptor/β-arrestin-GFP complexes based on a β-arrestin conjugated green fluorescent protein (GFP) reporter expressed in U2OS cells. The quinazoline compound 132 (Figure 27) was identified as the single hit from 62


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the HCS, which showed micromolar potency in the primary assay (EC50 = 5.9 µM). Optimization of this compound led to the discovery of ML314 (133), in which the cyclobutyl substituent was replaced by a cyclopropyl.157 ML314 showed higher potency in the HCS β-arrestin assay (EC50 = 2.0 µM), which was confirmed in the DiscoveRx β-arrestin recruitment assay (EC50 = 3.4 µM). Importantly, ML314 induced no response in Gq-mediated Ca2+ mobilization, indicating that it is fully biased toward β-arrestin signaling. In animal studies, ML314 attenuated amphetamine-like hyperlocomotion in dopamine transporter knockout mice and methamphetamine-induced hyperlocomotion.158

Figure 27. ML314 is a β-arrestin-biased agonist at NTR1. The Formyl Peptide Receptor 2 (FPR2). FPR2 (also known as lipoxin A4 receptor, ALXR) is a promiscuous GPCR that can be activated by an array of ligands, which include structurally unrelated lipids and peptides or proteins.159 It is a potential drug target because its involvement in a range of normal physiological processes and pathological diseases.159, 160 F2Pal10 (135, Figure 28) is a pepducin (lipidated peptide) for which the peptide moiety shares the amino acid sequence of 10 amino acids in the EL3 of FPR2, and its N-terminal was conjugated to palmitic acid.161 Comparing to the FPR2 peptide agonist WKYMVM (134), F2Pal10 was equally potent in Gq-mediated 63



calcium flux assay; but its activation of the receptor did not lead to β-arrestin recruitment, whereas WKYMVM was a potent agonist (EC50 = 19 nM).162 Although it appeared that FPR2 desensitization occurred independent of β-arrestins, F2Pal10-desensitized and WKYMVM-desensitized neutrophils differed in the reactivation of FPR2 through a cross-talk mechanism with other GPCRs.162 Moreover, the inability of F2Pal10 to recruit β-arrestins was found to be associated with a reduced rate of receptor internalization and impaired chemotaxis in neutrophils. Although the therapeutic advantage due to its biased properties is not yet reported, F2Pal10 serves as a useful tool for further study of FPR2 functions.

Figure 28. F2Pal10 is a G protein-biased agonist of FPR2. The amino acid sequences of each peptide are shown from the N-terminus (left) to the C-terminus (right). The Adenosine Receptors. Adenosine receptors are GPCRs for the endogenous molecule adenosine, for which four subtypes have been identified: A1AR, A2AAR, A2BAR and A3AR.163 Adenosine receptors play different roles in both the central and peripheral systems, and many ligands for them have been developed as potential therapeutics. Functional selectivity of previously known adenosine receptor ligands has been reviewed, which led to the conclusion that functional selectivity of synthetic ligands has been convincingly shown for the A1AR and A3AR.164 64


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A1AR is an important drug target for cardioprotection, but current A1AR drugs are limited for this indication because of the occurrence of bradycardia as a major adverse effect. A rationally designed bitopic A1AR agonist VCP746 (136, Figure 29) was recently reported by Valant et al., which is a hybrid molecule comprising adenosine linked to a positive allosteric modulator.165 Compound VCP746 is biased towards Gi-mediated inhibition of forskolin stimulated cAMP accumulation versus ERK1/2 phosphorylation, which was chosen as a noncanonical downstream convergent signaling pathway, because it can be both G protein-dependent and -independent. Using R-PIA (137) as the reference compound, the agonist transduction coefficient log(τ/KA) was used to evaluate compound bias properties. VCP746 showed a 33.9-fold bias for cAMP inhibition relative to ERK1/2 phosphorylation, while this bias was only 1.5- and 2.0-fold for adenosine and VCP900 (138) respectively. In animal tests, VCP746 was found to protect against ischemic insult in native A1AR-expressing cardiomyoblasts and cardiomyocytes and did not affect rat atrial heart rate.165 Given the undefined relationships between G protein pathway, β-arrestin pathway and ERK1/2 phosphorylation, future study is needed to better understand the roles that the distinct pathways play in the overall function of the receptor. VCP746 has also been subsequently characterized in further functional assays, which included cAMP inhibition, calcium mobilization, ERK1/2, Akt1/2/3, and cell survival.166 Using both cAMP inhibition and calcium mobilization to quantify bias properties, further studies around compound VCP746 were recently reported by the same group.167 Structural modifications were made to the orthosteric adenosine 65



pharmacophore,

the

linker,

and

the

allosteric

2-amino-3-benzoylthiophene

pharmacophore to study the SFSRs. As shown in Figure 29, compound VCP746 displays a bias toward cAMP signaling over calcium mobilization. Increasing the linker length by adding an additional methylene group afforded compound 139, for which the bias for cAMP signaling was enhanced compared to VCP746. However, when the 4-(trifluoromethyl)phenyl substituent of the thiophene was deleted from the allosteric moiety, compound 140 became unbiased. Furthermore, when all three substituents of the thiophene were removed as in compound 141, a reversal of the bias was observed (Figure 29). These results showed that the allosteric moiety of these bitopic agonists of A1AR is a very important contributor to their bias properties. This conclusion was supported by docking studies using an A1AR homology model based on an active A2AAR crystal structure, which indicated that interactions of the allosteric moiety and the linker region of compound VCP746 within the extracellular vestibule may underlie the initial trigger of its bias profile.

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Figure 29. VCP746 and analogues as biased A1AR bitopic agonists. The A3AR is another adenosine receptor that has been reported to play roles in a number of diseases such as cancer, inflammation and ischemia.168 Baltos et al. recently

reported

the

bias

properties

of

a

series

of

(N)-methanocarba

5’-N-methyluronamide nucleoside derivatives 143-148 (Figure 30).169 In their studies, the A3AR agonists were assessed for their ability to inhibit cAMP accumulation, 67



phosphorylate ERK1/2 and Akt, increase intracellular calcium concentrations and promote cell survival upon serum starvation. Using IB-MECA (142) as the reference agonist and pERK1/2 as the reference pathway, a significant positive correlation was observed between the C2 substituent length (in Å) of compounds and their bias toward cell survival.169 Molecular modeling suggested that extended C2 substituents promote an outward shift of TM2 of the receptor, which may underlie the SFSRs. Although it is not clear whether the ability of A3AR agonists to promote cell survival is G protein-dependent or G protein-independent, the SFSRs of these compounds proved that profiling of GPCR ligands at multiple pathways is necessary to better predict their therapeutic potential.

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I

HN N

H N

N

O N O

N

OH OH IB-MECA (142) pERK1/2: pEC50 = 9.6 (Emax = 106%) cAMP: pEC50 = 9.4 (Emax = 72%) bias factor = 1 survival: pEC50 = 9.2 (Emax = 85%) bias factor = 1 reference compound Cl

HN N

H N

O

N

N

N

H N

Cl

OH OH

Cl

HN N N

N

N

H N

N N

N O

OH OH

N

N

F F

MRS5698 (145) pERK1/2: pEC50 = 8.1 (Emax = 106%) cAMP: pEC50 = 8.3 (Emax = 89%) bias factor = 3.2 survival: pEC50 = 9.5 (Emax = 81%) bias factor = 20

Cl

HN H N

N O

N

N N

OH OH

OH OH MRS5704 (147)


N

Cl N

N

N

OH OH

HN H N

N O

N

MRS5655 (144) pERK1/2: pEC50 = 9.3 (Emax = 88%) cAMP: pEC50 = 9.1 (Emax = 78%) bias factor = 2.1 survival: pEC50 = 9.7 (Emax = 83%) bias factor = 8.1

pERK1/2: pEC50 = 9.8 (Emax = 94%) cAMP: pEC50 = 9.6 (Emax = 70%) bias factor = 1.4 survival: pEC50 = 10.2 (Emax = 85%) bias factor = 3.1

O

N O

Cl

HN

OH OH

MRS3558 (143)

H N

Cl

HN

pERK1/2: pEC50 = 6.2 (Emax = 107%) cAMP: pEC50 = 6.7 (Emax = 70%) bias factor = 2.6 survival: pEC50 = 7.9 (Emax = 79%) bias factor = 44


Figure 30. Biased agonists of A3AR. For the above A1AR and A3AR ligands, as well as the D2R agonists shown in Figure 12, comparisons have been made between the G protein signaling (cAMP assay) with other “indirect” downstream effects of the receptor (i.e. ERK phosphorylation, calcium mobilization and cell survival), rather than between G protein signaling and β-arrestin recruitment. Although these add to our understanding of the complexity of GPCR biased signaling, further study is needed to get a clearer 69



picture of all the events that will be initiated upon the activation of a GPCR. Most likely no universal rules could be applied to all the receptors, and profiling a GPCR ligand using various available assays is necessary for a better prediction of potential pharmacological effects in vivo. Biased Ligands of Class B GPCRs The majority of biased ligands reported so far target class A GPCRs, and literature on other classes of the GPCR family remains limited. Among them, class B is comprised of 15 GPCRs that respond to peptide hormones and are the targets of many drugs for the treatment of diabetes and osteoporosis. Class B GPCRs share the 7TM architecture common to all GPCRs, but they also feature a large extracellular N terminus, which is very important for ligand recognition and activation. Similarly as for some of the abovementioned class A GPCRs, reevaluation of existing ligands has revealed bias properties for a few class B receptors.170 For example, the peptide (D-Trp12, Tyr34)-PTH(7–34) (PTH-barr) has been reported as a biased agonist for the parathyroid hormone type 1 receptor (PTH1R), which activates β-arrestin but not the classic G protein signaling, therefore acts as a β-arrestin-biased agonist.171 In mice, PTH-barr induced anabolic bone formation, which can be abrogated in β-arrestin2-null mice. The glucagon-like peptide 1 receptor (GLP-1R) is a class B GPCR which plays very important roles in the pathophysiology of type 2 diabetes (T2D) and obesity, and receives extensive attention as a drug target.172 Several known agonists and positive allosteric modulators (PAMs) of this receptor have been reported as biased ligands 70


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after being evaluated in both the G protein and β-arrestin functional assays. For example, relative to the endogenous ligand GLP-1, both oxyntomodulin (151) and exendin-4 (152) are biased toward β-arrestin recruitment in ChoFlpIn cells (Figure 31).173 GLP-1R PAMs, such as BETP and Boc5, also display bias properties when both G protein-mediated and β-arrestin-mediated activities are tested.174 (A) GLP-1(1-37): (149)

HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

GLP-1(7-37): (150)

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Oxyntomodulin: (151)

HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNKNNIA

-arrestin-biased

Exendin-4: (152)

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS- NH2

-arrestin-biased

P5 (153):

ELVDNAVGGDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

G protein-biased

(B) GLP-1(7-36)-NH 2:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2 cAMP: pEC50 = 10.3 (Emax = 100%) -arrestin1: pEC50 = 8.4 (Emax = 100%) -arrestin2: pEC50 = 8.1 (100%)

peptide: (154) -arrestin-biased



HAEGTFTSDVSXYLEXQAAXEFIXWLVZGRG-NH2 cAMP: pEC50 = 8.8 (Emax = 97%) -arrestin1: pEC50 = 7.8 (Emax = 48%) -arrestin2: pEC50 = 7.9 (Emax = 47%)

HAEGTFTSDVSXYLEXQAAXEFIAWLVKGRG-NH 2 cAMP: pEC50 = 9.0 (Emax = 96%) -arrestin1: pEC50 = 7.7 (Emax = 43%) -arrestin2: pEC50 = 8.0 (Emax = 63%)

HAEGTFTSDVSXYLEXQAAXEFIXWLVKGRG-NH2 cAMP: pEC50 = 8.4 (Emax = 97%) -arrestin1: pEC50 = 7.5 (Emax = 41%) -arrestin2: pEC50 = 8.0 (Emax = 59%) O NH

O NH N H2

X

Z

Figure 31. Biased agonists of GLP-1R. The amino acid sequences of the peptides are 71



shown from the N-terminus (left) to the C-terminus (right), while a terminal “-NH2” indicates a primary amide at the C-terminus. A G protein-biased GLP-1R agonist, P5 (153, Figure 31), was discovered through a high-throughput autocrine-based screening of large combinatorial peptide libraries.175 It has been shown that P5 promotes G protein signaling comparable to GLP-1 and exendin-4, but exhibited a significantly reduced β-arrestin response. It is a weak insulin secretagogue, but demonstrated superior antihyperglycemic efficacy in both genetic and diet-induced obese mice.175 The structural difference between P5 and exendin-4 is at the N terminus, where the first eight residues of exendin-4 were removed (to give exendin9-39, which is a GLP-1R antagonist) and new amino acids were appended (Figure 31). Although the G protein-biased agonist P5 displays efficacy in animal models and supports the idea that G protein-biased agonists may serve as novel therapeutics for T2D, the recruitment of β-arrestin1 has been reported to promote β cell proliferation and protect β cells from apoptosis.176 Therefore, the discovery of β-arrestin-biased agonists of GLP-1R could also offer potential therapeutics for T2D. Through the modification of GLP-1 peptides by incorporating unnatural amino acids, a series of β-arrestin-biased agonists of GLP-1R have very recently been reported by Hager et al.177 As shown in Figure 31, the α → β replacements decrease peptide potency in both stimulation of cAMP production and β-arrestin recruitment, but for some replacement sets such as those in peptides 154, 155 and 156, cAMP production is more strongly affected than β-arrestin recruitment, therefore leading to β-arrestin-bias. 72


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Both SFSR and modeling results suggested that the central part of these peptides contributed to their biased properties. Compared to class A, class B GPCRs have been viewed as more challenging for ligand discovery, due to their large extracellular domain which is required for the activation of the receptor. The recently solved structures of GLP-1R have provided much detailed information on ligand recognition and activation mechanism of class B GPCRs and will facilitate future ligand design efforts targeting this important family of receptors.178-180 Further study on biased ligands and their therapeutic potential at class B GPCRs is warranted. Biased Ligands/Allosteric Modulators at Class C GPCRs The class C GPCRs consists of 22 receptors which include very important drug targets such as the metabotropic glutamate receptors (mGluR), the calcium-sensing receptors (CaSR), and the gamma-amino-butyric acid (GABA) type B receptors (GABAB), among others (Figure 2). The structures of class C GPCRs differ from class A in that the 7TM domain of these receptors is connected through a cysteine rich-region to a large bilobed extracellular aminoterminal domain termed the Venus flytrap domain (VFD), which binds the endogenous ligands.181 The mGluRs bind to the major excitatory neurotransmitter glutamate and modulate synaptic transmission in the CNS. Among the many effects glutamate has upon the activation of mGluRs, cytoprotective effects were recently reported in CHO cells that express the mGlu1R.182 Both glutamate (158) and the natural compound quisqualate (159) activate the mGlu1R as measured in the Gq-mediated PI hydrolysis assay, with 73



the latter being more potent (EC50 = 16 µM and 0.63 µM respectively, Figure 32). However, only glutamate displayed neuronal protective effects against apoptosis induced by conditions of trophic deprivation (EC50 = 153 µM).182 The cytoprotective effects were correlated to sustained ERK phosphorylation effects which were G protein-independent, but not with transient ERK phosphorylation, which was believed to be G protein-mediated. Additionally, further studies showed that β-arrestin1 plays a role in the cytoprotective effects of glutamate.182 Using the same method, Emery et al. also reported that like quisqualate, compound DHPG (159, Figure 32) is also a G protein-biased agonist of mGlu1R, which induced PI hydrolysis but not sustained ERK phosphorylation and cytoprotective effects, whereas glutaric acid (160) and succinic acid (161) are fully β-arrestin1-biased and they did not induce PI hydrolysis but displayed cytoprotective effects.183 These results support that β-arrestin-biased agonists of mGlu1R have the therapeutic potential as neuroprotective agents. By mutational analysis, it was found that residue Thr188 is critical for G protein-mediated signaling of mGlu1R, which interacts with the amino group of glutamate, quisqualate and DHPG. The removal of this amino group as in glutaric acid and succinic acid abolished this interaction and make both compounds β-arrestin1-biased (Figure 32).183

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Figure 32. Biased agonists of mGlu1R. However, all glutamate analogues have poor physiochemical properties that make them poor drug candidates. Therefore, the study of allosteric modulators of these receptors has become the main paradigm of current drug discovery efforts targeting mGluRs.184 It is worth noting that most allosteric modulators of class C GPCRs bind to a pocket formed by the transmembrane helices that resemble the orthosteric binding site of class A receptors.185,

186

PAMs of mGlu5R have shown promise for the

treatment of schizophrenia in both preclinical and clinical studies.187 One possible mechanism for these therapeutic effects has been believed to be the mGlu5R-induced potentiation of NMDAR (N-methyl-D-aspartate receptor) currents, but increased activation of NMDA receptors can also lead to excitotoxicity and neuronal death.188 Similar to other GPCRs, this likely “on-target” toxicity could potentially be avoided by biased ligands at this receptor. Recently, Rook et al. reported that the mGlu5R PAM VU0409551 (162, Figure 33) induced a robust potentiation of mGlu5R coupling to Gq-mediated calcium mobilization and other signaling pathways but does not enhance 75



mGlu5R modulation of NMDAR currents in hippocampal neurons.189 Importantly, VU0409551 maintains the robust, dose-dependent efficacy in preclinical rodent models of psychosis and cognitive function.189 Because the mGlu5R-induced potentiation of NMDAR currents in forebrain neurons is mediated by interactions of mGlu5R with NMDARs through adaptor proteins that are independent of signaling through Gq, this suggests that functionally selective mGlu5R PAMs (e.g., Gq-mediated signaling versus modulation of NMDAR currents) may provide safer drug candidates for further development. It has also been reported that another PAM of mGlu5R, NCFP (163, Figure 33), potentiates mGlu5R-mediated responses in the calcium mobilization and ERK1/2 phosphorylation assays, in both recombinant and native systems (rat cortical astrocytes), but does not potentiate responses involved in hippocampal synaptic plasticity (long-term depression and long-term potentiation).190 One possible reason for this distinct profile of NCFP might be that it binds to a different site on the receptor from the MPEP (2-methyl-6-(phenylethynyl)pyridine) binding site where most other mGlu5R PAMs bind.190 However, whether the regulation of hippocampal synaptic plasticity is related to unwanted side effects or is necessary for the therapeutic effects of mGlu5R PAMs remains to be demonstrated.

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Figure 33. Biased PAMs of mGlu5R. The CaSR is responsible for the regulation of extracellular Ca2+ concentrations in the body, and the activation of CaSR expressed in the parathyroid gland suppresses the secretion of parathyroid hormone (PTH). Various endogenous and exodogenous agonists can activate CaSR, including the divalent cations Ca2+ and Mg2+, polyamines, aminoglycosides and others.191 These orthosteric agonists have poor drug-like properties and are thus less explored as therapeutic agents. A number of such compounds have been studied for their activities in cellular effects including IP1 accumulation, cAMP inhibition and ERK1/2 phosphorylation, and they displayed varied biases toward one signaling pathway or another.192 Like the mGluRs, of more therapeutic relevance are the allosteric modulators of CaSR. The calcimimetic drug cinacalcet (164, Figure 34) was the first allosteric GPCR modulator that reached the pharmaceutical market, which has been approved for the treatment of hyperparathyroidism in 2004.193 This drug, however, causes the 77



adverse side effect hypocalcaemia, likely through the activation of CaSR in tissues other than the parathyroid gland. It has been hypothesized that the distinct therapeutic effects and side effects may be mediated by different signaling pathways associated with CaSR and biased PAMs may provide opportunities in discovering safer therapeutics targeting this receptor.194 Recently, Cook et al evaluated a number of structurally diverse calcimimetics for three key signaling effects associated with this receptor: Ca2+ mobilization, ERK1/2 phosphorylation and IP1 accumulation (Figure 34).194 The PAM cooperativity (αβ) of these compounds with the CaSR agonist Ca2+ was calculated as a parameter to evaluate the ligand bias for each signaling effects. It was discovered that cinacalcet shows a bias for Ca2+ mobilization and IP1 accumulation and away from pERK1/2.194 Compounds AC-265347 (165) and R,R-calcimimetic B (166) are biased for pERK1/2 and IP1 accumulation but away from Ca2+ mobilization, while compound S,R-calcimimetic B (167) biases CaSR for IP1 accumulation only and nor-calcimimetic B (168) is unbiased. It was proposed that compounds that bias CaSR signaling towards pERK1/2 may achieve tissue-selective suppression of PTH secretion while sparing calcitonin release.194 Given that the phosphorylation of ERK1/2 associated with the activation of CaSR can be both G protein- and β-arrestin-mediated,192 evaluating compounds in various downstream signaling effects is necessary rather than merely G protein binding and β-arrestin recruitment.

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Figure 34. Biased PAMs of CaSR (PAM cooperativity (αβ) was compared between compounds for quantification; pERK1/2 effects of CaSR can be mediated by both G protein and β-arrestin.192). The unique structural feature of class C GPCRs (i.e. the VFD domain), and the distinct binding sites for orthosteric and allosteric ligands (orthosteric agonists bind to the VFD while allosteric modulators bind to the TM domain), have made the structural basis for ligand bias at these receptors even more elusive. Some key residues related to the observed biased properties of compounds mentioned above have been identified through mutagenesis studies. For example in the mGlu1R case, Thr188 is critical for G protein-mediated signaling whereas Arg323 and Lys409 residues were required for β-arrestin1-mediated effects.183 For CaSR, a naturally occurring R680G mutation selectively enhances β-arrestin signaling by disrupting a salt bridge between Arg680 (on TM3) and Glu767 (on EL2).195 Furthermore, our understanding of the distinct physiological roles each signaling pathway plays is still at a very early stage. The discovery of more biased modulators with better functional 79



selectivity profiles will facilitate the functional study of each signaling pathways, and provide opportunities for the identification of better therapeutics. Challenges in the Discovery of Biased Ligands All the above examples have shown that biased ligands represent novel modalities that will potentially provide better therapeutics for a variety of diseases linked to GPCRs. Ligand bias, however, has also added another layer of challenges that medicinal chemists and pharmacologists have to confront when targeting a specific GPCR for drug discovery. We have summarized these main challenges below: (1) Mechanism of action. In most cases, the etiology of a specific disease is very complicated and can involve many receptors, proteins and signaling pathways. In cases where a GPCR is involved, it signals through multiple effectors and leads to various changes in downstream signaling events. As discussed above for most GPCRs, it is not clear which signaling pathway is therapeutically most important. For example, as showcased for the D2R, biased ligands with varied profiles (G protein- or β-arrestin-biased; agonist, partial agonist, or antagonist) have been reported, but it is not clear which combination would lead to the most desirable clinical outcomes. Genetic knockout of β-arrestin is a commonly used method for studying the roles of β-arrestin signaling for a given GPCR, and biased ligands serve as useful tools. Although biased ligands have been reported for many GPCRs as summarized in this Perspective, for most GPCRs the ideal tool compounds for the dissection of the signaling pathways is still missing.

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(2) Functional assays. Secondary messenger assays such as measuring calcium or cAMP levels have been widely used in drug discovery programs for the detection of G protein pathway activity, due to the ease of manipulation; and the β-arrestin activation is usually measured by the translocation of the protein. Most of the time, however, overexpression of engineered receptors on model cells (for example the CHO or HEK-293 cells) is necessary to magnify signals and to enable detection, which does not fully replicate the native biological conditions. Therefore, demonstration of ligand bias in additional cellular models (e.g. neuronal, cancer) may be necessary for the prediction of physiological conditions. One such example listed above is the CB1R ligands, which have been tested in a cell culture model of striatal medium spiny projection neurons.115,

116

Also, it has been recognized that ligand

kinetics have an impact on observed biased properties, for example as demonstrated for the 5-HT2B receptor.75, 196 Therefore, the readouts from functional assays may vary depending on detection time points. (3) Quantification of signaling bias. Bias factors are very important for SFSR studies of GPCR biased ligands, but they are often missing for reported ligands or not calculable as can be seen for many examples listed above. Arguably the most widely accepted method for the quantification of bias factors is the calculation based on transduction coefficient (τ/KA),12, 13 where the value of log(τ/KA) reflects the activity of a ligand for a specific pathway, which can be normalized to a reference compound to give ∆log(τ/KA), and further compared to its activity in another signaling pathway to give ∆∆log(τ/KA), from which a bias factor can be calculated as: bias factor = 81



10∆∆log(τ/KA). Importantly, the selection of a reference compound is critical, as the results can vary when a different reference compound is chosen. All of these issues add to the complexity in the data processing step for compound evaluations. (4) Multi-parameter optimization of GPCR ligands. For medicinal chemists, ligand bias would be an extra parameter that must be taken into account when working on GPCR ligands, in addition to ligand affinity, efficacy, off-target activity, physicochemical and ADMET properties, among others. As can be seen from the above examples, subtle structural modifications of a GPCR ligand often lead to significant changes in the functional selectivity of a compound. Compared to SAR data, SFSR conclusions are more difficult to summarize and to interpret, and add to the challenges for GPCR-targeted drug discovery. Medicinal Chemistry Strategies (1) Re-evaluating existing ligands in multiple functional assays. Prior to the introduction of the concept of ligand bias, GPCR ligands were often evaluated in a single, normally G protein-mediated second messenger assay, where results would define the intrinsic activity of a specific ligand (agonist, partial agonist, antagonist or inverse agonist). Re-evaluating such ligands for β-arrestin recruitment and other functional assays would, therefore, reveal their ligand bias properties. Therefore, known ligands of a specific GPCR serve as a reservoir of preliminary ‘hit’ compounds for the discovery of novel biased ligands. Such examples are the βAR, D1R, CB1R, CB2R, mGluR and CaSR.

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(2) Structure-based drug design. Numerous GPCR structures have been solved in the past decade, which provide valuable information for structure-based design targeting this important family of drug targets, but the structural basis for biased agonism is far from being understood. Nevertheless, for a limited number of GPCRs, for example the β2AR,27 D2R,74 D1R88 and 5-HT2B,75, 93 the molecular determinants that define biased agonism of GPCRs have been explored, where information could be further used for structure-based design of biased ligands. It is to be anticipated that the discovery of more biased ligands will be useful tools for the structural biological study of the receptors, which will in turn help future design of biased ligands. (3) The study of SFSRs. Just like that an extensive SAR study around a hit compound could be a powerful approach to the discovery of drug candidates with desirable drug-like properties, a careful study of SFSRs based on a biased compound can be applied in the discovery of biased ligands. This is also necessary in most cases given that a subtle structural modification could lead to significant changes to the bias properties of a specific ligand. Examples are the biased D2R ligands based on aripiprazole (Figures 10 and 11). (4) Measurement of binding kinetics. Binding kinetics should generally be taken into account as an additional parameter for medicinal chemists.197 Recently, it was reported that association rates at D2R determine the extrapyramidal side effects of antipsychotics.198 As for ligand bias, binding kinetics may influence or contribute to the bias properties of a specific ligand, for example in the abovementioned 5-HT2B receptor.75, 196 Therefore, this is an important aspect to consider in drug discovery 83



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targeting GPCRs, whereby the drug’s residence time at a given receptor may influence its duration and/or select signaling properties. CONCLUSIONS Besides G protein-dependent versus G protein-independent signaling pathways (e.g. β-arrestin recruitment), there have been also some cases where a specific ligand shows bias between different subtypes of G proteins or between different β-arrestins. For example, the aforementioned endocannabinoid AEA is biased for Gi over Gs at CB1R compared to the synthetic CB1 agonist WIN-55212.114 Bock et al. recently reported that the structural modifications of iperoxo led to the discovery of M2 acetylcholine receptor agonists biased for either Gαi or Gαs.199

In addition to the G

protein-biased opioid receptor agonists mentioned above, Pradhan et al. reported agonist-selective recruitment of different arrestin isoforms to DOR.200 Given the fact that specific subtypes of G proteins and arrestins may contribute differently to the overall physiology of a given cell type, these biased ligands are also very important for dissecting the roles that each effector plays and offer opportunities for the discovery of novel therapeutics. Biased agonism or functional selectivity provides opportunities for more precise regulations of GPCR functions, therefore affording therapeutics with enhanced efficacy and/or reduced adverse side effects. The advantages of biased ligands as a new generation of drugs have been exemplified at several GPCRs by investigational drugs in human clinical trials, such as TRV130, which is awaiting FDA approval. In fact, biased agonism has become the major paradigm in GPCR drug discovery. 84


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However, it is worth noting that the bias property of a ligand at a specific receptor may not always necessarily be therapeutically favorable, but nonetheless should be taken into account. From the above examples, it is clear that in most cases, a very small structural modification of a ligand could lead to a significant change or even the reversal of ligand bias properties. Underlying reasons for this include the relatively high conservative nature of GPCR ligand binding pockets that a given ligand samples, which may lead to favored active GPCR conformations, either a G protein activation-preferred state or β-arrestin recruitment-preferred state. As discussed, ligand bias represents a significant challenge for medicinal chemists, and as more efforts are aimed at targeting GPCRs for drug discovery, SFSR studies will serve to better characterize and benchmark drug-like compounds in biological assays. Although the number of GPCRs that have been crystallized is still rather limited, the development of biased ligands will lead to more accurate ‘capture’ of biased states of GPCRs, which in turn will lead to better advancements in biased ligand design. Progress in structural and functional aspects of GPCR signaling will lead to biased ligands as novel tools to dissect out signaling pathways relevant for disease-related GPCRs, and will ultimately lead to a new generation of GPCR-targeted drugs. AUTHOR INFORMATION Corresponding Authors *

J.D.M.: phone, +1 414-955-7635; email, [email protected].

85



*

J.C.: phone, +86 21 20685237; email, [email protected].

ORCID Jianjun Cheng: 0000-0001-6065-2682 John D. McCorvy: 0000-0001-7555-9413 Notes The authors declare no competing financial interest. Biographies Liang Tan received his Ph.D. in Medicinal Chemistry from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, in 2017. He is currently a postdoctoral researcher at iHuman Institute, ShanghaiTech University. His research focuses on the aminergic GPCRs, with the goal of identifying novel biased agonists with favorable drug-like properties and studying their therapeutic potential as antipsychotic drugs. Wenzhong Yan received his Ph.D. in Medicinal Chemistry in 2016 from East China University of Science and Technology in Shanghai. After a short period of working at Shanghai ChemPartner Co., Ltd., he joined iHuman Institute, ShanghaiTech University in 2017 as a postdoctoral research associate. His research focuses on the design and discovery of novel GPCR ligands with the goal of identifying novel drug candidates for the treatment of cancer and other diseases. John D. McCorvy obtained his Ph.D. from the department of Medicinal Chemistry and Molecular Pharmacology at Purdue University in 2012, and did his postdoctoral 86


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training at the University of North Carolina-Chapel Hill in the department of Pharmacology. Currently he is a Principal Investigator at Medical College of Wisconsin in the department of Cell Biology, Neurobiology, and Anatomy. His research focuses on structural determinants of GPCR activation that lead to biased signaling. Jianjun Cheng obtained his Ph.D. in Medicinal Chemistry from Shanghai Institute of Materia Medica, Chinese Academy of Sciences in 2010 and received his postdoctoral training in the department of Medicinal Chemistry and Pharmacognosy at University of Illinois at Chicago. He became a Principal Investigator, Research Associate Professor at iHuman Institute, ShanghaiTech University in 2016. His current research focuses on GPCR-targeted drug discovery, with special interests in biased ligands of GPCRs. ACKNOWLEDGMENTS We thank the Shanghai Municipal Government, ShanghaiTech University and the National Natural Science Foundation of China (81703361) (to J.C.) for financial support; and Dr. Raymond C. Stevens and Yekaterina Kadyshevskaya for their help in the preparation of Figure 2. ABBREVIATIONS USED 5-HT1BR, serotonin 1B receptor; 5-HT2BR, serotonin 2B receptor; 5-HT2CR, serotonin 2C receptor; 7TMR, seven transmembrane receptor; A1AR, adenosine A1 receptor; A2AAR, adenosine A2A receptor; A2BAR, adenosine A2B receptor; A3AR, adenosine A3 87



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receptor; AEA, anandamide; AHF, acute heart failure; AMPH, amphetamine; APJ, apelin receptor; ARB, angiotensin receptor blockers; AT1R, angiotensin II receptor type 1; β2AR, β2 adrenergic receptor; BRET, bioluminescence resonance energy transfer; cAMP, cyclic adenosine monophosphate; CaSR, calcium-sensing receptor; CBD, canabidiol; CB1R, cannabinoid receptor 1; CB2R, cannabinoid receptor 2; CNS, central nervous system; D1R, dopamine D1 receptor; D2R, dopamine D2 receptor; DAP, disubstituted aromatic piperazine; DOR, δ opioid receptor; EL, extracellular loop; EP2R, prostaglandin E2 receptor 2; ERK, extracellular signal-regulated kinase; ETA, endothelin A receptor; FDA, US Food and Drug Administration; FRET, fluorescence resonance energy transfer; FPR2, formyl peptide receptor 2; GABA, gamma-amino-butyric acid; GFP, green fluorescent protein; GLP-1R, glucagon-like peptide 1 receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor

kinase;

GTP,

guanosine-5'-triphosphate;

GTPγS,

guanosine

5’-O-[gamma-thio]triphosphate; H2R, histamine H2 receptor; H4R, histamine H4 receptor; HCS, high-content screening; HDL, high-density lipoprotein; hERG, human Ether-à-go-go-related

gene;

HTS,

high-throughput

screening;

IP1, inositol

monophosphate; mGluR, metabotropic glutamate receptor; MAPK, mitogen-activated protein kinases; MOR, µ opioid receptor; MPEP, 2-methyl-6-(phenylethynyl)pyridine; KOR, κ opioid receptor; NAM, negative allosteric modulator; NMR, nuclear magnetic resonance; NOPR, nociception receptor; NT1R, neurotensin receptor type 1; PAH, pulmonary arterial hypertension; PAM, positive allosteric modulator; PCP, phencyclidine; PGE2, Prostaglandin E2; PTHR1, parathyroid hormone 1 receptor; 88


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S1P1R, sphingosine-1-phosphate receptor 1; SAR, structure-activity relationship; SFSR, structure-functional selectivity relationship; THC, tetrahydrocannabinol; T2D, Type 2 diabetes; VFD, Venus flytrap domain. REFERENCES 1.

Lagerstrom, M. C.; Schioth, H. B. Structural diversity of G protein-coupled

receptors and significance for drug discovery. Nat. Rev. Drug Discov. 2008, 7, 339-357. 2.

Fredriksson, R.; Lagerstrom, M. C.; Lundin, L. G.; Schioth, H. B. The

G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 2003, 63, 1256-1272. 3.

Stevens, R. C.; Cherezov, V.; Katritch, V.; Abagyan, R.; Kuhn, P.; Rosen, H.;

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challenges

for

a

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novel

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121



Table of contents graphic 46x43mm (300 x 300 DPI)


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Figure 1. Biased signaling at a glance. 175x134mm (300 x 300 DPI)



Figure 2. Biased ligands have been reported for 30 GPCR targets. 215x200mm (300 x 300 DPI)


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Biased Ligands of G Protein-Coupled Receptors (GPCRs): Structure

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