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María L. López-Rodríguez , Bellinda Benhamú and Henar Vázquez-Villa
Organic Chemistry Department, Universidad Complutense de Madrid, Madrid, Spain

List of abbreviations:

7TM Transmembrane Helix Bundle
AM Allosteric Modulator
CNS Central Nervous System
CRF Corticotropin-Releasing Factor
CT Calcitonin
ECL Extracellular loop
FDA Food and Drug Administration
GCG Glucagon
HIV Human Immunodeficiency Virus
HTS High Throughput Screening
ICL Intracellular Loop
NAM Negative Allosteric Modulator
NMDA N-methyl-D-aspartate
PACAP Pituitary Adenylate Cyclase-Activating Polypeptide
PAM Positive Allosteric Modulator
PK Pharmacokinetic
SAM Silent Allosteric Modulator
VIP Vasoactive Intestinal Peptide
Wnt Wingless/Integrated

11.1. INTRODUCTION
Over one-third of currently marketed drugs modulate G-protein coupled receptors (GPCRs), though these exploited
therapeutic targets represent a small fraction of the possible druggable GPCRs in the human genome (Hauser et al., 2017;
Rask-Andersen et al., 2011). One of the multiple reasons for this is that several GPCR-targeted candidates have not
progressed into the clinic due to efficacy and/or safety issues related to off-target effects. In this context, GPCR allosteric
modulation appears to be an innovative targeting approach that confers specific advantages on distinguishing between
highly homologous receptor subtypes and is proving to be a viable drug discovery strategy, as evidenced by recent FDA
approvals and clinical trials (Lutjens and Rocher, 2017; Wootten et al., 2013a).

The term allostery was first coined to describe a newly identified protein function observed in enzymes composed
of various subunits (Monod et al., 1965). Nowadays, allosteric modulation is understood as a unifying mechanism for
functionality in many other protein types, including GPCRs (Canals et al., 2011, 2012; Changeux and Christopoulos,
2016). A GPCR allosteric modulator (AM) is defined as a ligand that binds to a spatially and topologically distinct
(allosteric) site, and does not occupy the orthosteric binding site of the endogenous ligand. Hence, AMs do not compete
with the endogenous ligand; the binding to the allosteric site produces conformational changes in the orthosteric site
that result in the modulation of the endogenous ligand response. Mechanistically, AMs can either increase or inhibit the

Chapter 11

GPCRs:Structure, Function, and Drug Discovery. https://doi.org/10.1016/B978-0-12-816228-6.00011-8
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functional response to an orthosteric agonist, acting as positive or negative AMs (PAMs or NAMs), respectively. Silent
AMs (SAMs) bind to an allosteric site but do not modulate the receptor function (Fig. 11.1). There have also been reports
of other ligands that bind to an allosteric site: ago-PAMs, which possess an intrinsic agonist profile even in the absence
of an orthosteric ligand, and allosteric agonists or antagonists that activate or block, respectively, the receptor but do not
potentiate or decrease responses to the orthosteric agonist (Christopoulos et al., 2014; Gentry et al., 2015).

The mode of action of GPCR AMs include: (i) fine-tuning the cellular response to the orthosteric ligand in a time
and spatially dependent manner, which mimics the physiological effect of the endogenous ligand (Conn et al., 2009); (ii)
“ceiling effect” or saturable nature of the binding, whereby the extent of their activity is dictated by the orthosteric ligand
concentration, potentially protecting against overdose concerns (Soudijn et al., 2004); (iii) receptor specificity due to the
reduced residue conservation in allosteric binding sites compared to that in highly homologous orthosteric sites (Jacoby
et al., 2006; Melancon et al., 2012); (iv) signaling bias (also termed functional selectivity), differentially modulating a
selected intracellular pathway(s) over other signaling cascades activated by the GPCR (Kenakin, 2012; Lane et al., 2013);
and (v) probe dependence, inducing different effects depending on the nature of the orthosteric ligand (Canals et al., 2012).
Altogether, the remarkably precise pharmacological modulation confers to GPCR AMs unique advantages over orthosteric
classical ligands (Wootten et al., 2013a), contributing to hold promise for central nervous system (CNS) and periphery
targets (Conn et al., 2009; Foster and Conn, 2017).

Allosteric drugs work by manipulating binding (allosteric) pockets away from (orthosteric) active sites to effect global
conformational changes, making them harder to predict. This brings a number of intense challenges to the discovery
of AMs from the standpoint of standard drug discovery programs, including, in particular, rational design (Wagner et
al., 2016). The recent breakthroughs in structural biology have made several structures of GPCRs with small-molecule
AMs available (Lu and Zhang, 2018). The complex crystal structures have revealed, indeed, the high diversity of
allosteric binding sites in extracellular loops (ECLs), the transmembrane helix bundle (7TM), and intracellular loops
(ICLs) of receptors. Knowledge of the key receptor−modulator interactions at the allosteric sites and the complementary
computational and biological approaches are expected to contribute to the structure-based design of GPCR allosteric drugs.

Allosteric sites on GPCRs represent novel drug targets because theoretical advantages over classic orthosteric ligands
confer an improved therapeutic action to AMs. Very recently the first AMs of GPCRs have been approved as drugs
(Fig. 11.2). These are ticagrelor (Brilinta, Brilique, Possia), an allosteric antagonist of the purinergic P2Y12 receptor (a
class A GPCR) that is used as a platelet aggregation inhibitor in antithrombotic therapy; maraviroc (Celsentry), an NAM
of the chemokine receptor CCR5 (a class A GPCR) used as a virus entry inhibitor in human immunodeficiency virus
(HIV) therapy; plerixafor (Mozobil), an NAM of the chemokine receptor CXCR4 (a class A GPCR) used for stem cell
mobilization for transplantation in patients with lymphoma and multiple myeloma; cinacalcet (Sensipar, Mimpara), a
PAM of the calcium-sensing receptor (a class C GPCR) used for the treatment of hyperparathyroidism; and vismodegib

FIGURE 11.1 Influence of AMs on orthosteric agonist function. The binding of orthosteric agonist results in the conformational changes of GPCRs,
which result in the activation of downstream signaling cascades. AMs bind to a topographically distinct site and induce conformational changes to a
receptor, which influence the orthosteric agonist function. Negative AM (NAM) decreases and positive AM (PAM) increases the affinity and/or efficacy
of orthosteric agonist. Silent AMs (SAMs) have no influence on the function of orthosteric agonist. Credit: Reprinted with permission from J. Med.
Chem. 2019, doi: org/10.1021acs.jmedchem.8b00368. Copyright (2019) American Chemical Society.



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FIGURE 11.2 AMs of GPCRs approved by the FDA as drugs. Ticagrelor (Brilinta, Brilique, Possia), used as a platelet aggregation inhibitor in
antithrombotic therapy; maraviroc (Celsentry), used as a virus entry inhibitor in HIV therapy; plerixafor (Mozobil), used for stem cell mobilization for
transplantation in patients with lymphoma and multiple myeloma; cinacalcet (Sensipar, Mimpara), used for the treatment of hyperparathyroidism; and
vismodegib (Erivedge), used for the treatment of basal cell carcinoma.

(Erivedge), a NAM of the smoothened receptor (a class F GPCR), used for the treatment of basal cell carcinoma
(Drugbank; Gpcrdb).

This chapter will focus on relevant medicinal chemistry advances for small-molecule AMs targeting GPCRs of class
A —rhodopsin-like receptors—, class B —the secretin family—, class C —metabotropic glutamate receptors—, and class
F —frizzled and smoothened receptors— (Foord et al., 2005; Lin et al., 2013). Herein, we provide an update on reported
AMs, their therapeutic relevance, and their binding sites.

11.2. ALLOSTERIC MODULATORS TARGETING CLASS A GPCRS
Class A represents the largest class of human GPCRs and makes up most of the current receptor drug targets (Roth and
Kroeze, 2015). Interest in class A GPCR AMs initially grew because of a theoretical improvement in target selectivity,
especially among receptor subtypes displaying high degrees of homology in the orthosteric site (Wild et al., 2014).
Since then, the development of AMs in class A has significantly improved and is now progressing candidates forward in
preclinical development and human clinical trials (Table 11.1) as well as in the market for clinical use (Fig. 11.2) (Wold
et al., 2018). A contributing factor to the optimization of allosteric ligands is the recent availability of 10 high resolution
crystal structures displaying allosteric binding sites and the corresponding ligand-receptor interactions (Lu and Zhang,
2018).

TABLE 11.1 Selected AMs of GPCRs currently in clinical trials.

GPCR Compound Indication (phase)

D1 LY3154207 (PAM) Parkinson disease, dementia (II)

M1 VU319 (PAM) Cognitive impairment (I)

FFAR1 MK-8666 (ago-PAM) Type 2 diabetes mellitus (I)

CXCR1 Reparixin (NAM) β-cell transplantation (III)

CXCR1/CXCR2 Ladarixin (NAM) Onset type 1 diabetes (II)

GABABR ADX71441 (PAM) Addiction (I)

mGlu4R Foliglurax (PAM) Parkinson disease (II)

mGlu5R Mavoglurant (NAM) Fragile X syndrome, alcohol drinking, cocaine-related disorder (II)

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Allosterically targeted class A GPCRs belong to β-adrenergic, serotonin, dopamine, muscarinic acetylcholine,
cannabinoid, free fatty acid, adenosine, purinergic P2Y, chemokine, opioid, and proteinase-activated receptor families.

11.2.1 β2-adrenergic receptor (β2AR)
The β2AR is the most extensively characterized member of adrenergic receptors or adrenoceptors, an aminergic family of
class A GPCRs whose endogenous signaling molecules are catecholamines. It is largely expressed in bronchial smooth
muscle and triggers bronchodilation upon activation (Lefkowitz, 2007). Therapeutically, β2AR agonists represent a large
class of drugs used to treat pulmonary disorders and asthma, while β2AR antagonists comprise selective and nonselective
β-blockers, widely used for the treatment of hypertension, cardiac arrhythmias, and other cardiovascular indications. At
present, nearly all known β-adrenergic ligands act orthosterically.

Kobilka and Lefkowitz (Ahn et al., 2017) have reported the first allosteric β-blocker, or β2AR NAM, compound 1 (Fig.
11.3), identified via a DNA-encoded small-molecule library screen comprising 190 million distinct compounds. In vitro
studies indicate the addition of compound 1 results in an inhibition of β2AR stimulated cAMP production and β-arrestin
recruitment. The cocrystal complex of this recently discovered NAM with β2AR (Fig. 11.4) reveals an intracellular binding
site formed by residues from helices I, II, and VI−VIII and the ICL1 of the β2AR, which is spatially distant from the
orthosteric site for binding of inverse agonist carazolol (Liu et al., 2017). This significant finding of a small-molecule
allosteric site on the intracellular surface of β2AR could extrapolate to additional class A GPCRs, opening new avenues
for allosteric drug discovery.

11.2.2 Serotonin 5-HT2C receptor (5-HT2CR)
The 5-HT2CR belongs to the aminergic family of class A GPCRs that are activated by the endogenous neurotransmitter
serotonin throughout the brain and the periphery. This subtype is critical for the anorectic effect of serotoninergic
activation (Sargent and Henderson, 2011) and several research efforts support the interest of a selective 5-HT2CR PAM
as a safer antiobesity drug devoid of potential hallucinogenic effects and cardiac valvulopathy associated to activation of
highly homologous 5-HT2A and 5-HT2B subtypes (Astrup, 2010; Dinicolantonio et al., 2014).

FIGURE 11.3 Representative allosteric ligands targeting aminergic β-adrenergic, serotonin, and dopamine GPCRs of class A.



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FIGURE 11.4 Cocrystal structure of the β2AR in a ternary complex with orthosteric inverse agonist carazolol and NAM compound 1 (see Fig. 11.3).
(A) Compound 1 is located in an intracellular binding site formed by residues from helices I, II, and VI−VIII and the ICL1 of the β2AR, which is
spatially distant from the orthosteric site for binding of carazolol. (B) In the allosteric binding site, compound 1 forms hydrogen bonding interactions
with the side chains of Asn692.40, Thr2746.36, Ser3298.47, and Asp3318.49 and a cation−π interaction with Arg63ICL1 via the bromobenzyl ring. The
cyclohexylmethylphenyl group is located inside a hydrophobic pocket surrounded by residues Val541.53, Ile581.57, Leu64ICL1, Ile722.43, Leu2756.37,
Tyr3267.53, and Phe3328.50 (PDB code 5X7D). Superscript represents residue number in accordance with Ballesteros−Weinstein nomenclature
(Ballesteros and Weinstein, 1995). Credit: Adapted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844.
Copyright (2019) American Chemical Society.

PNU-69176E (Fig. 11.3) was the first reported 5-HT2CR selective PAM, which was identified via screening of a
chemical library of Pharmacia (now Pfizer) (Ding et al., 2012; Im et al., 2003). However, the authors described the
behavior as an ago-PAM showing intrinsic activity in inositol phosphate release in HEK293 cells. Both the long alkyl
chain and polar moiety (α-D-galactopyranoside) in PNU-69176E are claimed to be essential to its function and may explain
anchoring to the membrane and binding to the allosteric site, respectively. CYD-1-79A (Fig. 11.3), also containing a
4-alkylpiperidine-2-carboxamide scaffold, has recently been reported to function as a pure 5-HT2CR PAM with no intrinsic
activity and a good preclinical pharmacokinetic (PK) profile, and to potentiate signaling in vivo in drug discrimination
assay (Wild et al., 2018), though no antiobesity properties have been described to our knowledge. Using the recent
high-resolution crystal structure of the 5-HT2CR (Peng et al., 2018), the authors performed molecular docking studies
that predict a bidentate hydrogen-bonding interaction between the 1,2-diol moiety of CYD-1-79A and the backbone
carbonyl of Leu209ECL2 residue in ECL 2, and between the ionizable N atom of the piperidine ring of the ligand and the
–OH side chain of Ser3346.58 in helix VI. These computational studies provide a possible explanation for the selective
profile of CYD-1-79A versus closely related 5-HT2AR and 5-HT2BR, since these residues are not conserved in the highly
homologous receptor subtypes (Wild et al., 2018).

Our group has recently contributed to highlight the promise of 5-HT2CR PAMs as antiobesity therapeutics
(García-Cárceles et al., 2017). In what is only the second reported synthetic small-molecule screening for 5-HT2CR PAMs,
an indole scaffold hit of Vivia Biotech chemical library was identified via an innovative automated flow-cytometry-based
screening system, the PharmaFlow platform (previously ExviTech platform) (Bennett et al., 2014). Further structural
modification led to the discovery of VA012 (Fig. 11.3), which exhibited dose-dependent enhancement of serotonin
efficacy, no significant off-target activities including 5-HT2 family members (5-HT2AR and 5-HT2BR), and low binding
competition against the endogenous agonist (5-HT) and other orthosteric ligands (mesulergine and clozapine).
Significantly, feeding rodent models indicate that PAM VA012 reduces both food intake and body weight gain without
causing taste aversion when acutely administered at 2 mg/kg (ip) (García-Cárceles et al., 2017).

11.2.3 Dopamine receptors (D1-D3Rs)
Two distinct families are distinguished among dopamine receptors: D1-like family —D1 and D5 subtypes— stimulate
cAMP production, and D2-like family —D2, D3, and D4 receptors— attenuate cAMP production. As a whole, dopamine
receptors play a substantial role in numerous neuropsychiatric disorders, including schizophrenia, Parkinson disease,
attention deficit hyperactivity disorder, and drug and alcohol dependence (Arnsten et al., 2017; Beaulieu et al., 2015;
Beaulieu and Gainetdinov, 2011).

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Stimulation of the D1-like receptors is challenging; despite the promise in preclinical studies, attempts to develop
D1R agonists for clinical use have so far not been successful. To date, two pharmaceutical companies have reported their
efforts toward a selective D1R activation by using the allosteric approach. Following a high-throughput screening (HTS)
of the Bristol-Myers Squibb chemical library, compounds 2 and 3 (Fig. 11.3) were identified as two distinct D1R PAM
chemotypes designated as piperazines and ethanoanthracenes, respectively, and represented the first described D1R PAMs
(Lewis et al., 2015).

At the same time, Lilly discovered the tetrahydroisoquinoline DETQ (Fig. 11.3) and demonstrated its activity in a broad
array of behavioral and neurochemical animal models that are thought to be predictive of therapeutic utility in Parkinson
disease, Alzheimer disease, schizophrenia, major depressive disorder, attention deficit disorder, and narcolepsy (Beadle et
al., 2014; Bruns et al., 2018; Svensson et al., 2017). At present, Lilly's molecule LY3154207, directly related analogue
of DETQ (Fig.11.3), is the first D1R potentiator that is being clinically studied (phase II) for the treatment of dementia
associated with Parkinson disease (Hall et al., 2019).

More recently, the discovery and characterization of two novel PAMs of D1R signaling, MLS1082 and MLS6585
(Fig. 11.3), have been reported (Luderman et al., 2018). Both compounds have no inherent agonist activity, but potentiate
receptor signaling stimulated by both the endogenous ligand dopamine and other D1R agonists. Using functional additivity
as well as mutational approaches, the authors suggest that MLS1082 and MLS6585 likely bind to diverse receptor sites.

Our group has also contributed with the development of biphenylsulfoximines as a new class of D1R PAMs as
promising safe agents for the treatment of CNS-related pathologies (López Rodríguez et al., 2018).

Due to the therapeutic potential of D2-like receptors, several AMs have been reported. The neuropeptide
Pro-Leu-Gly-NH2 (PLG, Fig. 11.3), initially isolated from brain tissue, was first characterized as an endogenous PAM of
the D2 and D4 receptors with potential for Parkinson disease treatment (Khan et al., 2010). However, the peptide nature of
PLG limited its development as a drug, and the rational design and modification strategies in the search for agents with
better PK properties have led to peptidomimetic analogs containing lactam, bicyclic and spiro-bicyclic scaffolds (Mann et
al., 2010; Verma et al., 2005). Interestingly, these extensive studies have produced both D2R PAMs and NAMs with minor
structural differences within the same series of peptidomimetics (Bhagwanth et al., 2013). Among them, analog PAOPA
(Fig. 11.3) has been characterized as a selective D2R PAM displaying 100- to 1000-fold higher potency than the parent
neuropeptide, a significantly improved PK and toxicological profile, and activity in preclinical models of schizophrenia
(Beyaert et al., 2013; Dyck et al., 2011; Tan et al., 2013a).

The atypical, allosteric antagonism of the tetrahydroisoquinoline derivative SB269652 (Fig. 11.3) at D2 and D3
receptors supported its characterization as the first small-molecule NAM of dopamine receptors (Silvano et al., 2010).
Further SAR studies revealed that SB269652 is a bitopic ligand that incorporates both an orthosteric site-binding
moiety (the tetrahydroisoquinoline “head”) and an allosteric site-binding moiety (“the indole tail”) separated by an alkyl
chain linker (Rossi et al., 2017; Shonberg et al., 2015). Interestingly, a trans-cyclopropylmethyl linker replacing the
trans-1,4-cyclohexylene linker while retaining the head and tail groups led to D3R preferring bitopic ligands (Kumar
et al., 2017). Fragmentation of bitopic ligand SB269652 was approached to target the putative allosteric site, and
indole-2-carboxamide derivatives of the allosteric fragment moiety were identified as new NAMs of the D2R (Mistry et
al., 2015).

Recently, a benzothiazole scaffold compound was reported as a D2R PAM both in vitro and in vivo, identified via
an HTS of 80,000 compounds, and provides another small-molecule hit for the development of AMs useful to treat
hypodopaminergic function (Wood et al., 2016).

A crystal structure of an AM in complex with its targeted dopamine receptor is certainly awaited; meanwhile, a recent
study of the structural basis for the allosteric mechanism of SB269652 by combining site-directed mutagenesis with
molecular modeling and simulations of D2R in complex with either SB269652 or its derivatives (Draper-Joyce et al.,
2018b) may aid the rational development to provide clinical candidates with improved in vitro and in vivo properties.

11.2.4 Muscarinic acetylcholine receptors (M1, M2, M4, and M5Rs)
Muscarinic acetylcholine receptors (mAChRs) are aminergic class A GPCRs activated by endogenous acetylcholine, and
they are broadly expressed in the CNS and other tissues in the periphery. They regulate a large body of peripheral and
central physiological functions in the human body and represent important drug targets for a number of diseases including
pain, Alzheimer disease, Parkinson disease, schizophrenia, diabetes, and obesity (Wess et al., 2007).

The muscarinic family of receptors is divided into five subtypes (M1–M5Rs) of which M1–M4Rs have been crystallized.
All subtypes possess at least one allosteric binding site, which is located in the extracellular region of the



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receptor on top of the ACh (i.e., orthosteric) binding site. The former can be specifically targeted by chemical compounds
(mostly small molecules); notably, the concept of allosteric modulation at GPCRs was initially described at muscarinic
receptors several decades ago (Mohr et al., 2013; Stockton et al., 1983). Today, a plethora of AMs exist for all five
muscarinic receptor subtypes (Bock et al., 2018).

mACh M1R. Benzylquinolone carboxylic acid BQCA (Ma et al., 2009), indole VU0108370 (Reid et al., 2011), and
isatin VU0119498 (Bridges et al., 2010) are the three initial hits identified (Fig. 11.5) by HTS campaigns that have been
used as representative scaffolds for optimization. Thus, highly selective M1R PAMs have been developed with improved in
vivo efficacy, in the search for novel therapeutics to counteract the negative cognitive symptoms associated with diseases
such as Alzheimer disease and schizophrenia. Structural modification of the quinolone ring of BQCA discovered by Merck
afforded new M1R PAMs with alternative scaffolds such as phenylpyridinone, phenylpyrimidinone, quinolizidinone, or
methoxynaphthalene (Kuduk et al., 2010b, 2011a, 2012, 2014; Mistry et al., 2016a, 2016b). Further optimization of the
quinolone core system was conducted (Abdul-Ridha et al., 2014; Kuduk et al., 2011b; Mistry et al., 2013; Yang et al.,
2010) to provide benzoquinazolinone MK-7622 (Fig. 11.5), which advanced into a phase II clinical trial in 2013 and was
terminated in 2016 for undisclosed reasons (Kuduk et al., 2010a). Interestingly, dibenzylpyrazoloquinolinone DBPQ (Fig.
11.5) is a tricyclic scaffold related to MK-7622 that was discovered as a hit from an HTS of the NIH (Han et al., 2015).

Indole and azaindole analogs of hit VU0108370 have been also described exhibiting improved nanomolar potency
(Davoren et al., 2016; Rook et al., 2017).

Among modifications around the third chemotype VU0119498 (Ghoshal et al., 2016; Melancon et al., 2013; Swahn
et al., 2010), the most recent is isoindolinone PF-06827443 (Fig. 11.5), a potent, low-clearance, orally bioavailable, and
CNS-penetrant M1R-selective PAM with minimal intrinsic agonist activity (Davoren et al., 2017).

Overall, preclinical studies have revealed significant improvements in PK and in vivo efficacy for selective mACh
M1R AMs. Importantly, PAM VU319 (undisclosed structure), discovered by a team of scientists at the Vanderbilt Center
for Neuroscience Drug Discovery, has recently entered phase I clinical trials for cognitive impairment associated with
Alzheimer disease.

FIGURE 11.5 Representative allosteric ligands targeting aminergic muscarinic GPCRs of class A.

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mACh M2R. The mACh M2R is distributed in both the CNS and periphery and has been predominately studied for
its role in regulating parasympathetic cardiac function. However, the general consensus is that M2R activation leads to
undesirable off-target effects for mAChR modulators (Moulton and Fryer, 2011). The importance of the M2R lies on the
pioneering cocrystal structure bound to PAM LY2119620 (Figs 11.5 and 11.6) that has contributed to the understanding
of mAChR, and GPCR, allosteric modulation (Croy et al., 2014; Kruse et al., 2013). LY2119620, a high-affinity PAM of
both M4R and M2R that potentiates the activity of agonist iperoxo, occupies a site above the orthosteric iperoxo binding
site on the extracellular side of helices II, VI, and VII forming extensive contacts with ECL2 and ECL3 (Kruse et al.,
2013).

mACh M4R. Several aminothienopyridine derivatives of LY2033298 (Fig. 11.5) identified as a selective M4R PAM
have been developed (Chan et al., 2008). However, a poor PK profile and/or a strong species bias have precluded their
preclinical development (Brady et al., 2008; Kennedy et al., 2009). An iterative optimization effort from Vanderbilt
Center for Neuroscience Drug Discovery improved in vivo PK properties in benzothiazole series such as ML293 (Fig.
11.5), though lowering allosteric potency (Salovich et al., 2012). Further optimization resulted in the recently reported
aminothienopyridazine VU6000918 (Fig. 11.5) that maintains high potency, good in vivo PK profile, and antipsychotic
efficacy in an established hyperlocomotion assay (Tarr et al., 2017).

mACh M5R. The therapeutic interest of drugs targeting the M5R subtype has currently upsurged with the discovery of
selective PAMs that are efficacious in relevant disease models including schizophrenia, Alzheimer disease, ischemia, and
migraine (Bock et al., 2018). Starting from nonselective mAChR PAM VU0119498 (Fig. 11.5), isatin scaffold was used
to optimize potency and selectivity that were achieved in ML326 (Fig. 11.5) (Gentry et al., 2013). However, a low CNS
exposure precluded its in vivo assessment. Non-isatin ML380, discovered during an HTS, displays selective M5R PAM
activity with submicromolar potency and markedly improved CNS penetration, representing a promising hit (Berizzi et al.,
2016).

With muscarinic receptor crystal structures at hand, many new scaffolds of AMs should be discovered by
structure-based drug design and virtual screening. First evidence comes from a very recent study at the M2R, which used
accelerated molecular dynamics simulations and has led to AMs with novel chemical scaffolds (Miao et al., 2016).

11.2.5 Cannabinoid CB1 receptor (CB1R)
Cannabinoid receptors (CB1 and CB2Rs) belong to the endocannabinoid system that is a ubiquitous neuromodulatory
system with wide-ranging actions (Pacher and Kunos, 2013). CB1R is widely distributed throughout the CNS and
is involved in the regulation of many physiological processes related to pain, metabolism, nociception, and

FIGURE 11.6 Cocrystal structure of the M2R in a ternary complex with orthosteric agonist iperoxo and PAM LY2119620 (see Fig. 11.5). (A) The
allosteric LY2119620 agonist binds to the extracellular vestibule of the receptor, which is directly above the orthosteric iperoxo binding site. The
allosteric binding site of LY2119620 is formed by residues from the extracellular side of helices II, VI, and VII and the ECL2 and ECL3. (B) In the
allosteric LY2119620 binding site, the piperidine group is engaged in a charge−charge interaction with Glu172ECL2; the amide oxygen and nitrogen make
hydrogen bonds with the side chains of Tyr802.61 and Asn419ECL3, respectively; the bridging oxygen accepts a hydrogen bond from the side chain of
Asn4106.58. In addition to polar contacts, the hydrophobic interactions between M2R and LY2119620 are mainly from the aromatic residues, including
Tyr802.61, Tyr832.64, Trp4227.35, Tyr4267.39, and Tyr177ECL2 (PDB code 4MQT). Credit: Adapted with permission from J. Med. Chem. 2019, https://doi.
org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.



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neurotransmission (Saleh et al., 2018). To date, CB1R orthosteric agonists and antagonists have not realized therapeutic
expectations largely due to adverse effects; for example, the orthosteric antiobesity antagonist rimonabant was withdrawn
from the market owing to neuropsychiatric adverse effects (Di Marzo, 2008). Alternatively, the development of both
NAMs and PAMs of the CB1R has been of high interest in recent years.

CB1R PAMs. The structural diversity of the small molecules reported as CB1R PAMs remains relatively small, mostly
containing 2-phenyl-1H-indole scaffold. Interestingly, GAT211 (Fig. 11.7) acts as an ago-PAM as its R-enantiomer, while
the S-enantiomer lacks intrinsic agonist activity (Laprairie et al., 2017). Structurally related ZCZ011 (Fig. 11.7) exhibited
broad allosteric activity across signaling pathways and multiple agonists, and reduced neuropathic pain in the mouse with
no psychoactive effects (Ignatowska-Jankowska et al., 2015).

CB1R NAMs. The past decade has seen the identification of multiple promising compounds as NAMs targeting the
CB1R, mainly comprising two scaffolds that have been extensively characterized: indolecarboxamides and the diarylurea
analogs. The discovery of Org27569, Org29647, and Org27759 (Fig. 11.7) displaying a reduction of CB1R agonists
efficacy demonstrated, for the first time, the existence of an allosteric binding site at the receptor that can be recognized
by synthetic small-molecule ligands (Price et al., 2005). Subsequent SAR studies have revealed both the 2-carboxamide
function and the 5-halogen substituent in the indole ring are required to maintain allosteric activity (Khurana et al., 2014;
Mahmoud et al., 2013; Nguyen et al., 2015; Piscitelli et al., 2012).

PSNCBAM-1 (Fig. 11.7), discovered by HTS, was the first CB1R NAM bearing a diarylurea scaffold (Horswill et al.,
2007). Numerous studies have now reported thorough SAR around diarylurea analogs (Baillie et al., 2013; Khajehali et al.,
2015). In particular, RTICBM-74 (Fig. 11.7) has shown a good PK profile and in vivo efficacy in a rat model of cocaine
withdrawal (Nguyen et al., 2017). Nevertheless, in vivo studies on promising CB1R NAMs are still limited and will need
to progress toward proof-of-concept studies to show therapeutic utility.

FIGURE 11.7 Representative allosteric ligands targeting lipidic cannabinoid and free fatty acid GPCRs of class A.

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11.2.6 Free fatty acid receptors (FFA1−FFA3Rs)
Free fatty acid receptors (FFARs) are a recently “deorphanized” family of receptors that are activated by nonesterified or
“free” fatty acids (FFAs), the members of which are now receiving substantial interest as novel targets for the treatment of
inflammatory and metabolic diseases (Milligan et al., 2017).

FFA1R (GPR40). The FFA1R, which was designated GPR40 prior to the discovery of endogenous signaling ligand,
is predominately expressed in pancreatic β cells and intestinal enteroendocrine cells and has been validated as a potential
target for the treatment of type 2 diabetes. In recent years, numerous pharmacologically diverse allosteric ligands of
FFA1R have been discovered and structural work has described their binding to distinct sites. The cocrystal complexes
of the structurally related ago-PAMs TAK-875 and MK-8666 (Fig. 11.7) with FFA1R reveal that they both bind to
a far uncommon site formed by helices III−V and the ECL2 and that is adjacent to the exterior lipid membrane
surface (Figs. 11.8 and 11.9) (Lu et al., 2017; Srivastava et al., 2014). In contrast, the ternary complex structure of
FFA1R-MK-8666-AP8 (Figs 11.7 and 11.9) reveals that the latter, a novel and more potent FFA1R ago-PAM, binds to a
lipid-facing pocket formed by helices II−V and ICL2 that is a site completely distinct from the allosteric binding site of
MK-8666 (Lu et al., 2017).

Importantly, TAK-875 (fasiglifam) was progressed into phase III clinical trials for the treatment of type 2 diabetes
mellitus, but the trial underwent early termination due to toxicity. Likewise, MK-8666 has recently been approved to
advance into a phase I clinical trial for the treatment of the same disease.

FFA2R (GPR43). FFA2 and FFA3 receptors have been found expressed in various cancer cells, including breast,
colon, and liver (Milligan et al., 2017). Phenylacetamide derivatives such as 4-CMTB4 (Fig. 11.7) were identified via
HTS and represent the first series of synthetic small molecules that display PAM-like effects at the FFA2R (Lee et al.,
2008). However, their poor PK properties have limited further development as preclinical candidates (Lee et al., 2008).
Recently, structurally related AZ1729 (Fig. 11.7) has been characterized as a FFA2R ago-PAM displaying an interesting
pharmacological biased profile (Bolognini et al., 2016).

FFA3R (GPR41). Hexahydroquinolone-3-carboxamide derivatives discovered by Arena Pharmaceuticals are the
predominantly reported allosteric FFA3R ligands (Leonard et al., 2006). Representative compound 4 (Fig. 11.7) is an
ago-PAM with modest potency and selectivity over FFA2R (Bolognini et al., 2016). However, this series displays a
complex pharmacology and considerable care must be taken to use their activity for defining specific roles of FFA3R in
either in vitro or in vivo settings.

FIGURE 11.8 Cocrystal structure of GPR40 in complex with ago-PAM TAK-875 (see Fig. 11.7). (A) TAK-875 is positioned outside the 7TM, in a
site formed by helices III−V and the ECL2; this site is adjacent to the exterior membrane surface. (B) In the allosteric binding site, the carboxylate group
of TAK-875 is engaged in salt bridge interactions with the side chains of Arg1835.39 and Arg2587.35 and hydrogen binding interactions with the side
chains of Tyr913.37 and Tyr2406.51. The dihydrobenzofuran group is encapsulated by residues Ala833.29, Phe873.33, Leu1384.57, Phe1424.61, Leu171ECL2,
and Trp174ECL2 defining a hydrophobic pocket. The benzyl group is situated between helices III and IV and forms hydrophobic interactions with residues
Val843.30, Leu1354.54, and Phe1424.61 (PDB code 4PHU). Credit: Reprinted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.
jmedchem.7b01844. Copyright (2019) American Chemical Society.



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11.2.7 Adenosine receptors (A1, A2A, A2B, and A3Rs)
Adenosine receptors (ARs) comprise a family of GPCRs that mediate the physiological actions of adenosine. The
four identified AR subtypes —A1, A2A, A2B, and A3Rs— have distinct localization and signal transduction pathways
(Sheth et al., 2014). Historically, both agonists and antagonists have been used to indiscriminately modulate adenosine
receptor subtypes and have been involved in the treatment of diseases that span cardiovascular disease, CNS disorders,
inflammatory and allergic disorders, and cancer. Allosteric modulation may provide multiple benefits for targeting subtype
selective ligands that are pursued to avoid side effects therapeutically.

A1R. Bruns et al. first developed a novel series of allosteric enhancers, represented by PD 81,723 (Fig. 11.10),
selectively enhancing the binding of agonist N6-cyclopentyladenosine (CPA) to the A1R (Bruns and Fergus, 1990; Bruns et
al., 1990). Following this pioneering work, multiple PAM derivatives were designed around 2-amino-3-benzoylthiophene
scaffold, and detailed SAR studies on the 4- and 5-positions of the thiophene ring have been conducted (Van Der Klein
et al., 1999). Within these series, analog T-62 (Fig. 11.10) was developed by King Pharmaceuticals and advanced into
clinical trials for the potential treatment of neuropathic pain associated with hyperalgesia and allodynia (Baraldi et al.,
2007). However, the program was terminated after failure to meet the end point for efficacy in phase IIb (Childers et al.,
2005; Kimatrai-Salvador et al., 2013).

To avoid observed intrinsic antagonist activity at high concentrations and moderate efficacy (Ferguson et al., 2008),
further chemical modifications and optimization generated a new series of 2-amino-3-benzoylthiophene derivatives
bearing an arylpiperazine ring linked to a methylene at the 4-position of the thiophene. Substantially higher efficacy than
PD 81,723 without significant antagonist activity at the A1R, A2R, or A3R was achieved in compound 5 (Fig. 11.10), a
representative analog of the series (Romagnoli et al., 2012).

As mentioned previously, some AMs of the A1R have displayed a complex pharmacological profile that results in
PAM activity toward orthosteric agonists while inhibiting the effect of orthosteric antagonists. While no structure of an
adenosine receptor bound to its AM has been disclosed to date, the understanding of the complex A1R pharmacology has

FIGURE 11.10 Representative allosteric ligands targeting nucleotide adenosine and P2Y GPCRs of class A.

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12 GPCRs:Structure, Function, and Drug Discovery

greatly benefited from the recent reports of solved structures of both the active and inactive state of A1R and cryo-electron
microscopy of active state of A1R (Draper-Joyce et al., 2018a; Glukhova et al., 2017). Together with previous mutagenesis
studies, this structural information provides additional insight for AM binding, and docking studies have identified ECL2
as a critical domain for PAM activity (Nguyen et al., 2016).

A2AR and A2BRs. Amiloride (Fig. 11.10) and derivatives thereof have been reported as adenosine A2AR PAMs, and
docking studies based on the high resolution crystal structure of agonist-bound A2AR describe their interactions in the
sodium ion pocket of the receptor (Gao and Ijzerman, 2000; Massink et al., 2016).

Compounds with a 1-benzyl-3-ketoindole scaffold, for example, compound 6 (Fig. 11.10), are the only reported AMs
for A2BR (Taliani et al., 2013; Trincavelli et al., 2014). These PAMs and NAMs may provide useful chemical probes to
elucidate the therapeutic potential of the least characterized adenosine subtype.

A3R. 1H-imidazo[4,5-c]quinolin-4-amine —for example, LUF6000— and 2,4-disubstituted quinoline —for example,
LUF6096— are the core scaffolds of the modest number of selective A3R AMs reported to date (Goblyos et al., 2006;
Heitman et al., 2009) (Fig. 11.10). Interestingly, LUF6000 shows significant potentiation of agonist efficacy and no
enhancing effect on agonist potency, whereas open-ring LUF6096 displays positive allosteric effects on both agonist
efficacy and potency.

11.2.8 P2Y receptors (P2Y1, P2Y2, and P2Y12Rs)
P2Y receptors are a family of purinergic GPCRs, stimulated by nucleotides such as ATP, ADP, UTP, UDP, and
UDP-glucose. To date, eight P2Y receptors have been cloned in humans —P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13,
and P2Y14— and the biological effects of their activation depend on how they couple to downstream signaling pathways,
either via Gi, Gq/11, or Gs protein (Von Kugelgen and Hoffmann, 2016).

Nonnucleotide allosteric antagonists of the P2Y1R such as BPTU (Fig. 11.10), discovered by Bristol-Myers Squibb,
and other diarylurea derivatives are also being considered as antithrombotic agents with improved safety profiles (Chao et
al., 2013; Qiao et al., 2013; Yang et al., 2014). In the recent cocrystal complex, BPTU is the first P2Y1R antagonist shown
to bind to an allosteric site entirely outside of the 7TM, not only outside of the orthosteric site. The allosteric binding site
of BPTU is situated adjacent to the lipid membrane and engages the ligand by mostly hydrophobic and aromatic residues
located in helices I−III as well as minor involvement of ECL1 (Fig. 11.11).

FIGURE 11.11 Cocrystal structure of P2Y1R in complex with allosteric antagonist BPTU (see Fig. 11.10). (A) BPTU binds to P2Y1R on the lipidic
interface of the transmembrane domain; this BPTU binding site is distinct from the endogenous ADP binding site and is formed by residues from
helices I−III and ICL1. (B) The allosteric binding pocket, formed by aromatic and hydrophobic residues, accommodates BPTU predominantly through
hydrophobic interactions. The pyridyl group of BPTU comes in contact with residues Ala1062.59 and F119ICL1. The benzene ring within the phenoxy
group of BPTU is situated in a hydrophobic subpocket formed by residues from helices II and III, including Thr1032.56, Met1233.24, Leu1263.27, and
Gln1273.28; by contrast, the tert-butyl substituent on the phenoxy group engages in a hydrophobic interaction with Leu1022.55. At the opposite side of
the ligand, the ureido phenyl ring is involved in aromatic−aromatic interactions with Phe623.27 and Phe661.47. The only polar interactions observed are
two hydrogen bonds between the NHs of the BPTU's urea group and the backbone carboxyl of Leu1022.55 (PDB code 4XNV). Credit: Reprinted with
permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.



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Compound 7 (Fig. 11.10), with a novel 4(1H)-quinolinone scaffold, is among the first nonnucleotide selective P2Y2R
AMs, characterized as an ago-PAM, and preclinical studies support their therapeutic potential for the treatment of
cardiovascular disorders (Sakuma et al., 2017).

The P2Y12R is activated by ADP to trigger glutamate release, facilitating thrombus formation, and has been validated
as a drug target for the treatment of thromboembolisms and other clotting disorders (Dorsam and Kunapuli, 2004). The
FDA approval of ticagrelor (AZD6140, Fig. 11.10), a P2Y12R allosteric antagonist discovered by AstraZeneca, has paved
the way for antithrombotic drugs in this class with safer bleeding profiles to emerge as therapeutics (Springthorpe et al.,
2007).

11.2.9 Chemokine receptors (CCR2, CCR4, CCR5, CCR9, CXCR1, CXCR2, CXCR3, and
CXCR4)
At least 18 chemokine GPCRs have been identified in humans that are classified in four subfamilies –CCR, CXCR,
CX3CR, and XCR— based on the relative positioning of conserved cysteine residues in the N-terminal domain of their
endogenous ligands (Bachelerie et al., 2014). Chemokine receptors are activated by more than 50 chemokine ligands in
a concerted manner in response to various immunologic or inflammatory events (Allegretti et al., 2016). Thus, probe
dependence may be a primary advantage for AMs of chemokine receptors. The development of AMs for chemokine
receptors represents a profound advance for AMs of class A GPCRs with marketed drugs (maraviroc, NAM of CCR5;
plerixafor, NAM of CXCR4, see Fig. 11.12) and structural studies of AMs binding to the receptors at high resolution
(for CCR2, CCR5, and CCR9) (Lu and Zhang, 2018). These structures have revealed allosteric modulation via binding
intracellular allosteric sites, which is still uncommon for class A GPCRs but may have important therapeutic implications,
especially for chemokine receptors.

CCR subfamily. CCR2 subtype is implicated in numerous inflammatory and neurodegenerative diseases (O'connor et
al., 2015). CCR2-RA-[R] (Fig. 11.12) is a selective NAM of CCR2 with an excellent PK profile. The cocrystal structure of
CCR2 in a ternary complex with CCR2-RA-[R] and orthosteric antagonist BMS-681 demonstrates that the former occupies
an intracellular allosteric binding site, as seen in other chemokine receptors (Fig. 11.13) (Zheng et al., 2016).

CCR4 subtype is crucial for recruiting T cells during the inflammatory response upon exposure to allergens, and has
thus been a target for the discovery of small-molecule therapeutics due to its central role in pathogenesis such as asthma,

FIGURE 11.12 Representative allosteric ligands targeting protein chemokine GPCRs of class A.

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14 GPCRs:Structure, Function, and Drug Discovery

FIGURE 11.13 Cocrystal structure of CCR2 in a ternary complex with orthosteric antagonist BMS-681 and NAM CCR2-RA-[R] (see Fig. 11.12).
(A) CCR2-RA-[R] occupies an allosteric site on the intracellular side, which is more than 30 Å away from the orthosteric site on the extracellular side
where BMS-681 is bound. The allosteric site of CCR2-RA-[R] is formed by residues from the intracellular ends of helices I−III and VI−VIII. (B) In
the allosteric site, the hydroxyl group of CCR2-RA-[R] forms a bifurcated hydrogen bond with the backbone amines of Glu3108.48 and Lys3118.49, and
the pyrrolone carbonyl group is hydrogen-bonded to the backbone amide of Phe3128.50. For the hydrophobic interactions, the phenyl group contacts
with Val631.53, Leu671.57, Leu812.43, Tyr3057.53, and Phe3128.50, and the cyclohexane ring interacts with Leu812.43, Leu1343.46, Ala2416.33, Val2446.36,
and Ile2456.37 (PDB code 5T1A). Credit: Adapted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright
(2019) American Chemical Society.

atopic dermatitis, cancer, and mosquito-borne tropical diseases. Representative indazole sulfonamide 8 (Fig. 11.12) is a
potent antagonist with a good PK profile that binds to the intracellular allosteric binding site of the CCR4 and has been
selected for further development (Procopiou et al., 2013). Subsequent studies suggest there are two additional allosteric
sites to which small molecules bind.

CCR5 subtype is widely implicated in the process of HIV type 1 infection (Maeda et al., 2012) and has been validated
as a drug target with the FDA approval of maraviroc (Fig. 11.12) as an anti-HIV agent. The cocrystal complex of CCR5
and maraviroc demonstrates that the allosteric ligand binds to the receptor occupying an extracellular site of the 7TM (Fig.
11.14) (Tan et al., 2013b). Recently, the first probe dependent CCR5 PAMs have been discovered based on 2-benzazepine,
bipyridine, and terpyridine scaffolds (Thum et al., 2017). The identification of probe dependent CCR5 PAM and NAM
ligands will broaden the biological knowledge of chemokine signaling and the therapeutic relevance.

FIGURE 11.14 Cocrystal structure of CCR5 in complex with NAM maraviroc (see Fig. 11.12). (A) Maraviroc occupies an extracellular cavity on
top of the 7TM from the helices I−III and V−VII and does not interact with the ECL2, which is among the major binding determinants for CCR5
chemokine agonists. (B) In the binding site, Glu2837.39 forms a salt bridge with the protonated nitrogen of the tropane group of maraviroc. Tyr2516.51

forms a hydrogen bond with the carboxamide nitrogen of the ligand. Tyr371.39 forms a hydrogen bond with the amine of the triazole group of the
ligand. Thr1955.39 and Thr2596.59 are involved in a hydrogen bond with one of the fluorines in the cyclohexane ring of the ligand. For the hydrophobic
interactions, the cyclohexane ring of the ligand is located inside the hydrophobic pocket formed by Gln1945.38, Ile1985.42, Leu2556.55, and Met2797.35;
the phenyl group protrudes into a deep hydrophobic cleft formed by a cluster of aromatic residues, Tyr1083.32, Phe1093.33, Phe1123.36, Trp2486.48, and
Tyr2516.51; and the triazole group is engaged in hydrophobic interactions with Trp862.60, Tyr892.63, and Met2877.43 (PDB code 4MBS). Credit: Reprinted
with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.



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CCR9 subtype is a member of the CC chemokine receptor subfamily implicated in inflammatory bowel disease.
Vercirnon (GSK1605786A, Fig. 11.12) is a selective allosteric antagonist of CCR9 that entered phase III clinical trials for
the treatment of Crohn disease, but the study was terminated due to the lack of efficacy (Wendt and Keshav, 2015). The
cocrystal structure of CCR9 with vercirnon shows that the allosteric ligand binds to the intracellular side of the receptor,
which is similar to that of CCR2-RA-[R] bound to CCR2 (Fig. 11.15) (Oswald et al., 2016).

CXCR subfamily. CXCR1 subtype is largely expressed on T lymphocytes and natural killer cells, playing a key role in
acute and chronic inflammatory conditions (Allegretti et al., 2016). Reparixin (Fig. 11.12) is a noncompetitive NAM for
CXCR1 that binds the receptor at an allosteric site between helices I, III, and VI and has been advanced into a phase III
clinical trial for pancreatic islet autotransplantation (Bertini et al., 2004). Also, a phase I study is ongoing to evaluate the
safety of orally administered reparixin in combination with paclitaxel in HER 2-negative metastatic breast cancer patients.
Ladarixin (Fig. 11.12) is a highly potent allosteric inhibitor of CXCR1/CXCR2 that has been advanced into clinical trials
for type 1 diabetes (Allegretti et al., 2016).

CCXR3 subtype is viewed as a promising drug target for a myriad of inflammatory diseases, such as rheumatoid
arthritis, multiple sclerosis, cancer, atherosclerosis, and allograft rejection. Azaquinazolinone derivatives such as
representative compound BD103 (Fig. 11.12) have been characterized as promising AMs of the CXCR3 endowed with
signaling bias and probe dependence (Bernat et al., 2015; Brox et al., 2018).

CXCR4 subtype is a chemokine receptor essential for hematopoietic stem cell colonization of fetal bone marrow
during development (Allegretti et al., 2016). Interestingly, plerixafor (Fig. 11.12), an NAM of CXCR4 with
tetraazacyclotetradecane scaffold, was initially in development as an anti-HIV drug but has been repurposed and is now
marketed for an indication of bone marrow transplantation for patients with non-Hodgkin lymphoma or multiple myeloma
(Miller et al., 2018).

11.2.10 Opioid receptors (μ-OR, δ-OR, and κ-OR)
Opioid GPCRs are widely distributed in the brain, the spinal cord, peripheral neurons, and the digestive tract. Opioid
receptors (ORs), especially the μ-OR subtype, have been targeted for the treatment of pain and related disorders for
thousands of years, and remain the most widely used analgesics in the clinic (Al-Hasani and Bruchas, 2011). Although
μ-OR activation produces profound analgesia, tolerance develops for opioid drugs and addiction can be severely
problematic (Morgan and Christie, 2011). Additionally, side effects such as respiratory depression, nausea, constipation,
and others have highlighted the urgent need for novel OR therapeutics that have a safer profile without the loss of

FIGURE 11.15 Cocrystal structure of CCR9 in complex with allosteric antagonist vercirnon (see Fig. 11.12). (A) Vercirnon binds to the intracellular
side of the CCR9, which is approximately 33 Å away from the orthosteric site. The allosteric site of vercirnon is formed by residues from the intracellular
ends of helices I−III and VI−VIII. (B) In the allosteric binding site, the sulfone group of vercirnon is engaged with hydrogen bonding interactions
with the backbone amine of Glu3228.48, Arg3238.49, and Phe3248.50. The pyridine-N-oxide group protrudes toward the intracellular face of the CCR9
and forms a bifurcated hydrogen bond with the side chains of Arg78ICL1 and Thr81ICL1. The ketone group is hydrogen bonded to the side chain of
Thr2566.37. For the hydrophobic interactions, the tert-butylphenyl group makes numerous contacts with the hydrophobic cleft formed by Val691.53,
Val721.56, Tyr731.57, Leu872.42, Tyr3177.53, and Phe3248.50. The chlorophenyl group is located in a narrow, hydrophobic cleft surrounded by residues
Leu872.43, Ile1403.46, Val2596.40, and Tyr3177.53, whereas the pyridine-N-oxide group is located in a polar cavity surrounded by residues Thr832.39,
Asp842,40, Arg1443.50, Arg3238.49, and Thr81ICL1 (PDB code 5LWE). Credit: Adapted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/
acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.

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16 GPCRs:Structure, Function, and Drug Discovery

efficacious analgesia. AMs may be suitable in this scenario, as they have a generally safer profile, owing to the ceiling
effect (Remesic et al., 2017). Both NAMs and PAMs have been explored for ORs, and while most work on allosteric
modulation has been directed toward the μ-OR, a few ligands have been reported as hits for the δ-OR (Remesic et al.,
2017).

Cannabidiol and salvinorin-A are among the earliest identified NAMs for the μ-OR and δ-OR (Kathmann et al., 2006;
Remesic et al., 2017). Recently, BMS-986121 and BMS-986122 (Fig. 11.16) were identified via an HTS as the first
selective PAMs for the μ-OR (Burford et al., 2013, 2015). Subsequent chemical SAR study around the latter led to the
discovery of BMS-986124 (Fig. 11.16) as an SAM (Burford et al., 2013, 2015). Additionally, diterpene alkaloid ignavine
(Fig. 11.16) has shown positive modulatory activity for μ-OR and an analgesic effect in vivo (Ohbuchi et al., 2016).

BMS-986187 (Fig. 11.16), with a chemically novel core compared to previous BMS series, was discovered as an
effective PAM at the δ-OR and at the κ-OR rather than the μ-OR (Livingston et al., 2018).

Allosteric modulation of ORs holds promise of delivering safer analgesics and other therapeutics to the clinic; however
significant optimization and development are still needed.

11.2.11 Proteinase-activated PAR2 receptor
PAR2 belongs to protease-activated receptors (PARs), a unique family of GPCRs that are not activated by endogenous
ligands but rather activated by the cleavage of their extracellular amino terminus at a specific site by proteases (Adams
et al., 2011). The newly formed N-terminus, also called a tethered ligand, subsequently binds to the receptor to initiate
receptor activation.

PAR2 has recently been recognized as an important modulator of coagulation and inflammatory responses
(Ramachandran et al., 2012). AZ3451 (Fig. 11.16) is a small-molecule allosteric antagonist of PAR2. The cocrystal
structure of PAR2 bound to AZ3451 shows that the ligand binds to a remote allosteric site outside the helical bundle (Fig.
11.17), which is distinct from the orthosteric site of antagonist AZ8838 located near the extracellular surface (Cheng et al.,
2017).

11.3. ALLOSTERIC MODULATORS TARGETING CLASS B GPCRS
Class B GPCRs, also known as the secretin family, are a subfamily of receptors that bind paracrine or endocrine peptide
hormones involved in the physiology and pathophysiology of major functions, and represent targets of particular interest
for the treatment of diseases such as type 2 diabetes mellitus, osteoporosis, hypercalcaemia, Paget disease, migraine,
depression, anxiety, Crohn disease, and cancer. Historically, the discovery and development of small-molecule orthosteric
ligands for class B receptors have proven to be challenging (Bortolato et al., 2014). More recently, the search for AMs
has become an intense field of research, as it represents an attractive strategy for the development of novel agents as an
alternative to peptides in clinical use or as first-generation therapeutics. The calcitonin (CT), corticotropin-releasing factor
(CRF), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and glucagon
(GCG) receptor families have been allosterically targeted, and their most relevant AMs will be reviewed in this section.

FIGURE 11.16 Representative allosteric ligands targeting protein opioid and proteinase-activated GPCRs of class A.



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FIGURE 11.17 Cocrystal structure of PAR2 in complex with allosteric antagonist AZ3451 (see Fig. 11.16). (A) The allosteric AZ3451 binding site
of PAR2 is located in the extracellular side, outside the 7TM, and lined by residues from helices II−IV. (B) The only hydrogen bond in the allosteric
binding site is formed between the benzimidazole nitrogen of AZ3451 and the side chain of Tyr2104.61. For the hydrophobic interactions, the central
benzimidazole moiety of AZ3451 engages in a hydrophobic interaction with Leu2034.54. The 1,3-benzodioxole ring is located inside a hydrophobic
pocket, which is defined by residues Ala1202.49, Leu1232.52, Phe1543.31, Ala1573.34, Cys1613.38, Trp1994.50, and Ile2024.53. The cyclohexyl ring protrudes
into the binding site and is positioned between the interface of the lipid layer and the receptor, which contacts with Leu1232.52. The benzonitrile group
with the cyclohexyl ring also sits at the interface of the lipid layer and the receptor and forms an aromatic−aromatic interaction with Tyr2104.61 (PDB
code 5NDZ). Credit: Reprinted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American
Chemical Society.

11.3.1 Calcitonin receptors (CT and CGRPRs)
The CT family of peptides includes CT, amylin, CT gene-related peptide (CGRP), and adrenomedullin, and their receptors
have therapeutic relevance for many pathologies, such as cardiovascular disease, migraine, osteoporosis, diabetes, obesity,
and lymphatic insufficiency (Poyner et al., 2002).

CTR. Agonists of the CT receptor (CTR) are of interest as biological tools and therapeutic agents in the treatment
of bone-related disorders such as osteoporosis and hypercalcaemia of malignancy, since CT peptide is involved in
maintenance of calcium homeostasis, mainly with respect to bone formation and metabolism. Few nonpeptide ligands have
been reported for CTR and among them, only a series of pyrazolopyridine derivatives has been deeply studied to define
the molecular mechanism of action (Boros et al., 2005; Dong et al., 2009). These ligands, represented by compound 9 (Fig.
11.18), weakly potentiated the cAMP production of the endogenous ligand and exhibited incomplete inhibition of [125I]
labeled CT, results that contributed to propose an allosteric mode of action. Importantly, mutagenesis and chimeric studies
supported that these molecules bind in TM1 and the extracellular domain of the receptor, a distinct site of action relative to
that of CT. These results represented the first identification of the juxtamembranous region of the amino-terminal domain
of the CTR as a critical site for allosteric drug action at class B GPCRs.

CGRPR. The CGRP is one of the most potent endogenous vasodilators and has been implicated in the pathogenesis
of migraine headache, which is directly related to dilatation of cranial vessels and activation of the trigemino-vascular
system (Brain et al., 2002). Therefore, blocking CGRP receptors (CGRPRs) has been suggested as a potential antimigraine
strategy. CGRPRs are heterodimeric complexes composed by the CT receptor-like receptor (CLR) and the receptor
activity modifying protein 1 (RAMP1) (Mclatchie et al., 1998). Although the complex structural nature of the CGRPR
has hampered the research, efforts within the pharmaceutical industry have led to the identification of small-molecule
inhibitors, which have turned out to act as AMs. Olcegepant (BIBN4096BS, Fig. 11.18) was the first potent and selective
CGRPR antagonist reported. It was developed from a series of dipeptide-like compounds identified by HTS (Doods et
al., 2000). Olcegepant inhibited induced neurogenic vasodilation in human brain vessels (Moreno et al., 2002) and its
efficacy in the acute treatment of migraine was confirmed (Olesen et al., 2004). Telcagepant (MK-0974, Fig. 11.18)
(Salvatore et al., 2008) is another highly selective nonpeptide antagonist that displayed good oral bioavailability. This
molecule resulted from a lead optimization process of a benzodiazepine CGRPR antagonist identified by HTS. In

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18 GPCRs:Structure, Function, and Drug Discovery

FIGURE 11.18 Representative allosteric ligands targeting calcitonin, corticotropin-releasing factor, and vasoactive intestinal peptide GPCRs of class
B.

vivo evaluation of telcagepant showed a concentration-dependent inhibition of dermal vasodilation, revealing its value in
the acute treatment of migraine (Ho et al., 2008).

In-depth research with olcegepant and telcagepant suggested an allosteric mechanism of antagonism as their plausible
mode of action (Hay et al., 2006; Miller et al., 2010). Both the CLR and RAMP1 components of the CGRPR have
extracellular domains, which interact with each other and together form part of the peptide-binding site. The
small-molecule binding site was suggested to be located on these extracellular domains. Mutagenic screen with CLR and
RAMP1 indicated that olcegepant and telcagepant share a similar binding site and occupy a region close to the interface of
the N-terminal domains of CLR and RAMP1.

11.3.2 Corticotropin-releasing factor receptor 1 (CRF1R)
The corticotropin-releasing factor receptors (CRF1 and CRF2R) are activated by the endogenous CRF peptide and the
related urocortin peptides, which are known to play important biological roles in the regulation of central and peripheral
stress responses.

It is well established that CRF1R antagonism represents an interesting mechanism for treating anxiety, depression,
and other stress-related disorders (Holsboer, 2001; Smagin et al., 2001), and allosteric modulation of this receptor has
proven to be a valuable approach for therapeutic intervention (Hoare, 2007). To date, many small-molecule, selective,
and high-affinity CRF1R ligands have been characterized as allosteric antagonists. CP-154526 (Schulz et al., 1996)
and antalarmin (Fig. 11.18) (Webster et al., 1996) were the first modulators reported as effective blockers of the
neuroendocrine effects and behavioral changes induced by CRF. Since then, as a result of HTS campaigns and subsequent
medicinal chemistry optimization programs, several compounds have been disclosed as selective CRF1R AMs and have
reached different stages of clinical evaluation, aimed at testing the hypothesis that CRF1 inhibitors could be used clinically
as antidepressant drugs (Kehne and Cain, 2010).



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Among a series of 2-aryloxy-4-alkoxypyridines derived from CP-154526 and developed by Pfizer, CP-316311 (Fig.
11.18) stood out as a potent and selective allosteric antagonist with oral efficacy in the CNS, which advanced to phase
II depression trials. Further optimization of these pyridine-based AMs addressing physicochemical properties led to
CP-376395 (Fig. 11.18) (Chen et al., 2008), which displayed improved potency over CP-316311 in relevant in vivo
models. CP-376395 was selected as backup candidate to CP-316311, entering also into clinical evaluation.

All these CRF1R agents have been described as NAMs rather than as competitive CRF1R antagonists. They keep the
receptor in an inactive conformation, and peptide and small-molecule ligands that bind to different conformational states
of the receptor do not compete for the same binding region (Chen et al., 2008; Grigoriadis et al., 2009; Hauger et al.,
2006). This binding mode has been confirmed by the crystal structure of CRF1R in complex with CP-376395 (Figs 11.18
and 11.19) (Hollenstein et al., 2013). The structure shows that the antagonist binds to an allosteric site very deep within
the 7TM toward the intracellular face of the receptor, inhibiting the binding and signaling of peptide-agonist ligands.
Importantly, this cocrystal structure of CRF1R has provided a model for class B GPCRs, revealing an unanticipated
antagonist-binding site and enabling future structure-based small-molecule drug discovery approaches.

11.3.3 Vasoactive intestinal peptide receptor 2 (VPAC2R)
The VPAC1, VPAC2, and PAC1 receptors (VPAC1, VPAC2, and PAC1Rs), mediated by neuropeptides VIP and PACAP,
have emerged as potentially useful therapeutic targets for inflammatory, metabolic, or circadian functions (Dickson and
Finlayson, 2009; Sherwood et al., 2000). They represent a clear example where the development of nonpeptide ligands
is of high interest since the endogenous agonists are not practical as therapeutics due to their short half-lives and lack
of oral bioavailability and brain penetration. However, small-molecule drug discovery for these class B GPCRs has
been certainly difficult. Compound 10 (Fig. 11.18) was the first specific antagonist reported for human VPAC2R (Chu
et al., 2010), and it was discovered via HTS of a 1.67 million-compound collection. This ligand is a specific NAM
that noncompetitively antagonizes VPAC2R-mediated cAMP accumulation and β-arrestin2 binding, with no activity on
VPAC1R or PAC1R. A structural similarity search starting from 10 yielded structurally related VPAC2R NAM 11 (Fig.
11.18), which also displayed some activity for VPAC1R and PAC1R. β–arrestin assays using chimera receptors indicated
that both compounds bind to an allosteric site in the 7TM of the receptor as opposed to the N-terminal extracellular domain,
where the endogenous ligand binds. The fact that compounds 10 and 11 are the only VPAC2R NAMs known to date makes
them a valuable tool for further study of VPAC2R and related receptors.

FIGURE 11.19 Cocrystal structure of CRF1R in complex with allosteric antagonist CP-376395 (see Fig. 11.18). (A) The allosteric CP-376395 binding
site of CRF1R is located in the intracellular half of the receptor, which is approximately 18 Å away from the orthosteric peptide agonist-binding site
located at the center of the large cavity presented to the extracellular side of the receptor. (B) CP-376395 binds in a deep site created by residues from
helices III, V, and VI. The pyridine nitrogen of CP-376395 in the allosteric binding site is involved in a hydrogen bond with the side chain of Asn2835.50.
For the hydrophobic interactions, the aryloxy moiety is located inside a hydrophobic pocket created by residues Phe2845.51, Leu2875.54, Ile2905.57,
Thr3166.42, Leu3196.45, and Leu3206.46. The central pyridine ring forms hydrophobic interactions with Met2063.47 and Val2795.46. The exocyclic
alkylamino group binds in a hydrophobic pocket defined by residues Phe2033.44, Leu2805.47, Leu3236.49, and Tyr3276.53 (PDB code 4K5Y). Credit:
Reprinted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.

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20 GPCRs:Structure, Function, and Drug Discovery

11.3.4 Glucagon receptors (GCG and GLPRs)
GCGR. The GCGR is considered an important drug target in the treatment of diabetes due to the key role of its endogenous
ligand in glucose homeostasis (Ahren, 2015). Despite many small-molecule GCGR antagonists from multiple chemical
classes being discovered, detailed studies about the nature of their inhibition mechanism are very scarce. Among early
disclosed antagonists, compound L-168049 (Fig. 11.20) was characterized as a noncompetitive antagonist. This pyrrole
derivative was identified from novel structural classes of nonpeptidyl ligands based on triarylimidazole and triarylpyrrole
scaffolds that showed significant binding affinity for the human GCGR (Cascieri et al., 1999). L-168049 behaves as a high
affinity and potent NAM that affects both affinity and activity of GCG. Site-directed mutagenesis experiments indicated
that the binding site of L-168049 is located within the 7TM, while the endogenous peptide binds to the amino-terminal
domain. Unfortunately, L-168049 displayed poor affinity for the different species orthologs of GCGR, hampering its
testing in preclinical animal models of disease.

NNC 25-2504 (Fig. 11.20) is another highly potent noncompetitive antagonist. This alkylidene hydrazide was found to
inhibit GCG-stimulated glucose production in isolated primary rat hepatocytes and to suppress exogenous GCG-induced
glucose increase in blood. NNC 25-2504 was orally bioavailable in dogs (Madsen et al., 2002).

MK-0893 (Fig. 11.20) (Xiong et al., 2012) was the first AM of GCGR entering clinical trials, and advanced to
phase IIa in diabetic patients. This NAM displayed good potency and selectivity profile, and was developed from an
optimization effort around a series of pyrazole-based antagonists, in which the N-heterocycle was found to be a good
replacement of a urea core present in a previously identified GCGR competitive antagonist (Lau et al., 2007). The
cocrystal structure of the complex GCGR-MK-0893 has been recently published (Fig 11.21) (Jazayeri et al., 2016). The
structure shows an unexpected binding site for the AM, located outside the 7TM, in a position between TM6 and TM7
and extending into the lipid bilayer, in contrast with the TM-inner site described for the CRF1R AM CP-376395 (Fig.
11.19). The role of the key residues of the allosteric site identified in the X-ray structure was confirmed by mutagenesis.

FIGURE 11.20 Representative allosteric ligands targeting glucagon GPCRs of class B.



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FIGURE 11.21 Cocrystal structure of GCGR in complex with allosteric antagonist MK-0893 (see Fig. 11.20). (A) MK-0893 binds to an allosteric
site outside the 7TM between helices VI and VII, extending into the lipid bilayer. (B) For polar interactions in the allosteric binding site, the terminal
anionic carboxylic acid group of MK-0893 forms a salt bridge with Arg3466.37, hydrogen bonds with the side chain of Asn4047.61 and the backbone
amine of Lys4057.62, and a water-mediated hydrogen bond with the side chain of Ser3506.44 and the backbone carbonyl of Leu3997.56. In addition, the
amide group of the ligand engages in hydrogen bonding interactions with the side chains of Lys3496.40 and Ser3506.41. For hydrophobic interactions,
the methoxynaphthalene moiety forms numerous hydrophobic interactions with residues Leu3295.61, Phe3456.36, Leu3526.43, Thr3536.44, and the alkyl
chain of Lys3496.40. The benzylpyrazole core forms hydrophobic interactions with residues Thr3536.44 and Leu3997.56 and a π−cation interaction with
Lys3496.40 (PDB code 5EE7). Credit: Reprinted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright
(2019) American Chemical Society.

The exact antagonist mechanism of MK-0893 remains unclear, but it has been suggested that the activation of the receptor
is hampered by restriction of the outward helical movement of TM6 required for G-protein coupling activation.

Competition binding assays with [3H]MK-0893 have been used to characterize the binding of previously reported
GCGR antagonists, chemically related or totally distinct to MK-0893. In this context, NAM NNC0640 (Fig. 11.20)
(Lau et al., 2007) was fully displaced by [3H]MK-0893, indicating a common allosteric binding site (Jazayeri et al.,
2016). Interestingly, the cocrystal structure of the full-length human GCGR in an inactive conformation in complex with
NNC0640 and an antigen-binding fragment of an inhibitory antibody has been recently published (Zhang et al., 2017).
This new structure has shown that NAM NNC0640 occupies a region on the external surface of the 7TM of GCGR and
binds in a site similar to that reported for MK-0893, which confirms previous radioligand binding assay results.

GLP-1R. Class B GLP-1R is activated by several endogenous GLP-1 peptide hormones and represents a key target
for type 2 diabetes mellitus, where it has a critical role in the potentiation of insulin secretion and suppression of GCG
secretion (Graaf et al., 2016). To date, several peptide GLP-1R agonists have demonstrated their utility for the treatment
of obesity and type 2 diabetes mellitus and have successfully reached the market (Tomlinson et al., 2016). However, the
discovery of nonpeptide small-molecule drugs remains highly desirable, since they may constitute a significant advance
due to their enhanced bioavailability, particularly oral absorption. In this sense, a number of nonpeptide GLP-1R agonists
have been reported and, interestingly, most of them behave as AMs that do not compete with GLP-1 in the orthosteric
site. Starting from a quinoxaline scaffold, a hit identified from a functional screening, Novo Nordisk developed the first
GLP-1R PAMs (Knudsen et al., 2007). Among them, compound 12 (Fig. 11.20) was the most potent agonist found and has
been extensively characterized. It acts as an ago-PAM, potentiating GLP-1-induced receptor activation in the presence of
GLP-1, and it is selective over other class B GPCRs. Interestingly, this PAM displays probe dependence for the effects on
peptide agonists of GLP-1R, such as exendin or extended GLP-1 peptides (Willard et al., 2012; Wootten et al., 2013b).
Compound 12 was capable of releasing glucose-dependent insulin from wild-type mouse islets, but not from GLP-1R
knockout mice. However, its poor PK properties precluded clinical development.

BETP (Fig. 11.20) is another remarkable GLP-1R PAM (Sloop et al., 2010). This compound was obtained by
structural modifications of a pyrimidine hit, which was identified from a small library generated from a three-dimensional
pharmacophore model. Despite key structural differences, BETP possesses a very similar biological profile in terms of
activation of receptor signaling and probe-dependence to that of compound 12.

An interesting aspect of both compound 12 and BEPT is their high electrophilic character, which allows them to
establish covalent interactions with free cysteine residues of the receptor (Eng et al., 2013; Nolte et al., 2014). This

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22 GPCRs:Structure, Function, and Drug Discovery

covalent mechanism of action was studied using a clickable analog of BETP named PETP (Fig. 11.20). Thus, Western
blot analysis of GLP-1R protein purified from CHO-K1 cells treated with PETP and subjected to click chemistry using
biotin-azide demonstrated the incorporation of the probe into GLP-1R, confirming the covalent modification of the
receptor. Additionally, mass-spectrometry-based proteomics of GLP-1R from cells treated with BEPT and subsequent
mutagenesis studies indicated that cysteine residue Cys347 was the specific residue covalently modified by the PAM. In
fact, adduct formed with Cys347 is critical for both the intrinsic efficacy and the cooperative allosteric effect. However,
this covalent modification is not enough to explain the differences in intrinsic efficacy and the cooperative effect of
compound 12 and BEPT.

Nonelectrophilic small-molecule ligands have been also reported to act as GLP-1R modulators. Among them, a
series of flavonoids represented by quercetin (Fig. 11.20) has been characterized as AMs that positively modulated
affinity and efficacy of GLP-1R peptide ligands, lacking intrinsic activity (Wootten et al., 2011). Quercetin displayed
functional selectivity and enhanced calcium signaling of endogenous truncated GLP-1 peptides and exendin, but not cAMP
production. However, polypharmacology and a flat SAR precluded further development of this family of compounds
(Perry et al., 2002).

VU0453379 (Fig. 11.20) is a CNS penetrant GLP-1R modulator (Morris et al., 2014) that represents an opportunity
for the assessment of the therapeutic potential of GLP-1 potentiation in the CNS, where the receptor has been linked
to neuroprotection, learning, neurogenesis, and memory (During et al., 2003; Perry et al., 2002). VU0453379 lacks
probe dependence, potentiating endogenous GLP-1 as well as synthetic peptide agonists exenatide and liraglutide.
Importantly, the compound exhibited efficacy in a haloperidol-induced catalepsy model, a preclinical model of Parkinson
disease, and current efforts are focused on improving both allosteric potency and PK profile to confirm Parkinson disease
as an exciting indication for GLP-1R PAMs.

Vivia Biotech has also contributed to the field of GLP-1R modulators with the identification of a series of oxadiazole
derivatives able to act as PAMs, enhancing the efficacy of GLP-1R stimulation dose-dependently and potentiating insulin
secretion (Rodriguez De Fonseca et al., 2012).

Although the main therapeutic strategy targeting GLP-1R has been the development of agonists for the treatment of
hyperglycemia, recent studies have revealed that antagonism of GLP-1R also plays an important role in the treatment of
acute and chronic stress as well as anxiety. The number of small-molecule GLP-1R antagonists identified to date is scarce.
T-0362 (Fig. 11.20), one of the first nonpeptide GLP-1R ligands disclosed, was described as an NAM, which binds to
GLP-1R, blocking GLP-1-induced cAMP production (Tibaduiza et al., 2001).

More recently, the first CNS penetrant and orally bioavailable NAM VU0650991 (Fig. 11.20) has been reported (Nance
et al., 2017). Molecular pharmacology assays showed that VU0650991 did not display probe dependence, affording similar
inhibitory activity for the endogenous agonist GLP-1 (7−36) amide and the synthetic agonist exendin-4. VU0650991
reduced blood insulin levels while increasing blood glucose levels in rats upon oral dosing, validating the role of GLP-1R
antagonism in vivo.

Crystal structures of the human GLP-1R were obtained in complex with the two nonselective NAMs PF-06372222
(Figs. 11.20 and 11.22) and NNC0640 (Figs. 11.20 and 11.23) (Song et al., 2017). These compounds had previously been
optimized as GCGR antagonists and, in fact, NNC0640 was reported as a GCGR NAM and its crystal structure in complex
with the GCGR was resolved (Zhang et al., 2017). The crystal structures have shown that both NAMs bind to the same
pocket located outside helices V–VII proximal to the intracellular half of the GLP-1R, similar to the site occupied by
MK-0893 in the crystal structure of the GCGR (Fig. 11.21).

Regarding the GLP-2R, this receptor subtype is an interesting therapeutic target for intestinal damage. In fact, a peptide
GLP-2R agonist, teduglutide, is under clinical development for short bowel syndrome and Crohn disease. However, unlike
the case of GLP-1R, there is only one report on compounds having allosteric activity toward human GLP-2R. In that work,
a series of thiophene derivatives represented by compound 13 (Fig. 11.20) was characterized as ago-PAMs of different
GLP-2 peptides in recombinant cells expressing the GLP-2R (Yamazaki et al., 2012). These modulators present a sulfanyl
group in their structures; however, it is not clear whether this electrophilic group establishes a covalent interaction with
the receptor, as described for GLP-1R PAMs BEPT and compound 12. Overall, although these results are preliminary they
may be useful for future development of small-molecule AMs of the GLP-2R.

11.4. ALLOSTERIC MODULATORS TARGETING CLASS C GPCRS
Class C GPCRs are characterized by two unique features: the large extracellular and ligand-binding N-terminal domain in
addition to the generic 7TM, and the formation of homo- and heterodimers where orthosteric and allosteric ligands can



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Chapter | 11 23

FIGURE 11.22 Cocrystal structure of GLP-1R in complex with allosteric antagonist PF-06372222 (see Fig. 11.20). (A) PF-06372222 binds to an
allosteric site located outside helices V−VII proximal to the intracellular half of the GLP-1R. (B) The terminal anionic carboxylic acid group of
PF-06372222 in the allosteric binding site is involved in hydrogen bonding interactions with the side chains of residues Ser3526.41 and Asn4067.61. The
amide group of the ligand forms hydrogen bonds with the side chains of Ser3526.41 and Lys3516.40, and the aminopyridine moiety engages a hydrogen
bond with the side chain of Ser3556.44. For hydrophobic interactions, the trifluoromethylpyrazole moiety of the ligand is located in a hydrophobic pocket
created by residues Ile3285.58, Val3315.61, Val3325.62, Leu3355.65, and Phe3476.36. The pyridine ring creates hydrophobic contacts with the alkyl chain of
Lys3516.40, and the phenyl ring establishes hydrophobic interactions with the alkyl chain of Lys3516.40 and Leu4017.56. Finally, the dimethylcyclobutane
group forms hydrophobic interactions with Ser3556.44, Met3976.38, and Leu4016.42 and protrudes into the lipid bilayer (PDB code 5VEW). Credit:
Adapted with permission from J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.

FIGURE 11.23 Cocrystal structure of GLP-1R in complex with allosteric antagonist NNC0640 (see Fig. 11.20). (A) NNC0640 binds to the same
allosteric site than PF-06372222, located outside helices V−VII proximal to the intracellular half of the GLP-1R. (B) The tetrazole group of NNC0640
in the allosteric binding site forms hydrogen bonds with the side chains of Ser3526.41 and Asn4067.61. The amide group of the ligand forms hydrogen
bonds with the side chains of Ser3526.41 and Lys3516.40, and the urea amine moiety of the ligand engages in a hydrogen bond with the side chain of
Ser3556.44. For hydrophobic interactions, the methylsulfonephenyl group of the ligand is located in a hydrophobic pocket defined by residues Phe3245.54,
Ile3285.58, Phe3476.36, Leu3546.43 and the alkyl chain of Lys3516.40. The tetrazolylbenzamide group creates hydrophobic contacts with the alkyl chains
of Arg3486.37 and Lys3516.40, Leu4017.56, and Val4057.60. The phenylcyclohexyl group forms hydrophobic interactions with Ser3556.44, Leu3596.48,
Phe3906.31, Met3976.38, and Leu4016.42 and protrudes into the lipid bilayer (PDB code 5VEX). Credit: Adapted with permission from J. Med. Chem.
2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.

occupy the same receptor or a canonical transmode. Another important characteristic of this class of receptors is that the
orthosteric binding site is not located within the 7TM.

Given the important (patho)physiological roles class C GPCRs play and their therapeutic potential, extensive drug
discovery efforts have been undertaken toward the identification of small-molecule ligands (Brauner-Osborne et al.,
2007). In particular, class C AM drug discovery has been fruitful, with an increasing number of selective PAMs and
NAMs discovered in the last two decades for the calcium-sensing receptor (CaSR), the GPCR class C group 6 subtype A

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24 GPCRs:Structure, Function, and Drug Discovery

receptor (GPRC6AR), the γ-aminobutyric acid type B receptor (GABABR), and the metabotropic glutamate receptors
(mGlu1-8Rs) (Table 11.1) (Engers and Lindsley, 2013; Leach and Gregory, 2017; Urwyler, 2011).

11.4.1 Calcium-sensing receptor (CaSR)
The CaSR is responsible for the maintenance of a stable blood Ca2+ concentration (Brown et al., 1993; Hofer and
Brown, 2003). It controls Ca2+ homeostasis by modulating the secretion of parathyroid hormone in parathyroid glands,
and regulating the reabsorption of Ca2+ in kidney and bone (Brown, 2013). The CaSR is able to activate many different
signaling pathways in a ligand- and tissue-specific manner, which allows it to play crucial roles in Ca2+ homeostasis
and in biological processes unrelated to calcium balance. Thus, the CaSR has become an important target for
hyperparathyroidism, hypocalcaemia, and hypercalcaemia of malignancy but also for diseases such as cancer, Alzheimer
disease, and diabetes mellitus (Ward et al., 2012).

Extracellular Ca2+ is the principal endogenous agonist of the CaSR and therefore mimicking it with selective drug-like
molecules is not obvious. However, the activation effect of Ca2+ and other direct agonists can be modulated and the use
of AMs, either agonists (calcimimetics) or antagonists (calcilytics), has been proposed as treatment for a variety of Ca2+

related diseases.
CaSR PAMs. Alkylbenzylamine NPS R568 and its dechloro-derivative NPS R467 (Fig. 11.24) were the first CaSR

PAMs reported and were obtained in a derivatization program starting from calcium channel blocker fendiline
(Hammerland et al., 1998; Nemeth et al., 1998). Both compounds increased the concentration of cytoplasmic Ca2+ and
inhibited parathyroid hormone secretion in cells. In a small clinical study, oral administration of NPS R568 lowered
parathyroid hormone and Ca2+ plasma levels in postmenopausal women with mild primary hyperparathyroidism
(Silverberg et al., 1997). However, both NPS R568 and NPS R467 caused hypocalcaemia due to a calcitonin-mediated
reduction in Ca2+ efflux from bone and activation of renal CaSRs.

Optimization of NPS R568 and NPS R467 resulted in the identification of cinacalcet (Fig. 11.24) (Nemeth et al.,
2004), with an improved metabolic profile, making this compound the first GPCR AM approved by FDA. Cinacalcet
is prescribed for the treatment of secondary hyperparathyroidism and hypercalcaemia in patients with parathyroid

FIGURE 11.24 Representative allosteric ligands targeting calcium-sensing, group 6 subtype A, and γ-aminobutyric acid type B GPCRs of class C.



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Chapter | 11 25

carcinoma. However, this drug also presents hypocalcaemia side effects and its use is restricted to severely affected
patients.

After the launch of cinacalcet, and with the aim of improving its side effect profile, the research focused on the selective
stimulation of the CaSR signaling in the parathyroid gland without affecting the CaSR in other tissues. These efforts led
to the identification of two novel PAMs, AC-265347 (Ma et al., 2011) and the dibenzylamine (R,R)-calcimimetic B (Fig.
11.24) (Henley et al., 2011). Both compounds decreased parathyroid hormone release in vivo at doses that did not promote
CT secretion, which resulted in nonsignificant reductions in serum Ca2+ levels. This activity profile has been explained
by the ability of these new PAMs to bias signaling toward inositol phosphate accumulation and ERK1/2 phosphorylation,
whereas alkylbenzylamine calcimimetics were biased toward allosteric modulation of Ca2+ immobilization and inositol
phosphate accumulation (Cook et al., 2015). Therefore, these results demonstrated that it was possible to normalize serum
parathyroid hormone and calcium levels by using CaSR PAMs without causing uncontrolled hypocalcaemia. Despite these
PAMs displaying good PK profiles, none of them have advanced to clinical trials.

CaSR NAMs. Although constant and high levels of parathyroid hormone stimulate bone resorption, short and
intermittent increase of serum parathyroid hormone can stimulate new bone formation and therefore improve bone
mineral density in osteoporosis patients. In this context, inhibition of CaSR via orally available NAMs could represent an
interesting alternative to current injected treatment with recombinant parathyroid hormone to achieve a burst of parathyroid
release. The first NAM reported was NPS-2143 (Fig. 11.24) (Gowen et al., 2000), which is the result of an optimization
process of a hit identified in a functional screening from the former SmithKline Beecham compound collection (Marquis
et al., 2009). NPS-2143 is a potent antagonist of the CaSR and promotes parathyroid hormone secretion in cells. When
tested in a rodent model of osteoporosis, the compound produced sustained elevations of plasma parathyroid hormone after
oral administration, which led to increased bone turnover. These results confirmed for the first time that in vivo treatment
with an NAM of the parathyroid cell CaSR may have a net bone forming effect and it represents a potential new treatment
for osteoporosis. However, NPS-2143 treatment did not produce any change in bone mass and density. This effect was
initially attributed to the long-lasting increase (>4 h) in parathyroid hormone levels, and subsequent research was focused
on the development of short-lived NAMs that would produce bursts of parathyroid hormone release and stimulate bone
formation.

Ronacaleret (Balan et al., 2009) and ATF936 (Fig. 11.24) (John et al., 2011) are NAMs that are rapidly metabolized
and display adequate PK and pharmacodynamic profiles. In fact, both compounds were able to trigger transient elevations
of endogenous parathyroid, but neither produced a significant improvement in bone mineral density. Further research
demonstrated that the caveat of these NAMs might be insufficient maximal parathyroid levels, and current work is focused
on the design of ligands that display the required pharmacological properties.

11.4.2 GPCR class C group 6 subtype a receptor (GPCR6AR)
The GPCR6AR, closely related to CaSR, is widely expressed in humans, with high expression levels in brain, skeletal
muscle, testis, leukocytes, liver, and kidney. This receptor is activated by L-amino acids, with a preference for L-Arg,
L-Orn, and L-Lys (Kuang et al., 2005; Wellendorph et al., 2005). These amino acids were found to directly activate or
positively modulate the receptor depending on its signaling pathway. Testosterone and osteocalcin, a hormone secreted
by osteoblasts, have been also reported to stimulate GPCR6AR (Pi et al., 2011). Early studies suggested that GPCR6AR
activation via L-amino acids or osteocalcin promote insulin release from β-islet cells, linking the receptor to metabolic
and endocrinological disorders (Oury et al., 2011). However, subsequent research has resulted in partly contradictory
(Rueda et al., 2016) and development of new specific GPCR6AR ligands, including AMs, which is highly desirable for
the elucidation of the physiological roles of the receptor.

The first GPRC6AR allosteric antagonists have been recently discovered by chemogenomic analysis using known
class A GPCR privileged structures. Thus, 2-arylindole derivatives were identified as AMs, which bind in a hydrophobic
pocket in the 7TM of the mouse GPRC6AR with inhibitory activity (Gloriam et al., 2011). Of these compounds,
only indole compound 14 (Fig. 11.24) was selective for the GPCR6AR, and it was selected as lead scaffold for a
medicinal chemistry program. Systematic modifications in each of the four regions of the pharmacophore afforded several
compounds displaying good activity and selectivity profiles (Johansson et al., 2015). Among them, compound 15 (Fig.
11.24) was characterized as the most potent GPRC6AR NAM, with improved selectivity over related class A and class
C GPCRs. Although further optimization of compound 15 is necessary for future pharmacological studies, this allosteric
antagonist constitutes an important tool to elucidate the physiological and therapeutic relevance of the GPRC6AR.

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11.4.3 γ-Aminobutyric acid type B receptor (GABABR)
The GABABR exists as a heterodimer of two subunits, GABAB1 and GABAB2. Activation of the receptor occurs via
agonist binding to the B1 subunit, which transactivates the B2 subunit, subsequently stimulating the Gi/o proteins (White
et al., 1998). GABABR is involved in the pathophysiology of many diseases such as muscle spasticity, pain, anxiety,
depression, epilepsy, drug addiction, schizophrenia, and neurodegenerative diseases (Froestl, 2010). Currently, there is
only one FDA-approved drug targeting the GABABR, baclofen, a selective agonist prescribed as a muscle relaxant
(Brogden et al., 1974). However, typical activation of the GABABR orthosteric site is associated to different adverse
effects, and baclofen causes dizziness, sedation, and mental confusion. In addition, baclofen also displays poor brain
penetration, short duration of action, rapid tolerance, and a narrow therapeutic margin (Bowery, 2006). Therefore, positive
allosteric modulation of the GABABR constitutes an attractive strategy to retain the benefits of receptor activation while
eliminating the adverse effects and limitations of the classic orthosteric approach (Filip et al., 2015).

The first characterized GABABR PAM was CGP7930 (Fig. 11.24) (Urwyler et al., 2001), which potentiated
GABA-induced signals. CGP7930 increased both potency and efficacy in cultured cells and rat brain membranes.
Later research showed that the compound is an ago-PAM of the GABABR that binds to an allosteric site located
on the heptahelical domain of the GABAB2 subunit, according to experiments with chimeric receptors (Binet et al.,
2004). CGP7930 showed antidepressant and anxiolytic properties in several rodent models, and was also able to reduce
drug-seeking behavior with substances such as alcohol, nicotine, and cocaine.

Optimization of CGP7930 structure by Hoffmann-La Roche led to BHFF (Fig. 11.24) (Malherbe et al., 2008),
a 3-hydroxybenzofuran-2-one derivative that increased the potency and the efficacy with which GABA stimulated
binding of [35S]GTP S. Furthermore, this PAM exhibited anxiolytic-like activity in a mouse model of stress-induced
hyperthermia.

New chemotype GS39783 (Fig. 11.24) and structurally related compounds were also reported as GABABR PAMs.
These ligands, as previously described PAMs, acted via a dual mechanism, enhancing both the affinity and the maximal
efficacy of GABA (Urwyler et al., 2003). GS39783 was a more potent PAM than CGP7930, and showed anxiolyticlike
effects in rodents (Cryan et al., 2004).

NVP-BHF177 (Fig. 11.24) is an optimized derivative of GS39783 where the nitro group has been removed.
Interestingly NVP-BHF177 is devoid of the genotoxicity side effects observed with GS39783. NVP-BHF177 exhibited
antinicotine and antialcohol effects as well as anxiolytic properties in mice (Paterson et al., 2008).

ADX71943 (Fig. 11.24) is a potent and selective GABABR PAM that exhibited consistent and target-related efficacy
in acute and chronic pain tests, while having no effect in studies associated with centrally mediated anxietylike reactivity
(Kalinichev et al., 2014). Further optimization of this compound led to ADX71441 (Fig. 11.24), which had outstanding
preclinical efficacy and tolerability in different rodent models of pain, anxiety, and addiction (Hwa et al., 2014).
ADX71441 is the first GABABR PAM entering clinical development, with phase I studies in addiction expected to start in
2018 (Addextherapeutics).

Overall, since the discovery of first GABABR PAM CGP7930, several new and potent scaffolds acting at the GABABR
allosteric site have been identified with interesting pharmacological activities. However, clinical translation of such effects
is now necessary to confirm their potential as therapeutic agents.

The first reported NAM of the GABABR, CLH304a (Fig. 11.24), was identified by an optimization process starting
from PAM CGP7930 (Chen et al., 2014). This compound decreased GABA-induced inositol phosphate production and
was selective over other GPCR class C members, such as Group I mGluRs. CLH304a negatively modulated GABABR
activity through the heptahelical domain of the GABAB2 subunit (Sun et al., 2016). However, CLH304a and its derivatives
presented several limitations in terms of their PK properties due to presence of an aromatic hydroxyl group and an
electrophilic α,β-unsaturated ketone, which may be responsible for toxicological liabilities.

11.4.4 Metabotropic glutamate receptors (mGlu1-5 and mGlu7Rs)
mGluRs are activated by glutamate, the main neurotransmitter in vertebrates, and modulate synaptic transmission. This
family of receptors includes eight members that are classified in three subgroups based on their sequence homology
and signaling pathway, namely, Group I (mGlu1 and mGlu5Rs), Group II (mGlu2-3Rs), and Group III (mGlu4 and
mGlu6-8Rs) (Niswender and Conn, 2010). mGluRs have a very rich pharmacology with broad therapeutic potential, mostly
in CNS-related pathologies such as pain, anxiety, depression, Parkinson disease, Alzheimer disease, cognition, addition,
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The traditional strategy to develop subtype selective ligands by targeting the glutamate binding site, the orthosteric
approach, has been highly challenging due to high conservation of this binding site across the eight mGlu subtypes.
Moreover, most orthosteric ligands are of peptidic nature and therefore display limited CNS exposure and poor oral
bioavailability. Therefore, allosteric modulation has emerged as an attractive approach for selectively targeting individual
mGluR subtypes (Engers and Lindsley, 2013; Gregory and Conn, 2015; Lindsley et al., 2016; Lutjens and Rocher, 2017).
In the next sections, we summarize key data and therapeutic potential for the most representative mGluR AMs.

mGlu1R. The mGlu1R has been described to play an important role in addiction, anxiety, epilepsy, pain, and psychotic
disorders. In this context, the positive allosteric modulation of the mGlu1R has been scarcely explored. Early PAMs
displayed high potency but suffered from poor CNS penetration and PK properties, limiting their utility in vivo (Knoflach
et al., 2001). Among them, only Ro-07-11401 (Fig. 11.25) presented acceptable CNS exposure and has been used as tool
compound for in vivo preclinical validation of mGlu1R activation in CNS disorders (D'amore et al., 2014). Interestingly,
Ro-07-11401 was found to potentiate the response to glutamate in schizophrenic mGlu1 mutant receptors, suggesting that
mGlu1R PAMs could represent a potential treatment for schizophrenic patients harboring these mutations (Cho et al.,
2014b).

In the search for in vivo tool compounds with improved properties, new PAMs were obtained by molecular switches
starting from an mGlu4R PAM (Garcia-Barrantes et al., 2015a). Among them, VU0486321 (Fig. 11.25) was characterized
as a highly potent, selective, and CNS-penetrant mGlu1R PAM. Moreover, VU0486321 does not induce epileptiform
activity and seizures in rats at drug concentrations far above its EC50, suggesting that the adverse effect liability of Group
I mGluR nonselective agonists is solely mediated by agonism at mGlu5R (Garcia-Barrantes et al., 2015b).

The development of mGlu1R NAMs has been an active area of research and structurally diverse ligands have been
described. CPCCOEt (Fig. 11.25) was the first mGlu1R NAM reported. The compound is a noncompetitive inhibitor with
moderate potency at the mGlu1R and good selectivity profile over the other mGluR subtypes, being a useful tool for
in vitro studies (Annoura et al., 1996; Hermans et al., 1998). More potent NAMs were subsequently reported (Owen,
2011; Urwyler, 2011). Among them, JNJ-16259685, FTIDC, and A-841720 (Fig. 11.25) displayed potencies in the low
nanomolar range. These molecules have been used to evaluate the role of mGlu1R in several diseases with mixed results.
Thus, although JNJ-16259685 and FTIDC showed anxiolytic-like activity in the lick suppression test, they were found to

FIGURE 11.25 Representative allosteric ligands targeting metabotropic glutamate receptors 1–2 GPCRs of class C.

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be inactive in the elevated plus maze (Satow et al., 2008; Steckler et al., 2005). Results in models of chronic pain were
more coherent; however, different side effects were found. Hence, A-841720 was active against Freund's adjuvant induced
inflammatory pain and reduced mechanical allodynia in sciatic nerve constriction and spinal nerve ligation models of
neuropathic pain, but significant motor side effects occurred at analgesic doses (El-Kouhen et al., 2006).

FITM (Fig. 11.25) is an mGlu1R NAM with excellent selectivity against the mGlu5R that has been cocrystallized bound
to the 7TM of the mGlu1R (Fig. 11.26) (Wu et al., 2014). FITM binds within the 7TM adjacent to the extracellular side.
Interestingly, study of the ligand-receptor interactions by docking of FITM analogs into the crystal structure predicted the
involvement of amino acid Leu757 in binding interactions, a residue that has been reported as critical for NAM activity
(Cho et al., 2014a).

mGlu2-3Rs. Most of the research on potentiation of Group II receptors has been focused on the development of
selective mGlu2R PAMs, since it was established that selective activation of mGlu2R is directly implicated in driving
the antipsychotic efficacy of orthosteric mGlu2/3R agonists. LY487379 (Johnson et al., 2003) and BINA (Galici et al.,
2006) (Fig. 11.25) are the prototypical mGlu2R PAMs. They have been used as preclinical tools and have recapitulated
much of the preclinical pharmacology of mGlu2/3R agonists, sparking the interest for selective PAMs (Ellaithy et al.,
2015). Therefore, in recent years, a number of mGlu2R PAMs has been reported. TASP0433864 (Fig. 11.25) is a
selective mGlu2R PAM, which has shown antipsychotic effects. It was able to modulate the N-methyl-D-aspartate (NMDA)
signaling pathway involved in pathophysiology of schizophrenia and to inhibit induced hyperlocomotion in rodents
(Hiyoshi et al., 2014). Janssen has contributed with different chemotypes to the mGlu2R PAM arena. JNJ-42153605 (Fig.
11.25) (Cid et al., 2012) is a highly optimized PAM with central activity in vivo by suppressing REM sleep stage in
the rat sleep-wake electroencephalogram paradigm, a phenomenon previously demonstrated to be mediated by mGlu2R.
JNJ-42153605 represented the first example of efficacy of an mGlu2R PAM in the conditioned avoidance response test, a
well-established antipsychotic model, inhibiting avoidance without impairing the escape response (Megens et al., 2014).
Interestingly, its 11C-labeled derivative compound 16 (Fig. 11.25) was used in PET studies, which confirmed its specific
and reversible binding to mGlu2R in vivo (Andrés et al., 2012).

JNJ-40411813 (ADX71149) (Fig. 11.25) is a pyridone-based CNS penetrant mGlu2R PAM with an attractive PK
profile, which was active in multiple antipsychotic animal models. JNJ-40411813 entered in an exploratory phase IIa
clinical study in patients with schizophrenia (Cid et al., 2014). It met the primary objectives of safety and tolerability and
demonstrated positive effect as adjunctive treatment to patients with residual negative symptoms. JNJ-40411813 was also

FIGURE 11.26 Cocrystal structure of mGlu1R in complex with NAM FITM (see Fig. 11.25). (A) FITM binds inside the 7TM adjacent to the
extracellular side. The allosteric FITM binding site of the mGlu1R partially overlaps with the orthosteric binding site for the class A GPCRs, but it is
distinct from its own orthosteric glutamate binding site at the N-terminal extracellular domain. The allosteric FITM binding site is formed by residues
from the ECL2, helices II, III, and V−VII. (B) The pyrimidineamine group of the FITM in the allosteric FITM binding site forms a hydrogen bond
with the side chain of Thr8157.38. For hydrophobic interactions, the p-fluorophenyl group of the ligand is situated deep into a pocket defined by residues
Ile7645.51, Thr7946.44, Ile7976.47, Trp7986.48, and Phe8016.51. The central thiazole linker of the ligand creates hydrophobic interactions with residues
Val6643.32, Pro7565.43, and Phe8016.51. The 5-N-isopropylpyrimidine group of the ligand establishes hydrophobic interactions with residues Leu6482.60,
Gln6603.28, Val6643.32, Thr748ECL2, Tyr8056.55, Thr8157.38 and the alkyl chain of Arg6613.29 (PDB code 4OR2). Credit: Reprinted with permission from
J. Med. Chem. 2019, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.



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evaluated in a phase II proof-of-concept study in patients with major depressive disorder with significant anxiety
symptoms. Although efficacy signals were met on some measures, the primary outcome measure was not significant, and
the development of the compound in anxious depression was discontinued.

A different mGlu2R PAM chemotype was developed by AstraZeneca. AZD8529 (Fig. 11.25) is an isoindolinone
derivative that advanced into a phase IIa proof-of-concept study in schizophrenic patients. However, although it had
demonstrated activity in several preclinical antipsychotic and anxiolytic models, the compound was discontinued due to
lack of efficacy.

Inhibition of Group II receptors has been linked to depression and cognition. Research in this field yielded a number
of mixed mGlu2/3R NAMs, which have proven to be valuable pharmacological tools. Thus, benzodiazepine derivatives
RO4491533 and RO4432717 (Fig. 11.27) have shown efficacy in rodent models of depression (Goeldner et al., 2013;
Woltering et al., 2010). Decoglurant (RO4995819, Fig. 11.27), a nonselective NAM, advanced to clinical phase II trial in
patients with major depressive disorder.

A new pyrazolo[1,5-a]quinazolin-5-one chemotype was recently proposed for the development of mGlu2/3R NAMs
(Mayer et al., 2014). The initial hit was identified from an FRET assay and it was optimized to compound 17 (Fig. 11.27),
a potent NAM with oral bioavailability and CNS exposure that improved spatial working memory in a dose-dependent
manner in mice challenged with scopolamine (Schann et al., 2013).

The search for selective mGlu2R NAMs has been elusive, although they are highly necessary to understand the
physiological roles of the individual subtypes of Group II. MRK-8-29 (Fig. 11.27) is a potent and selective mGlu2R
NAM discovered by Merck and its use contributed to establish the specific role of mGlu2R in long-term depression as
a major regulator of prefrontal cortex function and cognition (Walker et al., 2015). Scaffold-hopping around MRK-8-29
considering its similarity with a reported M1R PAM resulted in a new series of
4-oxo-1,4-dihydroquinoline-3-carboxamides represented by VU6001192 (Fig. 11.27), a potent and highly selective
mGlu2R NAM with an interesting PK profile (Felts et al., 2015).

Selective mGlu3R NAMs were developed at Vanderbilt University starting from an mGlu5R PAM, which displayed
weak mGlu3R NAM activity and selectivity over mGlu2R. Optimization of the initial hit afforded VU0469942 (ML337,
Fig. 11.27), selective against mGlu2 and mGlu5Rs, but displaying suboptimal CNS penetration and high protein binding

FIGURE 11.27 Representative allosteric ligands targeting metabotropic glutamate receptors 2–4 GPCRs of class C.

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in rats. However, VU0469942 could be used as an in vivo tool in mice to demonstrate the utility of a selective mGlu3R
NAM in a long-term depression model (Walker et al., 2015). Removal of the 1,2-diarylethyne fragment present in
VU0469942 and an extensive SAR exploration led to VU0650786 (Fig. 11.27). In this case, the introduction of a chlorine
atom in the molecule was key for good PK properties and VU0650786 displayed efficacy in anxiolytic and antidepressant
models.

mGlu4R. Among Group III mGluRs, mGlu4R research is the most advanced due to the implication of this receptor
subtype in Parkinson disease, both for symptomatic motor disturbances and for neuroprotection of dopaminergic neurons
(Bennouar et al., 2013; Valenti et al., 2005). In this context, research has been focused on the development of mGlu4R
PAMs due to the difficulty in identifying selective orthosteric ligands. (-)-PHCCC (Fig. 11.27) was the first mGlu4R PAM
disclosed. Although it is a relatively weak and nonselective PAM with a poor PK profile, (-)-PHCCC was able to relieve
motor symptoms in an animal Parkinson disease model after intracerebroventricular injection (Maj et al., 2003). Since then,
other mGlu4R PAMs with improved drug-like properties have been reported and contributed to validate the potential of
this approach for the symptomatic treatment of Parkinson disease. VU0364770 (ML292, Fig. 11.27) was active in several
Parkinson disease models when administered alone or in combination with inactive doses of L-DOPA (Jones et al., 2012).
mGlu4R PAMs Lu AF211934 (Bennouar et al., 2013) and ADX88178 (Fig. 11.27) (Le Poul et al., 2012) showed activity
in the 6-OHDA model in combination with L-DOPA, offering additional cotreatment options for Parkinson disease and
reducing the dosage of L-DOPA. Foliglurax (PXT002331, Fig. 11.27) is the first mGlu4R PAM that successfully completed
phase I trials, initiating a phase II study with Parkinson disease patients in 2017.

mGlu5R. Allosteric modulation of mGlu5R affords opportunities for the treatment of diverse CNS disorders and has
been extensively investigated, yielding a wealth of pharmacological tools as well as compounds that have entered clinical
trials. Below we cover key examples of mGlu5R AMs and recent advances in the field.

Multiple mGlu5R PAMs have been reported; however, activation of mGlu5R via PAMs has been limited due to severe
side effects found preclinically (Rook et al., 2013). These side effects have been associated to the level of intrinsic agonist
activity shown by the ligand, and therefore, compounds with ago-PAM activity should be avoided. Particularly, the adverse
effect liabilities of mGlu5R PAMs have been linked to NMDA receptor activation, and development of signal-biased
PAMs could be an alternative to circumvent these side effects. In this context, VU0409551 (Fig. 11.28) is a potent,

FIGURE 11.28 Representative allosteric ligands targeting metabotropic glutamate receptors 5–7 GPCRs of class C.



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selective, and orally bioavailable mGlu5R PAM that displayed robust antipsychotic and cognition enhancing efficacy,
without modulating NMDA receptor. Based on these results, VU0409551 was selected for further development, and
represents the first mGlu5R PAM preclinical candidate for schizophrenia, suggesting that modulation of NMDA receptor
activity may not be critical for in vivo efficacy (Conde-Ceide et al., 2015; Rook et al., 2015).

Compound 18 (Fig. 11.28) is a novel and safe PAM with activity in a preclinical model of cognition, wherein adverse
effect liability has been avoided by reducing maximal glutamate fold-shift, that is, cooperativity (Ellard et al., 2015).
Therefore, these examples demonstrated that target-related side effects can be overcome by different strategies.

Alkyne derivatives MPEP (Gasparini et al., 1999) and MTEP (Fig. 11.28) (Cosford et al., 2003) are prototypical
mGlu5R NAMs. These compounds exhibited high potency, selectivity, and brain penetrance and have helped to validate
preclinically therapeutic indications such as Parkinson disease (Gasparini et al., 2013). Since the discovery of MPEP and
MTEP, diverse NAM chemotypes have been developed and have shown preclinical efficacy for the treatment of disorders
associated with glutamatergic neurotransmission hyperactivity and/or dysfunction, with several of them entering clinical
trials (Li et al., 2013). Thus, mavoglurant (AFQ056) (Berg et al., 2011) and dipraglurant (Fig. 11.28) (Tison et al., 2016)
are mGlu5R NAMs that have shown efficacy in reducing levodopa-induced dyskinesia in Parkinson disease and have
progressed to phase II trials. Additionally, mavoglurant (Fig. 11.28) is currently in phase II studies for fragile X syndrome,
cocaine-related disorder, and alcohol drinking. More recently, basimglurant has reached clinical development for major
depressive disorder (Fig. 11.28) (Lindemann et al., 2015).

However, some mGlu5R NAMs have demonstrated adverse effects, including psychotomimetic-like effects in animals
and psychosis in humans, revealing a narrow therapeutic window (Emmitte, 2011). In this context, the identification of
mGlu5R partial NAMs represented a new mode of pharmacology that could limit potential adverse events associated to
complete blockade of receptor signaling with full NAMs. These new ligands fully occupy the allosteric site but only
partially block signaling, allowing to vary degrees of antagonist activity (Rodriguez et al., 2005). M-5MPEP, Br-5MPEPy,
and VU0477573 (Fig. 11.28) are partial mGlu5R NAMs with activity in selected preclinical models. Thus, M-5MPEP and
Br-5MPEPy exhibited antidepressan-tlike and anxiolytic-like activity, corresponding to in vivo mGlu5R occupancy, and
without enhancing hyperlocomotion, as is the case with MTEP (Gould et al., 2016). VU0477573, with an excellent PK
profile, displayed efficacy in rodent models of anxiolytic activity (Nickols et al., 2016). Therefore, the efficacy of partial
mGlu5R NAMs is similar to that observed with full NAMs but with a broader therapeutic window.

Regarding the binding site of these NAMs to mGlu5R, two crystal structures have been published. The cocrystal
structure of binary mGlu5R-mavoglurant complex has been recently reported showing that mavoglurant binds deep within
the 7TM (Figs. 11.28 and 11.29) (Dore et al., 2014). When compared to the crystal structure of mGlu1R in complex with

FIGURE 11.29 Cocrystal structure of mGlu5R in complex with NAM mavoglurant (see Fig. 11.28). (A) Mavoglurant is located in the extracellular
side, deep within the 7TM that is wrapped by residues from helices II, III, and V−VII. (B) The carbamate carbonyl of the mavoglurant in the allosteric
binding site forms a hydrogen bond with the side chain of Asn7475.47, and the hydroxyl group of the ligand forms hydrogen bonds with the side chains
of Ser8057.35 and Ser8097.39 as well as the backbone carbonyl of Ser8057.35. For hydrophobic interactions, the bicyclic ring of the ligand is located in the
main hydrophobic pocket created by residues Ile6513.36, Pro6553.40, Leu7445.44, Trp7856.50, Phe7886.53, Met8027.32, and Val8067.36. The 3-methylphenyl
ring fills a hydrophobic pocket created by residues Ile6252.46, Pro6553.40, Tyr6593.44, and Ala8107.40. The alkyne linker establishes contacts with residues
Pro6553.40, Tyr6593.44, Ser8097.39, and Val8067.36. The methoxy terminal portion of the carbamate creates hydrophobic contacts with residues Ile6513.36,
Pro7435.43, and Leu7445.44 (PDB code 4OO9). Credit: Reprinted with permission from J. Med. Chem. 201-, https://doi.org/10.1021/acs.jmedchem.
7b01844. Copyright (201-) American Chemical Society.

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FITM, the two structures are in agreement with a number of molecular features conserved across subtypes. Interestingly,
FITM is found higher in the mGlu1R allosteric site, highlighting the potential for different allosteric binding modes across
the same class of receptors. HTL14242 has also been cocrystalized with the mGlu5R (Figs. 11.28 and 11.30) (Christopher
et al., 2015). Despite lacking the alkyne function, the binding mode of HTL14242 is very similar to that one identified for
mavoglurant.

mGlu7R. Group III mGlu7R is widely distributed in the CNS, where it is thought to play a critical role in modulating
normal neuronal function and synaptic transmission (Dalezios et al., 2002). Thus, mGlu7R has been proposed as
therapeutic target for various CNS disorders, including Parkinson disease, autism, depression, bipolar disorder, and
schizophrenia (O'connor et al., 2010; Peterlik et al., 2016). The lack of small-molecule tool compounds with appropriate
selectivity and drug-like properties has hampered the progression of drug discovery across mGlu7R. In this context,
AMs have emerged as valuable tools with good potency, selectivity and physicochemical properties to study the receptor
therapeutic potential.

AMN082 was the first selective ligand reported for mGlu7R (Fig. 11.28) (Mitsukawa et al., 2005). This compound
acted as an allosteric agonist, which directly activated receptor signaling by binding to an allosteric site in the
transmembrane domain without affecting the affinity of orthosteric binders. AMN082 is orally active and brain penetrant,
and has been a valuable in vivo tool to unveil the role of the mGlu7R, mainly in stress-related CNS disorders. However,
the compound presented a rapid metabolism in rat liver microsomes toward a metabolite that displayed significant binding
affinity for the dopamine, serotonin, and norepinephrine transporters. Therefore, the in vivo activity of AMN082 may not
be purely mediated by mGlu7R, and other mechanisms cannot be discarded (Sukoff Rizzo et al., 2011).

Although different mGlu7R allosteric agonists have been described since the discovery of AMN082, the number of
pure PAMs has been scarce. VU0155094 and VU0422288 (Fig. 11.28) were disclosed as pan-Group III PAMs and, despite
their lack of selectivity, have represented useful tool compounds for the validation of the mGlu7R in different pathological
processes (Jalan-Sakrikar et al., 2014). VU0155094 was discovered in an HTS while looking for mGlu8R PAMs, and
potentiated mGluR subtypes 4, 6, 7, and 8, but not the other mGluRs. VU0422288, identified within an mGlu4R PAM
lead optimization program, is the most potent pan-Group III AM reported to date. VU0422288 and VU0155094 have
been studied by electrophysiological experiments in the hippocampus of adult rats, and data proved that both compounds
produced relevant and robust effects modulating synaptic transmission via a presynaptic mechanism, which confirmed that
mGlu7R can be modulated by a PAM (Jalan-Sakrikar et al., 2014).

A novel chemotype based on a pyrazolo[1,5-a]pyrimidine scaffold has yielded a new family of mGlu7R PAMs,
wherein VU6005649 (Fig. 11.28) stood out as an mGlu7R-preferring PAM with no activity at mGlu1-6Rs and four-fold
selectivity versus mGlu8R (Abe et al., 2017). VU6005649 was found to be CNS penetrant and showed modest but
significant efficacy in a mouse contextual fear conditioning model, exhibiting procognitive effects on associative

FIGURE 11.30 Cocrystal structure of mGlu5R in complex with NAM HTL14242 (see Fig. 11.28). (A) HTL14242 occupies an extracellular allosteric
pocket similar to that of mavoglurant in the mGlu5R receptor, lined by residues from helices II, III, and V−VII. (B) The pyridine nitrogen of HTL-14242
in the allosteric HTL-14242 binding site engages a hydrogen bond with the side chain of Ser8097.39, and the 5-cyano of the ligand is involved in a
water-mediated hydrogen bond with the backbone carbonyl oxygen of Val7405.40. For hydrophobic interactions, the pyridine ring of the ligand fits into
a hydrophobic pocket defined by residues Ile6252.46, Tyr6593.44, Ser8097.39, and Ala8107.40. The pyrimidine linker is situated in a hydrophobic pocket
created by residues Ser6543.39, Pro6553.40, and Val8067.36. The phenyl ring creates hydrophobic contacts with residues Trp7856.50 and Phe7886.53. The
cyano substituent establishes contacts with residues Leu7445.44, Phe7886.53, and Met8027.32. (PDB code 5CGD). Credit: Adapted with permission from J.
Med. Chem. 201-, https://doi.org/10.1021/acs.jmedchem.7b01844. Copyright (201-) American Chemical Society.



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learning. Although the compound also induced some level of sedation, which could contribute in diminishing mice
capacity for associative learning, this PAM constituted the first example of mGlu7/8R-mediated efficacy in this cognition
model.

Regarding mGlu7R antagonism, several allosteric antagonists and NAMs have been reported. MMPIP (Fig. 11.28)
is an allosteric antagonist selective for mGlu7R, orally bioavailable and CNS penetrant that has been used to study the
pharmacology of mGlu7R in rodents (Suzuki et al., 2007). The first potent and selective mGlu7R NAM, ADX71743 (Fig.
11.28), was reported by Addex Therapeutics (Kalinichev et al., 2013). It was developed from an HTS hit and its NAM
activity was confirmed in Schild plot experiments, where ADX71743 induced a concentration-dependent rightward shift
of reference agonist L-AP4 concentration response curve together with a decrease of its efficacy. This compound showed
good plasma exposures in rodents and was evaluated in several rodent behavioral models of psychosis, anxiety, and
depression. Specifically, ADX71743 was active in the marble burying and elevated plus maze tests in mice, suggesting an
anxiolytic-like effect mediated by mGlu7R (Klar et al., 2015). Last research published on mGlu7R NAMs corresponds to
the identification of VU6010608 (Fig. 11.28). This compound is the result of a challenging optimization program around
a hit identified from an HTS campaign. VU6010608 increased mGlu7R affinity and good selectivity over the other mGluR
subtypes, but it suffered from a poor PK profile. Nonetheless, this NAM represents a good tool compound for in vitro
studies thanks to its robust antagonist effect (Reed et al., 2017).

The growing pool of mGlu7R AMs available to date will spur further research around mGlu7R that should afford more
potent and selective ligands suitable for in vivo preclinical validation of the receptor.

11.5. ALLOSTERIC MODULATORS TARGETING CLASS F GPCRS
Recent research has shown that class F GPCRs, traditionally considered undruggable receptors, also possess allosteric
binding sites for exogenous ligands. This class includes the frizzled (FZD) and smoothened (SMO) receptors.

11.5.1 Frizzled receptors (FZD4R)
Human FZDRs comprise 10 members (FZD1-10Rs) that respond to the extracellular ligands of the Wingless/Integrated
(Wnt) family of lipo-glycoproteins (Dijksterhuis et al., 2014), and are implicated in many aspects of embryonic
development and in homeostasis of adult tissues (Clevers and Nusse, 2012). In this context, the FZD4R is of particular
interest since it regulates stemness during cellular development and in adult life, and misregulation of the receptor activity
is involved in cancer stem cell genesis in many types of malignancies and tumor proliferation.

In 2015, the first small molecules able to target the FZD4R and to inhibit Wnt signaling were reported (Generoso
et al., 2015). These compounds had been designed to behave as pharmacological chaperones for a misfolded mutant of
FZD4R, but they were indeed Wnt–β-catenin inhibitors. Among them, FzM1 (Fig. 11.31) stood out as an NAM that binds
to an allosteric site located near the ICL3 of the receptor and induces conformational changes that ultimately inhibit the
Wnt-β-catenin cascade. In that manner, FzM1 was able to affect the growth and differentiation state of U87MG and

FIGURE 11.31 Representative allosteric ligands targeting GPCRs of class F.

Allosteric modulators targeting GPCRs



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34 GPCRs:Structure, Function, and Drug Discovery

Caco-2 cells, two cells lines known to rely on an active Wnt pathway to maintain their undifferentiated proliferative state.
These results are of great importance, as they suggest the applicability of these compounds as antitumor drugs.

Further research conducted with this FZD4R NAM led to the identification of the first allosteric agonist of the FZD4R
(Riccio et al., 2018). Thus, the replacement of the thiophene ring present in FzM1 by a carboxylic acid group induced a

FIGURE 11.9 Cocrystal structure of GPR40 in a ternary complex with ago-PAMs MK-8666 and AP8 (see Fig. 11.7). (A) AP8 binds to a well-defined
lipid-facing pocket formed by helices II−V and ICL2, which is outside the intracellular halves of the 7TM, and this site is completely distinct from
that of MK-8666 located in the extracellular halves of the 7TM. (B) In the allosteric binding site, the carboxylate group of AP8 accepts three
hydrogen bonds from the side chains of Tyr442.42, Ser1234.42, and Tyr114ICL2. The methyl and cyclopropyl groups engage in hydrophobic and aromatic
interactions with residues Leu1063.52, Tyr114ICL2, Phe117ICL2, and Tyr1224.41. The piperidine-chroman group makes hydrophobic contacts with residues
Ala993.45, Ala1023.48, Val1264.45, and Ile1975.53. The terminal trifluoromethoxyphenyl group fits into a hydrophobic pocket formed by residues Ile1304.49,
Leu1334.52, Val1344.53, Leu1905.46, and Leu1935.47 (PDB code 5TZR). Credit: Adapted with permission from J. Med. Chem. 2019, https://doi.org/10.
1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.

FIGURE 11.32 Cocrystal structure of SMOR in complex with NAM vismodegib (see Fig. 11.31). (A) Vismodegib is bound to the 7TM adjacent to the
extracellular side. The vismodegib binding site is defined by residues from the linker domain, ECL2, ECL3, and helices I, II, and V−VII. (B) The pyridine
nitrogen and the amide NH of vismodegib in the allosteric vismodegib binding site are involved in a hydrogen bond with the side chain of Tyr394ECL2 and
Asp384ECL2, respectively. For hydrophobic interactions, the pyridinechlorophenyl moiety of the ligand is deeply buried, forming numerous hydrophobic
interactions with residues Met2301.32, Trp2812.58, Val386ECL2, Ser387ECL2, Ile389ECL2, Phe391ECL2, Tyr394ECL2, Arg4005.43, His4706.51, Glu518ECL3,
Leu5227.42, and Met5257.45. The methylsulfonephenyl moiety of the ligand creates hydrophobic contacts with residues Leu221 in the linker domain,
Tyr394ECL2, Gln4776.58, Trp4806.61, Phe4846.65, and Pro513ECL3 (PDB code 5L7I). Credit: Adapted with permission from J. Med. Chem. 2019, https://
doi.org/10.1021/acs.jmedchem.7b01844. Copyright (2019) American Chemical Society.



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molecular switch that transformed the molecule into an activator of Wnt signaling, the allosteric agonist FzM1.8 (Fig.
11.31). This ligand potentiated the β-catenin pathway in the absence of any Wnt ligand. In particular, FzM1.8 promoted
recruitment of heterotrimeric G proteins, and biased Wnt signaling toward a noncanonical route that involves
phosphatidylinositol-3-kinase. The treatment of a colon cancer line with FzM1.8 left unaltered the expression of colon
stem cell markers, indicating that FZD4R/phosphatidylinositol-3-kinase pathway stimulates proliferation and preserving
the stemness of the cells.

11.5.2 Smoothened receptor (SMOR)
The SMOR mediates signal transduction in the Hedgehog pathway, which is critical for development, differentiation,
growth, and cell migration (Arensdorf et al., 2016). The Hedgehog pathway plays key roles in a wide type of cancers (Wu
et al., 2017) and therefore, small molecules able to inhibit SMOR are under intensive development as antitumor agents.
In fact, several lead compounds are currently in clinical trials, including the SMOR NAM vismodegib (GDC-0449) (Fig.
11.31) (Robarge et al., 2009), which has been the first agent targeting the Hedgehog signaling pathway approved by the
FDA. Vismodegib was the result of an optimization program looking for improved potency, PK, and drug-like properties
by explorations around the amide moiety. This NAM produced complete tumor regression in a medulloblastoma allograft
mouse model that is wholly dependent on the Hedgehog pathway for growth. Vismodegib is prescribed for the treatment
of basal cell carcinoma.

Recently, the cocrystal structure of vismodegib bound to SMOR has been solved, revealing two allosteric binding sites
(Figs. 11.31 and 11.32) (Byrne et al., 2016). This structure clearly showed that vismodegib is bound to the 7TM adjacent
to the extracellular side. The second allosteric site, located in the extracellular domain, was occupied by cholesterol, and
appears to promote the binding of the natural agonist, the Hedgehog protein.

SANT-1 and SANT-2 (Fig. 11.31) are other SMOR antagonists whose mode of action has been characterized in
terms of allosteric modulation (Rominger et al., 2009). Both compounds exhibited partial competition of the radioligand
[3H]SAG-1.3 binding, but fully inhibited the activation of the Hedgehog pathway induced by agonist SAG-1.5 in a
β-lactamase reporter gene cellular assay. The crystal structure of the human SMOR in complex with SANT-1 has
been solved and revealed that the compound is bound within the 7TM (Wang et al., 2014), providing also a structural
explanation for the allosteric modulation exerted by SANT-1. Vismodegib and SANT-1 bind the same site of the SMOR,
although SANT-1 binds more deeply in the 7TM.

11.6. CONCLUSIONS
Studies in the past decade highlight the complexity for allosteric GPCR modulation but also call to attention the high
degree of specificity that can be achieved if probe dependence and signaling bias are therapeutically desired outcomes
based upon biological understanding. Therefore, utilizing allosteric modulation to precisely alter the function of the
receptors may enable the therapeutic targeting of previously intractable GPCRs or provide safer therapeutics for currently
targeted receptors. At present, GPCR allosteric modulation is a fundamental approach in drug discovery, and exploitation
of this paradigm has delivered FDA-approved therapies, multiple drug-candidates in the pipeline, and promises to provide
more precise and safer small-molecule therapeutics in the future.

A key challenge in the discovery of AMs targeting GPCRs includes the identification of nonconserved allosteric sites
where AMs can bind, as a prerequisite for discovering potent and selective drugs. The recent great progress in GPCR
structural biology using X-ray crystallography has solved distinct GPCRs in complex with their small-molecule AMs
across all human GPCR classes: A, B, C, and F. Knowledge of the detailed receptor−modulator interactions at the allosteric
sites pave the way for virtual screening to identify novel allosteric leads or structure-based drug design to improve the
binding affinity of existing AMs. This breakthrough is expected to contribute to the discovery of GPCR allosteric drugs
with an improved therapeutic action.

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