Fncel 16 812359

REVIEW
published: 13 April 2022

doi: 10.3389/fncel.2022.812359
Orexin Signaling: A Complex,

Multifaceted Process
Natasha C. Dale 1,2,3 , Daniel Hoyer 4,5,6 , Laura H. Jacobson 4,5,7 , Kevin D. G. Pfleger 1,2,3,8*
and Elizabeth K. M. Johnstone 1,2,3,9*
1
Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research and Centre for Medical Research,
The University of Western Australia, Nedlands, WA, Australia, 2 Australian Research Council Centre for Personalised
Therapeutics Technologies, Melbourne, VIC, Australia, 3 Australian Research Council Centre for Personalised Therapeutics
Technologies, Perth, WA, Australia, 4 Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia,
5
Department of Biochemistry and Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health
Sciences, The University of Melbourne, Parkville, VIC, Australia, 6 Department of Molecular Medicine, The Scripps Research
Institute, La Jolla, CA, United States, 7 Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental
Health, The University of Melbourne, Parkville, VIC, Australia, 8 Dimerix Limited, Nedlands, WA, Australia, 9 School
of Biomedical Sciences, The University of Western Australia, Nedlands, WA, Australia
The orexin system comprises two G protein-coupled receptors, OX1 and OX2 receptors
(OX1 R and OX2 R, respectively), along with two endogenous agonists cleaved from
a common precursor (prepro-orexin), orexin-A (OX-A) and orexin-B (OX-B). For the
receptors, a complex array of signaling behaviors has been reported. In particular, it
becomes obvious that orexin receptor coupling is very diverse and can be tissue-,
cell- and context-dependent. Here, the early signal transduction interactions of the
orexin receptors will be discussed in depth, with particular emphasis on the direct G
Edited by:
Terence Hébert, protein interactions of each receptor. In doing so, it is evident that ligands, additional
McGill University, Canada receptor-protein interactions and cellular environment all play important roles in the
Reviewed by: G protein coupling profiles of the orexin receptors. This has potential implications for
Karen J. Gregory,
Monash University, Australia
our understanding of the orexin system’s function in vivo in both central and peripheral
Juliana Dallagnol, environments, as well as the development of novel agonists, antagonists and possibly
McGill University, Canada
allosteric modulators targeting the orexin system.
*Correspondence:
Kevin D. G. Pfleger Keywords: orexin, orexin 1 receptor, orexin 2 receptor, signaling, G protein, GPCR (G protein coupled receptor),
[email protected] arrestin
Elizabeth K. M. Johnstone
[email protected]
INTRODUCTION
Specialty section:
This article was submitted to The orexin system was discovered in 1998 by two independent groups, De Lecea et al. (1998) and
Cellular Neurophysiology, Sakurai et al. (1998). De Lecea et al. (1998) described an mRNA located within the hypothalamus
a section of the journal that encodes for a peptide named prepro-hypocretin/orexin. Prepro-hypocretin/orexin, when
Frontiers in Cellular Neuroscience
cleaved, produces two highly conserved peptides termed hypocretin 1 and hypocretin 2. These
Received: 10 November 2021 peptides are also known as orexin-A (OX-A) and orexin-B (OX-B) respectively, and will be
Accepted: 07 March 2022
referred to using this terminology throughout this review. De Lecea et al. (1998) went on to show
Published: 13 April 2022
that Prepro-hypocretin/orexin was present specifically and exclusively in a limited population of
Citation: cells in the dorsal and lateral hypothalamus (3,000–7,000 in rodents, up to 70,000 in humans),
Dale NC, Hoyer D, Jacobson LH,
with immunoelectron microscopy studies indicating prepro-hypocretin/orexin associates with
Pfleger KDG and Johnstone EKM
(2022) Orexin Signaling: A Complex,
presynaptic vesicles. From these results, it was proposed that the OX-A and OX-B peptides
Multifaceted Process. were potential neurotransmitters, with their precursor’s localization in the lateral hypothalamus
Front. Cell. Neurosci. 16:812359. suggesting a role in energy homeostasis. Sakurai et al. (1998) also identified the presence of
doi: 10.3389/fncel.2022.812359 prepro-hypocretin/orexin mRNA within hypothalamic areas implicated in feeding behavior and
Frontiers in Cellular Neuroscience | www.frontiersin.org 1 April 2022 | Volume 16 | Article 812359

Dale et al. Orexin Signaling
demonstrated that administration of OX-A or OX-B into the discovery that the orexin system is a critical regulator of sleep-
lateral ventricles of rats resulted in increased food intake. wake cycles, with the loss of orexin neurons (Thannickal et al.,
However, Sakurai et al. (1998) first utilized reporter cell lines to 2000, 2009; Hara et al., 2001; Honda et al., 2009) and the resulting
de-orphanize G protein-coupled receptors (GPCRs), identifying loss of endogenous orexin (Nishino et al., 2000, 2001; Peyron
the OX-A and OX-B peptides and the cognate OX1 and OX2 et al., 2000; Ripley et al., 2001; Kanbayashi et al., 2002; Mignot
receptors (OX1 R and OX2 R, respectively) (Figure 1). This was et al., 2002) responsible for the rapid cycling between sleep
part of a larger GPCR de-orphanization program (which included and wake states, both during day and night, characteristic of
the orphan HFGAN72 sequence that corresponds to OX1 R, human narcolepsy. The underlying cause of orexin neuron loss
allowing identification of two related expressed sequence tags that in human narcolepsy is still unknown, however autoimmune-
correspond to OX2 R), conducted at the time in collaboration with related degenerative processes may play a role (Thannickal et al.,
Smith Kline Beecham, before their merger with Glaxo. Hence the 2000; Cvetkovic-Lopes et al., 2010; Lecendreux et al., 2017;
two groups discovered the orexin system by two complementary Ramberger et al., 2017; Latorre et al., 2018; Luo et al., 2018;
approaches, identification of either the neuropeptides or the Bassetti et al., 2019; Lippert et al., 2019; Mahoney et al., 2019;
receptors first and then their respective receptor or neuropeptide Pedersen et al., 2019).
counterparts in turn. Additionally, the role of the orexin system in sleep-wake
The discovery of OX-A and OX-B and the subsequent de- regulation was further validated when narcoleptic dogs were
orphanization of OX1 R and OX2 R forms the basis of the reported as having non-functional mutated OX2 R (Lin et al.,
orexin/hypocretin system as it is understood today. With regards 1999; Hungs et al., 2001). This is particularly interesting, as
to nomenclature, while hypocretin and orexin can be used similar receptor mutations have not been reported to cause
interchangeably, it is most widely accepted to refer to related narcolepsy in humans (Nishino et al., 2000, 2001; Peyron et al.,
genes by the hypocretin name (in agreement with HUGO) while 2000; Liblau et al., 2015). Indeed, NT1 is specifically linked
peptides and receptors are referred to by the orexin name (as to the loss of orexin producing cells in the hypothalamus and
recommended by IUPHAR, see Alexander et al. (2021), Coleman the resulting absence of measurable orexin in the cerebrospinal
et al. (2021), so as to recognize the contributions of both discovery fluid of NT1 patients. Interestingly, orexin receptor mutant mice
studies (De Lecea et al., 1998; Sakurai et al., 1998). show different sleep phenotypes depending upon the receptor(s)
As previously mentioned, upon initial discovery, the orexin mutated. OX1 R knockout results in almost normal sleep-wake
system was implicated in food intake regulation due to the patterns, OX2 R knockout results in a mild sleep phenotype
prevalence of prepro-hypocretin/orexin and orexin receptors with some sleep fractionation and very rare cataplectic events,
within the lateral hypothalamus – a brain region implicated in whereas the double receptor knockout replicates the orexin
the regulation of food intake (De Lecea et al., 1998; Sakurai peptide knockout (NT1) phenotype, with frequent cataplectic
et al., 1998) – and subsequently the obese phenotype of orexin attacks, fast transitions from wake to REM sleep and sleep
knockout mice. This localization evidence, combined with early fragmentation (Willie et al., 2003; Mang et al., 2012; Hoyer and
observations that intracerebroventricular (ICV) administration Jacobson, 2013; Jacobson et al., 2017, 2019). In particular, the
of orexins resulted in an increase in feeding behavior in rats, sleep phenotype of the OX2 R knockout mouse (Willie et al.,
heavily implicated the orexin system as a regulator of food 2003) did not replicate that of the orexin peptide knockout
intake (Sakurai et al., 1998; Edwards et al., 1999; Yamanaka (Diniz Behn et al., 2010), nor that of the double receptor
et al., 2000). Further antagonist studies also supported this, knockout, which both have fast transitions from wake to REM
with OX1 R antagonists decreasing the orexigenic effect of OX-A and cataplexy characteristic of NT1 in humans. The contributing
administration (Haynes et al., 2000). role of OX1 R to REM sleep modulation, was later confirmed in
Further functional characterization and neuronal circuitry multiple studies showing that dual orexin receptor antagonists
studies confirmed the orexin system as an important regulator (DORAs) in rodents and humans over proportionally trigger
of feeding behavior but also revealed orexin involvement in REM sleep, whether during the active or inactive phase (Betschart
the regulation of other homeostatic mechanisms, in particular et al., 2013; Hoyer and Jacobson, 2013; Hoyer et al., 2013). It
arousal and sleep-wake regulation (Peyron et al., 1998, 2000; has also become clear that DORAs have the ability to trigger
Chemelli et al., 1999; Date et al., 1999; Edwards et al., 1999; Lin cataplectic episodes in rodents, dogs and humans (Mahoney
et al., 1999; Nambu et al., 1999; Yamanaka et al., 2000; Muroya et al., 2020), and hence are contraindicated in patients suffering
et al., 2004; Sakurai and Mieda, 2011; Li and de Lecea, 2020). The from NT1. This is also evident from studies combining OX1 R
focus of research into the orexin system took a marked pivot upon and OX2 R antagonists in rodents (Dugovic et al., 2009), where
the observation that knockout of the prepro-hypocretin gene in REM sleep is more affected than with an OX2 R antagonist alone.
mice produced a phenotype that exhibited striking similarities Hence, there is contribution of both receptors to the phenotype
to human narcolepsy with cataplexy (Chemelli et al., 1999), also displayed, however, the mechanistic basis for each receptors’
known as type 1 narcolepsy (NT1) (Mahoney et al., 2019). At contribution is not yet understood, whether related to signaling
the time, narcolepsy had no known cause and as such, this and/or cellular/pathway crosstalk.
observation led to the rapid uptake of research into the orexin As these studies demonstrated that blockade/knockout of
system’s role in the regulation of sleep (Lin et al., 1999; Scammell both receptors had a more pronounced effect on sleep than
et al., 2000; Estabrooke et al., 2001; Saper et al., 2001; Sakurai, blockade/knockout of only one of the receptors, this led
2007; Tsujino and Sakurai, 2009). From this research came the pharmaceutical and biotech companies to develop DORAs as a

FIGURE 1 | The orexin system. The orexin system consists of two endogenous peptide agonists, OX-A and OX-B, cleaved from the common precursor peptide
prepro-orexin. Both agonists have affinity for the two orexin receptors, OX1 R and OX2 R, although it has been suggested that OX-B has some OX2 R selectivity,
whereas OX-A is not selective. Following agonist binding, an active conformation of the receptor is stabilized and effector proteins are recruited to induce a cellular
response.
novel treatment for insomnia (Brisbare-Roch et al., 2007; Cox 2012; Betschart et al., 2013; Hoyer and Jacobson, 2013; Hoyer
et al., 2010; Bettica et al., 2012; Coleman et al., 2012; Beuckmann et al., 2013; Dugovic et al., 2014; Bonaventure et al., 2015;
et al., 2017; Berger et al., 2020; Boss et al., 2020). The critical Clark et al., 2020). The DORA almorexant did not affect sleep-
role of OX2 R was further evidenced in subsequent receptor wake patterns in OX2 R knockout mice, and was completely
antagonism/agonism studies performed in various transgenic inactive in double receptor knockout mice, which incidentally
mice which led to the alternate hypothesis that OX2 R antagonism confirmed that almorexant’s hypnotic effects were mediated
may produce more physiological sleep than DORAs (Mang et al., exclusively by orexin receptors (Mang et al., 2012). Almorexant

had major hypnotic effects in OX1 R knockout mice, comparable activation in all recombinant systems. Early studies aimed to
to those seen in wildtype mice. However, a closer look at the elucidate the origin of the rise in cytoplasmic Ca2+ in order
polysomnography data suggested that almorexant’s effects on to give insight into the cellular mechanisms and transduction
sleep were even more pronounced in the OX1 R knockout (Mang cascades involved in orexin receptor signaling.
et al., 2012). Along the same lines, OX-A produced no effect in Van Den Pol et al. (1998) used the fluorescent free Ca2+
double receptor knockout but had stimulatory effects in OX1 R indicator Fura-2 to perform imaging on hypothalamic neurons
knockout mice, whereas it produced no obvious effects in OX2 R to characterize Ca2+ elevations upon stimulation with orexins.
knockout mice, emphasizing again a major role of the OX2 R in They observed that depletion of intracellular Ca2+ stores by
wake-related activities (Mang et al., 2012). One unusual feature pre-treatment with the endoplasmic reticulum Ca2+ ATPase
of a number of these DORAs is their very slow dissociation rates inhibitor thapsigargin had no significant effect on the orexin-
from the receptors, which result in sustained receptor occupancy induced response. However, stimulation of cells in the presence of
and explain their long duration of action on top of rather long a chelating agent (EGTA) abolished the rise in intracellular Ca2+
pharmacokinetic features (Mang et al., 2012; Callander et al., concentration in response to stimulation with orexins.
2013; Farkas, 2013; Hoyer and Jacobson, 2013; Jacobson et al., Smart et al. (1999) subsequently utilized a Fluorescence
2014). Imaging Plate Reader (FLIPR) along with CHO cells transfected
Since these initial studies revealing the orexin system’s role in with OX1 R or OX2 R to characterize the rise in intracellular Ca2+ .
critical homeostatic mechanisms, the system’s impact has been When OX1 R was stimulated with 1 µM OX-A, a rapid peak
further expanded to include roles in addiction, pain, anxiety, in intracellular Ca2+ followed by a slow decline in the elevated
panic, depression, binge eating and potentially post-traumatic Ca2+ level was observed. It is noted that similar results were
stress disorder (PTSD) among others (Nakamura et al., 2000; observed for OX-B at OX2 R, however these data were not shown.
Piccoli et al., 2012; Tsujino and Sakurai, 2013; Fitch et al., 2014; In contrast to Van Den Pol et al. (1998)’s findings, Smart et al.
Li et al., 2014; Roh et al., 2014; Sakurai, 2014; Abbas et al., 2015; (1999) observed a rise in intracellular Ca2+ in the absence of
Bonaventure et al., 2015, 2017; Bonnavion et al., 2015; Soya et al., extracellular Ca2+ , however also observed that the Ca2+ response
2017; Steiner et al., 2018; Kaufmann et al., 2020). Additionally, became more transient. Depletion of intracellular Ca2+ stores
a role for orexins and orexin receptors in peripheral tissues has abolished the OX1 R response to OX-A and OX-B. In addition,
also been suggested (Kirchgessner and Liu, 1999; Karteris et al., this study demonstrated that inhibition of phospholipase C (PLC)
2001, 2004, 2005; Mazzocchi et al., 2001; Randeva et al., 2001; by U73122 abolished the Ca2+ response. These results were
Patel et al., 2018). suggested to indicate that the orexins stimulate the release of
Mounting evidence has now demonstrated that classical G intracellular Ca2+ stores for the rapid peak response as well as
protein-dependent signaling at the plasma membrane is not the extracellular Ca2+ influx for the prolonged response, resulting in
only way most GPCRs transduce signals. β-arrestin-dependent the typical biphasic pattern.
signaling (Smith and Rajagopal, 2016), signaling from GPCRs Lund et al. (2000) however, support extracellular Ca2+
sequestered within subcellular compartments (Irannejad and von influx as the primary mechanism of intracellular Ca2+
Zastrow, 2014; Thomsen et al., 2018) and bias toward specific increase as a result of OX1 R stimulation. Lund et al. (2000)
signaling pathways due to ligand or cellular environment (Smith demonstrate OX1 R-mediated activation of cation channels and
et al., 2018) have all been observed. Additionally, with the use the resultant rise in intracellular Ca2+ acting synergistically
of newly developed biosensors to probe G protein coupling to with Gαq activation to increase PLC activation. These results
receptors [Wan et al., 2018; Laschet et al., 2019; Avet et al., suggest that inositol trisphosphate (IP3 )-mediated Ca2+ release
2020 (unpublished); Olsen et al., 2020], promiscuity in G protein from intracellular stores is secondary and dependent upon
coupling has been revealed to be more common than previously extracellular Ca2+ influx for OX1 R. This finding is reiterated
thought. Despite these shifts in dogma surrounding GPCR in Larsson et al. (2005) and Johansson et al. (2007), who also
signaling, investigation of these concepts in relation to the orexin implicate transient receptor potential cation channels (TRPCs) as
receptors has been very limited, with the understanding of their mediating Ca2+ influx. Interestingly, these studies demonstrate
signaling properties mostly confined to the classical perspective. the Ca2+ influx dependency of the response decreases at high
Here, the current understanding of orexin receptor interactions concentrations of OX-A (0.1 µM and above), however the
with direct effectors such as G proteins and β-arrestins, as well as physiological relevance of this is unclear.
the subsequent implications of these interactions for the signaling From these initial studies, subsequent works aimed to
of the orexin system, will be discussed. further elucidate the mechanisms underlying the robust Ca2+
response characteristic of orexin receptor activation. Studies in
recombinant cells largely support a primary role for extracellular
CALCIUM AND INTRACELLULAR Ca2+ influx (Peltonen et al., 2009; Putula et al., 2014; Wang et al.,
MESSENGERS 2014; Nagahara et al., 2015), however, divergences in function
have been described between these recombinant systems and
Calcium Ion Movement measurements in neurons. Indeed, there is conflicting evidence
Ever since the orexin system’s discovery, activation of the orexin between neuronal studies (Van Den Pol et al., 1998; Uramura
receptors has been strongly associated with a rise in intracellular et al., 2001; Kohlmeier et al., 2004, 2008; Muroya et al., 2004;
Ca2+ (Sakurai et al., 1998). This is a hallmark of the receptors’ Kukkonen and Leonard, 2014). In summary, it appears that

the orexin-induced intracellular Ca2+ increase is primarily a orexin receptors, with evidence of varying degrees of coupling
result of extracellular Ca2+ influx, with a small contribution to Gαq , Gαi/o and Gαs , depending upon the experimental
from intracellular Ca2+ release, with cell- and tissue-specific conditions. Given this variation in the reported coupled G
effects also likely. proteins, there is uncertainty around the relevance of these
reports to endogenous function as the literature contains multiple
Downstream Signaling Interactions accounts of conflicting evidence. A number of these discrepancies
Orexin receptor activation has been documented to result can be attributed, at least in part, to the varied cell and tissue
in an enormously varied set of downstream interactions, conditions under which orexin signaling has been studied. This
involving many key cascades within the signaling milieu. Studies possible tissue- and cell-dependent variation in the G protein
investigating responses downstream of G protein activation coupling indicates that the effector proteins may be influenced
indicate the involvement of multiple concurrent signaling by different environmental conditions (Randeva et al., 2001;
mechanisms. The activation of effectors classically thought of as Caillol et al., 2003; Woldan-Tambor et al., 2011). Other factors
directly downstream of G protein activation are often used as may also contribute to these perceived differences, including the
indicators of specific G protein activity, despite the two not always stimulating ligand used, ligand concentration and experimental
being directly correlated. The activation of PLC, the production method. Of particular note is the abundance of G protein
of diacylglycerol (DAG) and IP3 and an increase in intracellular coupling data that has been inferred based upon assays that
Ca2+ have been widely characterized upon stimulation of orexin detect the activation of downstream messengers of G proteins.
receptors. The activation of adenylate cyclase and production This has been historically necessary due to the lack of techniques
of cyclic adenosine monophosphate (cAMP) have also been to study G protein coupling directly. However, given the well-
characterized in some studies (Randeva et al., 2001; Tang et al., characterized promiscuity of the orexin receptors’ G protein
2008; Kukkonen, 2016a,b). coupling [Avet et al., 2020 (unpublished)], and the possibility for
Further downstream, activation of extracellular signal- cross-regulation between classical signaling cascades (Houslay
regulated kinases (ERKs) (Ammoun et al., 2006a), phospholipase and Kolch, 2000; Halls and Cooper, 2011), caution must be used
A2 (PLA2 ) (Turunen et al., 2012), phospholipase D (PLD) (Jäntti when applying these data to determine G protein coupling. Here,
et al., 2012) and p38 mitogen-activated protein kinases (Ammoun we will look in-depth at the evidence for G protein coupling to
et al., 2006b) among others (Ammoun et al., 2006a; Turunen each of the orexin receptors.
et al., 2012) have been reported, once again highlighting the
diverse signaling capabilities of the orexin system, the functional G Protein Coupling to Both Receptors
consequences of which are still being elucidated. To date, much In their discovery study, Sakurai et al. (1998) suggested that the
of this research has been conducted within recombinant cell orexin receptors couple to Gαq due to the generation of OX-
environments with a bias toward studying OX-A-induced OX1 R A or OX-B concentration-dependent increases in intracellular
function. Therefore an important part of future research will Ca2+ in CHO cells stably transfected with OX1 R or OX2 R.
be examining these aspects of orexin receptor signaling within Van Den Pol et al. (1998) subsequently gave indirect evidence
primary cell models and native tissue, as well as identifying how for coupling to Gαq , as inhibition of PKC using the inhibitor
much of the OX-A-induced OX1 R-derived signaling patterns can bisindolylmaleimide abolished orexin-induced intracellular Ca2+
be applied to OX2 R- and OX-B-induced signaling. Kukkonen increase. However, which specific orexin receptor was mediating
(2016b) began to address this, characterizing the signaling this effect in the hypothalamic neurons was not suggested
effectors activated by OX2 R in CHO cells and comparing the or investigated.
profile to that of OX1 R. They observed the involvement of the Randeva et al. (2001) detected OX-A-induced activation of
same effectors described for OX1 R with minor differences in Gαq , Gαs , and Gαi in human adult adrenal gland membrane
potency for some signaling pathways. However, they did not preparations using a GTP-azidoanilide-labeling and subsequent
investigate the effect of OX-B-induced stimulation and as such, immunoprecipitation technique. Subsequent functional analysis
any ligand-dependent effects at OX2 R on the signaling pathways showed a concentration-dependent increase in both cAMP and
investigated remain to be characterized in such detail. IP3 production upon treatment with OX-A. These findings are
Further information on the downstream effectors and in agreement with previous data detailing increased cAMP levels
signaling cascades implicated in orexin function is summarized in adrenal cells upon stimulation with orexins (Malendowicz
in Kukkonen and Leonard (2014) and Leonard and Kukkonen et al., 1999; Mazzocchi et al., 2001). Randeva et al. (2001)
(2014). also characterized the receptors present within the adrenal
tissue. Using RT-PCR, the expression of OX2 R, but not OX1 R,
was observed and this was confirmed using various additional
G PROTEINS methods, as was the presence of prepro-orexin. The orexin
expression pattern in adrenal tissue is contested however,
As a result of receptor activation leading to increased intracellular as discussed in the “Signaling in Peripheral Cell and Tissue
Ca2+ levels and PLC activation, the orexin receptors were initially Environments” section below.
thought to couple to Gαq with subsequent PLC-IP3 signaling Zhu et al. (2003) utilized neuronal hybrid cells (BIM cells)
(Sakurai et al., 1998; Lund et al., 2000; Holmqvist et al., 2002). stably expressing OX1 R-EGFP or OX2 R-EGFP to measure the
Further research expanded the G protein-coupling partners of the effect of orexin stimulation on cAMP and intracellular Ca2+

levels. From the trends observed in these pathways, Zhu et al. Gαs and Gαi involvement in orexin-induced ERK activation.
(2003) suggest coupling of both receptors to Gαq , OX2 R but not Even though involvement of all three G proteins in downstream
OX1 R coupling to Gαi , and coupling of neither receptor to Gαs . effector activation was detected, there was evidence for ligand-
However, as this study only investigated effectors downstream of specific bias in the activated G proteins and cascades. This is
G protein coupling, and had limited use of pathway modulators discussed in detail in the “Endogenous Ligands” section, along
[such as pertussis toxin (PTX) and cholera toxin (CTX)], these with potential limitations of the methodologies used in the study.
suggestions should be treated with caution. The lack of data These findings were in stark contrast to the findings of
on the effect of OX-B stimulation on cAMP in this study is Urbańska et al. (2012) in primary cultures of rat cortical neurons
also of note. A bias toward studying OX-A-induced responses is which showed no effect of orexin stimulation on cAMP levels,
present within the literature, with markedly less data available suggesting no direct activation via Gαs or any indirect activation,
on the effects of OX-B. This is unfortunate, as ligand-specific e.g., via PLC. They did, however, observe inhibition of forskolin-
effects have been reported for the orexin system and therefore induced cAMP accumulation with orexin stimulation, which was
important signaling events have potentially been missed due to completely reversed by PTX treatment, as well as inhibited by
the omission of OX-B in such studies. Ligand-specific effects that an OX2 R-selective antagonist (TCS OX2 29) but not an OX1 R-
have been elucidated are discussed in detail in the “Endogenous selective antagonist (SB 408124). These results suggest OX2 R-
Ligands” section below. mediated activation of Gαi is observed upon stimulation with
orexins in this particular cell model.
OX1 R G Protein Coupling Kukkonen (2016a) revisited the coupling partners of both
Multiple studies followed that aimed to investigate the coupling OX1 R and OX2 R in CHO cells by investigating the effect
of OX1 R to G proteins and the associated signaling cascades. of G protein inhibitors on downstream signaling effectors. It
Holmqvist et al. (2005) utilized CHO cells stably expressing was observed that for both receptors, Gαq was responsible
OX1 R to study the response to orexins of multiple downstream for the vast majority of responses, including intracellular
G protein effectors. CTX (which constitutively activates Gαs ) Ca2+ accumulation, which was abolished with UBO-QIC (Gαq
and PTX were used to probe for potential G protein mediation inhibitor) treatment. Their results also gave evidence for Gαs -
with both positive and negative regulation of cAMP production mediated adenylate cyclase activity with OX-A stimulation at
observed. Robust and concentration-dependent intracellular both receptors. Additionally, Kukkonen (2016b) suggested that
Ca2+ elevation and inositol phosphate production was also OX2 R has weaker coupling to Gαs relative to OX1 R in CHO
observed. From these results, the authors suggest that in CHO cells, as only weak adenylate cyclase activity could be detected
cells, OX1 R couples to a minimum of three G proteins: Gαi/o , upon stimulation with OX-A. Interestingly, UBO-QIC and PTX
Gαs , and Gα q . were found to reduce and abolish, respectively, the inhibitory
Magga et al. (2006) used immunoprecipitation of C-terminally effect of OX-A stimulation on adenylate cyclase for OX1 R and
FLAG-tagged OX1 R and subsequent immunoblotting with OX2 R, indicating this effect that has classically been attributed
Gα subunit-specific antibodies to investigate the G protein to Gαi may also be partially mediated by Gαq . It is noteworthy
coupling partners of OX1 R in HEK293 cells. Gαq showed that while these studies provide an excellent comparison of OX1 R
the strongest signal for co-immunoprecipitation with OX1 R- and OX2 R G protein coupling and downstream signaling, no
FLAG and this interaction was detected in all experiments. investigation with OX-B stimulation was pursued. Given that
Co-immunoprecipitation was also observed with Gαi and Gαs biased agonism of OX-A and OX-B to specific G protein signaling
but this was not detected in all experiments (6/11 and 2/3 cascades has been reported previously (Tang et al., 2008), this
independent experiments respectively) with no detection of Gαo would be an interesting aspect to investigate further.
(0/5). Subsequent functional analysis showed stimulation of More recently, Baker and Proudman (2021) reported a
HEK293 cells stably expressing OX1 R-FLAG with OX-A resulting biphasic cAMP response to OX-A and OX-B in CHO cells
in no change to cAMP levels from basal or when pre-treated with expressing human OX2 R but not in OX1 R expressing cells.
forskolin. From this study, it is not clear if OX1 R couples to Gαs The inhibitory, but not the stimulatory component, was PTX
or Gαi in HEK293 cells. While the co-immunoprecipitation data sensitive. Both OX-A and OX-B were highly potent activators
indicate weak coupling may be present, the functional data were of Ca2+ release, inositol phosphate (IP) accumulation and
unable to support this. ERK1/2 activity, with moderate or significant selectivity for OX-
Woldan-Tambor et al. (2011) observed robust cAMP B. Interestingly, in the same study compound C (Nagahara
production in rat cerebral cortex astrocyte cultures upon et al., 2015) was potent and OX2 R selective in the latter three
stimulation with OX-A. This effect was not seen upon treatment assays, but had no effect on cAMP accumulation, suggesting
with the moderately OX2 R selective agonist [Ala11 ,D-Leu15 ]-OX- biased signaling. Furthermore, the potency values of OX-B varied
B and was partially inhibited by an OX1 R-selective antagonist (SB between OX2 R-mediated Ca2+ release, IP accumulation, ERK1/2
40812), but not by the OX2 R-selective antagonist (TCS OX2 29). activity and cAMP inhibition and activation (pEC50 = 10.36,
9.84, 11.08, 9.68, and 7.85). Similar differences were observed
OX2 R G Protein Coupling for OX-A in the same assays (pEC50 = 9.72, 9.28, 10.47, 9.54,
Research focused specifically on OX2 R came somewhat later than and 7.72). The potency variations of OX-A and OX-B at the
that on OX1 R. Using dominant negative G proteins in HEK293 OX1 R were less marked. Altogether, these data would suggest that
cells stably expressing OX2 R, Tang et al. (2008) observed Gαq , natural and synthetic agonists can display pathway selectivity at

human OX2 R, with a range of potency and/or apparent efficacy these studies investigated OX-A-induced responses only, as OX-
values that are signaling dependent. Similar although less marked B was not tested.
differences were noticed at OX1 R, except that cAMP was not Contrasting results subsequently gave evidence for coupling
affected by any of the ligands tested. of Gαq , Gαs , Gαi as well as Gαo in rat adrenal cortex and
also the hypothalamus (Karteris et al., 2005), however, this may
represent a species-specific difference. Remarkably, this study
SIGNALING IN PERIPHERAL CELL AND also showed changes to the G protein activation profile upon OX-
TISSUE ENVIRONMENTS A stimulation following 24 h of food deprivation. Subsequent
work to further interrogate this change in G protein activation
Studies often characterize the presence of orexins and orexin would assist in understanding the implications of this finding, for
receptors within cells or tissue by utilizing techniques such as example, analysis of the contribution of each receptor. However,
RT-PCR, western blot and fluorescence in situ hybridization this study serves as yet another example of the potentially fluid
(FISH). In some cases, a predominant receptor subtype can be and highly variable nature of orexin signaling.
identified, allowing for signaling characteristics to be attributed Ramanjaneya et al. (2008) investigated orexin signaling in the
as predominantly OX1 R- or OX2 R-mediated. However, when H295R adrenocortical cell line. Steroidogenic acute regulatory
both receptors are detected, dissecting receptor-specific signaling protein (StAR) is a transporter protein important to acute
becomes more difficult and requires the use of receptor-specific steroid synthesis. Ramanjaneya et al. (2008) demonstrated that
antagonists or other tools. While orexin receptors can be located both orexin ligands upregulate expression of StAR in H295R
within the same areas of the CNS, they most often show distinct, cells. Using dominant negative G proteins, which it should
complementary distribution (Marcus et al., 2001; Sakurai and be noted can have issues of non-specificity (Kukkonen, 2004),
Mieda, 2011). As such, research using CNS-derived cells and they subsequently suggested the involvement of Gαq , Gαs , and
tissues rarely encounter this issue, with signaling observations Gαi in OX-A-induced StAR expression, but only Gαq and
often attributable to the one present orexin receptor subtype. Gαs involvement in OX-B-induced StAR expression. An OX1 R-
In contrast, expression of both receptors appears to be more selective antagonist was used to determine the role of each orexin
common within the periphery, and as such, techniques to receptor in these responses, with OX1 R found to be mediating
investigate the contribution of each receptor are necessary OX-A-induced coupling, and both receptors, but predominantly
in these studies. OX1 R, mediating the OX-B-induced response. In a second study,
In addition, there are conflicting data on the expression Ramanjaneya et al. (2009) investigated the regulation of mitogen-
of orexin receptors within peripheral environments, making activated protein kinase (MAPK) activation by the orexin system,
analysis of results in these studies difficult (Heinonen et al., 2008; showing distinct G protein coupling to ERK/MAPK cascade
Kukkonen, 2013). Perhaps the most prominent example of this regulation for both ligands. They suggested Gαq , Gαs , and
is in adrenal gland tissue and cells, in which some studies report Gαi involvement that was predominantly OX1 R-mediated, with
only OX2 R expression (Karteris et al., 2001; Randeva et al., 2001) minor input from OX2 R for both ligands’ effects. Although these
while others report expression of both OX1 R and OX2 R (Lopez studies did not investigate any possible activation or involvement
et al., 1999; Spinazzi et al., 2005). of Gαo within this cell model, they regardless give insight
These conflicting reports may be attributable to both lack into the potential complexity of orexin-G protein coupling
of binding to orexin receptors and lack of specificity of anti- within adrenal tissue.
orexin receptor peptide antibodies (Kukkonen, 2013), although Orexin receptors have been detected within many peripheral
inter-species and intra-organ (e.g., adrenal cortex vs. medulla) tissues (Johren et al., 2001; Heinonen et al., 2008), however G
differences may also contribute (Heinonen et al., 2008). As such, protein coupling of the orexin system within these tissues has not
while signaling behaviors are sometimes attributed to specific been widely investigated, with the most in-depth analysis found
receptors, this analysis should be approached with caution. in the adrenal glands as discussed above. Future research in a
range of peripheral tissues may shed light on the enigmatic orexin
Adrenal Tissue G protein coupling systems. Furthermore, an area of orexin
One of the earliest studies to investigate peripheral orexin research that has been reinvigorated by peripheral tissue studies
signaling and G protein coupling was the previously mentioned pertains to the roles of the two orexin ligands and the potential
study by Randeva et al. (2001) which gave evidence for OX2 R differences in their signaling and function. Further discussion of
coupling to Gαq , Gαs , and Gαi , but not Gαo in human adult this can be found in the “Endogenous Ligands” section.
adrenal tissue. A similar study in human fetal adrenal tissue
supported the finding of OX2 R, but not OX1 R expression
within human adrenal glands. However, interestingly, upon NON-G PROTEIN MODULATION
stimulation with OX-A, coupling of Gαs and Gαi but not
Gαq was detected by GTP-azidoanilide labeling (Karteris et al., While much attention has been given to G protein activation
2001). Why this difference between fetal and adult coupling and the subsequent signaling cascades, the role of other effector
was observed is unclear and warrants further investigation, proteins in orexin pharmacology has been less thoroughly
although it is not uncommon to observe differences in protein interrogated. Important receptor accessory proteins, such
expression between fetal and adult tissues. Also of note, both as arrestins, have the ability to impact signaling behavior,

and therefore understanding these interactions could reveal

important insights into the function of orexin receptors.
β-Arrestins
Both orexin receptors have been shown to interact robustly with
β-arrestins. Along with their classical role in signal cessation
and receptor internalization, β-arrestins also scaffold secondary
signaling platforms (Lefkowitz and Shenoy, 2005; DeWire et al.,
2007).
Evidence that β-arrestin2 plays a role in orexin signaling
through ERK pathways is given by Milasta et al. (2005).
Notably, ERK1/2 phosphorylation was reduced and the time-
scale of ERK1/2 phosphorylation shortened with a mutant OX1 R
that reduced agonist-induced β-arrestin2 recruitment. This was
despite no significant changes to the induction of increased
intracellular Ca2+ , indicating these changes do not result
from general reduced signaling capacity. Additionally, while β-
arrestin2-dependent internalization of the mutated receptor was
minimally impacted, colocalization of OX1 R and βarrestin-2
within endosomal compartments following internalization was
not observed with the mutant receptor, in contrast to the
wildtype OX1 R. This study did not investigate whether the same
patterns could be observed with OX2 R or OX-B treatment,
however subsequent studies suggest OX1 R and OX2 R have
distinct interaction profiles with β -arrestin2.
In COS-7 cells, Dalrymple et al. (2011) observed β-arrestins
to be recruited with a higher potency to OX2 R compared
to OX1 R in response to OX-A. Using a kinetic BRET
assay, OX1 R underwent relatively transient interactions with
arrestin-ubiquitin complexes compared to OX2 R that showed
FIGURE 2 | ERK1/2 phosphorylation data for OX1 R (OxR1 ) and OX2R (OxR2 )
more stable and prolonged interactions. Additionally, ERK1/2
stably transfected in HEK293 cells. (A) Stably transfected HEK293 cells were
phosphorylation in response to OX-A stimulation was similar for treated with OX-A (OxA) and measured over a 4-h period. Data were
both receptors in the short term (up to 10 min post-stimulation), normalized to time-matched vehicle treatments and are expressed as a
however, OX1 R exhibited lower levels of sustained ERK1/2 percentage of the maximal response induced at 2 min post-agonist treatment.
phosphorylation compared to OX2 R (Figure 2). Dalrymple et al. Data are expressed as mean ± SE of four independent experiments.
*p < 0.05 between OX1 R and OX2 R from 10 to 120 min post-agonist
(2011) suggested β-arrestins may form a more stable secondary
stimulation. (B) Concentration-response data were collected at 2- and 90-min
signaling platform with OX2 R than with OX1 R, indicating a post OX-A treatment of OX2 R-expressing cells. Data are expressed as a
mechanism that may play a role in the diverging functional percentage of the maximal response induced at the time point (mean ± S.E.
profiles of the two receptors. of four independent experiments). Reproduced from Dalrymple et al. (2011)
Possible structural determinants of the orexin-β-arrestin under a Creative Commons Attribution 4.0 International License. Full terms
provided at https://creativecommons.org/licenses/by/4.0/.
interaction profiles were investigated by Jaeger et al. (2014). Using
site-specific mutagenesis to isolate the contribution of serine
and threonine residues in the C-terminal tail of OX2 R, they Regardless, these foundational studies give insight into the role
showed that OX2 R has two C-terminal serine/threonine clusters of β-arrestin in orexin signaling and demonstrate the potential
that are phosphorylated by GRKs, creating a strong affinity for importance of non-G protein interactions.
the receptor with β-arrestin. This is in contrast to OX1 R which With the exception of arrestins, research investigating the
has only one C-terminal serine/threonine cluster important for potential role of accessory proteins in the control of orexin
GRK phosphorylation, resulting in the less stable interactions receptor signaling is sparse. Further in-depth investigation into
reported by Dalrymple et al. (2011). This difference in C-terminal the role of such proteins could uncover further modulators of
phosphorylation sites is also suggested to be the structural orexin signaling. This could ultimately aid in explaining the
determinant underpinning the slower recycling rate of OX2 R complex signaling and functional characteristics described for
observed by Dalrymple et al. (2011), with OX2 R requiring more the orexin system.
significant dephosphorylation post-internalization to dissociate
from β-arrestin, resulting in the receptor remaining within the Heteromerization
cell for longer. These results were produced using only OX-A Heteromerization has been suggested to play a functional role
stimulation. Whether different structural determinants may be in the orexin system, with particular focus on heteromerization
involved in OX-B-induced β-arrestin2 interactions is unknown. of orexin receptors with opioid and cannabinoid receptors

(Thompson et al., 2017). However, it is of note when considering Both orexins have a C-terminal amide group (Sakurai et al.,
these reports that these studies rely heavily on recombinant 1998). In their first report of hypocretin/orexin peptides, De
expression, which is prone to overexpression artifacts. Reported Lecea et al. (1998) reported the fast neuroexcitatory effects of
functional interactions between orexin receptors and other the native OX-B (1 µM) in various rat hypothalamic neuronal
GPCRs are listed in Table 1. cultures and the absence of effects of OX-B in four different
The effects of orexin receptor heteromerization include cross- hippocampal dentate granule neuronal cultures. In these early
activation and inhibition, as well as novel signaling mechanisms studies, OX-A activity was not tested.
that are different from those of the monomeric receptor. An In CHO and HEK293 cells, OX1 R shows preferential binding
example of this is seen with the kappa opioid receptor (κOR)- of OX-A over OX-B while at OX2 R, OX-A and OX-B have
OX1 R heteromer, in which signaling through Gαs is observed similar affinities in radioligand binding experiments (Sakurai
in response to OX-A or dynorphin A, with a reduction in et al., 1998). Similar trends are reflected in the potency of the
Gαi and Gαq signaling through κOR and OX1 R, respectively, ligands at the two receptors, with OX-A and OX-B showing
upon heteromerization (Chen et al., 2015). Heteromerization equivocal potency at OX2 R in IP production assays while OX-
resulting in novel signaling is strong evidence of a functional B shows significantly lower potency at OX1 R (Holmqvist et al.,
interaction between the two receptors that may result in 2002; Dalrymple et al., 2011; Kocan et al., 2011). Despite these
physiologically relevant applications. Given the wide range of characterizations, many nuanced ligand-specific effects have
reported heteromers from the orexin system, the formation of been reported, the roles of which remain to be elucidated.
higher order receptor oligomers is likely to be an important In other words, the putative receptor selectivity of OX-B
aspect of the orexin system’s complex in vivo functions, and may needs to be re-considered, depending on cellular environment
underlie some of the reported signaling variation in different and/or transduction mechanism considered. The data reported
cell and tissue types. Further research is needed to determine by different groups in a variety of cells (whether collected
the extent of the influence of heteromerization on these factors, from recombinant systems or from primary cultures) and/or
as well as further characterize the novel signaling properties of assays, demonstrate variations in apparent affinity, potency
specific orexin heteromers. and to some extent efficacy (see above, e.g., Section “OX2 R
G protein coupling”). Therefore, the purported “selectivities”
for endogenous and synthetic ligands, especially agonists, show
LIGANDS marked variations and the use of “selective” when describing such
ligands must be taken with caution. An obvious consequence
Endogenous Ligands of this, is that effects mediated by endogenous orexin receptors
The orexin system has two known endogenous ligands, OX-A need to be characterized using adequate pharmacological tools,
and OX-B, both resulting from a common precursor, prepro- preferably by measuring rank orders of potency of several
orexin. OX-A is a 33 amino acid peptide with two intrachain antagonists with various degrees of selectivity, rather than the
disulfide bonds, while OX-B is a 28 amino acid linear peptide. limited use of poorly selective agonists.
TABLE 1 | Select reported interactions between orexin receptors and other GPCRs in native and recombinant environments.
Orexin receptor Interacting receptor Reported findings
OX1 R Kappa opioid receptor OX1 R and κOR were suggested to co-express in rat hippocampal neurons. In HEK293 cells,
(κOR) colocalization/heteromerization lead to enhancement of Gαs , PKA and cAMP signaling (Chen et al., 2015). Another
study, however, suggested the interaction occurs at the level of downstream signaling pathways (Robinson and
McDonald, 2015).
OX1 R Corticotropin-releasing In HEK293 cells and in rat ventral tegmental area slices, CRF1 R-OX1 R heteromers caused negative crosstalk of OX-A
factor receptor 1 (CRF1 R) and CRF signaling. The heteromer also complexes with the cocaine target σ1 receptor. This promotes long-term
disruption of the OX-A and CRF negative crosstalk that modulates dendritic dopamine release (Navarro et al., 2015).
OX1 R Corticotropin-releasing In HEK293 cells, CRF2 R-OX1 R heteromers cross-antagonized one another; this was potentiated by amphetamine and
factor receptor 2 (CRF2 R) potentially involved formation of a higher order complex with σ1 and σ2 receptors. In rat ventral tegmental area slices,
amphetamine potentiated OX-A-induced dopamine and glutamate release, which was blocked by CRF2 R antagonism
(Navarro et al., 2019).
OX1 R Growth hormone In HEK293 cells, GHS-R1a-OX1 R heteromers cross-antagonized one another’s signaling, while formation of a trimeric
secretagogue receptor 1a complex with the leptin receptor abolished this antagonism. In primary cultures of hypothalamic neurons, agonist
(GHS-R1a; ghrelin receptor) responses resembled those mediated by the trimeric rather than the dimeric complexes (Medrano et al., 2018).
OX1 R Cholecystokinin A receptor In HEK293 cells, OX1 R and CCK1 formed heteromers. Dual receptor activation reduced G protein signaling and
(CCK1 ) migration, but not β-arrestin interactions, compared to single receptor activation (Bai et al., 2017).
OX2 R 5-hydroxytryptamine 1A OX2 R and 5-HT1A are suggested to colocalize in rat hippocampus slices and in the cell membrane of HEK293T cells.
receptor (5-HT1A ) Receptor co-expression lead to increased cAMP and Ca2+ signaling, and decreased ERK signaling (Wang et al., 2019).
OX1 R OX2 R Cannabinoid receptor type In Flp-In T-REx 293 cells, OX1 R and CB1 formed heteromers. OX-A induced internalization of CB1 , with increased
1 (CB1 ) potency compared to OX-A-induced internalization of OX1 R (Ward et al., 2011). BRET evidence for formation of
CB1 -OX1 R, CB1 -OX2 R and OX1 R-OX2 R heteromers (Jäntti et al., 2014).

TABLE 2 | Described orexin ligands.
Name Selectivity Peptide or Chemical name/Peptide sequence

Non-peptide
Antagonists
SB-334867 OX1 R selective antagonist Non-peptide 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea
SB-408124 OX1 R selective antagonist Non-peptide 1-(6,8-difluoro-2-methylquinolin-4-yl)-3-(4-dimethylaminophenyl)urea
SB-410220 OX1 R selective antagonist Non-peptide 1-(5,8-difluoroquinolin-4-yl)-3-(4-dimethylaminophenyl)urea
SB-674042 OX1 R selective antagonist Non-peptide [5-(2-Fluorophenyl)-2-methyl-4-thiazolyl][2(S)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)methyl-
1-pyrrolidinyl]methanone
ACT-335827 OX1 R selective antagonist Non-peptide (2R)-2-[(1S)-1-(3,4-Dimethoxybenzyl)-6,7-dimethoxy-3,4-dihydro-2(1H)-isoquinolinyl]-N-
isopropyl-2-phenylacetamide
ACT-539313 OX1 R selective antagonist Non-peptide ((4-methyl-2-[1,2,3]triazol-2-yl-phenyl)-[(R)-
3-(3-[1,2,3]triazol-2-yl-benzyl)-morpholin-4-yl]-methanone) (Kaufmann et al., 2020)
Nagase 71 OX1 R selective antagonist Non-peptide (E)-N-((4R,4aS,7R,7aR,12bS)-3-{[2-(Dimethylamino)phenyl]sulfonyl}-4a-hydroxy-9-
methoxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-
yl)-N-methyl-3-(pyridin-2-yl)acrylamide (Nagase et al.,
2017)
GSK-1059865 OX1 R selective antagonist Non-peptide [5-bromo-N-({1-[(3-fluoro-2-methoxyphenyl)carbonyl]-5-methylpiperidin-2-
yl}methyl)pyridin-2-amine]
JNJ-54717793 OX1 R selective antagonist Non-peptide (1S,2R,4R)-7-([(3-fluoro-2-pyrimidin-2-ylphenyl)carbonyl]-N-[5-(trifluoromethyl)pyrazin-2-
yl]-7-azabicyclo[2.2.1]heptan-2-amine) (Bonaventure et al.,
2017)
TCS-OX2-29 OX2 R selective antagonist Non-peptide (2S)-1-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-3,3-dimethyl-2-(pyridin-4-
ylmethylamino)butan-1-one
EMPA OX2 R selective antagonist Non-peptide N-ethyl-2-[(6-methoxypyridin-3-yl)-(2-methylphenyl)sulfonylamino]-N-(pyridin-3-
ylmethyl)acetamide
JNJ-10397049 OX2 R selective antagonist Non-peptide N-(2,4-dibromophenyl)-N0 -[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl]-urea
JNJ-42847922 OX2 R selective antagonist Non-peptide ([5-(4,6-dimethyl-pyrimidin-2-yl)-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl]-(2-fluoro-6-
(Seltorexant) [1,2,3]triazol-2-yl-phenyl)-methanone) (Bonaventure et al.,
2015)
IPSU OX2 R selective antagonist Non-peptide 2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one
MK-1064 OX2 R selective antagonist Non-peptide 5-(5-chloropyridin-3-yl)-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2-pyridin-2-ylpyridine-3-
carboxamide
MK-3697 OX2 R selective antagonist Non-peptide N-[(5,6-dimethoxypyridin-2-yl)methyl]-2-(5-methylpyridin-3-yl)-5-(1,3-thiazol-2-
yl)pyridine-4-carboxamide
SDM-878 OX2 R selective antagonist Non-peptide (2-(3-(2-(1H-pyrazol-1-yl)nicotinoyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-3-
methoxyisonicotinonitrile) (Maehara et al.,
2020)
LSN2424100 OX2 R selective antagonist Non-peptide N-biphenyl-2-yl-4-fluoro-N-(1H-imidazol-2-ylmethyl) benzenesulfonamide HCl (Fitch
et al., 2014)
MK-4305 (Suvorexant) Dual receptor antagonist Non-peptide [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-
1,2,3-triazol-2-yl)phenyl]methanone
MK-6096 (Filorexant) Dual receptor antagonist Non-peptide [(2R,5R)-5-[(5-fluoropyridin-2-yl)oxymethyl]-2-methylpiperidin-1-yl]-(5-methyl-2-
pyrimidin-2- ylphenyl)methanone (Winrow et al.,
2012)
E-2006 (Lemborexant) Dual receptor antagonist Non-peptide (1R,2S)-2-[(2,4-dimethylpyrimidin-5-yl)oxymethyl]-2-(3-fluorophenyl)-N-(5-fluoropyridin-
2- yl)cyclopropane-1-carboxamide (Yoshida et al.,
2015)
SB-649868 Dual receptor antagonist Non-peptide N-([(2S)-1-([5-(4-fluorophenyl)-2-methyl-4-thiazolyl]carbonyl)-2-piperidinyl]methyl)-4-
benzofurancarboxamide
TCS-1102 Dual receptor antagonist Non-peptide N-[1,10 -Biphenyl]-2-yl-1-[2-[(1-methyl-1H-benzimidazol-2-yl)thio]acetyl-2-
pyrrolidinedicarboxamide
ACT-078573 Dual receptor antagonist Non-peptide (2R)-2-[(1S)- 6,7-Dimethoxy-1-{2-[4-(trifluoromethyl)ph-3,4-dihydroisoquinolin-2(1H)-yl]-
(Almorexant) N-methyl-2-phenylacetamide
ACT-462206 Dual receptor Antagonist Non-peptide (2S)-N-(3,5-dimethylphenyl)-1-(4-methoxyphenyl)sulfonylpyrrolidine-2-carboxamide
ACT-541468 Dual receptor Antagonist Non-peptide [(2S)-2-(5-Chloro-4-methyl-1H-benzimidazol-2-yl)-2-methylpyrrolidin-1-yl]-[5-methoxy-
(Daridorexant) 2-(triazol-2-yl)phenyl]methanone (Treiber et al.,
2017)
(Continued)

TABLE 2 | Continued
Name Selectivity Peptide or Chemical name/Peptide sequence

Non-peptide
Agonists
Orexin A (Hypocretin-1) Dual receptor endogenous agonist Peptide H-DL-Pyr-Pro-Leu-Pro-Asp-Cys(1)-Cys(2)-Arg-Gln-Lys-Thr-Cys(1)-Ser-Cys(2)-Arg-Leu-
Tyr-Glu-Leu-Leu-His-Gly-Ala-Gly-Asn-His-Ala-Ala-Gly-Ile-Leu-Thr-Leu-NH2
Orexin B (Hypocretin-2) Dual receptor endogenous agonist Peptide H-Arg-Ser-Gly-Pro-Pro-Gly-Leu-Gln-Gly-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Ala-Ser-Gly-
Asn-His-Ala-Ala-Gly-Ile-Leu-Thr-Met-NH2
[Ala11 ,D-Leu15 ] OX2 R selective agonist Peptide H-Arg-Ser-Gly-Pro-Pro-Gly-Leu-Gln-Gly-Arg-Ala-Gln-Arg-Leu-D-Leu-Gln-Ala-Ser-Gly-
-OX-B Asn-His-Ala-Ala-Gly-Ile-Leu-Thr-Met-NH2 (Asahi et al.,
2003)
Nag 26 OX2 R selective agonist Non-peptide 40 -methoxy-N,N-dimethyl-30 -[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl
sulfamoyl]-(1,10 -biphenyl)-3-carboxamide (Nagahara et al., 2015)
YNT-185 OX2 R selective agonist Non-peptide 2-(dimethylamino)-N-[2-[3-[[5-[3-(dimethylcarbamoyl)phenyl]-2-
methoxyphenyl]sulfonylamino]anilino]ethyl]benzamide
TAK-925 OX2 R selective agonist Non-peptide methyl (2R,3S)-3-[(methylsulfonyl)amino]-2-{[(cis-4-
phenylcyclohexyl)oxy]methyl}cpiperidine-1-carboxylate (Yukitake et al.,
2019)
Yan 7874 Weak partial non-selective agonist Non-peptide 1-(3,4-dichlorophenyl)-2-[2-imino-3-(4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-
1-yl]ethan-1-ol (Turku et al.,
2017)
From observations on the effects of both ligands on In contrast, OX-B was observed to be more potent in
second messenger cascades in CHO cells, Holmqvist et al. stimulating IP3 production in human testicular membrane
(2005) demonstrated that OX-A and OX-B do not exhibit preparations (Karteris et al., 2004). While it is suggested this
the canonical affinity-efficacy relationship at OX1 R that had indicates a predominantly OX2 R-mediated effect, no further
been previously described. While the relative potencies of investigation was conducted.
the ligands were as expected for the Gαq -mediated cascades, The possible implications of such differences are highlighted
for the Gαs -mediated cascades the relative potencies of the by Patel et al. (2018) in which OX-B, but not OX-A, was
two ligands were observed to be similar. Tang et al. (2008) found to produce contractile shortening through OX2 R in
also observed biased agonism of OX-A and OX-B in their cardiomyocytes. It was further observed that OX-B exerted
activation of G proteins in HEK293 cells stably expressing a cardioprotective effect in rat heart models. The G protein-
OX2 R. In HEK293 cells, OX-B exhibited bias toward Gαq - based signaling mechanisms underlying these effects were
mediated pathways while OX-A seemed to favor Gαs - and Gαi - not thoroughly investigated in this study, however the
mediated pathways of ERK activation. Neither OX-A nor OX-B phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) and
showed total bias to a specific pathway for ERK activation, ERK1/2/MAPK pathways were implicated as mediators of the
with activity detected for all three G protein cascades that OX-B-induced cardioprotective effects observed.
were screened with both OX-A and OX-B. However, it is Experiments further investigating these ligand-dependent
also of note that this study utilized dominant-negative G biases, including effects on other cellular machinery and
proteins, which have been reported to have non-specific effects translation of findings into higher-order cell or tissue
(Kukkonen, 2004). environments, would give great insight into the relative
Gorojankina et al. (2007) also noted differences in the roles of OX-A and OX-B.
action of the orexin ligands within an immortalized cell line
of olfactory sensory neuron lineage (Murrell and Hunter, Exogenous Ligands
1999) transfected with OX1 R-EGFP or OX2 R-EGFP. In this The majority of drug discovery efforts targeting the orexin
cell line, OX-B produced a synergistic increase in cAMP system have been focused on the development of antagonists
production with co-treatment of forskolin, however, this was as treatments for insomnia and other sleep and mental health
not observed with treatment of OX-A. This was observed disorders. This has led to the development of selective and
with both receptors. potent small molecule orexin receptor antagonists as shown
A differential role for OX-A and OX-B in peripheral in Table 2. Antagonist research has focused on DORAs which
tissues, in particular adrenal tissue, was postulated early have been, at least initially, preferred for clinical applications,
on, when OX-A but not OX-B stimulation resulted in a since double receptor knockout mice had the most pronounced
concentration-dependent increase in basal cAMP production sleep phenotypes (Mang et al., 2012). However, there is
(Mazzocchi et al., 2001). Initially this was thought to be due evidence that dual antagonism can promote cataplexy-like
to the presence of OX1 R with only low level detection of symptoms in rodents (Black et al., 2013) and the Food and
OX2 R (Mazzocchi et al., 2001), however further research Drug Administration (FDA) has issued a warning about such
conflicted with these findings (Randeva et al., 2001). events in humans (Farkas, 2013; Hoyer and Jacobson, 2013).

Suvorexant and lemborexant are the only orexin-targeted drugs Ca2+ elevation and β-arrestin2 recruitment, the endogenous
on the market for the treatment of insomnia, with the New ligands had equal potency while the potency of TAK-925
Drug Application for Idorsia’s daridorexant (ACT-541468) just was 30–50- fold lower; all ligands had the same maximum
being approved by the FDA. Multiple other DORAs and response. No data were presented on the relative potency
selective orexin receptor antagonists (SORAs), are currently of the agonist on non-Gαq mediated effects. As more is
under development/in clinical trials for the treatment of a revealed about the interplay of multiple G proteins in orexin
variety of CNS disorders, such as depression, anxiety, panic signaling, the potential for biased agonists may become a
or PTSD (Fitch et al., 2014; Bonaventure et al., 2015, 2017; particularly interesting consideration for the therapeutic
Boss et al., 2020; Kaufmann et al., 2020; Maehara et al., 2020; targeting of this system.
Zammit et al., 2020b). Alongside the development of antagonists The development of orexin-targeted therapeutics, especially
for clinical use, highly selective and potent orexin antagonists agonists, is still in the early stages with much more work needed
have also been produced for research applications as these to explore their therapeutic potential. Interest in orexin-targeted
compounds are highly desirable for probing receptor-specific therapeutics has remained strong since initial research began in
effects (Nagase et al., 2017). the early 2000’s. Significant efforts have been made in recent
In contrast to antagonist research that has focused on non- years to produce novel small molecule orexin agonists and
selective compounds, much of the research relating to orexin antagonists which hold exciting potential for future therapeutics.
receptor agonists is focused on OX2 R-selective agonists. This is The translation of these compounds to clinical use has been
in part due to evidence indicating that stimulation of OX1 R in recently demonstrated with Lemborexant being approved in the
the ventral tegmental area increases dopaminergic transmission, US and Japan, and the FDA approving daridorexant in January
raising concerns that OX1 R-targeted agonists may result in 2022, as well as other compounds, e.g., seltorexant, being in late
dependency issues (Nakamura et al., 2000; Yukitake et al., stage clinical development (Dauvilliers et al., 2020; Hoyer et al.,
2019). For the agonists that have been developed (Nagahara 2020; Kaufmann et al., 2020; Muehlan et al., 2020a,b; Zammit
et al., 2015; Turku et al., 2016, 2019; Leino et al., 2018; Rinne et al., 2020a,b).
et al., 2018), comprehensive analysis of their pharmacological
profiles has not been conducted. Studies have mainly relied
upon Ca2+ measurements to determine receptor selectivity and CONCLUSION
agonist activity, since until recently, FLIPR has been a traditional
workhorse for drug screening in industry. Given the apparent Research into orexin pharmacology, while fragmented, presents
differences between OX-A and OX-B at both orexin receptors, a body of interesting and exciting research, which demonstrates
detailed pharmacological profiling of these new synthetic agonists many complex signaling behaviors that could hold vital insights
will hopefully assist in developing our understanding of the into orexin system function and GPCR pharmacology as a whole.
pharmacological function of the orexin receptors. The recent development of various biophysical assay panels
Initial reports indicate further optimization may be (Wan et al., 2018; Avet et al., 2020; Olsen et al., 2020) allows
needed before some of the proposed agonists are suitable for monitoring of the effect of receptor stimulation on a wide
for clinical applications (Irukayama-Tomobe et al., 2017), range of GPCR effectors, in live cells and in real-time. Applying
however the OX2 R-selective agonist YNT-185 has shown technologies such as these to study orexin receptor signaling
positive results in ameliorating morphine-induced sedation will allow investigation of orexin receptor pharmacology with
without affecting analgesia in a rat model (Toyama et al., greater detail and precision. Indeed, the elucidation of orexin
2018). Additionally, the OX2 R-selective agonist TAK-925 function in peripheral environments, and the development of
has shown wake-promoting effects in wild-type mice, but novel, small molecule ligands in recent years demonstrates
not OX2 R-knockout mice, indicating target-specific effects that there is undoubtedly much still left to be discovered
in an in vivo model (Yukitake et al., 2019) as was expected about this system.
from original studies performed in a variety of orexin
receptor knockout mice (Willie et al., 2003; Mang et al.,
2012).
AUTHOR CONTRIBUTIONS
As the orexin system has been demonstrated to stimulate All authors listed have made a substantial, direct, and intellectual
multiple downstream signaling pathways, there is exciting contribution to the work, and approved it for publication.
potential for therapeutically harnessing any existing ligand-
induced bias. An example of this potential is seen in Yukitake
et al. (2019) in which the OX2 R-selective agonist TAK-925 was FUNDING
characterized using an array of methods.
With respect to phospholipase C activity, TAK-925 and This work was supported by an Australian Government Research
the endogenous ligands had equal potency and maximum Training Program Scholarship and a University of Western
response. For cAMP response element-binding protein Australia Baillieu Research Scholarship (ND), an Australian
(CREB) and ERK phosphorylation, the potencies for all Research Council Industrial Transformation Training Centre
three ligands were essentially the same but the maximum IC170100016 Postdoctoral Fellowship (EJ), and an NHMRC
response to TAK-925 was somewhat lower. With respect to Ideas Grant APP20033770 (DH and LJ).

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published: 13 April 2022

Orexin Signaling: A Complex,

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and therefore understanding these interactions could reveal

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Orexin receptor Interacting receptor Reported findings

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TABLE 2 | Described orexin ligands.

Name Selectivity Peptide or Chemical name/Peptide sequence

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Name Selectivity Peptide or Chemical name/Peptide sequence

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