Cardiac action of the first G protein biased small molecule apelin agonist

Article abstract of DOI:10.1016/j.bcp.2016.07.018

Apelin peptide analogues displaying bias towards G protein signalling pathways have beneficial cardiovascular actions compared with the native peptide in humans in vivo. Our aim was to determine whether small molecule agonists could retain G protein bias.

Full text of DOI:10.1016/j.bcp.2016.07.018

Accepted Manuscript
Cardiac action of the first G protein biased small molecule apelin agonist
Cai Read, Christopher M. Fitzpatrick, Peiran Yang, Rhoda E. Kuc, Janet J.
Maguire, Robert C. Glen, Richard E. Foster, Anthony P. Davenport
PII:
S0006-2952(16)30195-2
http://dx.doi.org/10.1016/j.bcp.2016.07.018
BCP 12598
DOI:
Reference:
Biochemical Pharmacology
To appear in:
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Accepted Date:
26 May 2016
25 July 2016
Please cite this article as: C. Read, C.M. Fitzpatrick, P. Yang, R.E. Kuc, J.J. Maguire, R.C. Glen, R.E. Foster, A.P.
Davenport, Cardiac action of the first G protein biased small molecule apelin agonist, Biochemical Pharmacology
(2016), doi: http://dx.doi.org/10.1016/j.bcp.2016.07.018
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Cardiac action of the first G protein biased small molecule apelin agonist
Running title: Biased Small Molecule Apelin Agonist
Cai Reada*, Christopher M. Fitzpatrickb*, Peiran Yanga*, Rhoda E. Kuca, Janet J.
Maguirea†, Robert C. Glenc, d†, Richard E. Fosterb†, Anthony P. Davenporta†
*These authors contributed equally
†These authors were joint principal investigators
aExperimental Medicine and Immunotherapeutics, Addenbrooke’s Hospital,
University of Cambridge
bSchool of Chemistry and Astbury Centre for Structural Biology, University of Leeds
cCentre for Molecular Informatics, Department of Chemistry, University of Cambridge
dBiomolecular Medicine, Department of Surgery and Cancer, Imperial College
London, London, UK
Author for Correspondence
Dr Anthony P Davenport
Tel: 01223 336899
Fax: 01223 762576
Experimental Medicine and Immunotherapeutics
Level 6 Centre for Clinical investigation
Box 110 Addenbrooke’s Hospital
Cambridge, CB2 0QQ, UK
Email: apd10@medschl.cam.ac.uk

1. Introduction
The apelin receptor is a class A G protein-coupled receptor first cloned in 1993 [1]
and was deorphanised in 1998 when its cognate ligand, apelin, was identified from
bovine stomach extracts [2]. The predominant isoform in the human cardiovascular
system is [Pyr1]apelin-13 [3]. Infusion of apelin leads to vasodilatation, in humans in
vitro [3] and in vivo [4] and in rodents in vivo [5], as well as cardiac inotropy in vitro
[3,6,7] and in vivo in rats [8-10], mice [11] and humans [12] without hypertrophy.
These properties of apelin signalling have made it of particular interest as a potential
therapeutic for a number of diseases such as pulmonary arterial hypertension (PAH)
and heart failure (HF) [13]. Moreover, there is significant evidence to suggest that
while the apelin peptide is downregulated in both PAH [14-16] and HF [17-19],
expression of the receptor remains unchanged and responsive to apelin [15, 20, 21].
This provides an opportunity to replace the downregulated endogenous agonist in a
therapeutic setting. Previous work has shown that infusion of apelin is beneficial in a
number of disease models of PAH [22] and HF [8-10, 23-25], but long term
therapeutic efficacy is limited by a short half-life of a few minutes. Oral therapy for
these chronic diseases would necessitate a compound with longer half-life.
Furthermore, upon activation of the receptor, bound apelin is rapidly internalised
through the β-arrestin pathway with the rate of recycling back to the plasma
membrane highly dependent on the ligand [26, 27]. Therefore, agonist induced
desensitisation may limit clinical efficacy.
We hypothesise that a G protein biased small molecule apelin agonist could provide
a solution to this limitation. We have previously designed apelin peptide analogues
that are G protein biased and this strategy resulted in an improved duration of action
2

and increased efficacy [4]. Alteration of a specific serine residue (Ser348) in the apelin
receptor has been shown to abolish the G protein receptor kinase/ β-arrestin pathway
signalling whilst preserving signalling through the G protein pathway. It has also been
reported that removal of the C-terminal phenylalanine of apelin-17 can induce bias
towards G protein signalling [28]. These reports suggest that the apelin receptor is
tractable to biased signalling [29]. Here we identify CMF-019, a small molecule,
which binds to the human apelin receptor with high affinity, exerts a biased response
through the G protein pathway and mimics the beneficial cardiovascular actions of
apelin in rodents.
3

2. Methods
2.1 Materials
Chemicals were obtained from Sigma Aldrich Co. Ltd (Poole, UK) unless otherwise
stated. [Pyr1]apelin-13 (Glp-RPRLSHKGPMPF) was from Severn Biotech
(Kidderminster, UK), [Glp65,Nle75,Tyr77][125I]apelin-13, was from Perkin Elmer (MA,
USA). CMF-019 was synthesised in the School of Chemistry, University of Leeds.
Human tissues were obtained with informed consent from the Papworth Hospital
Research Tissue Bank (08/H0304/56) and ethical approval (05/Q104/142) and
conformed to the principles outlined in the declaration of Helsinki. Animal
experiments were performed in accordance with guidelines from the local ethics
committee (University of Cambridge) and the Home Office (UK) under the Scientific
Procedures Act (1986).
2.2 Homology Modelling of the Apelin Receptor/Apelin-13 and Ligand Receptor
Docking of CMF-019
A homology model was constructed from the 2.5Å resolution crystal structure of the
human CXCR4 chemokine receptor. MODELLER9v8 was used to generate
homology models of apelin which were subsequently refined using molecular
dynamics. A more detailed description of the procedure is given in Brame et al.
(2015) [4]. Water and ions were removed in preparation for docking studies. The
model of CMF-019 (carboxylic acid ionized) was constructed using Sybyl 7.3 and
energy minimized in vacuo using the TriposFF and Gasteiger-Huckel charges.
Docking was performed using the program GOLD (CCDC 2015) [30, 31].
It was hypothesized that the agonist, CMF-019, occupied a site in close proximity to
the site occupied by apelin-13 within the pocket formed by the seven transmembrane
4

helices of the receptor. However, initial docking of CMF-019 into this long pocket
formed from the receptor model resulted in a number of possible solutions. To
constrain the system, we utilized the previously determined homology model of
bound apelin-13 [4], selecting the region containing the serine-histidine-lysine (SHK)
sequence of apelin-13 as the most promising region for binding of CMF-019 (which is
adjacent to the important residue Tyr88 identified from mutagenesis studies). A
receptor cavity composed of residues up to 12Å from the SHK region of apelin-13
was used for docking. The ChemPLP scoring function was used with standard GOLD
settings.
2.3 Synthesis of CMF-019
CMF-019 ((S)-3-[1-(1-Ethyl-propyl)-2-thiophen-2-ylmethyl-1H-benzoimidazole-5-
carbonyl]-amino-5-methyl-hexanoic acid) has previously been prepared (compound
number 107 from patent US20140094450, 2014) via a minor modification of the
literature procedure (Figure 2) [32]. For in vivo studies we used the potassium salt of
the compound (Figure 1) dissolved in saline at pH9 as it showed better solubility than
the parent compound. Thermodynamic solubility (potassium salt) and an in vitro
murine microsomal stability of CMF-019 were performed at Cyprotex (Macclesfield,
UK).
2.4 Competition Radioligand Binding
Initial binding assays were performed by Cerep (Celle L’Evescault, France) on
Chinese Hamster Ovary (CHO-K1) cells expressing the human apelin receptor.
Further experiments were performed on human homogenised left ventricle (HLV,
1.5mg/ml) and rat and mouse whole heart homogenates to determine any species
variability prior to in vivo experiments. Competition binding experiments were carried
5

out using [Glp65,Nle75,Tyr77][125I]apelin-13 (0.1nmol/L) as described previously [4],
with unlabelled CMF-019 (2pmol/L-10µmol/L) as the competitive agonist. Binding in
the presence of 2µmol/L [Pyr1]apelin-13 was considered non-specific. Experiments
were performed in triplicate and data analysed using GraphPad Prism 6 (GraphPad
Software, Inc. La Jolla, USA). Binding affinities were calculated by the Cheng-Prusoff
methodology using measured IC50 values.
2.5 Cell-Based Assays
β-Arrestin recruitment, β-arrestin mediated internalisation and cAMP assays were
performed according to the manufacturer’s instructions (DiscoverX®, Fremont, CA)
and as described previously [4]. Agonist responses, measured in relative light units,
were expressed as a percentage relative to the EMAX of [Pyr1]apelin-13 for β-arrestin
and internalisation experiments and as a percentage relative to the forskolin
response for cAMP experiments. Data were fitted to a four parameter model using
GraphPad Prism 6 and the pD2 (-log10 of the EC50 (the concentration producing 50%
of the maximum response)) and maximum response (EMAX) values were calculated
and compared.
2.6 Rat Heart Catheterisation and Jugular Vein Cannulation for in vivo
Assessment of CMF-019
Male Sprague-Dawley rats (273 6g; Charles River Laboratories, Margate, UK) were
induced to anaesthesia with inhalation of 3% isoflurane and then maintained under
1.5% isoflurane via a face mask. Temperature was monitored throughout the surgical
procedure using a rectal probe. Prior to the start of surgery, it was ensured that pain
reflexes had ceased by use of a hind-paw pinch test. The surgery was completed as
previously described [33]. In brief, the right external jugular vein was cannulated,
6

flushed with heparin solution (2%, 0.9% saline, pH5, Macopharma) and the right
common carotid artery located. The catheter (Millar Inc., SPR-869) was calibrated
using the MPVS Ultra system (ADIstruments) before being inserted into the carotid
and advanced to the left ventricle. Once a stable pressure-volume loop could be
observed, the catheter was left in place for ten minutes. Three cumulative bolus
doses of CMF-019 (50-5000nmol, 0.5mL, 0.9% saline, pH9, Macopharma, n=7-9)
were then administered intravenously, followed by a saline flush (0.9%, 0.1mL, pH5)
via the cannula at ten minute intervals, so that a stable baseline was reached before
the next injection. The effects on pressure and volume were measured and the heart
rate monitored to determine and maintain a suitable depth of anaesthesia. Control
animals were injected with saline (0.9%, 0.5mL, pH9) and a flush of equal volume
(0.9%, 0.1mL, pH5) to distinguish the drug effects from any volume effects (n=3-5).
The effects of [Pyr1]apelin-13 (50, 400nmol, 0.5mL, 0.9% saline, pH5, Macopharma,
n=8) were studied for comparison. Animals were randomly chosen to receive CMF-
019, saline or [Pyr1]apelin-13 injection. Data were acquired using the MPVS Ultra
system (ADIstruments) and analysed using LabChart 8 (ADIstruments). Values for
the maximal change in left ventricular systolic pressure (LVSP), stroke volume,
cardiac output, contractility (dP/dtMAX) and lusitropy (dP/dtMIN) from baseline were
calculated from the raw data and compared. Following completion of the
measurements the animal was euthanised by exsanguination under high flow
isoflurane.
2.7 Analysis of Plasma Samples by Mass Spectrometry
Analysis of plasma samples was performed by Peakdale Molecular (Chapel-en-la-
Frith, UK). End-point blood samples were taken from rats (n=6) following completion
of three cumulative doses of CMF-019 (as described before), approximately 10
7

minutes after the last intravenous administration. The blood was collected in heparin-
coated vials and spun at low speed (2000g, 5min), the plasma supernatant was
removed and frozen. Quantitative analysis was performed using LC-MS/MS with
metaprolol as an internal standard and CMF-019 to quantify CMF-019 in the
samples.
2.8 Computational Assessment of Oral Drug Properties
CMF-019 was assessed for drug-like properties using FAF-Drugs3 [34]. This
compared the properties of CMF-019 to a library of oral drugs and predicted its
physiochemical properties according to Lipinski’s and Verber’s rules. In addition,
CMF-019 was analysed using a principal component analysis (PCA) of 15 key
physiochemical properties and compared to the properties of oral drugs in current
use, extracted as sublibraries from the databases, eDrugs and DrugBank.
2.9 Statistical Analysis
All data are expressed as mean SEM values. Binding and cell-based experiments
were performed in triplicate. For cell based assays, n-values are given as the number
of replicates/number of experiments. Bias calculations were performed, as previously
described [35], to obtain values for relative effectiveness of CMF-019 compared to
[Pyr1]apelin-13 within each cell-based assay and bias factors calculated to compare
the relative activities of the agonists between different pathways. For the in vivo
study, values for the cardiovascular parameters measured in saline and CMF-019
treated animals were compared using a two-tailed student’s t-test (GraphPad Prism
6). Statistical significance was taken as 5%.
8

3. Results
3.1 Computational Docking of CMF-019
CMF-019 bound into a mainly hydrophobic cavity on the apelin receptor near the
SHK region of bound apelin-13. The carboxylic acid of CMF-019 was predicted to
form a strong hydrogen bond (2.3Å) with the side-chain of Arg168. The thiophene,
which is important for maintenance of good potency, formed a π-stacking interaction
with residue Tyr88. A ligand/protein plot of these close interactions between CMF-019
and the apelin receptor was generated (Figure 3A), as well as a close up view of the
ligand receptor interactions (Figure 3B). To compare the similarity in the expected
binding regions of CMF-019 and the predicted apelin-13 docked structure, they were
overlaid (Figure 3C).
3.2 CMF-019 Binds to the Human Apelin Receptor with High Affinity
CMF-019 bound to the human apelin receptor expressed in CHO cells with
nanomolar affinity (pKi=7.61 0.14). In human left ventricle, CMF-019 showed
monophasic competition and a ten-fold higher binding affinity to the native apelin
receptor (pKi=8.58 0.04, Figure 4A). Similar binding was observed in rat and mouse
heart (pKi=8.49 0.04, Figure 4B; pKi=8.71 0.06, Figure 4C respectively).
3.3 CMF-019 Displays G Protein Bias
CMF-019 induced β-arrestin recruitment (Figure 5B) to the apelin receptor
(pD2=6.65 0.15, n=13/4) and subsequent receptor internalisation (Figure 5C)
(pD2=6.16 0.21, n=6/2) but was less potent than [Pyr1]apelin-13 (pD2=8.65 0.10,
n=12/4; pD2 = 9.28 0.10, n=6/2 respectively). In contrast, CMF-019 potently inhibited
Gαi-mediated cAMP production (Figure 5A) (pD2=10.00 0.13, n=11/4), comparable to
9

[Pyr1]apelin-13 (pD2=9.34 0.15, n=8/4). In all assays, EMax values for CMF-019 were
similar to those obtained for [Pyr1]apelin-13 suggesting that the compound acted as a
full agonist at the apelin receptor.
To quantify the degree to which CMF-019 displayed bias compared to the reference
agonist [Pyr1]apelin-13, relative effectiveness (RE) and bias factors (BF) were
calculated as described previously [35] and are shown in Tables 1 and 2
respectively.
3.4 CMF-019 is a Positive Inotrope in vivo
Injection of CMF-019 caused a dose dependent increase in the dP/dtMAX, a measure
of cardiac contractility, with significance at 500nmol of 606 112mmHg/s (p<0.001)
and at 5000nmol of 833 152mmHg/s (p<0.01) (Figure 6A). [Pyr1]apelin-13 also
caused a dose dependent increase with a significant response at 400nmol of
3025 680mmHg/second (P<0.01) (Figure 6B). [Pyr1]apelin-13 dose dependently
caused a significant increase in stroke volume and cardiac output up to a response at
400nmol of 9.18 0.86RVU (P<0.0001) and 3989 537RVU/min (P<0.001)
respectively (Figure S1B,D). CMF-019 similarly increased these parameters with
smaller responses detected. Changes at 500nmol of 2.62 0.31RVU (P<0.05) and
1210 170RVU/min (P<0.01) respectively were significant (Figure 7A,C). Neither
[Pyr1]apelin-13 nor CMF-019 altered the dP/dtMIN, a measure of lusitropy (Figure 7E-
F). [Pyr1]apelin-13 caused a significant dose dependent drop in the LVSP with a
maximal drop of 15.7 1.8mmHg detected at 400nmol (P<0.01) (Figure 7G), however,
CMF-019 resulted in a dose dependent increase in pressure with a significant
increase of 6.22 1.98mmHg at 5000nmol (P<0.05) (Figure 7H). A limitation of this
study was the relatively low solubility of CMF-019 (potassium salt) in saline (2.09
10

mg/mL at pH7.4) that did not allow higher doses to be infused. No adverse effects
were observed at any of the concentrations administered.
3.5 CMF-019 is Present in Rat Plasma Samples
CMF-019 had a half-life of 38 minutes in the in vitro microsomal stability experiment.
Analysis of the plasma samples taken from rats treated with CMF-019 (potassium
salt) demonstrated a mean plasma concentration of 25.4 2.5µM (heparinised
samples, n=6).
3.6 CMF-019 Demonstrates Good Physiochemical Properties for an Oral Drug
Analysis of CMF-019 using FAF-Drugs3 [34] demonstrated a good physiochemical
profile when compared to an oral drug library for Lipinski’s rules (Figure 8A), Verber’s
rules (Figure 8B) and in a PCA analysis of 15 physiochemical descriptors (Figure
8C). CMF-019 had only one Lipinski’s Rule-of-Five violation which was for logP (the
octanol-water partition coefficient).
11

4. Discussion
4.1 CMF-019 is the First Evaluated Biased Small Molecule Agonist at the Apelin
Receptor
We have characterised and quantified the pharmacological properties of a small
molecule, CMF-019, predicted to bind to the apelin receptor in a hydrophobic cavity
near the SHK region of bound apelin-13 in computational models. CMF-019
demonstrated high affinity binding to native human, rat and mouse cardiac apelin
receptors, suggesting no evidence for species differences. In cells expressing the
human apelin receptor, CMF-019 had similar Gαi activity to [Pyr1]apelin-13 (RE=4.19),
however, for β-arrestin recruitment and internalisation, it was much less potent
(RE=0.01 and 7.19x10-4 respectively). CMF-019, thus, displayed significant bias
compared to the endogenous agonist [Pyr1]apelin-13. This bias was towards the G
protein signalling pathway, with a bias factor of ~400 compared to apelin receptor
mediated β-arrestin recruitment and ~6000 compared to β-arrestin mediated receptor
internalisation. This is predicted to be beneficial in a therapeutic setting as reduced
receptor internalisation would allow for maintained apelin signalling with repeated
drug administration. Indeed, our previously published work has shown that MM07, a
novel biased peptide agonist, induced reproducible dilatation in the human forearm
[4]. This work provided proof of concept for the merits of a biased agonist and our
current work has focussed on the investigation of a small molecule agonist with this
desirable pharmacological profile. CMF-019 is one of a larger series of apelin
agonists based on the benzimidazole scaffold [32] and these additional molecules
may also display a similar bias profile.
To our knowledge this is the first report of bias for a small molecule apelin agonist.
To date, previous synthetic apelin agonists lack desirable characteristics required of
12

drug-like molecules. E339-3D6, a reported peptidomimetic agonist did exhibit
vasoactivity in rat aorta preconstricted with noradrenaline and in vivo prevented
vasopressin release when injected intracerebroventricularly in water-deprived mice
[36]. However, the high molecular weight (1400Da) suggests it would be an
unsuitable drug molecule, particularly for oral dosing. Moreover, it was later shown to
be a mixture of polymethylated species from which a number of analogues were
subsequently purified [37]. These molecules, displayed low micromolar binding
affinities for the apelin receptor compared to CMF-019 which binds in the nanomolar
range. ML233, a small molecule of molecular weight of 359Da, was limited by low
solubility in saline at room temperature [38]. Moreover, the structure suggests that it
would likely be a pan-assay interference compound (PAINS) and have toxicity,
including being a Michael acceptor and having an activated quinone [34]. In contrast
to these previously reported compounds, CMF-019 is more drug-like and shows a
good physiochemical profile when compared to an oral drug library for Lipinski’s rules
(Figure 8A), Verber’s rules (Figure 8B) and in a PCA analysis of 15 physiochemical
descriptors (Figure 8C). CMF-019 has one Lipinski’s Rule-of-Five violation (logP),
although this is ameliorated by the ionisation of the carboxylic acid at physiological
pH, reducing the logD to a more acceptable range (logD=2.74 at pH7.4). Following
the completion of the in vivo studies, we detected a mean plasma concentration of
~25µM suggesting that CMF-019 is relatively stable with significant compound
remaining in the plasma after ten minutes. In contrast, the plasma half-life of
[Pyr1]apelin-13 in rat has previously been measured to be ~2 minutes [4].
4.2 CMF-019 Affects Cardiac Action in vivo
We examined the effects of CMF-019 in vivo when administered to male Sprague-
Dawley rats. We used the potassium salt of the compound dissolved in saline at pH9
13

that showed better solubility than the parent compound. CMF-019 caused a dose
dependent increase in dP/dtMAX, an index of increased cardiac contractility. A similar
response was observed for [Pyr1]apelin-13 and is consistent with its role as an
inotropic agent through the apelin receptor. [Pyr1]apelin-13 also caused a decrease in
LVSP, whereas, CMF-019 caused an increase. The blood pressure response to
[Pyr1]apelin-13 can be explained by its action as a vasodilatory agent. The fact that
CMF-019 did not mirror [Pyr1]apelin-13 may simply reflect its limited solubility and a
higher concentration might be required to observe an effect on the vasculature.
Indeed, it was previously demonstrated that MM07, a novel G protein biased apelin
agonist, causes vasodilatation in forearm blood flow studies in human volunteers [4].
Alternatively, the cardiac action of CMF-019 may mask any vasodilator response.
However, there is a report that modified apelin-17 peptide fragments biased towards
the β-arrestin pathway are more able to induce decreases in blood pressure [39] and
this could explain the limited effects of CMF-019 on the vasculature. [Pyr1]apelin-13
also increased cardiac output due to an increased stroke volume rather than an
increased heart rate. CMF-019 similarly showed a trend to increase stroke volume
and cardiac output (with a significant effect observed at 500nmol) without a change in
heart rate. [Pyr1]apelin-13 and CMF-019 did not alter the dP/dtMIN, suggesting that
neither had lusitropic effects. Overall, the responses to CMF-019 showed a similar
profile to [Pyr1]apelin-13 and are consistent with it acting at the apelin receptor in
vivo, corroborating the in vitro data. Future studies will look at the development of
novel compounds to maintain the beneficial bias profile whist improving solubility.
14

4.3 CMF-019 is Predicted to Bind to a Highly Conserved Region of the Apelin
Receptor
Analysis of the predicted binding region of CMF-019 within the hydrophobic cavity
near the SHK region of bound apelin-13 in computational models revealed close
proximity to the only reported naturally occurring mutation in the apelin receptor to
date. In the zebrafish, Danio rerio, a single allele of a recessive mutation, grinchs608
leads to a Trp85 to Leu85 amino acid change in the second transmembrane domain
and results in a complete loss of apelin binding. As a consequence of this loss of
function, in the most severely affected mutants the heart fails to develop [40].
,
Interestingly, in the SHK region of apelin-13 in our model is the almost adjacent Tyr88
residue. Importantly, both residues have been conserved in humans and all species
where apelin receptor sequences have been reported, suggesting CMF-019 binds in
a critical region of the receptor. Interestingly, in support of our proposed model, a
series of small molecule apelin agonists have been reported [41] that share some
structural similarity to CMF-019, having two hydrophobic substituents extending from
a heterocyclic core, suggesting that they may bind to the same site of the apelin
receptor. It is not yet known if these compounds also display receptor bias.
4.4 Conclusions
CMF-019 is the first biased small molecule apelin agonist reported and binds with
high affinity to the apelin receptor in human heart homogenate. Binding of CMF-019
was predicted through molecular modelling to occur to a region in the apelin receptor
that, by comparison with other species, is highly conserved and is crucial for
interaction with the SHK amino acid sequence of apelin-13. In rats, in vivo, CMF-019
15

produced a dose dependent increase in cardiac contractility, corroborating its in vitro
activity.
Importantly, acute apelin infusion is beneficial in PAH and HF and it is predicted that
apelin agonists would display efficacy in treating these conditions. We have
previously designed apelin peptide analogues that display bias towards the G protein
signalling pathway and have beneficial cardiovascular actions compared with the
native peptide in humans in vivo. Retention of bias towards G protein signalling over
β-arrestin recruitment/internalisation in a small molecule should prove beneficial in a
therapeutic setting by reducing receptor downregulation following chronic use,
thereby preventing the need for dose escalation or patients becoming refractory to
treatment. Consequently, compounds based on the structure of CMF-019 with an
improved pharmacokinetic profile could provide a novel and much needed therapy to
treat PAH and HF.
Acknowledgements
We thank the Papworth Hospital Research Tissue Bank and NIHR Cambridge
Biomedical Research Centre.
Conflict of Interests
None declared.
Funding
This work was supported by the British Heart Foundation [FS/14/59/31282 to CR];
Wellcome Trust [WT107715/Z/15/Z to APD, REK], Wellcome Trust Programme in
Metabolic and Cardiovascular Disease [096822/Z/11/Z to PY]; Medical Research
Council [MRC WT107715/Z/15/Z to APD, JJM, RCG, PY]; Pulmonary Hypertension
16

Association UK; Cambridge Biomedical Research Centre Biomedical Resources
Grant University of Cambridge [099156/Z/12/Z]; Engineering and Physical Sciences
Research Council [EP/M506552/1 to CMF]; and the Biomedical Health Research
Centre, University of Leeds [CMF, REF].
References
1.
2.
O'Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, Shi X,
Petronis A, George SR, Nguyen T. A human gene that shows identity with
the gene encoding the angiotensin receptor is located on chromosome 11.
Gene 1993;136:355-360.
Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX,
Kawamata Y, Fukusumi S, Hinuma S, Kitada C, Kurokawa T, Onda H,
Fujino M. Isolation and characterization of a novel endogenous peptide
ligand for the human APJ receptor. Biochem Biophys Res Commun.
1998;251:471-476.
3.
4.
Maguire JJ, Kleinz MJ, Pitkin SL, Davenport AP. [Pyr1]apelin-13 identified
as the predominant apelin isoform in the human heart: vasoactive
mechanisms and inotropic action in disease. Hypertension 2009;54:598-
604.
Brame AL, Maguire JJ, Yang P, Dyson A, Torella R, Cheriyan J, Singer M,
Glen RC, Wilkinson IB, Davenport AP. Design, Characterization, and First-
In-Human Study of the Vascular Actions of a Novel Biased Apelin Receptor
Agonist. Hypertension 2015;65:834-40
5.
6.
Tatemoto K, Takayama K, Zou MX, Kumaki I, Zhang W, Kumano K,
Fujimiya M. The novel peptide apelin lowers blood pressure via a nitric
oxide-dependent mechanism. Regul Pept. 2001;99:87-92.
Szokodi I, Tavi P, Foldes G, Voutilainen-Myllyla S, Ilves M, Tokola H,
Pikkarainen S, Piuhola J, Rysä J, Tóth M, Ruskoaho H. Apelin, the novel
endogenous ligand of the orphan receptor APJ, regulates cardiac
contractility. Circ Res 2002;91:434-440.
7.
Perjés Á, Skoumal R, Tenhunen O, Kónyi A, Simon M, Horváth IG, Kerkelä
R, Ruskoaho H, Szokodi I. Apelin increases cardiac contractility via protein
kinase Cε- and extracellular signal-regulated kinase-dependent
mechanisms. PLoS One. 2014;9:e93473.
8.
9.
Berry MF, Pirolli TJ, Jayasankar V, Burdick J, Morine KJ, Gardner TJ, Woo
YJ. Apelin has in vivo inotropic effects on normal and failing hearts.
Circulation 2004;110:187-193.
Atluri P, Morine KJ, Liao GP, Panlilio CM, Berry MF, Hsu VM, Hiesinger W,
Cohen JE, Woo YJ. Ischemic heart failure enhances endogenous
17

myocardial apelin and APJ receptor expression. Cell Mol Biol Lett
2007;12:127-138.
10.
11.
Jia YX, Pan CS, Zhang J, Geng B, Zhao J, Gerns H, Yang J, Chang JK,
Tang CS, Qi YF. Apelin protects myocardial injury induced by isoproterenol
in rats. Regul Pept 2006;133:147-154.
Ashley EA, Powers J, Chen M, Kundu R, Finsterbach T, Caffarelli A, Deng
A, Eichhorn J, Mahajan R, Agrawal R, Greve J, Robbins R, Patterson AJ,
Bernstein D, Quertermous T. The endogenous peptide apelin potently
improves cardiac contractility and reduces cardiac loading in vivo.
Cardiovasc Res. 2005;65:73-82.
12.
Japp AG, Cruden NL, Barnes G, van Gemeren N, Mathews J, Adamson J,
Johnston NR, Denvir MA, Megson IL, Flapan AD, Newby DE. Acute
cardiovascular effects of apelin in humans: potential role in patients with
chronic heart failure. Circulation 2010;121:1818-1827.
13.
14.
15.
Yang P, Maguire JJ, Davenport AP. Apelin, Elabela/Toddler, and biased
agonists as novel therapeutic agents in the cardiovascular system. Trends
Pharmacol Sci 2015;36:560-567.
Goetze JP, Rehfeld JF, Carlsen J, Videbaek R, Andersen CB, Boesgaard
S, Friis-Hansen L. Apelin: a new plasma marker of cardiopulmonary
disease. Regul Pept 2006;133:134-138.
Kim J, Kang Y, Kojima Y, Lighthouse JK, Hu X, Aldred MA, McLean DL,
Park H, Comhair SA, Greif DM, Erzurum SC, Chun HJ. An endothelial
apelin-FGF link mediated by miR-424 and miR-503 is disrupted in
pulmonary arterial hypertension. Nat Med 2013;19:74-82.
Alastalo TP, Li M, Perez Vde J, Pham D, Sawada H, Wang JK, Koskenvuo
M, Wang L, Freeman BA, Chang HY, Rabinovitch M. Disruption of
PPARgamma/beta-catenin-mediated regulation of apelin impairs BMP-
induced mouse and human pulmonary arterial EC survival. J Clin Invest
2011;121:3735-3746.
Földes G, Horkay F, Szokodi I, Vuolteenaho O, Ilves M, Lindstedt KA,
Mäyränpää M, Sármán B, Seres L, Skoumal R, Lakó-Futó Z, deChâtel R,
Ruskoaho H, Tóth M. Circulating and cardiac levels of apelin, the novel
ligand of the orphan receptor APJ, in patients with heart failure. Biochem
Biophys Res Commun 2003;308:480-485.
16.
17.
18.
19.
Chong KS, Gardner RS, Morton JJ, Ashley EA, McDonagh TA. Plasma
concentrations of the novel peptide apelin are decreased in patients with
chronic heart failure. Eur J Heart Fail 2006;8:355-360.
Francia P, Salvati A, Balla C, De Paolis P, Pagannone E, Borro M, Gentile
G, Simmaco M, De Biase L, Volpe M. Cardiac resynchronization therapy
increases plasma levels of the endogenous inotrope apelin. Eur J Heart
Fail 2007;9:306-309.
20.
21.
Andersen CU, Markvardsen LH, Hilberg O, Simonsen U. Pulmonary apelin
levels and effects in rats with hypoxic pulmonary hypertension. Respir Med
2009;103:1663-1671.
Barnes GD, Alam S, Carter G, Pedersen CM, Lee KM, Hubbard TJ, Veitch
S, Jeong H, White A, Cruden NL, Huson L, Japp AG, Newby DE.
Sustained cardiovascular actions of APJ agonism during renin-angiotensin
18

system activation and in patients with heart failure. Circ Heart Fail
2013;6:482-491.
22.
Falcao-Pires I, Goncalves N, Henriques-Coelho T, Moreira-Goncalves D,
Roncon-Albuquerque R, Jr., Leite-Moreira AF. Apelin decreases
myocardial injury and improves right ventricular function in monocrotaline-
induced pulmonary hypertension. Am J Physiol Heart Circ Physiol
2009;296:2007-2014.
23.
24.
Koguchi W, Kobayashi N, Takeshima H, Ishikawa M, Sugiyama F,
Ishimitsu T. Cardioprotective effect of apelin-13 on cardiac performance
and remodeling in end-stage heart failure. Circ J 2012;76:137-144.
Wang M, Gupta RC, Rastogi S, Kohli S, Sabbah MS, Zhang K, Mohyi P,
Hogie M, Fischer Y, Sabbah HN. Effects of acute intravenous infusion of
apelin on left ventricular function in dogs with advanced heart failure. J
Card Fail 2013;19:509-516.
25.
26.
27.
Pang H, Han B, Yu T, Zong Z. Effect of apelin on the cardiac
hemodynamics in hypertensive rats with heart failure. Int J Mol Med
2014;34:756-764.
Zhou N, Fan X, Mukhtar M, Fang J, Patel CA, DuBois GC, Pomerantz RJ.
Cell-cell fusion and internalization of the CNS-based, HIV-1 co-receptor,
APJ. Virology 2003;307:22-36.
Lee DK, Ferguson SS, George SR, O'Dowd BF. The fate of the
internalized apelin receptor is determined by different isoforms of apelin
mediating differential interaction with beta-arrestin. Biochem Biophys Res
Commun 2010;395:185-189.
28.
29.
Ceraudo E, Galanth C, Carpentier E, Banegas-Font I, Schonegge AM,
Alvear-Perez R, Iturrioz X, Bouvier M, Llorens-Cortes C. Biased signaling
favoring gi over beta-arrestin promoted by an apelin fragment lacking the
C-terminal phenylalanine. J Biol Chem 2014;289:24599-24610.
Chen X, Bai B, Tian Y, Du H, Chen J. Identification of serine 348 on the
apelin receptor as a novel regulatory phosphorylation site in apelin-13-
induced G protein-independent biased signaling. J Biol Chem
2014;289:31173-31187.
30.
31.
32.
Jones G, Willett P, Glen RC. Molecular recognition of receptor sites using
a genetic algorithm with a description of desolvation. J Mol Biol
1995;245:43-53.
Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and
validation of a genetic algorithm for flexible docking. J Mol Biol
1997;267:727-748.
Hachtel S, Wohlfart P, Weston J, Müller M, Defossa E, Mertsch K, Weng J,
Robert A. Binnie, Abdul-latif F, Bock WJ, Walser A. Benzoimidazole-
carboxylic acid amide derivatives as apj receptor modulators. Google
Patents, 2014; WO2014044738 A1
33.
Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass DA.
Measurement of cardiac function using pressure-volume conductance
catheter technique in mice and rats. Nat Protoc 2008;3:1422-1434.
19

34.
35.
Lagorce D, Sperandio O, Baell JB, Miteva MA, Villoutreix BO. FAF-Drugs3:
a web server for compound property calculation and chemical library
design. Nucleic Acids Res 2015;43:200-207.
van der Westhuizen ET, Breton B, Christopoulos A, Bouvier M.
Quantification of ligand bias for clinically relevant beta2-adrenergic
receptor ligands: implications for drug taxonomy. Mol Pharmacol
2014;85:492-509.
36.
Iturrioz X, Alvear-Perez R, De Mota N, Franchet C, Guillier F, Leroux V,
Dabire H, Le Jouan M, Chabane H, Gerbier R, Bonnet D, Berdeaux A,
Maigret B, Galzi JL, Hibert M, Llorens-Cortes C. Identification and
pharmacological properties of E339-3D6, the first nonpeptidic apelin
receptor agonist. Faseb j 2010;24:1506-1517.
37.
38.
Margathe JF, Iturrioz X, Alvear-Perez R, Marsol C, Riche S, Chabane H,
Tounsi N, Kuhry M, Heissler D, Hibert M, Llorens-Cortes C, Bonnet D.
Structure-activity relationship studies toward the discovery of selective
apelin receptor agonists. J Med Chem 2014;57:2908-2919.
Khan P, Maloney PR, Hedrick M, Gosalia P, Milewski M, Li L, Roth GP,
Sergienko E, Suyama E, Sugarman E, Nguyen K, Mehta A, Vasile S, Su Y,
Stonich D, Nguyen H, Zeng FY, Novo AM, Vicchiarelli M, Diwan J, Chung
TDY, Pinkerton AB, Smith LH. Functional Agonists of the Apelin (APJ)
Receptor. Probe Reports from the NIH Molecular Libraries Program 2010.
El Messari S, Iturrioz X, Fassot C, De Mota N, Roesch D, Llorens-Cortes
C. Functional dissociation of apelin receptor signaling and endocytosis:
implications for the effects of apelin on arterial blood pressure. J
Neurochem 2004;90:1290-1301.
39.
40.
41.
Scott IC, Masri B, D'Amico LA, Jin SW, Jungblut B, Wehman AM, Baier H,
Audigier Y, Stainier DY. The g protein-coupled receptor agtrl1b regulates
early development of myocardial progenitors. Dev Cell 2007;12:403-413.
Pinkerton AB, Smith LH. Agonists of the apelin receptor and methods of
use thereof. Google Patents 2015; WO2015184011 A3
20

Figure Legends
Figure 1: The structure of the potassium salt of CMF-019.
Figure 2: The synthetic pathway for the production of the potassium salt of CMF-
019. Intermediates: (1) 4-fluoro-3-nitrobenzoic acid, (2) Methyl-4-fluoro-3-
nitrobenzoate, (3) 3-(3-aminopentane)-4-nitrobenzylmethanoate, (4) Methyl-3-amino-
4-(pentan-3-ylamino)benzoate, (5) Methyl-1-(pentan-3-yl)-2-(thiophen-2-ylmethyl)-
1H-benzo[d]imidazole-5-carboxylate, (6) 1-(Pentan-3-yl)-2-(thiophen-2-ylmethyl)-1H-
benzo[d]imidazole-5-carboxylic acid. Reagents and conditions: (a) SOCl2, MeOH, (b)
K2CO3, 3-aminopentane, DMF, (c) 5% Pd/C, H2 (15bar), MeOH, (d) 2-thiophene
acetic acid, SOCl2, DCM, (e) acetic acid, 140°C (microwave), (f) NaOH, MeOH,
110°C (microwave), (g) (i) HCTU, DIPEA, DMF, (ii) NaOH, MeOH, (h) KOH, 1,4-
dioxane.
Figure 3: Computational Docking of CMF-019. A 2D interaction map (A) showing the
binding site and interactions between CMF-019 and the APJ receptor. The key
interactions are π-stacking (thiophene to Tyr88, green line) and ionic (carboxylate to
Arg168, purple lines) bonds. CMF-019 (pale blue sticks) docked via GOLD into the
APJ receptor (green) (B). The key binding interactions between the ligand and APJ
receptor (at Arg168 and Tyr88) are highlighted (dotted yellow lines) with intermolecular
distances shown. The favoured overlay of apelin 13 (red lines) with CMF-019 (grey
lines) (C) places CMF-019 at the SHK region, suggesting that the SHK sequence of
apelin-13 is the most promising region for binding of CMF-019 to the APJ receptor.
Figure 4: Competition binding experiments in heart tissue homogenates. The
specific binding of CMF-019 to human left ventricular homogenate (A, ●), rat whole
heart homogenate (B, ■) and mouse whole heart homogenate (C, ▲).
21

Figure 5: Pathway selectivity of CMF-019 determined from cell-based assays. The
1
●) and CMF
■) in cell
-019 (
concentration response curves for [Pyr ]apelin-13 (
-based
●n=8/4, ■n=11/4
●n=12/4, ■n=13/4
), β-arrestin (B, ) and internalisation (C,
cAMP (A,
●n=6/2, ■n=6/2) assays. Bias was calculated as described previously [35], relative
effectiveness (RE) and bias factors (BF) are shown in Tables 1 and 2 respectively.
Figure 6: The cardiac contractility of CMF-019 in vivo. Dose dependent increases in
left ventricular contractility in anaesthetised rats to (A) intravenous CMF-019
potassium salt (▲, n=7-9) and (B) [Pyr1]apelin-13 (apelin, ■, n=8) compared to saline
(●, n=3-5) control. Each dose was compared by a Student’s t-test to its
corresponding saline control as doses were administered cumulatively (*p<0.05,
**p<0.01, ***P<0.001).
Figure 7: Cardiovascular actions of CMF-019 in vivo. Graphs showing changes in
stroke volume (A-B), cardiac output (C-D), relaxation (dp/dtMIN) (E-F) and left
▲, n=7
ventricular systolic pressure (LVSP) (G-H) for CMF-019 potassium salt (
-9,
A,C,E,G) and [Pyr1]apelin-13 (apelin, ■, n=8, B,D,F,H) compared to saline (●, n=3-5,
A-H) when injected intravenously into anaesthetised rats. Each dose was compared
by a Student’s t-test to its corresponding saline control as doses were administered
cumulatively (*p<0.05, **p<0.01, ***P<0.001, ****P<0.0001).
Figure 8: Assessment of drug-like properties of CMF-019 using FAF-Drugs3 [34].
(A) The properties of CMF-019 (HBD; H-bond donors; HBA, H-bond acceptors; logP,
Octanol-water partition coefficient; MW, molecular weight) are shown as a dark blue
line. A compound library of oral drugs was also assessed for these characteristics to
illustrate the desirable physiochemical properties for a drug-like molecule according
to Lipinski’s rules (light blue line). The red area denotes where it was not possible to
22

have a molecule with those properties, or where there was a lack of data in the
library.
(B) An oral absorption estimation for CMF-019 (dark blue line) (HBD; H-bond donors;
HBA, H-bond acceptors; RotB, rotatable bonds, logP, Octanol-water partition
coefficient; MW, molecular weight; tPSA, topological polar surface area). A
compound library of oral drugs was also assessed for these characteristics to
illustrate the desirable physiochemical properties for a drug-like molecule according
to Verber’s rules (dark green line). The red area denotes where it was not possible to
have a molecule with those properties, or where there was a lack of data in the
library.
(C) An oral property space obtained by applying a Principal Component Analysis
(PCA) of 15 physicochemical descriptors of CMF-019 (shown as the red dot),
compared to properties of oral drugs in current use, extracted as sublibraries from
the databases, eDrugs (blue) and DrugBank (orange).
23

Table 1. Values of ∆LogR and Relative effectiveness (RE) for CMF-019 compared to
[Pyr1]apelin-13 in cAMP, β-arrestin and internalisation assays.
ΔLogR
RE
β-arrestin
-1.98±0.18
0.01
7.19x10-4
4.19
Internalisation -3.14±0.19
cAMP 0.62±0.16
∆LogR is Log10(τ/KA) where τ is a measure of agonist efficacy and KA a measure of
functional affinity [35]. n values for each of the assays are as indicated in Figure 5.
24

Table 2. ∆∆LogR and bias factors (BF) for CMF-019 compared [Pyr1]apelin-13 in
cAMP, β-arrestin and internalisation assays.
ΔΔLogR
BF
β-arrestin
n/a
Internalisation
1.17±0.26
15
β-arrestin
2.60±0.24
398
3.77±0.25
5828
cAMP
∆∆LogR is the difference between ∆LogR values in the different pathways [35].
25

26

27

28

29