Structure-kinetic relationship studies of cannabinoid CB2 receptor agonists reveal substituent-specific lipophilic effects on residence time

Article abstract of DOI:10.1016/j.bcp.2018.03.018

A decade ago, the drug-target residence time model has been (re-)introduced, which describes the importance of binding kinetics of ligands on their protein targets. Since then, it has been applied successfully for multiple protein targets, including GPCRs, for the development of lead compounds with slow dissociation kinetics (i.e. long target residence time) to increase in vivo efficacy or with short residence time to prevent on-target associated side effects. To date, this model has not been applied in the design and pharmacological evaluation of novel selective ligands for the cannabinoid CB₂ receptor (CB₂R), a GPCR with therapeutic potential in the treatment of tissue injury and inflammatory diseases. Here, we have investigated the relationships between physicochemical properties, binding kinetics and functional activity in two different signal transduction pathways, G protein activation and β-arrestin recruitment. We synthesized 24 analogues of 3-cyclopropyl-1-(4-(6-((1,1-dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazoleidine-2,4-dione (LEI101), our previously reported in vivo active and CB₂R-selective agonist, with varying basicity and lipophilicity. We identified a positive correlation between target residence time and functional potency due to an increase in lipophilicity on the alkyl substituents, which was not the case for the amine substituents. Basicity of the agonists did not show a relationship with affinity, residence time or functional activity. Our findings provide important insights about the effects of physicochemical properties of the specific substituents of this scaffold on the binding kinetics of agonists and their CB₂R pharmacology. This work therefore shows how CB₂R agonists can be designed to have optimal kinetic profiles, which could aid the lead optimization process in drug discovery for the study or treatment of inflammatory diseases.

Full text of DOI:10.1016/j.bcp.2018.03.018

Accepted Manuscript
Structure-kinetic relationship studies of cannabinoid CB2 receptor agonists re-
veal substituent-specific lipophilic effects on residence time
Marjolein Soethoudt, Mark W.H. Hoorens, Ward Doelman, Andrea Martella,
Mario van der Stelt, Laura H. Heitman
PII:
S0006-2952(18)30123-0
https://doi.org/10.1016/j.bcp.2018.03.018
BCP 13095
DOI:
Reference:
Biochemical Pharmacology
To appear in:
Received Date:
Accepted Date:
9 January 2018
16 March 2018
Please cite this article as: M. Soethoudt, M.W.H. Hoorens, W. Doelman, A. Martella, M. van der Stelt, L.H. Heitman,
Structure-kinetic relationship studies of cannabinoid CB2 receptor agonists reveal substituent-specific lipophilic
effects on residence time, Biochemical Pharmacology (2018), doi: https://doi.org/10.1016/j.bcp.2018.03.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Structure-kinetic relationship studies of cannabinoid CB2 receptor
agonists reveal substituent-specific lipophilic effects on residence time
Marjolein Soethoudt,1,2 Mark W.H. Hoorens,2 Ward Doelman,2 Andrea Martella,2
Mario van der Stelt,2 Laura H. Heitman1
1Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research,
University of Leiden, Einsteinweg 55, 2333 AR, The Netherlands
2Department of Molecular Physiology, Leiden Institute of Chemistry, University of
Leiden, Einsteinweg 55, 2333 AR, The Netherlands
Author for correspondence: Laura H. Heitman, Tel.: +31 71 5274558,
l.h.heitman@lacdr.leidenuniv.nl
Key words: Target residence time, Physicochemical properties, Cannabinoid CB2
receptor, Functional potency
1

Abstract
A decade ago, the drug-target residence time model has been (re-)introduced, which
describes the importance of binding kinetics of ligands on their protein targets. Since
then, it has been applied successfully for multiple protein targets, including GPCRs,
for the development of lead compounds with slow dissociation kinetics (i.e. long
target residence time) to increase in vivo efficacy or with short residence time to
prevent on-target associated side effects. To date, this model has not been applied in
the design and pharmacological evaluation of novel selective ligands for the
cannabinoid CB2 receptor (CB2R), a GPCR with therapeutic potential in the treatment
of tissue injury and inflammatory diseases. Here, we have investigated the
relationships between physicochemical properties, binding kinetics and functional
activity in two different signal transduction pathways, G protein activation and β-
arrestin recruitment. We synthesized 24 analogues of 3-cyclopropyl-1-(4-(6-((1,1-
dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazoleidine-2,4-dione
(LEI101), our previously reported in vivo active and CB2R-selective agonist, with
varying basicity and lipophilicity. We identified a positive correlation between target
residence time and functional potency due to an increase in lipophilicity on the alkyl
substituents, which was not the case for the amine substituents. Basicity of the
agonists did not show a relationship with affinity, residence time or functional activity.
Our findings provide important insights about the effects of physicochemical
properties of the specific substituents of this scaffold on the binding kinetics of
agonists and their CB2R pharmacology. This work therefore shows how CB2R
agonists can be designed to have optimal kinetic profiles, which could aid the lead
optimization process in drug discovery for the study or treatment of inflammatory
diseases.
2

1.
Introduction
Traditionally, in drug discovery, the affinity or potency of a drug candidate for a given
target was considered a key determinant for in vivo activity, but later it was found that
these parameters do not correlate as well as originally thought.[1, 2] In contrast, the
binding kinetics of a ligand for a given target, in particular slow dissociation kinetics
and therefore a long target residence time, may be a better predictor of in vivo
efficacy in specific cases,[3-6] as emphasized by several excellent reviews.[7-9] For
example, a correlation was found between long residence time of Fab-l enoyl
reductase inhibitors and their in vivo activity in a mouse model of tularemia infection,
leading to prolonged survival of the mice.[3, 4] Recently, this “drug-target residence
time model” has aided several clinical-stage drug development programs[10, 11] by
selecting compounds with high efficacy,[12] or reduced on-target toxicities.[13]
However, the association rate is increasingly recognized as well as an important
factor in determining a ligand’s functional activity. For example, slowly associating
ligands may decrease on-target related side effects by preventing high target
occupancy and fast target activation,[14] while fast associating ligands may have an
influence in prolonged activity if rebinding occurs.[15]
Retrospective analysis of marketed drugs for G protein-coupled receptors
(GPCRs), an important class of drug targets, revealed that the beneficial effects of
some of these drugs may be attributed to their long drug-target residence times.[8]
Interestingly, in case of GPCR agonists, a positive correlation was also found
between long residence time and in vitro efficacy for the Adenosine A2A receptor[16]
and the Muscarinic M3 receptor.[17] For the latter, it was also shown that long target
residence time of an antagonist, i.e. tiotropium, resulted in so-called kinetic selectivity
3

over the other muscarinic receptor subtypes, thereby reducing off-target side
effects.[18]
The cannabinoid CB1 and CB2 receptor (CB1R and CB2R) are class A GPCRs
and both part of the endocannabinoid system. This signaling system comprises the
receptors as well as their endogenous ligands, anandamide (AEA) and 2-
arachidoylglycerol (2-AG), which are called endocannabinoids.[19] The CB1R is
mainly found within the central nervous system,[20] which is therefore mainly
responsible for the psycho-active effects of 9-tetrahydrocannabinol (THC), the main
active substituent in cannabis.[21] In contrast, the CB2R is predominantly abundant in
immune cells, is involved in cell migration and immunosuppression,[22, 23] and is
upregulated during pathophysiological conditions.[24] CB2R activity has been
associated with therapeutic benefits in inflammatory or immune system related
pathologies.[24, 25] Selective activation of the CB2R is therefore associated with
therapeutic benefits and may prevent CB1R-mediated adverse side effects.
Recently,
our
group
reported
on
3-cyclopropyl-1-(4-(6-((1,1-
dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazoleidine-2,4-dione
(LEI101) (Figure 1), a promising CB2R partial agonist.[26] LEI101 showed in vivo
efficacy in preclinical models of neuropathic pain and cis-platin-induced
nephrotoxicity.[26],[27] The CB2R kinetic profile of LEI101 is unknown, therefore we
were interested to systematically investigate the binding kinetics and functional
activity of this chemical series.
To this end, we synthesized a library of 24 compounds based on the scaffold
of LEI101 (Figure 1), in which we systematically varied their basicity and lipophilicity
(pKa and LogP) of the R1 (amine) and R2 (alkyl) substituents and determined their
equilibrium binding affinity and Kinetic Rate Index (KRI), a high-throughput measure
4

as an indication for ligand-receptor kinetics.[28] In addition, the full kinetic profile, as
well as functional potency and efficacy in G protein activation and β-arrestin
recruitment, was measured for 14 of these compounds. Correlation analysis of the
data identified a relationship between target residence time and potency in both
signal transduction pathways due to increased lipophilicity specifically on the R2
position. This work provides important insights in the impact of divergent binding
kinetics of LEI101-based agonists on CB2R pharmacology and the role of
physicochemical properties therein. In turn, these insights show how CB2R agonists
can be designed to have optimal kinetic profiles, which will aid the lead optimization
process in drug discovery for the study or treatment of inflammatory diseases.
2.
Materials and Methods
2.1
Chemical and reagents
All common reagents were purchased from commercial sources and used as received. The agonist
library was synthesized as described previously in van der Stelt et al, 2011,[27] with only small
modifications (see Figure 2). After purification, all compounds had more than 95% purity as
determined by Liquid Chromatography Mass Spectroscopy (LCMS), by measuring UV absorbance at
254 nm and were fully characterized using 1H-NMR and 13C-NMR. High resolution mass spectra were
recorded on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electrospray ion
source (ESI) in positive mode. The spectrometer was calibrated prior to each measurement with a
calibration mixture (Thermo Finnigan). Molecules are drawn with ChemDraw Professional 16.0. Full
details regarding synthetic procedures and compound characterization can be provided upon request
from the corresponding author. Cell culture medium components (Ham’s F12 Nutrient Mixture,
gllutamine and antibiotics penicillin, streptomycin, hygromycin and geneticin), bovine serum albumin
(BSA), polyethylenedimide (PEI), guanosine diphosphate (GDP), dithiothreitol (DTT) and cannabinoid
receptor ligands CP55940 and AM630 were purchased from Sigma Aldrich (St. Louis, MO).
[3H]CP55940 (specific activity 141.2 Ci/mmol), [35S]GTPγS (specific activity 1250 Ci/mmol) and GF-
B/GF-C filters were purchased from Perkin Elmer (Waltham, MA). Bicinchoninic acid (BCA) and BCA
5

protein assay reagent were obtained from Pierce Chemical Company (Rochford, IL). The PathHunter®
CHO-K1 CNR1 (CHOK1hCB1_bgal) and CNR2 (CHOK1hCB2_bgal) β-Arrestin Cell Lines and the
PathHunter® detection kit were obtained from DiscoveRx (Fremont, United States). Cell culture plates
were purchased from Sarstedt and 384-well white walled assay plates from Perkin Elmer. All buffers
and solutions were prepared using Millipore water (deionized using a MilliQ A10 BiocelTM, with a 0.22
µm filter) and analytical grade reagents and solvents. Buffers are prepared at room temperature and
stored at 4°C, unless stated otherwise.
2.2
Cell culture
CHOK1hCB2_bgal cells were cultured in Ham’s F12 Nutrient Mixture, supplemented with 10% fetal
calf serum, 1 mM glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, 300 mg/mL hygromycin and
800 μg/mL geneticin in a humidified atmosphere at 37°C and 5% CO2, as reported previously.[29]
Cells were subcultured twice a week at a ratio of 1:20 on 10-cm diameter plates by trypsinization. For
membrane preparation the cells were subcultured 1:10 and transferred to 15-cm diameter plates. Cells
were passaged no longer than 25 times or 3 months.
2.3
Membrane preparation
Per batch of membranes, cells on thirty 15-cm ø plates were detached from the bottom by scraping
them into 5 mL phosphate-buffered saline (PBS), collected in 12 mL Falcon tubes and centrifuged for
5 minutes at 200 g (3,000 rpm). The pellets were resuspended in ice-cold 50 mM Tris-HCl buffer and 5
mM MgCl2 (pH 7.4). An Ultra Thurrax homogenizer (Heidolph Instruments, Schwabach, Germany) was
used to homogenize the cell suspension. The membranes and cytosolic fractions were separated by
centrifugation at 100,000 g (31,000 rpm) in a Beckman Optima LE-80 K ultracentrifuge (Beckman
Coulter Inc., Fullerton, CA) at 4°C for 20 minutes. The pellet was resuspended in 10 mL of Tris-HCl
buffer and 5 mM MgCl2 (pH 7.4) and the homogenization and centrifugation steps were repeated.
Finally, the membrane pellet was resuspended in 10 mL 50 mM Tris-HCl buffer and 5 mM MgCl2 (pH
7.4) and aliquots of 250 μL were stored at -80°C. Membrane protein concentrations were measured
using the BCA method.[30]
6

2.4
[3H]CP55940 equilibrium displacement assay
[3H]CP55940 displacement assays were used for the determination of affinity (IC50) values of
unlabeled ligands. Membrane aliquots containing 1.5 μg of membrane protein were incubated in a
total volume of 100 μL assay buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2 and 0.1% BSA) at
25°C for 2 hours in presence of ~1.5 nM [3H]CP55940. Ten different concentrations of competing
ligand were used for determination of IC50 values, and nonspecific binding was determined in the
presence of 10 μM AM630. Incubations were terminated and samples harvested as described by the
96-wells harvest procedure (see below).
2.5
96-wells harvest procedure
Samples were harvested on 96-wells GF/C filters, precoated with 25 μL 0.25% (v/v) PEI per well, with
rapid vacuum filtration, to separate the bound and free radioligand, using a Perkin Elmer 96-wells
harvester (Perkin Elmer, Groningen, The Netherlands). Filters were subsequently washed ten times
with ice-cold assay buffer on the 96-well plate and 5 times on a wash plate. Filter plates were dried at
55°C for ~45 min, then 25 μL Microscint was added per well (Perkin Elmer, Groningen, The
Netherlands). After 3 hours, the filter-bound radioactivity was determined by scintillation spectrometry
using a Microbeta2® 2450 microplate counter (Perkin Elmer, Boston, MA).
2.6
[3H]CP55940 Association Assay
To determine association kinetics of [3H]CP55940, it was incubated at a concentration of ~1.5 nM with
1.5 μg of membrane protein in a total volume of 100 μL of assay buffer at 25°C or 10°C for a range of
timepoints (90, 60, 30, 25, 20, 15, 10, 5, 3 and 1 minutes). For the assay at 10°C, an additional time
point at 120 min was added. Nonspecific binding was determined in the presence of 10 μM AM630.
Incubations were terminated and samples harvested as described by the 96-wells harvest procedure
(see above).
2.7
[3H]CP55940 Dissociation Assay
To determine dissociation kinetics of [3H]CP55940, it was incubated at a concentration of ~1.5 nM with
1.5 μg of membrane protein in a total volume of 100 μL of assay buffer at 25°C or 10°C for 2 hr.
Dissociation was then initiated at a range of timepoints (25°C: 90, 30, 20, 15, 10, 8, 5, 3, 1 min; 10°C:
7

360, 300, 240, 180, 120, 90, 60, 30, 10 and 5 min) by addition of 5 μL of AM630 (final assay
concentration: 10 μM). Nonspecific binding was determined by addition of 10 μM AM630 from the start
of the assay. Incubations were terminated and samples harvested as described by the 96-wells
harvest procedure (see above).
2.8
[3H]CP55940 Dual-point Competition Association Assay
For fast determination of the relative kinetics of the agonist library, the KRI was determined using a
dual-point competition association assay.13 The agonists were incubated at their IC50 concentration (as
determined at 25°C) with 1.5 nM of [3H]CP55940 and 1.5 μg membrane protein in assay buffer in a
total volume of 100 μL, for either 1 or 2 hours at 10°C (t1 and t2, respectively). Nonspecific binding was
determined by addition of 10 μM AM630 from the start of the assay. Incubations were terminated and
samples harvested as described by the 96-wells harvest procedure (see above).
2.9
[3H]CP55940 Full Competition Association Assay
To determine the kon and koff values of unlabeled competing ligands. Ligands were incubated at their
IC50 concentration (see 2.12 Data Analysis) in presence of ~1.5 nM [3H]CP55940 and with 1.5 μg of
membrane protein in a total volume of 100 μL of assay buffer at 10 °C for a range of timepoints (120,
90, 60, 30, 25, 20, 15, 10, 5, 3 and 1 minutes). Nonspecific binding was determined in the presence of
10 μM AM630. Incubations were terminated and samples harvested as described by the 96-wells
harvest procedure (see above).
2.10
[35S]GTPγS assay
G protein activation as a measure for receptor activity was determined by the binding of radiolabeled
non-hydrolyzable GTP ([S35]GTPγS) to the receptor.[29, 31] To homogenized CHOK1CB2R_bgal
membranes (5 µg) in 20 µL assay buffer (50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 150 mM NaCl,
1 mM EDTA, 0.05% BSA and 1 mM DTT, freshly prepared every day), 5 µg saponin and 1 µM GDP
were added (final assay concentration). To determine the pEC50 and Emax values of the agonist library,
the membranes were directly incubated for 30 min at room temperature with various concentrations of
the ligands of interest. The basal level of [S35]GTPγS binding was measured in untreated membrane
samples, and the maximal level of [S35]GTPγS binding was measured by treatment of the membranes
8

with 10 µM CP55940. Subsequently, [S35S]GTPγS (0.3 nM) was added and the samples were
o
incubated for 90 minutes at 25 C on a shaking platform in a total sample volume of 100 µL.
Incubations were terminated and samples harvested as described by the 96-wells harvest procedure
(see above). Here, samples were harvested on 96-wells GF/B filters and washed using buffer
containing 50 mM Tris HCl, pH 7.4 and 5 mM MgCl2.
2.11
PathHunter® β-Arrestin Recruitment Assay
The assay was performed using the PathHunter® CHOK1CB2R_bgal cells and β-arrestin recruitment
assay kit (DiscoveRx Corporation, Fremont, CA), as published before.[29, 32] Briefly, PathHunter®
CHOK1hCB2R_bgal cells were seeded at a density of 5000 cells per well of solid white walled 384-
well plates (Perkin Elmer, MA, USA) in 20 μL HAM’s F12 Nutrient Mixture culture medium and
incubated overnight in a humidified atmosphere at 37°C and 5% CO2. The cells were stimulated with 5
μL of 50 μM (10 μM final assay concentration) of each agonist (single point assay) or 10 increasing
concentrations of each agonist and incubated for 90 minutes in a humidified atmosphere at 37°C and
5% CO2. The DMSO concentration was the same in each well. The activity of β-galactosidase was
determined using the PathHunter® Detection Kit (DiscoveRx Corporation, Fremont, CA), following the
supplier’s protocol. In short, the cells were loaded with 12 μL detection reagent (DiscoveRx
Corporation, Fremont, CA) and incubated for 1 hour in the dark at room temperature. Luminescence
(400-700 nm), indicated as relative light units (RLU), was measured on an EnVision multilabel plate
reader (Perkin Elmer, MA, USA), using a Luminescence 700 emission filter.
2.12
Data Analysis
cLogP and pKa values were calculated using ChemDraw® Professional 16.0 (Perkin Elmer). All
experimental data were analyzed using the nonlinear regression curve fitting program GraphPad
Prism 7 (GraphPad Software, Inc., San Diego, CA). From displacement assays at 25°C, the non-linear
regression analysis for one site - Fit Ki was used to obtain logKi values, which are provided by Prism
by direct application of the Cheng-Prusoff equation:[33] Ki = IC50 / (1 + ([L]/KD)) in which [L] is the
exact concentration of [3H]CP55940 determined per experiment (i.e. ~1.5 nM). The kinetic KD (1.24 ±
0.10 nM) of [3H]CP55940 was calculated using the formula KD = koff/kon. The kon (1.6 ± 0.1 x 106 M-1 s-
1) and koff (2.0 ± 0.1 x 10-3 s-1) of [3H]CP55940 at this temperature were determined using an
9

association and dissociation assay, respectively (three experiments performed in duplicate, data not
shown). The logKi values were converted manually to pKi values (Table 1). For the kinetic
experiments, a concentration equal to the IC50 value of each agonist was used, as determined from
the non-linear regression analysis for “one site - Fit logIC50”. For non-linear regression analysis “one
site - Fit Ki” and “one site - Fit logIC50” the top and bottom of the curve were constrained at 100 and 0,
respectively. From association assays, the association rate constant (kon) of [3H]CP55940 was
calculated using the formula kon=(kobs-koff)/[L], in which [L] is the exact concentration of [3H]CP55940
determined per experiment. The observed association rate (kobs) was determined with Prism’s “one-
phase exponential association” analysis that uses the following formula: Y = Y0 + (Plateau – Y0) * (1 –
exp(-kobs* t), where Y0 is the specific radioligand binding at time 0 (constrained at 0), Plateau
represents the maximum specific [3H]CP55940 binding at equilibrium, kobs is the observed association
rate in min-1 and t is the time in minutes. From dissociation assays, the dissociation rate constant (koff)
of [3H]CP55940 was determined using Prism’s “one-phase exponential decay” analysis using the
following formula: Y = (Y0 – NSB) * exp(-koff * t) + NSB, where koff is the dissociation rate constant in
min-1 and where Y0 is the specific radioligand binding at time 0 (constrained at 100). From competition
association assays, the kon and the koff of cold ligands were obtained by non-linear regression analysis
“kinetics of competitive binding” that uses the following equation:[34]
[RL] = Q*((k4DIFF)/(KFKS)) + ((k4 – KF)/KF)*exp(-KFt) – ((k4 – KS)/KS)*exp(-KSt), using the following
variables:
KA = k1[L](10-9) + k2
KB = k3[I](10-9) + k4
S = √((KA – KB)2 + 4*k1k3[L][I](10-18))
KF = 0.5 * (KA + KB + S)
KS = 0.5 * (KA + KB – S)
DIFF = KF - KS
Q = (Bmaxk1[L](10-9))/DIFF
Where [RL] is the amount of receptor-ligand complex, [L] is the concentration [3H]CP55940 in nM per
experiment (~1.5 nM), [I] depicts the used concentration of unlabeled competitor in nM, KA and KB are
the observed association rates (kobs) of [3H]CP55940 and the unlabeled competitor, respectively, k1
and k3 the association rate constants (kon in M-1min-1) of [3H]CP55940 (determined per experiment)
10

and the unlabeled competitor, respectively, k2 and k4 the dissociation rate constants (koff in min-1) of
[3H]CP55940 (0.0115 min-1, determined using three independent dissociation experiments) and the
unlabeled competitor, respectively and t is the time in minutes. The kon (M-1min-1) and koff (min-1)
provided by Prism were converted manually to kon (M-1s-1) and koff (s-1). Receptor residence time (RT,
in min) was calculated by taking the reciprocal of the dissociation rate as follows room temperature =
1/(60*koff), as koff is in s-1. ß-Arrestin recruitment and GTPγS data were analyzed by Prism’s nonlinear
regression analysis “log (agonist) vs. response – variable slope” to obtain potency (EC50) and efficacy
(Emax) values of ligands. The efficacy of all agonists was normalized to the effects of 10 μM CP55940.
The bottom of the curves were constrained at 0. All data was obtained from three separate
experiments performed in duplicate, unless stated otherwise. The correlation between two
independent variables or data sets was calculated using a two-tailed Pearson correlation analysis.[35]
A P-value of less than 0.05 was considered significant.
3.
Results
3.1
Equilibrium binding affinity of the LEI101-library
The affinities of the 24 newly synthesized compounds were determined in a
radioligand displacement assay using [3H]CP55940 as the radiolabeled competitor at
a temperature of 25°C. The structure, affinity (pKi) and physicochemical properties of
the library are presented in Table 1. All compounds showed concentration-
dependent displacement of [3H]CP55940. Compounds 6, 7, 8, 17, 21, 22 and 23,
carrying a propyl or isobutyl group at the R2 position, displayed the highest affinities
within the library (pKi > 7.5). In contrast, compounds 3, 11, 16, and 20, carrying a
more bulky methoxyethyl or butyl group at the R2 position, displayed ~10- to 100-fold
lower affinities, ranging from 5.35 ± 0.04 (compound 11) to 6.56 ± 0.13 (compound
20). Compounds 1, 9 and 18, without a substituent at R2, had the lowest affinities (pKi
= ~5.5) of the library. On the R1 position, compounds with a morpholine substituent
(compounds 10-17) or a piperazine (compound 24) generally had lower affinities
11

compared to corresponding dioxidethiomorpholino agonists with the same substituent
at the R2 position (e.g. compound 11 vs. 2 and 20, 12 vs. 3 and 21 or 15 vs. 6 and
24).
3.2
High throughput kinetic screening of LEI101-library
Next, the binding kinetics of all compounds were determined using the high
throughput dual-point competition association assay, yielding Kinetic Rate Index
(KRI) values that describe the relative (dissociation) kinetics of the agonist library
compared to the radioligand used, [3H]CP55940. These experiments, and all the
following kinetic experiments, were performed at a reduced temperature of 10°C to
increase the ‘resolution’ of the assay, enabling us to examine the influence of
different physicochemical properties on the relative binding kinetics of the
compounds within the library. Firstly, we validated that the affinities of the molecules
were similar (particularly in rank order) at 10°C as compared to 25°C using a
selection of 8 representative agonists with low, moderate and high affinity (data not
shown). Subsequently, we used a single concentration of the compounds (1.0 x IC50)
for determination of the KRI values (Table 1). Most compounds had a KRI value
lower than 1.0, which indicates a residence time (RT) shorter than that of
[3H]CP55940. Compounds 2, 4 and 6 had the lowest KRI values (0.53 ± 0.06, 0.52 ±
0.09 and 0.51 ± 0.05, respectively), whereas only 7, 22 and 23 had a KRI value
larger than 1.0 (1.06 ± 0.11, 1.21 ± 0.07 and 1.03 ± 0.08, respectively). These three
compounds all have an isobutyl moiety at the R2 position, the most lipophilic
substituent in this series, but have different R1 substituents, a dioxidethiomorpholine
(7), a piperidine (22), or a methylpiperidine (23).
3.3
Full kinetic profiling of the LEI101-library
12

Based on the results from the KRI screen, twelve agonists were selected for further
kinetic characterization. These compounds contained a dioxidethiomorpholine at the
R1 position (group A, compounds 1-7) or an isobutyl group at the R2 position (group
B, compounds 7, 8, 15, 17, 22, 23). Of note, compound 7 belongs to both groups.
The molecules comprised a wide range of KRI values between 0.51 and 1.21,
respectively the lowest and highest KRI measured in this agonist library. Together
this allowed a comprehensive investigation of structure-kinetic relationships at the
CB2R. We used a competition association assay with [3H]CP55940 that yielded the
association- and dissociation rate constants (kon and koff values, respectively) of the
compounds (Table 2). A significant correlation between the KRI values and koff
values was found (Figure 3A). The association of [3H]CP55940 alone and in
presence of a fast dissociating compound (2; KRI = 0.53 ± 0.06) and a slow
dissociating compound (7; KRI = 1.06 ± 0.11) is shown in Figure 3B. The association
of [3H]CP55940 (koff = 1.9 ± 0.1 x 10-4 s-1, data not shown) in competition with 7 (koff =
2.4 ± 0.1 x 10-4 s-1), resulted in a small overshoot after which it reached a plateau at
~20%. In contrast, association of [3H]CP55940 in competition with 2 (koff = 1.2 ± 0.6 x
10-2 s-1) resulted in a gradual increase of [3H]CP55940 binding over time. The kon
values varied between 2.2 ± 1.0 x 103 M-1 s-1 (1) and 1.9 ± 0.8 x 105 M-1 s-1 (15).
Moreover, the variety in kon and koff values was visualized using a kinetic map (Figure
3C), created by plotting the kon values against koff values. The diagonals represent
the ‘kinetic’ KD value (KD = koff/kon) and show that compounds with similar KD values
can have different combinations of koff and kon values. For example, 2 and LEI101 (4)
have a similar KD value (KD = 10-7 M), but have a more than 0.5 log-difference both in
koff and kon values. Of note, compounds with a KD ≤ 10-8 M (black circle) all have
dissociation rates slower than 10-3 s-1, while compounds with a dissociation rate
13

between 10-3 and 10-2 s-1 (dashed circle) predominantly had a KD between 10-7 and
10-8 M, due to a small variety in their kon values. Of note, 1 (R2 = H) had a 10-fold
smaller kon value compared to the other compounds, thereby making it an outlier in
the kinetic map (Figure 3C).
The kinetic KD values of all compounds (Table 2) were compared to the
equilibrium affinities (Ki values) (Figure 4A). A statistically significant correlation was
found between the negative logarithm of the kinetic KD (10°C) and the equilibrium pKi
(25°C). Of note, the pKD values were all 0.5 log unit (~3-fold) higher than the pKi
values. A correlation between pKD and koff or residence time was also identified
(Figure 4B,C), but not between pKD and kon values (Figure 4D).
3.4
Structure-Kinetics Relationships
The kinetic profile of the compounds was used to derive structure-kinetics
relationships. The longest residence times (RT > 30 min) were displayed by
compounds with a propyl (6, RT = 32 ± 9 min) or isobutyl group at the R2 position (7,
8, 17, 22 and 23, RT = 71 ± 3, 37 ± 5, 72 ± 8, 69 ± 2 and 31 ± 6 min, respectively).
Compounds 2 and 3 displayed the shortest residence times (RT = 2.2 ± 0.8 and 3.6 ±
1.5 min, respectively). Interestingly, the residence time of 1 was similar as LEI101 (4)
(RT = 14 ± 6 and 8.8 ± 1.6 min for 1 and 4, respectively), despite a 10-fold lower
binding affinity (pKi = 5.55 ± 0.08 and 6.51 ± 0.09 for 1 and 4, respectively), which
was due to the very low kon value of 1 (2.2 ± 1.0 x 103 M-1s-1).
3.5
Influence of physicochemical properties on affinity and binding kinetics
Next, we analyzed the effects of physicochemical properties on equilibrium affinity
and binding kinetics. Hence, the cLogP (Table 1) of the compounds with varying alkyl
14

R2 substituents (group A) and the basicity (pKa) with varying amine R1 substituents
(group B) were plotted against equilibrium affinity, association rate kon and residence
time. The basicity of group B did not correlate with any of the measured parameters
(pKi: Pearson r: 0.02328, p-value = 0.9064; kon: Pearson r: -0.2213, p-value = 0.6735;
RT: Pearson r: 0.3944, p-value = 0.5112; graphs not shown). In case of group A, a
near-significant correlation was identified with their lipophilicity and equilibrium affinity
(Pearson r: 0.692, p-value = 0.0542, Figure 5A), but not with kon (Pearson r: 0.1452,
p-value = 0.7561, Figure 5B). Interestingly, cLogP of group A was highly correlated
with residence time (Pearson r: 0.8869, p-value = 0.0078, Figure 5C). Noteworthy,
this correlation was not observed with the R1 substituents of group B (Figure 6).
3.6
Influence of binding kinetics on functional activity
Finally, the influence of residence time on functional activity of the compound library
was investigated. To this end, both groups were characterized in two functional
assays: GTPγS binding and β-arrestin recruitment (Table 2). All compounds
displayed partial agonism in both assays relative to CP55940. The highest intrinsic
efficacy was observed for 5 in the G protein activation assay (Emax = 79 ± 14%),
whereas agonist 2 had the highest efficacy in the β-arrestin recruitment assay (Emax
=
76 ± 15%). The lowest efficacy was observed for 1 in both functional assays (β-
arrestin: Emax = 25 ± 2%; GTPγS: Emax = 48 ± 7%). Generally, agonists showed a
lower efficacy for β-arrestin recruitment, except for agonists 2, 6 and 15 (Emax β-
arrestin: 76 ± 15, 62 ± 8 and 64 ± 10 compared to Emax GTPγS: 54 ± 13, 50 ± 2 and
65 ± 12, respectively), although these differences were not significant. Indeed, no
correlation was observed between the efficacies of the compounds in the two
functional assays (Pearson r: 0.4247, p-value = 0.1688, correlation graphs not
15

shown). In addition, no correlation between residence time and in vitro efficacy was
identified (GTPγS Pearson r: 0.00621, p-value: 0.9895; β-arrestin Pearson r: 0.1053,
p-value 0.8222, graphs not shown). For example, the long residence time of agonists
6, 7, 8, 17, 22 and 23 did not have a higher efficacy than the other agonists in either
functional assay. In fact, agonist 2 with the shortest residence time (2.2 ± 0.8 min)
had a very moderate efficacy in GTPγS (54 ± 13%), and the highest efficacy of all
agonists for β-arrestin recruitment (76 ± 15%).
The potencies ranged from 6.06 ± 0.27 (3) to 7.94 ± 0.24 (7) in the GTPγS
assay, whereas in the β-arrestin recruitment assay the potencies ranged from 6.12 ±
0.23 (1) to 8.14 ± 0.08 (22). In contrast to efficacy, the potency of the compounds
was similar and highly correlated in the two functional assays (Pearson r: 0.8445, p-
value < 0.0005). In general the compounds showed a higher potency in β-arrestin
recruitment assays. For example, 22 showed a 17-fold higher potency for β-arrestin
recruitment compared to G protein activation (pEC50 = 8.14 ± 0.08 and 6.91 ± 0.32,
respectively).
Notably, nanomolar potency (pEC50 > 7.5) was only displayed by agonists with
a residence time of at least 30 min as exemplified by compounds 6, 8 and 23 (with
residence times of 32 ± 9, 37 ± 5 and 31 ± 6 min, respectively) and compounds 7, 17
and 22 (RT = 71 ± 3, 72 ± 8 and 69 ± 2 min, respectively). A statistically significant
correlation was found of the residence times of group A with functional potency for
both G protein activation and β-arrestin recruitment (Figure 7A-B). Interestingly, the
residence times of group B did not correlate with either potency or efficacy (GTPγS
Emax Pearson r: 0.0021, p-value: 0.9968; pEC50 Pearson r: 0.3391, p-value: 0.5108;
β-arrestin Emax Pearson r: -0.2591, p-value: 0.6200; pEC50 Pearson r: 0.6586, p-
value: 0.1549, graphs not shown).
16

4.
Discussion
4.1
Kinetic characterization of LEI101-based agonists
Recently, drug discovery research has focused on the development of selective
CB2R agonists for the treatment of tissue injury and inflammatory diseases that avoid
inducing CB1R-mediated psychoactive side effects. CB2R knockout mice show
enhanced pathology in various inflammatory disease models, including heart, liver or
kidney injury and inflammatory pain, thereby supporting the notion that CB2R plays
an essential role in these conditions. Despite compelling proof-of-concept data
obtained in preclinical pain models, several CB2R agonists lacked efficacy in phase 2
clinical trials for unknown reasons.[29, 36]
Drug-target binding kinetics and their influence on functional activity are
increasingly considered in drug discovery because it may aid in the design of lead
compounds.[2] Therefore, we have investigated the relationships between functional
activity and binding kinetics of a series of agonists, based on the CB2R-selective
agonist LEI101, which showed in vivo efficacy in the treatment of neuropathic pain
and inflammation-induced tissue damage.[26, 27]
In this study, radioligand binding assays were performed with [3H]CP55940, an
agonistic radioligand commonly used to determine CBR pharmacology,[37] including
binding kinetics.[38, 39] Recently, Sykes et al. showed the importance of using
physiological concentrations of sodium when determining binding kinetics at the
muscarinic M3 receptor.[40] However, in this study sodium ions were absent in all
assays where the agonist [3H]CP55940 was used to prevent that the receptor
population was forced into a predominantly inactive state, i.e. for which an agonist
would have a low affinity.[41] In addition, in our system we have never observed a
17

biphasic interaction for agonists, which would prohibit the use of the the Motulsky-
Mahan mathematical model as it describes binding of a ligand to a single site, e.g.
receptor.[34, 42] Hence, we also did not apply GTP to force the receptor population
in a single (inactive) state. Importantly, we believe that it is unlikely that the omission
of sodium salts and/or GTP would result in a different rank order of binding kinetics of
the agonist library. This line of thought is further corroborated by the study on
tiotropium and NVA237 in presence of sodium ions that resulted in shorter residence
times, but the same rank order.[40]
The measured equilibrium binding affinities corresponded to previously
determined structure-activity relationships (SAR) for this scaffold.[27] Using a high-
throughput kinetic screening assay, based on its equivalent for the Adenosine A1
receptor,[28] agonists with R1 = dioxidethiomorpholine (group A) and R2 = isobutyl
(group B) were selected for full kinetic characterization (Figure 3A). We found that
the kinetic profile of the agonists had smaller variations in kon values, but larger
variations in koff values, which were visualized using a kinetic map of the agonist
library (Figure 3C). For this series of compounds, binding affinity was mostly
influenced by their dissociation rate, as illustrated by a significant correlation with koff
values, but not with kon values. (Figure 4C,D). This observation was similar as
reported for the the adenosine A2A receptor,[16, 17] but in contrast to reports on β2
adrenergic receptors and the hERG channel, for which the association rate was
found to be the main driving force in ligand affinity.[43, 44]
4.2
The role of physicochemical properties on binding kinetics and functional
activity
18

Previously, it has been shown that controlling physicochemical properties such as
lipophilicity and basicity can lead to ‘tuned drug-target binding kinetics.[8, 45, 46]
Therefore we divided our library into two groups in which we systematically varied
either the lipophilicity or the basicity at different locations of the scaffold. This way,
we could investigate the relationships between physicochemical properties, binding
kinetics and functional activity of these agonists, for which two independent signaling
pathways were used; G protein activation and β-arrestin recruitment.
A significant correlation was found between increasing lipophilicity at the R2
position of the LEI101 scaffold and residence time (group A agonists, Figure 5C), but
not for the R1 position (group B agonists, Figure 6). By dividing our compound library
in two parts, we showed that there is a lipophilic binding domain in the receptor
targeted by the R2 substituents. Occupying this pocket increases binding affinity due
to decreased dissociation rate. Hence, it is not the overall lipophilicity of a molecule
that determines its dissociation rate, but rather the lipophilicity at a specific position of
the scaffold.[45] These findings fit well with the observation that any relationships
between physicochemical properties and binding kinetics are both ligand and target
specific and constitute the molecular underpinning of the lipophilic efficiency
index.[47]
Currently, there is no CB2R crystal structure available to validate the
positioning of this lipophilic binding domain, but a lipophilic binding domain was
identified in the active site of CB1R, formed by six amino acid residues,[48, 49] of
which four (i.e. Val1143.32, Tyr1915.39, Leu1925.40 and Met2756.55) are conserved in
the CB2R active site.[50] This indicates that these residues may also play a role in
the formation of a lipophilic binding domain responsible for the increased residence
time of LEI101-based agonists with lipophilic R2 substituents.
19

All compounds were identified as partial agonists in two signaling pathways, G
protein activation and β-arrestin recruitment relative to CP55940 that behaved as a
full agonist.[51] From these so-called ‘end-point’ assays no obvious biased agonism
was observed, although these molecules have different binding kinetics. However,
follow up studies with regard to the influence of assay time and readout should be
performed to investigate the role of kinetic context on biased agonism.[52] On a
similar note, these functional assays were performed at different temperatures (i.e.
25°C and 37°C), which may influence the potency and efficacy values of the
compounds tested. Although this will probably not result in a difference in rank order,
it may influence the observed lack of biased signaling.[52]
Interestingly, nanomolar potency for G protein activation and β-arrestin
recruitment was associated with compounds having a residence time longer than 30
min, as a significant correlation between dissociation rate and functional potency for
both assays was identified (Figure 7). Again, this observation was specific for group
A agonists. No correlation between residence time and functional efficacy was
identified, as was reported for the Adenosine A1 receptor.[53] This observation is in
contrast with the previously reported positive correlation found between residence
time and efficacy, but not potency, for the Adenosine A2A receptor and Muscarinic M3
receptor.[16] Of note, for the Adenosine A2A receptor these molecules showed
significant longer residence times than the LEI101-based agonist library.
4.3
Target-specific binding kinetics in drug discovery
Previously, the CB2R binding kinetics of CP55940, as well as some other synthetic
cannabinoid ligands (e.g. JWH133, HU308) and endocannabinoids were
reported.[42] Because CP55940 was measured in both studies, we could use its
20

binding kinetics as reference to compare the binding kinetics of the ligands tested in
the two different studies. Interestingly, the kinetic profile of this agonist library shows
remarkable differences compared to the reported binding kinetics of some structurally
different synthetic ligands for CB2R, like JWH133 and SR144528 and
endocannabinoids anandamide (AEA) and noladin ether (NE), which all had
divergent, but relatively fast kinetics.[54] For these molecules, the association rate
was the main driving force for their affinity. Knowledge of the kinetic binding
parameters of a target’s endogenous ligands is important for two reasons: 1) it is an
indication of the ligand binding kinetics necessary to maintain homeostasis and 2)
these play a major role in defining the pharmacological effect of a drug, as they have
to compete with the endogenous ligands for binding to the active site.[55, 56]
Notably, LEI101, identified to be in vivo active in the treatment of neuropathic pain
and inflammation-induced tissue damage,[26, 27] has similar binding kinetics as 2-
AG, relative to CP55940 (10-fold slower kon, 10-fold faster koff).[54] This may indicate
that slow association plays a role in the in vivo efficacy of LEI101. Interestingly,
HU308 and JWH133, also in vivo active CB2R-selective agonists,[29, 57, 58] had
slower association rates,[54] but a similar dissociation rate, relative to CP55940 (20-
50-fold slower kon, similar koff). This may indicate that the optimal kinetic profile of in
vivo active CB2R agonists is flexible, or may be dependent on disease type and/or
progression. Although, it is noted that species differences between mouse and
human CB2R have not been taken into account.
Interestingly, slowly associating ligands may decrease on-target related side
effects by preventing high target occupancy and fast target activation.[14] This could
be important, because prolonged activation of CB2R is hypothesized to interfere with
the ECS homeostasis.[54],[59] Specifically, local, transient activation of CB2R by
21

endocannabinoids may lead to immunosuppression in the early phases of the
immune response, perhaps via apoptotic mechanisms.[60, 61] Rapid restoration of
cellular activity might also be required to counteract potential infectious threats.[62]
This indicates that the optimal kinetic profile of novel molecules needs to be
established according to their functional activity, and should always be a combination
of association and dissociation rates, resulting in an optimal level of receptor
occupancy in vivo.[63]
4.4
Conclusion
In summary, we have reported the structure kinetics relationship of LEI101-based
agonists of the cannabinoid CB2 receptor. We identified the lipophilicity of R2 position
as important feature to increase receptor residence time, which correlated with
increased potency, but not with efficacy, in two signaling pathways: G protein
activation and β-arrestin recruitment. The findings of this study provide important
insights into how CB2R agonists can be designed with desired kinetic profiles for the
future development of novel treatments of inflammatory diseases.
Acknowledgments
This work was supported by an ECHO-STIP grant from the Netherlands Organisation
for Scientific Research. The authors would like to thank Hans van den Elst, Fons
Lefeber and Karthick Babu Sai Sankar Gupta for technical assistance with HRMS
and NMR measurements, respectively.
Conflicts of interest
None
22

Authorship contributions
Participated in research design: Soethoudt, van der Stelt, Heitman
Conducted experiments: Soethoudt, Hoorens, Doelman, Martella
Performed data analysis: Soethoudt, Hoorens, Doelman
Wrote or contributed to the writing of the manuscript: Soethoudt, van der Stelt,
Heitman
References
[1] R. Zhang, F. Monsma, Binding kinetics and mechanism of action: toward the
discovery and development of better and best in class drugs, Expert Opin Drug
Discov 5(11) (2010) 1023-9.
[2] R.A. Copeland, D.L. Pompliano, T.D. Meek, Drug-target residence time and its
implications for lead optimization, Nat Rev Drug Discov 5(9) (2006) 730-9.
[3] H. Lu, K. England, C.A. Ende, J.J. Truglio, S. Luckner, B.G. Reddy, et al., Slow-
Onset Inhibition of the Fabl Enoyl Reductase from Francisella tularensis: Residence
Time and in Vivo Activity, Acs Chemical Biology 4(3) (2009) 221-231.
[4] F. Daryaee, A. Chang, J. Schiebel, Y. Lu, Z. Zhang, K. Kapilashrami, et al.,
Correlating drug-target kinetics and in vivo pharmacodynamics: long residence time
inhibitors of the FabI enoyl-ACP reductase, Chem Sci 7(9) (2016) 5945-5954.
[5] L.T. Gooljarsingh, C. Fernandes, K. Yan, H. Zhang, M. Grooms, K. Johanson, et
al., A biochemical rationale for the anticancer effects of Hsp90 inhibitors: slow, tight
binding inhibition by geldanamycin and its analogues, Proc Natl Acad Sci U S A
103(20) (2006) 7625-30.
[6] R.A. Copeland, J.M. Williams, J. Giannaras, S. Nurnberg, M. Covington, D. Pinto,
et al., Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H
synthase, Proc Natl Acad Sci U S A 91(23) (1994) 11202-6.
[7] R.A. Copeland, The drug-target residence time model: a 10-year retrospective,
Nat Rev Drug Discov 15(2) (2016) 87-95.
[8] D. Guo, J.M. Hillger, I.J. AP, L.H. Heitman, Drug-target residence time--a case for
G protein-coupled receptors, Med Res Rev 34(4) (2014) 856-92.
[9] G. Vauquelin, S.J. Charlton, Long-lasting target binding and rebinding as
mechanisms to prolong in vivo drug action, Brit J Pharmacol 161(3) (2010) 488-508.
[10] K.P. Cusack, Y. Wang, M.Z. Hoemann, J. Marjanovic, R.G. Heym, A.
Vasudevan, Design strategies to address kinetics of drug binding and residence time,
Bioorg Med Chem Lett 25(10) (2015) 2019-27.
[11] G.S. Van Aller, M.B. Pappalardi, H.M. Ott, E. Diaz, M. Brandt, B.J. Schwartz, et
al., Long residence time inhibition of EZH2 in activated polycomb repressive complex
2, ACS Chem Biol 9(3) (2014) 622-9.
[12] M.R. Dowling, S.J. Charlton, Quantifying the association and dissociation rates
of unlabelled antagonists at the muscarinic M3 receptor, Br J Pharmacol 148(7)
(2006) 927-37.
23

[13] G. Vauquelin, S. Bostoen, P. Vanderheyden, P. Seeman, Clozapine, atypical
antipsychotics, and the benefits of fast-off D2 dopamine receptor antagonism,
Naunyn Schmiedebergs Arch Pharmacol 385(4) (2012) 337-72.
[14] D.A. Sykes, H. Moore, L. Stott, N. Holliday, J.A. Javitch, J.R. Lane, et al.,
Extrapyramidal side effects of antipsychotics are linked to their association kinetics at
dopamine D-2 receptors, Nature Communications 8 (2017).
[15] W.E.A. de Witte, M. Danhof, P.H. van der Graaf, E.C.M. de Lange, In vivo Target
Residence Time and Kinetic Selectivity: The Association Rate Constant as
Determinant, Trends in Pharmacological Sciences 37(10) (2016) 831-842.
[16] D. Guo, T. Mulder-Krieger, I.J. AP, L.H. Heitman, Functional efficacy of
adenosine A(2)A receptor agonists is positively correlated to their receptor residence
time, Br J Pharmacol 166(6) (2012) 1846-59.
[17] D.A. Sykes, M.R. Dowling, S.J. Charlton, Exploring the mechanism of agonist
efficacy: a relationship between efficacy and agonist dissociation rate at the
muscarinic M3 receptor, Molecular pharmacology 76(3) (2009) 543-51.
[18] C.S. Tautermann, T. Kiechle, D. Seeliger, S. Diehl, E. Wex, R. Banholzer, et al.,
Molecular Basis for the Long Duration of Action and Kinetic Selectivity of Tiotropium
for the Muscarinic M3 Receptor, Journal of Medicinal Chemistry 56(21) (2013) 8746-
8756.
[19] A. Ligresti, L. De Petrocellis, V. Di Marzo, From Phytocannabinoids to
Cannabinoid Receptors and Endocannabinoids: Pleiotropic Physiological and
Pathological Roles Through Complex Pharmacology, Physiol Rev 96(4) (2016) 1593-
659.
[20] K. Mackie, Cannabinoid receptor homo- and heterodimerization, Life Sci 77(14)
(2005) 1667-73.
[21] R. Mechoulam, L.O. Hanus, R. Pertwee, A.C. Howlett, Early phytocannabinoid
chemistry to endocannabinoids and beyond, Nat Rev Neurosci 15(11) (2014) 757-64.
[22] B.K. Atwood, A. Straiker, K. Mackie, CB(2): therapeutic target-in-waiting, Prog
Neuropsychopharmacol Biol Psychiatry 38(1) (2012) 16-20.
[23] S. Galiegue, S. Mary, J. Marchand, D. Dussossoy, D. Carriere, P. Carayon, et
al., Expression of central and peripheral cannabinoid receptors in human immune
tissues and leukocyte subpopulations, Eur J Biochem 232(1) (1995) 54-61.
[24] P. Pacher, R. Mechoulam, Is lipid signaling through cannabinoid 2 receptors part
of a protective system?, Prog Lipid Res 50(2) (2011) 193-211.
[25] J. Guindon, A.G. Hohmann, Cannabinoid CB2 receptors: a therapeutic target for
the treatment of inflammatory and neuropathic pain, Br J Pharmacol 153(2) (2008)
319-34.
[26] P. Mukhopadhyay, M. Baggelaar, K. Erdelyi, Z. Cao, R. Cinar, F. Fezza, et al.,
The novel, orally available and peripherally restricted selective cannabinoid CB2
receptor agonist LEI-101 prevents cisplatin-induced nephrotoxicity, Br J Pharmacol
173(3) (2016) 446-58.
[27] M. van der Stelt, J. Cals, S. Broeders-Josten, J. Cottney, A.A. van der Doelen,
M. Hermkens, et al., Discovery and optimization of 1-(4-(pyridin-2-
yl)benzyl)imidazolidine-2,4-dione derivatives as a novel class of selective
cannabinoid CB2 receptor agonists, J Med Chem 54(20) (2011) 7350-62.
[28] D. Guo, E.J. van Dorp, T. Mulder-Krieger, J.P. van Veldhoven, J. Brussee, A.P.
Ijzerman, et al., Dual-point competition association assay: a fast and high-throughput
kinetic screening method for assessing ligand-receptor binding kinetics, J Biomol
Screen 18(3) (2013) 309-20.
24

[29] M. Soethoudt, U. Grether, J. Fingerle, T.W. Grim, F. Fezza, L. de Petrocellis, et
al., Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target
activity, Nat Commun 8 (2017) 13958.
[30] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D.
Provenzano, et al., Measurement of protein using bicinchoninic acid, Anal Biochem
150(1) (1985) 76-85.
[31] C. Harrison, J.R. Traynor, The [35S]GTPgammaS binding assay: approaches
and applications in pharmacology, Life Sci 74(4) (2003) 489-508.
[32] M. Soethoudt, N. van Gils, M. van der Stelt, L.H. Heitman, Protocol to Study
beta-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors, Methods Mol
Biol 1412 (2016) 103-11.
[33] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and
the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction, Biochem Pharmacol 22(23) (1973) 3099-108.
[34] H.J. Motulsky, L.C. Mahan, The kinetics of competitive radioligand binding
predicted by the law of mass action, Molecular pharmacology 25(1) (1984) 1-9.
[35] K. Pearson, Determination of the Coefficient of Correlation, Science 30(757)
(1909) 23-5.
[36] T. Ostenfeld, J. Price, M. Albanese, J. Bullman, F. Guillard, I. Meyer, et al., A
randomized, controlled study to investigate the analgesic efficacy of single doses of
the cannabinoid receptor-2 agonist GW842166, ibuprofen or placebo in patients with
acute pain following third molar tooth extraction, Clin J Pain 27(8) (2011) 668-76.
[37] A. Cooper, S. Singh, S. Hook, J.D.A. Tyndall, A.J. Vernall, Chemical Tools for
Studying Lipid-Binding Class A G Protein-Coupled Receptors, Pharmacological
reviews 69(3) (2017) 316-353.
[38] L. Xia, H. de Vries, E.B. Lenselink, J. Louvel, M.J. Waring, L. Cheng, et al.,
Structure-Affinity Relationships and Structure-Kinetic Relationships of 1,2-
Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor
Antagonists, Journal of medicinal chemistry 60(23) (2017) 9545-9564.
[39] L. Xia, H. de Vries, X. Yang, E.B. Lenselink, A. Kyrizaki, F. Barth, et al., Kinetics
of human cannabinoid 1 (CB1) receptor antagonists: Structure-kinetics relationships
(SKR) and implications for insurmountable antagonism, Biochem Pharmacol (2017).
[40] D.A. Sykes, M.R. Dowling, J. Leighton-Davies, T.C. Kent, L. Fawcett, E. Renard,
et al., The Influence of receptor kinetics on the onset and duration of action and the
therapeutic index of NVA237 and tiotropium, The Journal of pharmacology and
experimental therapeutics 343(2) (2012) 520-8.
[41] V. Katritch, G. Fenalti, E.E. Abola, B.L. Roth, V. Cherezov, R.C. Stevens,
Allosteric sodium in class A GPCR signaling, Trends in Biochemical Sciences 39(5)
(2014) 233-44.
[42] A. Martella, H. Sijben, A.C. Rufer, U. Grether, J. Fingerle, C. Ullmer, et al., A
Novel Selective Inverse Agonist of the CB2 Receptor as a Radiolabeled Tool
Compound for Kinetic Binding Studies, Molecular pharmacology 92(4) (2017) 389-
400.
[43] D.A. Sykes, S.J. Charlton, Slow receptor dissociation is not a key factor in the
duration of action of inhaled long-acting ss(2)-adrenoceptor agonists, Brit J
Pharmacol 165(8) (2012) 2672-2683.
[44] Z. Yu, J.P.D. van Veldhoven, J. Louvel, I.M.E. 't Hart, M.B. Rook, M.A.G. van der
Heyden, et al., Structure-Affinity Relationships (SARs) and Structure-Kinetics
Relationships (SKRs) of K(v)11.1 Blockers, Journal of Medicinal Chemistry 58(15)
(2015) 5916-5929.
25

[45] D.C. Miller, G. Lunn, P. Jones, Y. Sabnis, N.L. Davies, P. Driscoll, Investigation
of the effect of molecular properties on the binding kinetics of a ligand to its biological
target, Medchemcomm 3(4) (2012) 449-452.
[46] G. Tresadern, J.M. Bartolome, G.J. Macdonald, X. Langlois, Molecular
properties affecting fast dissociation from the D2 receptor, Bioorg Med Chem 19(7)
(2011) 2231-41.
[47] D. Guo, L.Z. Xia, J.P.D. van Veldhoven, M. Hazeu, T. Mocking, J. Brussee, et
al., Binding Kinetics of ZM241385 Derivatives at the Human Adenosine A(2A)
Receptor, Chemmedchem 9(4) (2014) 752-761.
[48] T. Hua, K. Vemuri, S.P. Nikas, R.B. Laprairie, Y. Wu, L. Qu, et al., Crystal
structures of agonist-bound human cannabinoid receptor CB1, Nature 547(7664)
(2017) 468-+.
[49] L. Xia, H. de Vries, E.B. Lenselink, J. Louvel, M.J. Waring, L. Cheng, et al.,
Structure–Affinity Relationships and Structure–Kinetic Relationships of 1,2-
Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor
Antagonists, Journal of Medicinal Chemistry (2017).
[50] R.D. Zhang, D.P. Hurst, J. Barnett-Norris, P.H. Reggio, Z.H. Song, Cysteine
2.59(89) in the second transmembrane domain of human CB2 receptor is accessible
within the ligand binding crevice: Evidence for possible CB2 deviation from a
rhodopsin template, Molecular Pharmacology 68(1) (2005) 69-83.
[51] G. Griffin, P.J. Atkinson, V.M. Showalter, B.R. Martin, M.E. Abood, Evaluation of
cannabinoid receptor agonists and antagonists using the guanosine-5 '-O-(3-[S-
35]thio)-triphosphate binding assay in rat cerebellar membranes, Journal of
Pharmacology and Experimental Therapeutics 285(2) (1998) 553-560.
[52] C. Klein Herenbrink, D.A. Sykes, P. Donthamsetti, M. Canals, T. Coudrat, J.
Shonberg, et al., The role of kinetic context in apparent biased agonism at GPCRs,
Nat Commun 7 (2016) 10842.
[53] J. Louvel, D. Guo, M. Agliardi, T.A.M. Mocking, R. Kars, T.P. Pham, et al.,
Agonists for the Adenosine A(1) Receptor with Tunable Residence Time. A Case for
Nonribose 4-Amino-6-aryl-5-cyano-2-thiopyrimidines, Journal of Medicinal Chemistry
57(8) (2014) 3213-3222.
[54] A. Martella, H. Sijben, A. Rufer, J. Fingerle, U. Grether, C. Ullmer, et al., A novel
selective inverse agonist of the CB2 receptor as a radiolabeled tool compound for
kinetic binding studies, Mol Pharmacol (2017).
[55] I. Nederpelt, D. Bleeker, B. Tuijt, I.J. AP, L.H. Heitman, Kinetic binding and
activation profiles of endogenous tachykinins targeting the NK1 receptor, Biochem
Pharmacol 118 (2016) 88-95.
[56] I. Nederpelt, M. Kuzikov, W.E.A. de Witte, P. Schnider, B. Tuijt, S. Gul, et al.,
From receptor binding kinetics to signal transduction; a missing link in predicting in
vivo drug-action, Sci Rep-Uk 7 (2017).
[57] H.P. Xu, C.L. Cheng, M. Chen, A. Manivannan, L. Cabay, R.G. Pertwee, et al.,
Anti-inflammatory property of the cannabinoid receptor-2-selective agonist JWH-133
in a rodent model of autoimmune uveoretinitis, J Leukocyte Biol 82(3) (2007) 532-
541.
[58] M. Rajesh, H. Pan, P. Mukhopadhyay, S. Batkai, D. Osei-Hyiaman, G. Hasko, et
al., Pivotal Advance: Cannabinoid-2 receptor agonist HU-308 protects against
hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory
response, and apoptosis, J Leukocyte Biol 82(6) (2007) 1382-1389.
26

[59] V. Di Marzo, The endocannabinoid system: its general strategy of action, tools
for its pharmacological manipulation and potential therapeutic exploitation,
Pharmacol Res 60(2) (2009) 77-84.
[60] A.C. Herring, W.S. Koh, N.E. Kaminski, Inhibition of the cyclic AMP signaling
cascade and nuclear factor binding to CRE and kappa B elements by cannabinol, a
minimally CNS-Active cannabinoid, Biochemical Pharmacology 55(7) (1998) 1013-
1023.
[61] T.K. Eisenstein, J.J. Meissler, Q. Wilson, J.P. Gaughan, M.W. Adler,
Anandamide and Delta(9)-tetrahydrocannabinol directly inhibit cells of the immune
system via CB(2) receptors, J Neuroimmunol 189(1-2) (2007) 17-22.
[62] R. Pandey, K. Mousawy, M. Nagarkatti, P. Nagarkatti, Endocannabinoids and
immune regulation, Pharmacological Research 60(2) (2009) 85-92.
[63] I. Bot, N.V. Ortiz Zacarias, W.E. de Witte, H. de Vries, P.J. van Santbrink, D. van
der Velden, et al., A novel CCR2 antagonist inhibits atherogenesis in apoE deficient
mice by achieving high receptor occupancy, Sci Rep 7(1) (2017) 52.
27

Tables
Table 1. Overview of chemical structures, physicochemical properties, equilibrium affinity and Kinetic
Rate Index (KRI) of the LEI101-based agonist library.
Physicochemical properties
Binding affinity
pKi ± SEMa
Kinetic Rate Index
KRI ± SEMb
Nr.
1
R1
R2
MW (Da)
cLogP
pKa
H
432
0.4
5.1
5.55 ± 0.08
0.62 ± 0.06
2
3
447
491
0.4
0.8
5.1
5.1
6.61 ± 0.18
6.25 ± 0.04
0.53 ± 0.06
0.67 ± 0.03
4
LEI101
473
0.9
5.1
6.51 ± 0.09
0.52 ± 0.09
5
6
461
475
0.9
1.5
5.1
5.1
7.06 ± 0.12
7.66 ± 0.08
0.51 ± 0.05
0.71 ± 0.05
7
8
489
1.9
5.1
7.74 ± 0.08
1.06 ± 0.11
475
384
398
442
424
412
426
441
440
4.4
1.3
1.4
1.7
1.9
1.9
2.4
2.8
3.0
6.1
6.3
6.3
6.3
6.3
6.3
6.3
6.3
6.3
7.48 ± 0.10
5.65 ± 0.05
6.06 ± 0.24
5.35 ± 0.04
6.17 ± 0.03
6.84 ± 0.25
7.13 ± 0.15
7.07 ± 0.10
6.21 ± 0.20
0.79 ± 0.05
0.72 ± 0.08
0.71 ± 0.06
0.77 ± 0.05
0.67 ± 0.12
0.71 ± 0.08
0.57 ± 0.08
0.67 ± 0.05
0.62 ± 0.12
9
H
10
11
12
13
14
15
16
17
18
475
382
3.5
2.6
6.9
8.3
7.67 ± 0.14
5.48 ± 0.06
0.85 ± 0.13
0.68 ± 0.07
H
28

19
20
21
22
396
440
425
439
2.6
3.0
3.7
4.1
8.3
8.3
8.3
8.3
6.70 ± 0.05
6.56 ± 0.13
7.92 ± 0.06
7.56 ± 0.02
0.60 ± 0.08
0.78 ± 0.05
0.66 ± 0.14
1.21 ± 0.07
23
24
453
454
4.6
3.3
8.3
8.8
7.61 ± 0.22
5.45 ± 0.09
1.03 ± 0.08
0.67 ± 0.01
apKi ± SEM was obtained from a [3H]CP55940 equilibrium displacement assay at 25°C, on membrane fractions of CHOK1B2R cells, and determined in three independent
experiments performed in duplicate (N=3 in duplicate)
bKRI ± SEM was obtained from a [3H]CP55940 dual point competition association assay at 10°C, on membrane fractions of CHOK1CB2R cells (N=3 in duplicate)
29