![The co-crystal structure of tiotropium in the rat M3 receptor.[84] This figure was generated with ICM Browser v3.7 (Molsoft) from PDB code: 4DAJ. Tiotropium (black) binds within the pocket. Three tyrosine residues, Y1483.33, Y5066.51, and Y5297.39, together prevent the ligand from moving out of the receptor. N5076.52 interacts with the carbonyl and hydroxyl groups through H-bonds, while the ligand's typical quaternary ammonium group interacts with D1473.32.](https://www.researchgate.net/profile/Dong-Guo-14/publication/260252504/figure/fig5/AS:267597023281193@1440811510668/The-co-crystal-structure-of-tiotropium-in-the-rat-M3-receptor84-This-figure-was_Q320.jpg)
Content uploaded by Dong Guo
Author content
All content in this area was uploaded by Dong Guo on Apr 05, 2018
Content may be subject to copyright.
Drug-Target Residence Time—A Case
for G Protein-Coupled Receptors
Dong Guo, Julia M. Hillger, Adriaan P. IJzerman, and Laura H. Heitman
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box
9502,2300 RA Leiden, the Netherlands
Published online 18 February 2014 in Wiley Online Library (wileyonlinelibrary.com).
DOI 10.1002/med.21307
䉲
Abstract: A vast number of marketed drugs act on G protein-coupled receptors (GPCRs), the most
successful category of drug targets to date. These drugs usually possess high target affinity and selectivity,
and such combined features have been the driving force in the early phases of drug discovery. However,
attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a
demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that
their beneficial effects in patients may be attributed to their long drug-target residence times (RTs).
Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on-target
side effects. Hence, the compounds’ kinetics behavior might in fact be the guiding principle to obtain a
desired and durable effect in vivo. We therefore propose that drug-target RT should be taken into account
as an additional parameter in the lead selection and optimization process. This should ultimately lead
to an increased number of candidate drugs moving to the preclinical development phase and on to the
market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo
efficacy and summarizes methods for assessing drug-target RT. C2014 Wiley Periodicals, Inc. Med. Res. Rev.,
34, No. 4, 856–892, 2014
Key words: residence time; GPCR; dissociation rate; drug discovery
1. INTRODUCTION
Drug-target residence time (RT) is an emerging concept in the drug research community. Its
definition, coined by Copeland in 2006, stands for an experimental measure of the lifetime of
a drug-target complex, reflected by its dissociation rate (koff).1This is an important pharma-
cological feature as a drug is only effective when bound to, and influencing the activity of its
physiological target.1, 2 Recently, several notable reviews including Swinney et al.,2–5 Copeland
et al.,1, 6, 7 Vauquelin et al.,8–11 and Zhang and Monsma,12,13 to name a few, have emphasized
Contract grant sponsor: Netherlands Research Organization (NWO); Contract grant number: 11188.
Correspondence to: Adriaan P. IJzerman, Gorlaeus Lab/LACDR, Department of Medicinal Chemistry, Room
L072, Leiden University, Einsteinweg 55, 2333 CC Leiden, the Netherlands. E-mail: ijzerman@lacdr.leidenuniv.nl
Medicinal Research Reviews, 34, No. 4, 856–892, 2014
C2014 Wiley Periodicals, Inc.
DRUG-TARGET RESIDENCE TIME r857
the pivotal role of RT in the early phases of drug discovery. These discussions led to increasing
recognition that drug-target RT may even be of greater importance for a drug’s effect in the
patient than its affinity, since the body, unlike an in vitro setting, is an open system where the
concentration of free drug fluctuates over time.13, 14 The dynamic flow in vivo—absorption, dis-
tribution, metabolism, and excretion—often prevents the free drug from reaching equilibrium
conditions that are otherwise readily obtained in a test tube.13,14 In this sense, optimizing drug
candidates based on equilibrium-derived parameters alone, such as affinity (Ki)/potency (IC50),
may not be ideal.15 Drug-target RT may be a useful additional parameter as it is thought to
represent a surrogate marker of drug clinical efficacy: the longer the drug occupies the receptor,
the more profound the drug may exert its effect.1, 12 Although this reasoning most certainly is
too simplistic, there is substantial evidence, as witnessed in a survey of 50 drugs on 12 different
drug targets, that about 70% of long RT therapeutics displayed higher efficacy than comparable
faster dissociating drugs.5
It needs to be pointed out that the criteria for “long” or “short” RT may vary for different
targets and for divergent clinical indications.16 For therapies requiring prolonged target occu-
pancy, a long RT drug offers advantages as it remains bound to the target and continuously
exerts its pharmacological effect even when most of the free drug has already been eliminated
from circulation.17 Importantly, this simultaneously implies that a slowly dissociating drug will
only offer a noticeably increased effect over a faster dissociating drug, when its dissociation
half-life (t1/2) outlasts its pharmacokinetic half-life, or when there are large fluctuations in the
endogenous competitor (e.g., hormone or neurotransmitter) concentrations.11 Further proof
for the importance of RT comes from numerous examples of drugs that are best-in-class due
to slow dissociation from their target receptor. One example is the well-known muscarinic M3
receptor antagonist, tiotropium, which is a long-acting, 24 hr, anticholinergic bronchodilator
used in the management of chronic obstructive pulmonary disease (COPD).29 On the other
hand, there are cases where the mechanism-based toxicity outweighs the therapeutic advan-
tages by long receptor occupancy. In this situation, a fast dissociating compound displaying
short-lived intervention is preferred. This may be the case for the antipsychotic D2dopamine
receptor antagonists, where long RT is not desired due to on-target toxicity associated with
strong extrapyrimidal motor effects. We will discuss this in a later part of this review.
2. MATHEMATICAL DEFINITIONS OF DRUG-TARGET RT
Drug-target RT is equal to the reciprocal of the dissociation rate constant (RT =1/koff).1Its
value is interchangeable with the dissociation half-life (t1/2 =ln2/koff) with a factor of ln2, that
is, RT =t1/2/ln2. Both can be used to represent the duration of the drug-target binary complex.
For the convenience of discussion, we chose to use the dissociation half-lifeto describe the drug-
target RT in the following part of this review, since most of the kinetic values in literature were
reported with t1/2 values.
As mentioned above, it is necessary to determine the dissociation rate constant to calculate
the RT or t1/2. However, the definition of koff depends on the specific mechanism of ligand–
receptor interaction.1,14 In general, three situations are well known. Equation 1 represents
a simple one-step association and dissociation process, where the receptor (R) and ligand
(L) encounter each other to form a binary complex (RL) with association rate k1(kon)and
dissociation rate k2(koff), respectively.
R+Lk1
k2
RL (1)
Medicinal Research Reviews DOI 10.1002/med
858 rGUO ET AL.
In contrast, Equation 2 represents a more complicated process. Upon the binding of the
ligand to the receptor, the initial complex (RL) undergoes a conformation isomerization lead-
ing to a much higher affinity final complex (RL*). This is also known as the “induced-fit”
mechanism. In this case, koff entails both forward (k3) and reverse steps (k4) in receptor iso-
merization as well as the on- and off-rate constants for the free ligand bound to and unbound
from the free receptor, such that koff =k2k4/(k2+k3+k4). This isomerization step enhances
the interaction between the ligand and the target, hence resulting in an extended overall t1/2.
R+Lk1
k2
RL k3
k4
RL∗(2)
A third mechanism is represented in Equation 3, where the receptor is in equilibrium
between two conformational states (R and R*) in the absence of ligand. Of these conformational
states, the ligand specifically binds to one, R*, rather than the other (R), to form the R*L
complex. This is also known as the “conformation-selection” mechanism. The interconversion
between conformational states R and R* is slow relative to the binding of the ligand to the R*
state, thus it represents the rate-limiting step for the formation of the binary complex. For the
dissociation rate constant, its value is equal to k4.
Rk1
k2
R∗k3·[L]
k4
R∗L(3)
Among these three mechanisms, the first situation is most commonly encountered in ligand-
GPCR (where GPCR is G protein-coupled receptor) binding kinetics, while the second mech-
anism can be the case for high-affinity ligand–receptor interactions, especially for enzyme in-
hibitors and receptor antagonists1,6, 14 ; the third situation is rarely observed in ligand–receptor
interactions.14
3. EXPERIMENTAL STRATEGIES TO ASSESS DRUG-TARGET RT
Measurement of drug-target complex lifetime, reflected by koff or t1/2, is not a new phenomenon
per se. To date, there are several strategies available to examine the binding kinetics of ligand–
GPCR interactions. The advantages and disadvantages/limitations of these experimental strate-
gies to access drug-target RT are briefly outlined in Table I. Of these approaches, the most
common and straightforward method is to radiolabel a compound of interest with high affinity
and directly measure its on- and off-rate in a kinetic setup of a radioligand binding experiment.
Two examples, in the context of drug-target RT, are radiolabeled aprepitant at the neurokinin
1(NK
1) receptor and tiotropium at the muscarinic M3receptor (M3R). Both compounds have
been well characterized by using this method.19,20 Alternatively, a ligand can be fluorescently
labeled to calculate its drug-target RT. For instance, Keppler and co-workers have developed a
so-called Tag-lite Rplatform, a combination of a Homogeneous Time-Resolved Fluorescence
(HTRF R) detection method with a covalent labeling technology called SNAP-tag R.21 One
application note from Cisbio describes fluorescently labeled spiperone binding at the Tag-lite
dopamine D2receptor.22 Nevertheless, the process of radiolabeling or fluorescent labeling is
costly and labor intensive, which, as a consequence, confines kinetics determinations to a lim-
ited scope. Therefore, alternative approaches are highly desired (see Fig. 1 for experimental
schemes and representative results).
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r859
Table I. The Advantages and Disadvantages/Limitations of Experimental Strategies to Access Drug-
Target Residence Time
Assay Advantages Disadvantages/limitations Ref.
Direct kinetic
radioligand binding
assay
Straightforward; robust;
quantitative measurement
Ligand of interest needs to be
radiolabeled; low-throughput
assay format
19–21
Indirect kinetic
radioligand binding
assay
Only one labeled ligand
necessary; quantitative
measurement; adjustable for
high-throughput screening
Need for suitable radiolabeled
ligand; intermediate filtration
and/or “washout” steps are
needed
23–26
Competition association
assay
Only one labeled ligand
necessary; quantitative
measurement
Need for suitable radiolabeled
ligand; low-throughput assay
format
27–34
Dual-point competition
association assay
Only one labeled ligand
necessary; high-throughput
assay format; easy readout for
longer or shorter residence
time compounds
Need for suitable radiolabeled
ligand; qualitative
measurement
18, 33
Dual-point IC50/Ki
value determination
Only one labeled ligand
necessary; adjustable for
high-throughput screening
Need for suitable radiolabeled
ligand; only selects long
residence time compounds
30, 34
Functional reversibility
assay
No need for labeled ligands;
adjustable for
high-throughput screening
Qualitative measurement; not
suitable for binding kinetics at
low temperatures
35–46
Label-free kinetic
measurement
No need for labeled ligands;
quantitative measurement
Need for protein purification and
immobilization;
low-throughput assay format
47–55
A. Indirect Kinetic Radioligand Binding Assays
Contrary to the above-mentioned, one-step, direct measurement of radiolabeled ligands’ dis-
sociation rates, indirect approaches usually contain two consecutive parts (Fig. 1A). First,
the receptor-bearing material is preincubated with a saturating concentration of unlabeled
ligand (to occupy the majority of receptors), followed by an intermediate filtration and/or
“washout” step—methods vary in different labs23–26—to initiate ligand dissociation. Second,
a fixed concentration of radioligand is added and incubated thereafter for a short period
before assay termination. Thus, the unlabeled competitor’s dissociation rate could be in-
ferred from the recovery of radioligand binding. Furthermore, this method can be adopted
and modified for high-throughput screening; an example is at the dopamine D2receptor
(D2R).26
B. Competition Association Assay
The competition association assay has been shown to be highly accurate in determining the
binding kinetics of unlabeled ligands at the β-adrenergic receptor,27, 28 and more recently,
at the muscarinic M3receptor,29 gonadotropin-releasing hormone (GnRH) receptor,30 his-
tamine H1receptor,31 and adenosine receptors,32,33 to name but a few. This method is based
on a framework developed by Motulsky and Mahan,34 where an unlabeled competitor is
co-incubated with a radioligand during a kinetic association experiment (Fig. 1B). If the com-
petitor dissociates faster from its target than the radioligand (k2<k4), the specific binding
Medicinal Research Reviews DOI 10.1002/med
860 rGUO ET AL.
Figure 1. Biochemical assays to evaluate drug-target residence time. (A) Indirect kinetic radioligand binding
assays: the receptor-bearing material is preincubated with a saturating concentration of unlabeled ligand, fol-
lowed by an intermediate filtration and/or “washout” step to initiate ligand dissociation. Subsequently, a fixed
concentration of radioligand is added and incubated thereafter for a period before assay termination. This
scheme is adapted from Vauquelin et al.35 In this assay, a slowly dissociating compound () induces an ap-
parent decrease in the amount of free receptor, compared to a fast dissociating compound (), which allows
the binding of a subsequently added radioligand; control: no preincubation (•); (B) (dual-point) competition
association assay: an unlabeled competitor is co-incubated with a radioligand in a kinetic association experi-
ment, and followed for two or more time points. a: a radioligand association curve without co-incubation of an
unlabeled competitor. b: a co-incubated unlabeled competitor dissociates slower than the radioligand used.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r861
of the radioligand will slowly and monotonically approach its equilibrium in time (Fig. 1B,
c). However, when the competitor dissociates slower (k2>k4), the association curve of the
radioligand will consist of two phases starting with a typical “overshoot” followed by a decline
until a new equilibrium is reached (Fig. 1B, b).34 In contrast, the association rates of either
the competitor (k3) or the radioligand (k1) are usually less influential on the pattern of the
curves.34 Recently, the competition association assay has also been adopted and modified by
Guo et al. for high-throughput kinetic screening studies at the adenosine A1receptor.33 Briefly,
in a so-called dual-point competition association assay, two time points are selected to mea-
sure radioligand binding (1) at which the radiolabeled ligand just reaches equilibrium under
control conditions in the absence of an unlabeled ligand (Fig. 1B, aand t1), and (2) at which
the incubation time is long enough for the labeled and unlabeled compound to equilibrate
with the target (Fig. 1B, t2). Next, the ratio of the binding at the first time point (Bt1)and
that at the second time point (Bt2) is calculated, which Guo et al. defined as the “kinetic rate
index” (KRI). In this manner, the compounds that quickly dissociate from their target will
have a KRI below or equal to 1 (Fig. 1B, c). Conversely, compounds that dissociate slowly
from their target compared to the radioligand will have a KRI value larger than 1, resulting
from the typical “overshoot” in the association curve (Fig. 1B, b). Notably, the dual-point
assay and KRI value can be applied to screen for candidate compounds with either long
or short RT. This suggests the general applicability of this assay for other drug targets as
well.
C. Dual-Point IC50/KiValue Determination
Another method enabling kinetic screening was reported by Heise et al. They used a scintil-
lation proximity assay to measure an antagonist’s affinity after 30 min or 10 hr of incuba-
tion and examined their ratio (Ki30 min/Ki10 hr) (Fig. 1C).30 The theory behind this is based
on the observation that displacement curves will shift leftward over time until a state of
equilibrium is reached; the time to reach that state is determined by the dissociation rate
of the slower unbinding process.34 In other words, a slowly dissociating compound requires
longer incubation times to reach equilibrium than a fast dissociating one, which is often ne-
glected. This translates into markedly decreased experimental IC50 or Kivalues over time.
In this sense, the ratio of the dual-point IC50 or Kivalues represents a surrogate of overall
RT. However, the principle of this approach also simultaneously implies its limited appli-
cation, that is, in screening long RT candidate compounds only, since for short RT com-
pounds the state of equilibrium is quickly established and the Kiratio derived thereof is hardly
changed.
c: a co-incubated unlabeled competitor dissociates faster than the radioligand used. Bt1: specific radioligand
binding at the first time point (t1); Bt2: specific radioligand binding at the second time point (t2). Kinetic rate index
(KRI) is defined as Bt1/Bt2 . This scheme is adapted from Guo et al.33; (C) dual-point IC50 /Kivalue determination:
an unlabeled competitor is co-incubated with a radioligand for either a short time (in this case, e.g., 30 min) or a
long time (in this case, e.g., 10 hr) and their corresponding IC50 or Kivalues are calculated. In the representative
result, a slowly dissociation compound (and ) will have a more pronounced affinity shift than a fast dissociation
compound (and ); (D) functional reversibility experiment: in the upper panel, tissues/cells are preincubated
with an antagonist before challenge with an agonist. A slowly dissociating antagonist () will cause a rightward
shift of the concentration-response curve and a concomitant decline in maximal response of the agonist; a fast
dissociating antagonist () will cause a rightward shift of the concentration-response curve without affecting the
maximal response of the agonist. If as in the lower layer an antagonist is co-incubated with an agonist, both
slowly and fast dissociating competitive antagonists will cause a rightward shift of the concentration-response
curve without affecting the maximal response of the agonist. Otherwise they are noncompetitive to the agonist;
control: agonist only (•). This scheme is adapted from Vauquelin et al.35
Medicinal Research Reviews DOI 10.1002/med
862 rGUO ET AL.
D. Functional Reversibility Assay
A ligand’s binding kinetics profile could also be inferred from circumstantial evidence, which is
commonly based on functional assays resembling the classical “organ-bath” experiment. More
specifically, it requires preincubating tissues/cells with antagonists (to allow them sufficient time
to reach binding equilibrium conditions) before their challenge with an agonist (Fig. 1D).35
As a result, fast dissociating antagonists can only cause a rightward shift of the agonist’s
concentration-dependent curve, since the receptor can quickly be liberated from the antago-
nist’s blockade and thus readily respond to the subsequently administered agonist. These types
of antagonists are categorized as “surmountable” (Fig. 1D, preincubation experiment).9, 36, 37
In contrast, slowly dissociating antagonists or, at the extreme, irreversible binders can induce
not only a rightward shift but also a concomitant decline in maximal response of the ag-
onist, due to sustained receptor blockade which, on a macroscopic scale, reduces the total
receptor population available for an agonist’s response.38, 39 Such antagonists are denoted as
insurmountable (Fig. 1D, preincubation experiment).9,36, 37, 40 Notably, such an insurmountable
pattern can also be caused by a number of different mechanisms, such as allosteric interactions,
receptor internalization, or postreceptor functional response blockade.41–44 None of these re-
sult from competitive antagonism. Nevertheless, these mechanisms can be distinguished in a
co-incubation experiment, where a depressed maximal response can only be observed by a
noncompetitive mechanism, but not for competitive antagonists, no matter how fast or slowly
they dissociate from the target (Fig. 1D, co-incubation experiment). Thus, it is always necessary
to perform a co-incubation experiment along with the preincubation experiment to clarify the
mechanism of the insurmountable antagonism.37,45, 46 It is important to mention that the extent
of the insurmountable effect, for example, a decreased maximal response, is time dependent.
During a short period of agonist exposure (following preincubation with a slowly dissociating
antagonist), not all the receptor sites are liberated before the response is measured, a state of
so-called “hemi-equilibrium”.38, 39, 44 However, if the agonist exposure lasts longer, the occu-
pancy of the receptors will readjust with time until, ultimately, both the agonist-receptor and
the antagonist-receptor interactions reach equilibrium.44 Evidently, the insurmountability of
such slowly dissociating compounds will change if the response is measured at different time
points.
E. Label-Free Kinetic Measurements
One of the most well-established label-free measurements for kinetics is surface plasmon res-
onance (SPR) measurement. In SPR, the protein is immobilized on a coated gold chip, which
is then exposed to the compound of interest under continuous flow. Ligand binding to the
receptor immobilized on the chip’s surface induces a real time change in the refractive index
on the sensor surface. As such a change is linear to the number of molecules bound, it allows
quantitative characterization of binding kinetics (for review, see16,47, 48 ).
Another emerging label-free technology enabling kinetics measurement is by surface acous-
tic wave (SAW) biosensor.49 This methodology captures real-time mass changes on the surface,
which result in a shifted phase and/or changed amplitude of the sound wave signal.50 Be-
sides these two biophysical assays, other equally valid technologies have also been reported
elsewhere.51
However, these biophysical techniques have not been thoroughly tested on GPCRs. The
difficulty is that these receptors are integral membrane proteins, and rapidly deteriorate when
taken out of their natural environment, which is often a prerequisite for these newer biophys-
ical approaches. Several strategies were developed to overcome this inherent difficulty. One
of the successful examples is the application of a protein engineering method to generate a
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r863
stabilized receptor (StaR), for example, the adenosine A2A receptor, minimally modified for
thermostability.52–54 The stabilized receptors can be purified in large quantities, while retaining
correct folding, thus generating materials suitable for a broad range of structural and bio-
physical studies.53 Another technique is to solubilize a given GPCR from whole cell pellets
and capture it on antibody surfaces for analysis, exemplified by an SPR study on the CCR5
and CXCR4 chemokine receptors.55 These solubilized receptor preparations could be useful
materials for binding kinetics studies.
4. REVISIT KINETICS DATA ON GPCRs
To date, nearly 800 genes encoding GPCRs have been identified, representing a substantial
share of the human genome.56 In general, all these receptors possess structurally conserved
heptahelical transmembrane motifs (7TM) connected by alternating intracellular and extracel-
lular loops, with the amino terminus located at the extracellular side and the carboxyl terminus
at the intracellular side.57 Despite these common features in their molecular structure, GPCRs
are characterized by a relatively low overall sequence identity (less than 20%). According to
phylogenetic classification, these receptors can be divided into five main families—Rhodopsin
(Class A), Secretin (Class B), Glutamate (Class C), Adhesion,andFrizzled/Taste2.56,58 Recep-
tors from Class A to C have been most addressed in drug discovery efforts. The natural ligands
of GPCRs are highly variable ranging from small ions and photons to large glycoproteins,
which indicates the important and versatile roles these proteins play in a vast number of biolog-
ical processes. These are also the reasons why GPCRs have developed into a very “druggable”
class of targets for more than 30% of currently available drugs.59 However, in the introduc-
tion we also mentioned the tremendous attrition along R&D pipelines, and suggested that
detailed mechanism-based studies of drug-target interaction should be introduced in the early
phases of drug discovery to prevent “fail late, fail expensive” scenarios. For this we examined
the gigantic “pool” of GPCR ligands and retrospectively analyzed the “kinotype” (kinetics
features) of some cases. Without claiming to be exhaustive, we have summarized a series of
prototypic GPCR ligands that have known kinetics information (Table II). We listed one rep-
resentative (candidate) drug per target (26 targets in total) including their clinical indications.
Several conclusions can be drawn from this table. First, the existing kinetics data for GPCR
ligands are nowhere near the number of affinity-based evaluations in the early phases of drug
discovery. The sum of targets presented here is far behind the total population of “druggable”
GPCRs, estimated to be 300.60, 61 Clearly, ligand–receptor binding kinetics has been overlooked
and less investigated in the past. Second, the range of drug-target RTs, reflected by t1/2 in
Table II, varies from seconds to hours. Most of these are in line with the clinical applications
of the related drug. For instance, patients with allergy can benefit from the long-lasting effect
by desloratidine (t1/2 >6 hr) in relieving nasal congestion, or patients with COPD from taking
tiotropium (t1/2 =27 hr) for a durable bronchodilatory effect. In both cases, their clinical in-
dications favor sustained target occupancy. On the contrary, for some marketed drugs, serious
adverse effects could go along with the desired effects due to unwanted kinetic rates. This is
exemplified by clopidogrel, which is an antiplatelet agent used to inhibit the formation of blood
clots. However, its irreversible inhibitory effect may prolong or exacerbate bleeding.62 Another
case is represented by lofentanil for analgesia. Although its longer duration of action has an
advantage for certain types of analgesia,63 it is still unsuitable for most practical applications.64
Instead, short-acting derivatives such as sufentanil or remifentanil are preferred for medical
use in human surgical procedures.65,66 One can speculate that such clinical limitations could be
avoided or at least foreseen if kinetics were to be incorporated in the early phase of candidate
selection.
Medicinal Research Reviews DOI 10.1002/med
864 rGUO ET AL.
Table II. Overview of GPCR Ligand Kinetics
Cmpd Targeta
Dissociation
half-life (t1/2)bClinical indications StatuscRef.
Ritanserin 5-HT2receptor 160 min (37◦C) Cocaine-related
disorders
Phase II 160
Risperidone 5-HT7receptor Irreversible (37◦C) Schizophrenia
Alcohol abuse
Marketed 161
Clonidine α2A-Autoreceptor 27.6 sec (37◦C) Attention-deficit
hyperactivity
disorder
Marketed 99
Bopindolol β1-Adrenoceptor >4 hr (rat) Hypertension Marketed 162
Olodaterol β2-Adrenoceptor 17.8 hr Chronic obstructive
pulmonary disease
Marketed 163
Lofentanil μReceptor 260 min (37◦C) Analgesia Marketed 164
Candesartan AT1receptor 120 min (37◦C) Hypertension Marketed 43
UK432,097 A2A receptor 173 min (5◦C) Chronic obstructive
pulmonary disease
Phase II
(terminated)d
32
Taranabant CB1receptor 122 min (37◦C) Obesity Phase III
(terminated)e
25
Maraviroc CCR5 >136 hr (4◦C) HIV Marketed 165
SCH527123 CXCR2 22 hr Asthma Psoriasis Phase II 166
PF-4850890 CRF1receptor 6.79 hr (rat) Depression Anxiety
Stress disorders
Preclinical 153
Clozapine D2receptor 14.5 sec (37◦C) Schizophrenia Marketed 135
AM432 DP2receptor 90 min (37◦C) Chronic obstructive
pulmonary disease
Preclinical 167
Uracil 3 GnRH receptor >43 hr Reproductive-
endocrine axis
disorders
Preclinical 168
Desloratidine H1receptor >6 hr Allergy Marketed 169
JNJ7777120 H4receptor 62 min Inflammation Preclinical 170
Tiotropium M3receptor 27 hr Chronic obstructive
pulmonary disease
Marketed 80
Aprepitant NK1receptor 154 min Chemotherapy-
induced
emesis
Marketed 19
Ibodutant NK2receptor 28 min Irritable bowel
syndrome
Phase II 171
Osanetant NK3receptor 10 min Schizophrenia Phase II 172
Almorexant OX2receptor 139 min Chronic primary
insomnia
Phase III 173
Clopidogrel P2Y12 receptor Irreversible (37◦C) Stroke Heart attack
Severe chest pain
Marketed 62
Apafant PAF receptor 50 min (guinea
pig)
Arrhythmias Breast
cancer
Inflammation
Preclinical 174
GSK1562590 UT receptor >1 hr (rat) Hypertension Preclinical 175
SCH500946 Y5receptor 71 min Obesity Preclinical 176
aOfficial IUPHAR receptor name. bDissociation half-life at room temperature and on human receptors
unless mentioned otherwise. cClinical status obtained from ClinicalTrials.gov.179 dDiscontinued due
to lack of efficacy. eDiscontinued due to high level of central nervous system side effects.177,178
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r865
5. STRUCTURE–KINETICS AND STRUCTURE–AFFINITY RELATIONSHIPS
Thus far the above-mentioned analyses in retrospect are still largely based on “hindsight”
information; hardly any structure–kinetics relationship (SKR) studies, in prospect, have been
reported previously. Therefore, we should like to propose a strategy for compound optimization
that combines an extensive SKR study with a classical structure–affinity relationship study
(SAR). It is envisioned that this added metric, that is, binding kinetics or the “kinotype” of a
given compound, offers several advantages over the traditional and simple SAR paradigm:
1. A better understanding of the molecular mechanism of action. This includes detailed
characterization of not only the bound states under equilibrium conditions (SAR) but
also the entire drug-target interaction that comprises metastable intermediate states and
transition states (SKR).14,67 Such gained knowledge offers rationales for efficient drug
design.
2. A more steered drug design and development. Different targets may have distinct kinetics
preferences. Aiming for long or short RT compounds is a crucial consideration in early
phase discovery to triage and advance the best leads toward clinical applications.16
3. An efficient translation from in vitro to in vivo settings. This may decrease the prevalence
of the “fail late, fail expensive” scenario, especially considering that lack of efficacy is one
of the major reasons for drug attrition.68, 69
In addition, a detailed SKR study also describes the association rate of the ligand–receptor
interaction, which has been less investigated and is often thought to be diffusion-limited.14 As
a consequence, this could lead to the assumption that optimizing for a long RT is tantamount
to optimizing the binding affinity, since KD=koff/kon and kon reaches an (identical) limit.
However, Vilums et al. have recently reported a thorough SKR study on the CCR2 receptor, in
which it was shown that this general assumption is not necessarily valid.18 They documented
that the compounds’ on-rates varied significantly from 0.01 to 0.0003 nM−1·min−1—more than
three orders of magnitude from the diffusion-limited value (approximately 1010 M−1·min−1).1
It is also notable that the affinity difference of two diastereomers in that paper with Kivalues
of 3.6 and 289 nM, was predominantly caused by their 18-fold difference in the on-rate, rather
than their rather similar RTs (135 and 77 min). Such valuable information can only be obtained
by a detailed SKR study, which has not been reported for many GPCRs so far.
Evidently, a detailed SKR offers added value to the traditional metric of affinity. This
new strategy of combining SAR and SKR can be expected to further enhance the success of
drug discovery and development. To further illustrate the importance of incorporating SKR
into the pipeline of early phases of drug research, in the following part of this review we will
primarily address small molecules targeting class A and B GPCRs, which have been reported
to have optimized binding kinetics, more specifically dissociation kinetics. Specific evidence will
be given for three “long RT” targets and one “short RT” target; muscarinic M3, tachykinin
NK1and CRF1receptor, and dopamine D2receptors, respectively, since these targets have been
(most) extensively studied in this area.
6. DRUG-TARGET RT FOR CLASS A GPCRs
A. Muscarinic Receptors
Muscarinic receptors, or muscarinic acetylcholine receptors (mAchRs), are a group of class
A GPCRs, which are endogenously activated by acetylcholine.70 To date, five subtypes of
Medicinal Research Reviews DOI 10.1002/med
866 rGUO ET AL.
Figure 2. Structures of small molecule M3muscarinic receptor ligands.
muscarinic receptors are identified, namely M1–M5.71,72 These receptors can be further cate-
gorized into two broad groups based on their primary coupling efficiency to G proteins; M2
and M4muscarinic receptors couple to the pertussis toxin-sensitive Gi/o proteins, while M1,
M3, and M5muscarinic receptors to Gq/11 proteins.70,73 Of all five muscarinic subtypes, M3
receptor is located in the smooth muscles of blood vessels throughout the body as well as in the
lung. Hyperactivation of the lung M3receptor can cause overconstriction of smooth muscle,
such as that observed during bronchoconstriction. Thus, antagonizing M3receptor can lead to
relaxation of airway smooth muscles and result in symptom relief of COPD.74, 75 Additionally,
for the treatment of chronic illness, long duration of action (preferably 24 hr) is an important
feature of drugs that enables both prolonged efficacy and patient compliance.76 Therefore,
the treatment of COPD can benefit from a long-acting muscarinic antagonist (LAMA), as
evidenced by tiotropium and other M3antagonists summarized below.
1. Tiotropium
Tiotropium is a long-acting anticholinergic bronchodilator, which is currently marketed under
the trade name SpirivaTM (Fig. 2, Compound 1).77–79 Given its long in vivo duration of action,
several groups, in retrospect, performed in vitro time-dependent IC50 value analysis to obtain
the accurate affinity and binding kinetics of tiotropium.29, 80 It was found that equilibrium is
reached only after 18–20 hr in a radioligand binding assay. This phenomenon is consistent with
the theory by Motulsky and Mahan that a long incubation time is needed for a radioligand to
reach equilibrium in the presence of a slowly dissociating competitor.34 Thus, it also explains
that different affinities were published by different groups; after a period of 20 hr incubation,
the Kivalue of tiotropium was 8 pM,29,80 while after only 4 hr incubation, its KDvalue was
0.33 nM.78 The t1/2 of [3H]-tiotropium was 34.6 hr at room temperature, which was much longer
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r867
Table III. Binding Kinetics of M3Muscarinic Receptor Ligands
Nr CmpdaAffinity (Kior pKi)bDissociation half-life (t1/2)cRef.
1. Tiotropium 8 pM 34.6 hr 29, 78
2. Ipratropium 0.69 nM 0.26 hr 78
3. Aclidinium 18 pM 10.7 hr 80
4. PF-335659 132 pM >24 hr 96
5. Glycopyrronium 10.04 6.3 hr 80
6. Acetylcholine 4.87 7.8 sec 98
7. Carbachol 4.09 6.3 sec 98
8. Methacholine 4.52 6.0 sec 98
9. Oxotremorine-M 4.61 7.2 sec 98
10. Oxotremorine 5.61 2.6 sec 98
11. Bethanechol 3.71 3.7 sec 98
12. Pilocarpine 4.78 3.0 sec 98
aCompound names and synonyms used in original references. bAffinity, expressed as Kivalues
or pKias reported in original references; result on human receptor unless mentioned otherwise.
cDissociation half-life at room temperature.
than ipratropium (Fig. 2, Compound 2; t1/2 =0.26 hr, Table III), a relatively short-acting agent
requiring up to four doses per day.74, 78 Similarly, a kinetics study performed by Dowling and
Charlton using a competition association assay has also demonstrated that tiotropium has a
very long t1/2 (7.7 hr) at room temperature,29 which, however, is shorter than the value (34.6
hr) reported by Disse et al.78 This may be due to the difference in their buffer for membrane
preparation and/or the binding buffer for kinetic measurements, for instance, the presence of
sodium ions. Likewise, tiotropium has an even shorter dissociation half-life (46.2 min) under
mock physiological conditions at 37◦C, where the ionic strength (including Na+,Ca
2+,K
+,
Mg2+,andCl
−ions) of the assay buffer was increased.81 This example furthermore highlights
the importance of careful choices in assay development for kinetic measurements. Another
important pharmacological feature of tiotropium is its “kinetic receptor subtype selectivity,”
namely a longer dissociation half-life at the M3receptor, 34.7 hr, than at the M1and M2
receptors (14.6 and 3.6 hr, respectively).78 In contrast, tiotropium’s affinities at these receptors
were similar and in the subnanomolar concentration range.78 Taken together, these features
make tiotropium the most widely used LAMA worldwide so far.81, 82
To understand the molecular basis of the exceedingly long RT of tiotropium, Tautermann
et al. performed a “kinetic mapping” of its binding sites and the “exit” channel from the
M3receptor by testing the kinetics of tiotropium at mutant M3receptors.83 They found that
the slowly dissociating profile of tiotropium was attributed to interactions in the binding site
particularly with N5086.52 (N5076.52 on the rat M3receptor, see Fig. 3). Importantly, this residue
appeared to “anchor” tiotropium at the receptor via directly interacting with its hydroxyl
group and ester group so as to restrict the movement of the ligand toward the exit channel.
Consequently, tiotropium’s RT was 45-fold longer on the wild-type M3receptor than that of
its analogue lacking the hydroxyl group, while little difference was observed at the N5086.52A
mutant.
Furthermore, a recent structural biology study of the M3receptor (from rat) co-crystallized
with tiotropium (PDB code: 4DAJ) provided a clear image for the molecular mechanism behind
its long RT profile.84 As presented in Figure 3, tiotropium binds deeply in the receptor core
and is covered by an aromatic “lid” comprising three conserved tyrosines—Y1483.33, Y5066.51 ,
and Y5297.39. This aromatic “lid” nearly occludes the ligand from the solvent thus preventing it
Medicinal Research Reviews DOI 10.1002/med
868 rGUO ET AL.
Figure 3. The co-crystal structure of tiotropium in the rat M3receptor.84 This figure was generated with ICM
Browser v3.7 (Molsoft) from PDB code: 4DAJ. Tiotropium (black) binds within the pocket. Three tyrosine residues,
Y1483.33, Y5066.51 , and Y5297.39, together prevent the ligand from moving out of the receptor. N5076.52 interacts
with the carbonyl and hydroxyl groups through H-bonds, while the ligand’s typical quaternary ammonium group
interacts with D1473.32.
from being “wetted” by water molecules and then “washed” off the binding pocket. In addition,
the above-mentioned N5076.52 interacts with the carbonyl and hydroxyl groups of tiotropium
through H-bonds, while the ligand’s typical quaternary amine interacts with D1473.32.
The elucidation of the co-crystal structure of tiotropium in the rat M3receptor also facili-
tated molecular dynamic simulations to characterize the pathway by which tiotropium binds to
and dissociates from the rat M3receptor.84 In contrast to a simple one-step dissociation process,
the ligand appeared to first leave the orthosteric ligand binding pocket and then “pause” at
an extracellular “vestibule,” forming a loosely connected intermediate state, before tiotropium
finally dissociated from the receptor. During this process, the first step formed a large energy
barrier that involved the aromatic “lid” (Y1483.33, Y5066.51, and Y5297.39 ) around the charged
ligand head group opening up, while the second step posed a smaller energetic barrier. In this
sense, the RT of tiotropium is greatly determined by the first step, which is rate limiting for the
ligand dissociation process.
As expected from the kinetic binding profile of tiotropium, an insurmountable antagonist
behavior was observed in a functional reversibility experiment. This was detailed by Casarosa
et al. during the preclinical evaluation of several long- and short-acting muscarinic antagonists,
where tiotropium was preincubated for 15 min before the addition of the agonist muscarine
in the inositol phosphate accumulation assay.80 Fitting the dose ratios over a large range of
tiotropium concentrations (10−10–10−5M) generated a Schild plot with a slope of 1.29, higher
than unity. Such a value indicated the insurmountable M3R antagonism of tiotropium, which
was a result of a hemiequilibrium due to its long M3R RT profile.37
Taken together, the in vitro results mentioned above combined with further evidence unveil
the molecular basis of the long RT profile of tiotropium. These results correlate with this
compound’s long in vivo duration of bronchoprotection (>24 hr) as determined in a model
of acetylcholine-induced bronchoconstriction in anesthetized dogs.80 Moreover, despite the
possible influence of drug metabolism and pharmacokinetic factors,85 the clinical profile of
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r869
tiotropium in COPD highlights an inextricable link between long receptor RT and long duration
of action that enables a simple, once-daily dosage regime.77,86, 87
2. Aclidinium
Aclidinium (Fig. 2, Compound 3) is a long-acting, inhaled muscarinic antagonist recently ap-
proved by the Food and Drug Administration (FDA) as a maintenance treatment for COPD.88
The synthesis and biological evaluation of aclidinium and its analogues were elaborately re-
ported by Prat et al. and Gavalda et al.20, 89 This antagonist was specifically designed for a
maintained bronchodilatory efficacy with fewer unwanted systematic anticholinergic effects,
such as dry mouth (reported in 11.6% of patients treated with tiotropium bromide vs. 3.5%
of patients treated with placebo), glaucoma, constipation, increased heart rate, and urinary
retention.90 According to Prat et al., aclidinium had a rapid hydrolysis in human plasma, hence
minimizing the class-related systematic side effect. However, such a fast pharmacokinetics pro-
file of aclidinium did not influence its long duration of action (over 24 hr), as evaluated in an
animal model.20 This phenomenon was attributed to aclidinium’s slowly dissociating profile
from the M3receptor.
Compared to tiotropium, aclidinium had a similar affinity for the M3receptor (Ki=
18 pM), while its t1/2 (10.7 hr) was approximately twofold shorter (Table III).80 In another
study, Gavalda et al. used radiolabeled aclidinium and found a KDvalue of 0.32 nM, combined
with t1/2 of 29.24 hr, which was shorter than [3H]-tiotropium (62.19 hr).20
In vitro functional activity and in vivo duration of bronchoprotection of aclidinium was
also examined by Gavalda et al.20 More specifically, they used the isolated guinea pig trachea
and treated it with carbachol to generate dose-dependent contraction. A 60 min pretreatment
with aclidinium or tiotropium shifted the concentration-response curves of carbachol to the
right. Along with the potency shift, tiotropium also suppressed the maximal agonist effect while
aclidinium did not. Such a result is indicative of the relatively shorter RT of aclidinium than
that of tiotropium at the M3receptor.
In addition to the characterization of aclidinium’s functional activity, the onset of action of
this compound was also studied in the carbachol-induced contraction assay. Notably, aclidinium
displayed a faster onset of action (t1/2 =6.8 min, tmax =35.9 min) than tiotropium (t1/2 =
13.6 min, tmax =61.2 min). For the in vivo duration of bronchoprotection, the effect half-life
of aclidinium (defined as the time taken to reduce the maximal bronchoconstriction by 50%)
was 29 hr. This duration of action was considerably longer than that of ipratropium (8 hr)
yet shorter than tiotropium (64 hr). Likewise, Casarosa et al. reported that aclidinium and
tiotropium displayed different levels of bronchoprotection at 24 hr after equieffective dose
administration (tiotropium =35%, aclidinium =21%).80 Clearly, these in vivo results are in line
with the in vitro binding kinetics.
Furthermore, compared to tiotropium’s once-daily dose regime, aclidinium required a
twice-daily dose regime for a 24 hr bronchodilation effect in clinical trials.91–95 Such a difference
appears to be derived from their divergent RTs at the M3receptor. In addition, aclidinium’s
faster onset of action, mostly due to its fast association rate, can provide quicker symptom
relief as compared to tiotropium.
3. PF-3635659
The synthesis and biological evaluation of PF-3635659 (Fig. 2, Compound 4) and its analogues
was reported by Glossop et al.96 This study nicely exemplifies a systematic combination of both
a SAR and SKR study for early-phase drug design and development. More specifically in this
study, Glossop et al. demonstrated that a gem-dimethyl substitution adjacent to the basic amine
moiety of PF-3635659 exerted a profound effect on the dissociation rate. As evidence, the t1/2
Medicinal Research Reviews DOI 10.1002/med
870 rGUO ET AL.
of the initial gem-dimethyl-free compound (38 min) was extended to over 1440 min, indicating
the importance of the gem-dimethyl substitution for attaining ligand–receptor RT. Further
chemical modifications on the heterocyclic ring and the right side of the phenyl ring induced
corresponding changes in ligand affinity and kinetics, which eventually led to the discovery of the
candidate compound, that is, PF-3635659 (Compound 47 in the original paper). Following-up
binding kinetics characterization of this compound in a competition association assay revealed
that it had an on-rate of 1.05 ×107M−1·min−1, slower than tiotropium and ipratropium (kon
=2.12 ×108and 1.00 ×109M−1·min−1) and an off-rate of 1.39 ×10−4min−1, translating into
at1/2 of more than 24 hr (Table III).
Importantly, the in vivo onset and duration of action data for PF-3635659 were in line with
its in vitro binding kinetics. The profiling of PF-3635659 in a conscious dog model of bron-
choconstriction illustrated a slower onset of action compared to ipratropium and tiotropium,
with yet a longer duration of action (>24 hr) than ipratropium.96 This compound has now
progressed into a phase II clinical trial. Data from healthy volunteers demonstrated that PF-
3635659 provided efficacious bronchodilation over 24 hr from a single inhaled dose, thus
confirming the suitability of PF-3635659 as a novel once-daily inhaled muscarinic M3receptor
antagonist for the treatment of COPD.
4. Glycopyrronium
Glycopyrronium (Fig. 2, Compound 5) is the cation and active moiety of glycopyrronium
bromide, of which the dry-powder formulation is also known as NVA237.81 In vitro binding
kinetics profiling of this compound in a radioligand competition association assay revealed
a dissociation rate of 0.11 ±0.02 hr−1from the M3receptor, equal to a dissociation t1/2 of
6.3 hr (Table III). This value was much longer than the short-acting M3antagonist ipratropium
(0.22 hr) but shorter than tiotropium (27 hr).80 However, glycopyrronium had a more rapid
rate of onset of action (three- to 4.8-fold) than tiotropium, as determined in an in vitro calcium
assay and rat tracheal strip assay.81 This profile was similar to that obtained with ipratropium
bromide in preparations of guinea pig and human airways.97
Notably, glycopyrronium also displayed higher equilibrium binding and kinetic selectivity
for M3versus M2receptors as compared to both tiotropium and ipratropium.80,81, 97 Its kinetic
selectivity resulted from a 16.5-fold longer receptor RT at the M3receptor than at the M2
receptor, which was higher than tiotropium (10.4-fold) and ipratropium (7.3-fold). Such a
property may provide a better therapeutic window.
5. M3Receptor Agonists
The RTs of seven M3agonists (Fig. 2, Compound 6–12) were recently reported by Sykes et al.98
These agonists are acetylcholine, carbachol, methacholine, oxotremorine-M, oxotremorine,
bethanechol, and pilocarpine, and are not very selective. The data, however, were only collected
on the M3receptor subtype. Among the seven agonists, oxotremorine displayed the shortest
t1/2 at the M3receptor (2.6 sec), while acetylcholine had the longest receptor RT (t1/2 =7.8 sec).
In general, these agonists’ dissociation rates were much faster than that of the above-mentioned
M3antagonists (Table III). Further in vitro functional characterization of the M3agonists
demonstrated that they had different relative agonist efficacies. Notably, Sykes et al. found
that there was no relationship between agonist efficacy and the equilibrium binding affinity of
each agonist. Interestingly, when efficacy was compared with the dissociation rate constant, the
two were highly correlated. This result suggests a relationship between the duration of agonist
binding at the receptor and the intrinsic efficacy, a finding also reported by Guo et al. and
Hoeren et al. at the adenosine A2A receptor and at the α2A-adrenoceptor.32, 99 Importantly,
such a finding again emphasizes that equilibrium models and assays alone are not sufficient to
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r871
Table IV. Binding Kinetics of NK1Receptor Antagonists
Nr CmpdaAffinity (Ki,nM)
bDissociation half-life (t1/2)cRef.
1 Aprepitant 0.019 154 min (22◦C) 19
2 L-742,694 0.037 27 min (22◦C) 111
5 Vestipitant 0.027 59.7 min (37◦C) 119
aCompound names and synonyms used in original references. bAffinity, expressed as Kivalues
that were reported in original references; results on human receptor unless mentioned otherwise.
cDissociation half-life at different temperatures indicated in brackets.
describe a dynamic signaling system; instead, a combination of kinetic models alongside the
traditional affinity determination might be preferred to better capture the nature of efficacy
and to develop efficacious drugs for GPCRs.98
In summary, several case studies of long-acting M3receptor antagonists suggest their
sustained pharmacological effect lies in these compounds’ long receptor occupancy time. The
design and development of long RT antagonists may provide further candidate drugs with once-
daily application for COPD. We also included the kinetics data of several M3agonists, since
they provide important information that agonist efficacy may be correlated to its drug-target
RT rather than the commonly tested equilibrium affinity. This further indicates the importance
of combining both binding and kinetics information in the early phase of drug design and
development.
B. Tachykinin Receptors
Tachykinin receptors comprise three different subtypes, namely the NK1R, NK2R, and NK3R,
which can be activated by tachykinin peptides, including substance P (SP), neurokinin A
(NKA), and neurokinin B (NKB).100–102 The three members of the neurokinin receptor class
are differentiated by their varying affinities for these three tachykinins, which are SP >NKA
>NKB for the NK1R, NKA >NKB >SP for NK2R, and NKB >NKA >SP for NK3R
(for reviews, see100–102). All three subtypes are functionally coupled to Gq/11, which leads to
the activation of phospholipase C-IP3/DAG signaling and results in elevation of intracellular
Ca2+levels.102 The localization of the tachykinin receptors varies between subtypes in the
human body: the NK1R and NK3R are prominent both in the central nervous system (CNS)
and in peripheral tissues, while the NK2R is widely expressed in the smooth muscle of the
gastrointestinal, respiratory, and urinary tracts and to a much lesser extent in the CNS.102–104 A
large body of preclinical evidence has suggested that pharmacological or gene manipulation of
the NK1R has potential clinical utility in the treatment of chronic pain, migraine, neurogenic
inflammation, and emesis.105–107 These diseases could benefit from therapeutics with a long
duration of NK1R antagonism.
1. Aprepitant
Aprepitant, also known as L-754,030, MK0869, or Emend (Fig. 4, Compound 1),19,108–110 is the
only tachykinin receptor antagonist on the market to date. It is a selective, slowly dissociating
NK1antagonist with a t1/2 of 154 min at 22◦C(TableIV).
19 In comparison, it was found by
Cascieri et al. that the dissociation rate of the radiolabeled NK1’s natural ligand SP (i.e., 125I-
[Tyr8]-SP) was too rapid to be measured accurately at a comparable temperature.111 At 15◦C,
this peptide dissociates with t1/2 =46 and 0.2 min for the G protein-coupled and -uncoupled
receptor states, respectively.112 Evidently, aprepitant had a much longer RT on the NK1receptor
Medicinal Research Reviews DOI 10.1002/med
872 rGUO ET AL.
Figure 4. Structures of small molecule NK1receptor antagonists.
than the natural ligand, which has been speculated to be the reason for its high potency and in
vivo efficacy.
As expected from a slowly dissociating ligand, aprepitant’s activity was found to be in-
surmountable in functional assays compared to other, fast dissociating NK1R antagonists.
This functional effect was associated with its long-lasting in vivo efficacy, a finding shared by
many authors.19, 108, 110 One of the most elaborate studies reported on aprepitant was conducted
by Lindstr¨
om et al.108 Several functional experiments (i.e., preincubation, co-incubation, and
washout experiments) were used to reaffirm that aprepitant was a competitive yet pseudoirre-
versible antagonist with slow functional reversibility. Next to the measurement of its functional
interactions in vitro, the authors also examined the pharmacokinetic/pharmacodynamics re-
lationship of aprepitant in vivo by using the gerbil foot tap (GFT) assay, a model reflecting
central NK1R activation.113 It was found that the duration of GFT inhibition by aprepitant
outlasted the time course of its plasma or brain concentrations, indicating that its long du-
ration of action was indeed more likely due to a sustained receptor blockade rather than a
slow pharmacokinetic half-life. In contrast, two other structurally unrelated NK1antagonists,
CP-99994 and ZD6021, demonstrated rapid functional reversibility that was in line with their
pharmacokinetic profile.108 Furthermore, these authors also examined ex vivo receptor occu-
pancy by autoradiography, reaffirming that time-dependent inhibition of GFT by aprepitant
correlated well with NK1occupancy in the gerbil brain, a finding that was shared by Duffy
et al. in 2002.109 Taken together, all in vitro and in vivo results provide strong evidence that
aprepitant is a slowly dissociating NK1R antagonist.
2. L-742,694
Aprepitant was originally developed from L-742,694 (Fig. 4, Compound 2),109,111, 112 which
was shown to be a pseudoirreversible yet competitive antagonist by Cascieri et al. in 1997.111
The t1/2 of [3H]-L-742,694 (27 min, Table IV) at 22◦C is considerably faster than the half-life of
aprepitant but slower than the natural ligand SP.19, 111, 112 Its functional interaction was further
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r873
characterized by Cascieri et al. by using both human and rat NK1receptor.111 Interestingly, its
nature of NK1receptor antagonism was species related as L-742,694 was insurmountable on
the hNK1receptor but not on the rNK1receptor. A follow-up mutational analysis revealed that
mutating H1975.39 in TM5 into Ser reduced L-742,694 affinity and simultaneously abolished its
functional insurmountability at the hNK1receptor, making it a key amino acid for L-742,694
interaction and function. When testing several structural analogues of L-742,694, the authors
found that the addition of the triazoline moiety (as in L-742,694) converted a surmountable
antagonist into an insurmountable one, possibly by slowing the dissociation rate.111
3. Compound 22
Aprepitant has formed the basis for the discovery of more potentially long-acting NK1R
antagonists. Young et al. developed a series of pyrrolidine-carboxamides and oxadiazoles based
on aprepitant, aiming for feasible clinical candidates for a once-daily dosing regimen.114 Their
screening strategy was to find compounds with enduring functional activity in vitro and long
duration of action in vivo at lower drug levels as to obtain an improved safety profile. Overall,
Compound 22 (Fig. 4, Compound 3) was found to be the best compound with efficacious
functional activity in an IP-1 functional assay and long duration of action (over 24 hr) in
the GFT model. During the course of compound optimization, the authors were also able
to establish a kinetically informed structure–activity relationship. They concluded that both
receptor binding affinity and functional insurmountability are highly sensitive to both absolute
and relative stereochemistry,114 which was reaffirmed for another compound series by Morriello
et al.115
4. Compound 20
Morriello et al. employed virtually the same screening technique as described by Young
et al.114, 115 to develop both a series of long-acting 5,5-fused pyrrolidines115–117 and later a
series of 5,5,5-fused tricyclic antagonists with even slower dissociation rates, with cyclopen-
tane Compound 20 (Fig. 4, Compound 4) being the most active.118 The authors found that
increasing the overall compound rigidity as well as modifying some other specific molecular
components, such as by the addition of a substituent on the pyrrolidine nitrogen, increased
functional insurmountability.
5. Vestipitant and Its Analogues
Vestipitant (Fig. 4, Compound 5) has a t1/2 of 59.7 min (Table IV, 37◦C) from the human
NK1R(TableIV).
119 It is an insurmountable yet competitive antagonist with long duration
of action (over 8 hr) in the in vivo GFT model.119 Compared to other structural analogues of
vestipitant, it appeared that certain features such as the benzylic substitution patterns and its
stereochemistry were important for vestipitant’s mode of antagonism,119 which was reaffirmed
in another publication by Di Fabio et al.120 Surprisingly, even a small, hydrophobic substituent,
for instance a methyl substituent, played an important role in determining the magnitude of an
antagonist’s insurmountability and duration of action.120
6. MEN1149
This peptidomimetic compound (Fig. 4, Compound 6) set the example that long-acting small
molecule antagonists can be developed from short-acting peptide antagonists, in this case from
FK888, by simple structural modifications. The main modification introduced, originally to in-
crease metabolic stability, was the β-aminocycloalkyl carboxylic acid residue replacing FK888’s
prolyl group.121 Interestingly, these modifications turned the purely surmountable FK888 into
the insurmountable MEN1149 with much slower reversibility during organ bath washout assays
Medicinal Research Reviews DOI 10.1002/med
874 rGUO ET AL.
and a longer duration of action in vivo of over 3 hr.121 The authors speculated that introduction
of the β-amino cycloalkyl carboxylic acid caused a slower rate of dissociation that was likely
to be responsible for the change in type antagonism, but only provided circumstantial evidence
and no direct kinetics measurements.121
Overall, the examples summarized above provide a strong indication that NK1Rare
amongst the targets for which slowly dissociating antagonists could offer therapeutic advan-
tages.
C. DOPAMINE RECEPTORS
Dopamine receptors are a group of class A GPCRs, which are named after their endoge-
nous ligand dopamine.122 To date, five subtypes of dopamine receptors have been identified,
namely D1to D5.123 These receptors can be further categorized into D1-like (D1and D5)
and D2-like (D2,D
3and D4) subfamilies, based on their similarities in sequence, pharmacol-
ogy, and ability to stimulate or inhibit adenylyl cyclase activity: the D1-like subfamily couples
to Gαsand mediates excitatory neurotransmission, while the D2-like (D2,D
3, and D4) sub-
family couples to Gαi/o and mediates inhibitory neurotransmission.124 Dopamine receptors,
particularly D1and D2receptors, are prominent in the vertebrate CNS,123,125 indicating their
functional importance in neuronal signaling (for reviews, see126–131). To date, numerous drugs
targeting the D2receptor are available on the market. Antagonizing the dopamine D2recep-
tor has been reported as the molecular foundation of schizophrenia treatment.132 Currently,
all known marketed antischizophrenia drugs bind to the D2receptor and are supposed to
modulate dopamine hyperfunction.26, 133 In general, these drugs can be categorized into two
groups. The first generation or the so-called typical antipsychotics (e.g., haloperidol) can effec-
tively treat positive schizophrenia symptoms (i.e., hallucination and delusions), yet they invoke
concomitant on-target adverse effects, such as extrapyrimidal symptoms (EPS) and hyperpro-
lactinaemia, through excessive blockade of the D2receptor. The second generation, also known
as “atypical” antipsychotics, are defined according to their “clozapine-like” properties, that is,
low incidence of the on-target/mechanism-based side effects, which are mostly attributed to
their short RTs at the D2receptor.134 Other theories have also been proposed to explain “atyp-
icality.” One of these is the multireceptor hypothesis or polypharmacology.180 Many atypical
antipsychotics were found to have high affinity at other neurotransmitter receptors in addi-
tion to D2receptors, in particular at serotonin 5-HT2A receptors, while typical antipsychotics,
like haloperidol, bind more specifically to D2receptors.135 However, fully occupying serotonin
5-HT2A receptors with different antipsychotics at clinically relevant doses did not induce EPS
tothesamelevel.
134 Hence, the atypicality is more likely connected to the compounds’ short
RTs at the D2receptor than their antagonism at the 5-HT2A receptors.
Additionally, it is worth mentioning that interaction of atypical antipsychotics with
additional receptors may cause undesired off-target side effects—distinct from its on-
target side effect (e.g., EPS and hyperprolactinaemia), such as 5HT2C receptors (weight
gain), α1-adrenoceptors (orthostatic hypotension, reflex tachycardia, and hypnosedation), α2-
adrenoceptors (tachycardia), histamine H1receptors (sedation and weight gain), and muscarinic
receptors (blurred vision, dry mouth, constipation, and cognitive impairment).135 These side
effects can logically be minimized by decreasing the compound’s affinity or RT on the off-targets.
Contrary to aforementioned cases of other GPCRs, where long RT ligands are desired
for optimal clinical outcomes, short RT D2receptor antagonists are preferred, since they will
supposedly induce less on-target “mechanism-based toxicology”.25, 35 In the following part of
this section, we will focus on published binding kinetics data of dopamine D2antagonists and
the related benefit from short RT in clinical applications.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r875
Figure 5. Structures of small molecule D2receptor antagonists.
1. Haloperidol and Other Slowly Dissociating D2Receptor Antagonists
Haloperidol (Fig. 5, Compound 1) was approved by the U.S. FDA in 1967 and marketed as
Haldol in United States. and other countries. Haloperidol exhibited high-affinity antagonism
for human dopamine D2receptor (1.8 nM) with a koff of 0.01 sec−1, equal to a t1/2 of 72.4 sec
(Table V).135 Notably, its t1/2 was substantially changed when the kinetics were tested at rat
striatal membranes (i.e., t1/2 =42 min), indicating significant species differences.134 One might
argue that the absolute value of haloperidol’s t1/2 is much smaller than other classical slowly
dissociating GPCR antagonists, such as aforementioned NK1R antagonist aprepitant (t1/2 =
154 min). It should be realized that the time scale for endogenous dopamine concentration
burst can be within a millisecond to second range, which is much shorter than the t1/2 of
haloperidol.136 In this sense, the receptor blockade by haloperidol is already too lengthy to allow
normal physiological responses to fast and small changes in the neurotransmitter concentration.
Next to the result from the direct kinetic binding assay at both human and rat D2receptor
preparations, haloperidol was found to have long-lasting D2blockade by using an indirect
kinetic measurement. Only 48% (lower than that of clozapine) of the total receptor population
was recovered and able to bind to subsequently added [3H]-spiperone after preincubating
haloperidol for 1 hr with the receptor.135
Medicinal Research Reviews DOI 10.1002/med
876 rGUO ET AL.
Table V. Binding Kinetics of D2Receptor Antagonists
Nr CmpdaAffinity (Ki,nM)
bDissociation half-life (t1/2)cRef.
1 Haloperidol 1.8 72.4 sec 135
2 Nemonapride 0.025 (rat) 355 min (rat) 134
3 Spiperone 0.1 (rat) 200 min (rat) 134
4 Sertindole 1.2 (rat) 47 min (rat) 134
5 Chlorpromazine 1.3 (rat) 35 min (rat) 134
6 Raclopride 1.6 (rat) 28 min (rat) 134
7 Olanzapine 6.4 (rat) 18 min (rat) 134
8 Clozapine 137 14.5 sec 135
9 Quetiapine 67.6 (rat) 23 sec (rat) 139
10 JNJ-37822681 158 6.5 sec 135
aCompound names and synonyms used in original references. bAffinity, expressed as Kivalues that
reported in original references; result on human receptor unless mentioned otherwise. cDissociation
half-life at room temperatures and on human receptors unless mentioned otherwise.
The binding kinetics of other slowly dissociating D2receptor antagonists were elaborately
studied by Kapur and Seeman.134 The authors investigated the affinity and the kinetics of nine
typical and atypical antipsychotics (main results are included in Table V; compound structures
in Fig. 5). Among these compounds, nemonapride and spiperone had a t1/2 of 355 and 200 min,
respectively, indicative of D2receptor blockade for more than 3 hr; these two compounds were
followed by sertindole (47 min), haloperidol (40 min), chlorpromazine (35 min), raclopride
(28 min), and olanzapine (18 min) in terms of their t1/2 values. In contrast, the two atypical
antipsychotics clozapine and quetiapine displayed faster dissociation half-lives, that is, less than
1 min. Furthermore, it is important to note that the koff values varied a 1000-fold from 0.002
to 3.013 min−1, yet less variation was found in kon. Apparently, the rate at which antipsychotic
agents come off the receptor (koff) causes the variation in their affinity for the D2receptor,
while differences in kon do not account for that. Moreover, since atypical antipsychotics display
relatively faster dissociation than typical ones, it can be inferred that the atypical antipsychotic
effects are mostly attributed to their high koff values for the D2receptor.
2. Clozapine
Clozapine was the first atypical antipsychotic drug on the market (Fig. 5, Compound 8).
Compared to haloperidol, clozapine had a relatively low affinity for the D2receptor (Ki=
137 nM) and a fivefold shorter t1/2 of approximately 14.5 sec (Table V).135 This (partially)
explained the observation by Kapus and Seeman that clozapine reached equilibrium 100 times
faster than haloperidol in the displacement assay.134 Such a fast molecular interaction enabled
clozapine to have a fast-on and then fast-off blockade of the D2receptor. Therefore, the
receptor was readily responsive to the endogenous dopamine burst upon physical stimulation,
hence lowering the risk of EPS or hyperprolactinaemia. This mechanism was further supported
by the in vivo observation that clozapine caused a surmountable blockade of striatal dopamine
receptors in contrast to cataleptogenic neuroleptics (the insurmountable control).137 Moreover,
clinical positron emission tomography (PET) analysis of a subject receiving clozapine (400 mg)
revealed transiently high D2receptor occupancy (i.e., 80%) that rapidly declined to low levels
(i.e., 15%) within 48 hr. In comparison, D2receptor occupancy by haloperidol (5 mg) peaked at
approximately 80%, but it declined more slowly over time, showing D2occupancy of over 10%
even 3–5 days later.138 This reaffirmed that fast kinetics may be favorable for a low incidence of
mechanism-based, on-target, toxicology.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r877
3. Quetiapine
Quetiapine (Fig. 5, Compound 9), also known as Seroquel, Xeroquel, or Ketipinor, is another
marketed atypical antipsychotic approved for schizophrenia treatment. Similar to the molecular
properties of clozapine, Caboni et al. found that quetiapine displayed low D2receptor affinity
(Ki=67.6 nM) and a fast dissociation rate (1.8 min−1), equal to a t1/2 of 23 sec, at a rat striatum
homogenate (Table V).139 In the subsequent in vivo profiling, Caboni et al. performed time-
dependent analysis of plasma prolactin levels, a surrogate of peripheral D2receptor blockade,
after orally treating rats with vehicle or different dose of quetiapine or haloperidol. They found
that compared to haloperidol treatment quetiapine displayed a short-lived increase of prolactin
level, which was in line with its short RT profile at the D2receptor. Moreover, serial PET
scans evaluating the D2receptor occupancy of quetiapine demonstrated that this compound
dissociated very rapidly from the D2receptor.140 Taken together, all in vitro and in vivo results
provided strong evidence that quetiapine is a fast dissociating D2receptor antagonist.
4. JNJ-37822681
JNJ-37822681 (Fig. 5, Compound 10) was developed by Janssen Pharmaceuticals. One of the
most elaborate studies on JNJ-37822681 has been reported by Langois et al.135 The compound
was initially selected from an indirect kinetic screening assay (for method, see23, 135 ), where
JNJ-37822681 was estimated to have a koff similar to clozapine. Subsequently, a kinetic [3H]-
JNJ-37822681 binding experiment revealed that this compound had an extremely short t1/2
(6.5 sec, Table V) at the D2receptor, which was approximately two- and 11-fold shorter than
clozapine and haloperidol, respectively. In the follow-up in vivo profiling, JNJ-37822681 dis-
played a larger specific margin (compared with haloperidol) between the inhibition and block-
ade of apomorphine-induced behaviors, which represents a model for psychosis. This result
was further reaffirmed in phase IIb clinical trials, where JNJ-37822681 displayed an efficacious
antipsychotic effect but low propensity of EPS and prolactin liability.141, 142
5. Overall Structural Determinants for Binding Kinetics at the Dopamine D2Receptor
Compared to the RTs of M3RorNK
1R antagonists, D2receptor antagonists are preferred to
have a short RT to circumvent on-target side effects. It is therefore highly desired to analyze the
structural determinants that increase ligand dissociation from the receptor for future rational
drug design. For this reason, Tresadern et al. performed a comprehensive statistical analysis of
the binding kinetics of over 1800 D2receptor antagonists and examined their physicochemical
properties that potentially discriminate fast dissociating compounds from slowly dissociating
ones.26 They discovered that faster dissociating antagonists were in general less lipophilic and
had lower molecular weight. This seems to hold true for other fast dissociating GPCR ligands
as well, a finding shared by Miller et al.143 However, it is also important to note that other
descriptors, for instance polar surface area, partial positive charge, or the amount of hydrogen
bond donor and acceptor groups, caused different effects on the ligand’s dissociation, depending
on the particular cluster of chemicals that was analyzed. This suggested that local effects of a
specific chemical scaffold on the binding kinetics could be dominant over other physiochemical
descriptors. Thus, instead of intuitively modifying a compound’ lipophilicity, molecular weight
or other molecular properties, a more specific SKR study is needed to direct future drug design
and discovery.
In summary, examples presented here demonstrate the benefit of fast dissociating D2
receptor antagonists for lowering the risk of mechanism-based toxicology. Such an advantage
over long RT antagonists is supported by evidence from preclinical and clinical aspects of the
typical and atypical antipsychotics. This case study on the D2receptor further emphasizes the
Medicinal Research Reviews DOI 10.1002/med
878 rGUO ET AL.
necessity of characterizing a ligand’s binding kinetics profile in the early phases of candidate
drug selection.
7. DRUG-TARGET RT FOR CLASS B GPCRs
A. Corticotrophin Releasing Factor Type-1 Receptor (CRF1)
Corticotrophin releasing factor (CRF) is a 41 amino acid peptide that exerts its effects through
activation of CRF receptors, which are members of the family B GPCRs.144 The CRF receptor
subfamily has two subtypes, namely CRF1and CRF2. Both receptors are primarily coupled to
Gαsprotein, resulting in the activation of adenylate cylase and increased cAMP levels.145, 146
Since CRF acts on the pituitary gland to regulate the hypothalamic–pituitary–adrenal axis and
the CNS to modulate behavior responses to stress, it has caught considerable attention in drug
discovery for psychiatric diseases.144–147 In particular, antagonism of the CRF1receptor has
been proposed for rebalancing a dysfunctional stress axis.147–149 Although highly potent in vitro
and efficacious in animal models, early reported compounds such as CP-154,526 and NBI-27914
are highly lipophilic, which leads to compound accumulation in tissues or long elimination half-
life, and thus high risks of toxicity.150 For this reason, efforts have been specifically directed
toward discovering less lipophilic, fast clearance CRF1antagonists to decrease the incidence
of toxicity but with maintained drug efficacy. A long RT CRF1receptor antagonist is therefore
highly desired, which could provide the benefit of a long duration of action and at the same time
less accumulation on the unwanted targets. Such compounds would exemplify new mechanisms
for depression treatment beyond classical monoamine modulators.148, 151
1. The Binding Kinetics of CRF1R Antagonists and Their In Vivo Pharmacodynamics
One of the most elaborate binding kinetics studies at the CRF1receptor was performed by
Fleck et al.151 In this article, the authors investigated a series of CRF1receptor antagonists
regarding their in vitro binding kinetics and in vivo pharmacodynamics. They found that the
koff and kon values of 12 CRF1receptor compounds varied 170-fold and 13-fold, respectively,
which resulted in a maximal change of around 510-fold in the kinetically derived affinity (KD
=koff/kon)at37
◦C. Notably, this finding was in stark contrast with the previous reported
Kivalues measured in radioligand displacement assays, where little difference was observed
between compounds. This discrepancy was mostly attributed to insufficient incubation time to
reach equilibrium for compounds with long RT profile. For instance, less than 40% of NBI
30775 dissociated from the CRF1receptor after 2 hr, far from equilibrium, hence the Kivalues
were underestimated. Furthermore, several lead compounds displayed divergent dissociation
half-lives; NBI 35965 with 16 min, NBI 34041 with 53 min, and NBI 30775 with 130 min (Fig. 6,
Compound 1–3; Table VI). This ranking of their binding kinetics, in fact, was consistent with
the ranking of their in vivo pharmacodynamic profiles, at least in adrenalectomized rats, where
NBI 34041 and NBI 30775 produced sustained suppression of adrenocorticotropin for 4 and 6
hr, respectively, while NBI 35965 induced a more transient effect (less than 2 hr). In addition, all
three compounds (NBI 35965, NBI 34041, and NBI 30775) had similar pharmacokinetics. This
confirmed that the unique sustained adrenocorticotropin suppression evoked by NBI 30775
was derived from its prolonged CRF1receptor occupancy rather than its pharmacokinetics.
Functional reversibility assays were also performed to kinetically profile CRF1receptor
antagonists.152, 153 Briefly, cells expressing recombinant CRF1receptor were incubated with
CRF in either the presence or absence of an antagonist to measure the suppression of the
maximal cell response to CRF by the tested antagonist. Although CRF binds to a different
site on the receptor than the nonpeptide small molecule antagonists, the insurmountability
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r879
Figure 6. Structures of small molecule CRF1receptor antagonists.
Table VI. Binding Kinetics of CRF1Receptor Antagonists
Nr CmpdaAffinity (Ki,nM)
bDissociation half-life (t1/2)cRef.
1. NBI 35965 2.3 16 min (37◦C) 151
2. NBI 34041 1.7 53 min (37◦C) 151
3. NBI 30775 0.36 130 min (37◦C) 151
4. DMP-904 1.5 5.5 hr 152
5. R121919 12 12.8 hr 152
6. Cmpd 25 (Pfizer) 11 10.7 hr 152
7. SN003 3.64 (rat) 0.35 hr (rat) 153
8. PF-4325743 8.22 (rat) 0.37 hr (rat) 153
aCompound names and synonyms used in original references. bAffinity, expressed as Kivalues
that were reported in original references; result on human receptor unless mentioned otherwise.
cDissociation half-life at room temperature and on human receptor unless mentioned otherwise.
of the noncompetitive antagonists still correlated to their off-rates. In particular, Miller et al.
plotted the antagonists’ dissociation half-lives against their suppression of the maximal CRF1
receptor response to its endogenous ligand. They found a good correlation between the two
parameters, although the exact molecular mechanism of this link is yet to be discovered.152 By
using this assay, at least in a qualitative manner, Miller et al. investigated several other long RT
compounds reported in literature. They found that DMP-904 and R121919 almost completely
suppressed the maximal CRF response (Fig. 6, Compounds 4 and 5). Both compounds were
found to have slow dissociation half-lives, that is, 5.5 and 12.8 hr, respectively, which were
calculated from koff values determined in a competition association assay.152 In comparison,
shorter RT CRF1receptor antagonists (Fig. 6, Compounds 7 and 8) such as SN003 (t1/2 =
0.35 hr, Table VI) or PF-4325743 (t1/2 =0.37 hr, Table VI) did not significantly decrease the
maximal CRF response.153
Medicinal Research Reviews DOI 10.1002/med
880 rGUO ET AL.
Figure 7. (A) Diagram of the putative functional groups implicated in SKRs of nonpeptide antagonists. This
diagram was adapted from Gilligan et al., Kehne and De Lombaert, and Fleck et al.,160, 162, 163 based on the
comprehensive analysis of ligand SAR. (B) The co-crystal structure of CP-376395 in the human CRF1receptor.
This figure was generated with ICM Browser v3.7 (Molsoft) from PDB code: 4K5Y. CP-376395 (black) binds deeply
within the pocket. Two aromatic residues, F2033.44 and Y3276.53, together prevent the ligand from moving out
of the receptor. The pose of the compound’s trimethylphenoxy moiety is rotated to fit in a hydrophobic pocket
formed by F2845.51, L2875.54 , T3166.42 , L3196.45, and L3206.46 with its pyridine nitrogen at the core to form a
hydrogen bond with N2835.50.156
2. Overall Structural Determinants for Binding Kinetics at the CRF1Receptor
A detailed SKR study was performed by Fleck et al., in which the authors performed a
retrospective binding kinetics investigation on a set of CRF1receptor ligands including the
initial, low-affinity chemical starting points up to the lead compounds. Key features of the high
affinity or long RT CRF1receptor ligand are represented in Figure 7A (adapted from the models
developed by Gilligan et al., Kehne and De Lombaert, and Fleck et al.151, 154, 155) and the SKRs
are summarized as follows: (1) ortho-position substitution (R1) on the low ring was necessary to
enforce a twisted bioactive conformation to enhance ligand–receptor interactions. This can be
illustrated by the conformation of CP-376395 in the recently elucidated CRF1R crystal structure
(PDB code: 4K5Y, Fig. 7B). Apparently, the pose of the compound’s trimethylphenoxy moiety
is rotated to fit in a hydrophobic pocket formed by F2845.51, L2875.54, T3166.42 , L3196.45,and
L3206.46 with the pyridine nitrogen at the core to form a hydrogen bond with N2835.50.156 In
contrast, the association rate of an ortho-substitutent free analogue decreased 33-fold compared
to its chloro-substituted analogue, suggesting a substantial barrier for the free rotating ligand
to fit into the binding pocket. (2) Adding a methyl moiety at R2resulted in an increased kon and
decreased koff, indicating a raised probability of successful collision between this group and a
rigid binding pocket on the receptor, combined with a stabilized ligand–receptor conformation.
This finding was in agreement with other published results that suggested that the methyl-group
interacted with a hydrophobic pocket in the receptor.154, 155, 157, 158 (3) Chemical modifications
on the upper aliphatic groups at R3demonstrated varying effects on the binding kinetics
depending on their size, branching pattern, and the stereochemical configuration, which may
affect the compound’s interaction with F2033.44 and Y3276.53, two aromatic residues gating the
small access channel from the extracellular part (Fig. 7B).156
In another SKR study, Miller et al. started from a bicyclic purine core and performed a series
of chemical modifications on the substitution patterns at the low ring and the upper branched
groups. Follow-up functional investigations in vitro led to the identification of Compound 25
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r881
(Fig. 6, Compound 6), which displayed 79% suppression of the maximal CRF response. Binding
kinetics characterization of this compound revealed that it had a slow t1/2 of 10.7 hr, correlating
well with its insurmountable antagonist behavior.152
8. CONCLUDING REMARKS
We reviewed the existing kinetics data for a number of GPCRs. The examples presented make a
strong case for the value of measuring drug-target RT, or, more generally speaking, the kinetic
profile of interaction for compound optimization in the early phases of the drug discovery
process. Thus, a strategy is warranted that comprises both a SKR study and a classical SAR
study. This may offer added value to the traditional metric of affinity-based drug evaluation.
This review further summarized the factors influencing drug-target RT, in particular,
to find a universal molecular determinant or a certain “hot spot” on GPCRs for designing
kinetics-favorable ligands. Several kinetics-related statistical analyses indicated that manipu-
lating physicochemical properties such as lipophilicity or molecular weight can lead to tuned
drug-target RTs.26,143 Alternatively, this could be achieved by modulating the rigidity/flexibility
of a drug candidate.117 However, these “intuitive,” “one size-fits-all” approaches are very likely
too simple and may increase the concern of binding to collateral targets, thereby resulting in
unwanted side effects.67, 159 In this sense, fine-tuning drug-target RT should probably be more
specific. Despite this challenge, progress can still be made by gaining knowledge from both the
ligand and target perspectives, such as by ligand modifications with subtle changes to probe
relevant pharmacophores, mutation studies, co-crystallization, and molecular dynamic studies
to establish the corresponding “anchoring” sites on the receptor.
We also summarized the kinetic methodologies in this review. Several screening schemes
are available and new assays are emerging, which enable quick identification of compounds
of interest. Quantitative characterization of a compound’s binding kinetics can be obtained in
a kinetic binding assay or competition association assay. Functional reversibility experiments
prevail in drug discovery as well, which offer an alternative way to interpret a ligand’s kinetics
profile. In addition, ex vivo receptor occupancy experiments or in vivo duration of action
experiments can translate the in vitro binding kinetics into the effect in an in vivo setting, which
provides further evidence for a compound’s drug-target RT. Notably, it is also necessary to take
into account the pharmacokinetics (PK) profile of the candidate compound, particular for one
aiming at long drug-target RT, since RT can only prolong the duration of action only when the
RT outlasts the PK.2, 15
The compounds summarized in the present review are predominantly GPCR antagonists.
This reflects a lack of kinetic information on agonists in the literature. However, more kinet-
ics studies focused on the latter are emerging. For instance, the case studies at the M3and
A2A receptors explored the molecular basis of the compounds’ intrinsic efficacy from a kinetic
perspective.32, 98 In addition to examining an agonist’s mechanism of action, one can also specif-
ically optimize an agonist’s kinetic properties for therapeutic benefit. This can be exemplified
by the administration of long-acting agonists at the GnRH receptor to intentionally desensitize
the GnRH receptor and therefore antagonize gonadotropin secretion in hormone-dependent
cancers.181 Such a strategy could be a therapeutic substitute for targets where no antagonist is
available. On the other hand, one needs to take into consideration that agonist-induced desen-
sitization or internalization by prolonged or repeated administration can cause drug tolerance,
for instance opiate drugs targeting μ-opioid receptors.182 In such a case, it might be tempting
to search for shorter RT agonists to prevent their target’s internalization. Thus, a study that
compares agonist binding kinetics and GPCR desensitization/internalization kinetics would
be of great interest.
Medicinal Research Reviews DOI 10.1002/med
882 rGUO ET AL.
Taken together, a rapid expansion of the field of GPCR binding kinetics may be anticipated
in the near future, which hopefully will lead to the identification of novel and better-in-class
drugs for clinical applications.
ACKNOWLEDGMENTS
The authors thank Zhiyi Yu for fruitful discussions and helpful comments on the manuscript.
This project was financially supported by the Innovational Research Incentive Scheme of the
Netherlands Research Organization (NWO; VENI-Grant 11188 to L.H.).
REFERENCES
1. Copeland RA, Pompliano DL, Meek TD. Drug-target residence time and its implications for lead
optimization. Nat Rev Drug Discov 2006;5(9):730–739.
2. Swinney DC. Can binding kinetics translate to a clinically differentiated drug? From theory to
practice. Lett Drug Des Discov 2006;3(8):569–574.
3. Swinney DC. Biochemical mechanisms of new molecular entities (NMEs) approved by United States
FDA during 2001–2004: Mechanisms leading to optimal efficacy and safety. Curr Top Med Chem
2006;6(5):461–478.
4. Swinney DC, Anthony J. How were new medicines discovered? Nat Rev Drug Discov
2011;10(7):507–519.
5. Swinney DC. Biochemical mechanisms of drug action: What does it take for success? Nat Rev Drug
Discov 2004;3(9):801–808.
6. Copeland RA. Evaluation of Enzyme Inhibitors in Drug Discovery:A Guide for Medicinal Chemists
and Pharmacologists. New York: John Wiley & Sons; 2005. p 153.
7. Copeland RA. Conformational adaptation in drug-target interactions and residence time. Future
Med Chem 2011;3(12):1491–1501.
8. Vauquelin G, Charlton SJ. Long-lasting target binding and rebinding as mechanisms to prolong in
vivo drug action. Br J Pharmacol 2010;161(3):488–508.
9. Vauquelin G, Fierens F, Verheijen I, Vanderheyden P. Insurmountable AT1receptor antago-
nism: The need for different antagonist binding states of the receptor. Trends Pharmacol Sci
2001;22(7):343–344.
10. Vauquelin G, Morsing P, Fierens FL, De Backer JP, Vanderheyden PM. A two-state receptor model
for the interaction between angiotensin II type 1 receptors and non-peptide antagonists. Biochem
Pharmacol 2001;61(3):277–284.
11. Vauquelin G, Van Liefde I. Slow antagonist dissociation and long-lasting in vivo receptor protection.
Trends Pharmacol Sci 2006;27(7):356–359.
12. Zhang R, Monsma F. The importance of drug-target residence time. Curr Opin Drug Discov Dev
2009;12(4):488–496.
13. Zhang R, Monsma F. Binding kinetics and mechanism of action: Toward the discovery and devel-
opment of better and best in class drugs. Expert Opin Drug Discov 2010;5(11):1023–1029.
14. Tummino PJ, Copeland RA. Residence time of receptor-ligand complexes and its effect on biological
function. Biochemistry 2008;47(20):5481–5492.
15. Dahl G, Akerud T. Pharmacokinetics and the drug-target residence time concept. Drug Discov
Today 2013;18(15–16):697–707.
16. Nunez S, Venhorst J, Kruse CG. Target-drug interactions: First principles and their application to
drug discovery. Drug Discov Today 2012;17(1–2):10–22.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r883
17. Swinney DC. The role of binding kinetics in therapeutically useful drug action. Curr Opin Drug
Discov Dev 2009;12(1):31–39.
18. Vilums M, Zweemer AJ, Yu Z, de Vries H, Hillger JM, Wapenaar H, Bollen IA, Barmare F,
Gross R, Clemens J, Krenitsky P, Brussee J, Stamos D, Saunders J, Heitman LH, IJzerman AP.
Structure-kinetic relationships—An overlooked parameter in hit-to-lead optimization: A case of
cyclopentylamines as chemokine receptor 2 antagonists. J Med Chem 2013;56(19):7706–7714.
19. Hale JJ, Mills SG, MacCoss M, Finke PE, Cascieri MA, Sadowski S, Ber E, Chicchi GG, Kurtz
M, Metzger J, Eiermann G, Tsou NN, Tattersall FD, Rupniak NM, Williams AR, Rycroft W, Har-
greaves R, MacIntyre DE. Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)
phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent,
orally active, long-acting morpholine acetal human NK-1 receptor antagonist. J Med Chem 1998;
41(23):4607–4614.
20. Gavalda A, Miralpeix M, Ramos I, Otal R, Carreno C, Vinals M, Domenech T, Carcasona C,
Reyes B, Vilella D, Gras J, Cortijo J, Morcillo E, Llenas J, Ryder H, Beleta J. Characterization
of aclidinium bromide, a novel inhaled muscarinic antagonist, with long duration of action and a
favorable pharmacological profile. J Pharmacol Exp Ther 2009;331(2):740–751.
21. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K. A general method for the
covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 2003;21(1):86–89.
22. Pierre N. Determination of Association (kon) and Dissociation (koff) Rates of Spiperone on the
Dopamine D2Receptor Using a Platform for GPCR Applications, Vol. 2013. Bedford: American
Laboratory; 2011.
23. Leysen JE, Gommeren W. The dissociation rate of unlabelled dopamine antagonists and agonists
from the dopamine-D2 receptor, application of an original filter method. J Recept Res 1984;4(7):817–
845.
24. Malany S, Hernandez LM, Smith WF, Crowe PD, Hoare SR. Analytical method for simultaneously
measuring ex vivo drug receptor occupancy and dissociation rate: Application to (R)-dimethindene
occupancy of central histamine H1receptors. J Recept Signal Transduct Res 2009;29(2):84–93.
25. Packeu A, Wennerberg M, Balendran A, Vauquelin G. Estimation of the dissociation rate of un-
labelled ligand-receptor complexes by a ‘two-step’ competition binding approach. Br J Pharmacol
2010;161(6):1311–1328.
26. Tresadern G, Bartolome JM, Macdonald GJ, Langlois X. Molecular properties affecting fast dis-
sociation from the D2receptor. Bioorg Med Chem 2011;19(7):2231–2241.
27. Affolter H, Hertel C, Jaeggi K, Portenier M, Staehelin M. (-)-S-[3H]CGP-12177 and its use to
determine the rate constants of unlabeled beta-adrenergic antagonists. Proc Natl Acad Sci USA
1985;82(3):925–929.
28. Contreras ML, Wolfe BB, Molinoff PB. Kinetic analysis of the interactions of agonists and antag-
onists with beta adrenergic receptors. J Pharmacol Exp Ther 1986;239(1):136–143.
29. Dowling MR, Charlton SJ. Quantifying the association and dissociation rates of unlabelled antag-
onists at the muscarinic M3receptor. Br J Pharmacol 2006;148(7):927–937.
30. Heise CE, Sullivan SK, Crowe PD. Scintillation proximity assay as a high-throughput method to
identify slowly dissociating nonpeptide ligand binding to the GnRH receptor. J Biomol Screen
2007;12(2):235–239.
31. Slack RJ, Russell LJ, Hall DA, Luttmann MA, Ford AJ, Saunders KA, Hodgson ST, Connor HE,
Browning C, Clark KL. Pharmacological characterization of GSK1004723, a novel, long-acting
antagonist at histamine H1and H3receptors. Br J Pharmacol 2011;164(6):1627–1641.
32. Guo D, Mulder-Krieger T, IJzerman AP, Heitman LH. Functional efficacy of adenosine A2A
receptor agonists is positively correlated to their receptor residence time. Br J Pharmacol
2012;166(6):1846–1859.
33. Guo D, van Dorp EJ, Mulder-Krieger T, van Veldhoven JP, Brussee J, IJzerman AP, Heitman LH.
Dual-point competition association assay: A fast and high-throughput kinetic screening method for
assessing ligand-receptor binding kinetics. J Biomol Screen 2013;18(3):309–320.
Medicinal Research Reviews DOI 10.1002/med
884 rGUO ET AL.
34. Motulsky HJ, Mahan LC. The kinetics of competitive radioligand binding predicted by the law of
mass action. Mol Pharmacol 1984;25(1):1–9.
35. Vauquelin G, Bostoen S, Vanderheyden P, Seeman P. Clozapine, atypical antipsychotics, and the
benefits of fast-off D2dopamine receptor antagonism. Naunyn Schmiedebergs Arch Pharmacol
2012;385(4):337–372.
36. Gaddum JH, Hameed KA, Hathway DE, Stephens FF. Quantitative studies of antagonists for
5-hydroxytryptamine. Q J Exp Physiol Cogn Med Sci 1955;40(1):49–74.
37. Kenakin T, Jenkinson S, Watson C. Determining the potency and molecular mechanism of action
of insurmountable antagonists. J Pharmacol Exp Ther 2006;319(2):710–723.
38. Paton WD, Waud DR. The margin of safety of neuromuscular transmission. J Physiol 1967;191(1):
59–90.
39. Paton WDM, Rang HP. A kinetic approach to the mechanism of drug action. In: Harper NJ,
Simmonds AB, Eds. Advances in Drug Research. London & New York: Academic Press; 1998. p
57–80.
40. Rang HP. The kinetics of action of acetylcholine antagonists in smooth muscle. Proc R Soc Lond
B Biol Sci 1966;164(996):488–510.
41. Fierens FL, Vanderheyden PM, De Backer JP, Vauquelin G. Insurmountable angiotensin
AT1receptor antagonists: The role of tight antagonist binding. Eur J Pharmacol 1999;372(2):
199–206.
42. Morsing P, Vauquelin G. How can the differences among AT1-receptor antagonists be explained?
Cell Biochem Biophys 2001;35(1):89–102.
43. Vauquelin G, Fierens FL, Verheijen I, Vanderheyden PM. Distinctions between non-peptide an-
giotensin II AT1-receptor antagonists. J Renin-Angio-Aldo Syst 2001;2(1 Suppl):S24–S31.
44. Vauquelin G, Van Liefde I, Birzbier BB, Vanderheyden PM. New insights in insurmountable an-
tagonism. Fundam Clin Pharmacol 2002;16(4):263–272.
45. Vauquelin G, Van Liefde I, Vanderheyden P. Models and methods for studying insurmountable
antagonism. Trends Pharmacol Sci 2002;23(11):514–518.
46. Vauquelin G, Szczuka A. Kinetic versus allosteric mechanisms to explain insurmountable antago-
nism and delayed ligand dissociation. Neurochem Int 2007;51(5):254–260.
47. Shiau AK, Massari ME, Ozbal CC. Back to basics: Label-free technologies for small molecule
screening. Comb Chem High T Scr 2008;11(3):231–237.
48. Rich RL, Myszka DG. Higher-throughput, label-free, real-time molecular interaction analysis. Anal
Biochem 2007;361(1):1–6.
49. Gronewold TMA, Baumgartner A, Hierer J, Sierra S, Blind M, Schafer F, Blumer J, Tillmann T,
Kiwitz A, Kaiser R, Zabe-Kuhn M, Quandt E, Famulok M. Kinetic binding analysis of aptamers
targeting HIV-1 proteins bya combination of a microbalance array and mass spectrometry(MAMS).
J Proteome Res 2009;8(7):3568–3577.
50. Gronewold TMA. Surface acoustic wave sensors in the bioanalytical field: Recent trends and chal-
lenges. Anal. Chim. Acta 2007;603(2):119–128.
51. Rich RL, Myszka DG. Grading the commercial optical biosensor literature—Class of 2008: ‘The
Mighty Binders’. J Mol Recogn 2010;23(1):1–64.
52. Zhukov A, Andrews SP, Errey JC, Robertson N, Tehan B, Mason JS, Marshall FH, Weir M,
Congreve M. Biophysical mapping of the adenosine A2A receptor. J Med Chem 2011;54(13):
4312–4323.
53. Robertson N, Jazayeri A, Errey J, Baig A, Hurrell E, Zhukov A, Langmead CJ, Weir M, Marshall
FH. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug
discovery. Neuropharmacology 2011;60(1):36–44.
54. Congreve M, Andrews SP, Dore AS, Hollenstein K, Hurrell E, Langmead CJ, Mason JS, Ng IW,
Tehan B, Zhukov A, Weir M, Marshall FH. Discovery of 1,2,4-triazine derivatives as adenosine
A(2A) antagonists using structure based drug design. J Med Chem 2012;55(5):1898–1903.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r885
55. Navratilova I, Dioszegi M, Myszka DG. Analyzing ligand and small molecule binding activity of
solubilized GPCRs using biosensor technology. Anal Biochem 2006;355(1):132–139.
56. Bjarnadottir TK, Gloriam DE, Hellstrand SH, Kristiansson H, Fredriksson R, Schioth HB. Com-
prehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and
mouse. Genomics 2006;88(3):263–273.
57. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol
2002;3(9):639–650.
58. De Amici M, Dallanoce C, Holzgrabe U, Trankle C, Mohr K. Allosteric ligands for G protein-
coupled receptors: A novel strategy with attractive therapeutic opportunities. Med Res Rev
2010;30(3):463–549.
59. Overington JP, Al-Lazikani B, Hopkins AL. Opinion—How many drug targets are there? Nat Rev
Drug Discov 2006;5(12):993–996.
60. Russ AP, Lampel S. The druggable genome: An update. Drug Discov Today 2005;10(23–24):1607–
1610.
61. Chen L, Jin L, Zhou N. An update of novel screening methods for GPCR in drug discovery. Expert
Opin Drug Discov 2012;7(9):791–806.
62. Post JM, Alexander S, Wang YX, Vincelette J, Vergona R, Kent L, Bryant J, Sullivan ME, Dole WP,
Morser J, Subramanyam B. Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition
of platelet aggregation (II): pharmacodynamic and pharmacokinetic characterization. Thromb Res
2008;122(4):533–540.
63. Foldes FF. Pain control with intrathecally and peridurally administered opioids and other drugs.
Anaesthesiol Reanim 1991;16(5):287–298.
64. Laduron PM, Janssen PFM. Axoplasmic-transport and possible recycling of opiate receptors labeled
with H-3-labeled lofentanil. Life Sci 1982;31(5):457–462.
65. Savoia G, Loreto M, Gravino E. Sufentanil: an overview of its use for acute pain management.
Minerva Anestesiol 2001;67(9 Suppl 1):206–216.
66. Patel S, Spencer C. Remifentanil. Drugs 1996;52(3):417–427.
67. Pan AC, Borhani DW, Dror RO, Shaw DE. Molecular determinants of drug-receptor binding
kinetics. Drug Discov Today 2013;18(13–14):667–673.
68. Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov
2004;3(8):711–715.
69. Kola I. The state of innovation in drug development. Clin Pharmacol Ther 2008;83(2):227–230.
70. Caulfield MP, Birdsall NJ. International Union of Pharmacology. XVII. Classification of muscarinic
acetylcholine receptors. Pharmacol Rev 1998;50(2):279–290.
71. Bonner TI, Buckley NJ, Young AC, Brann MR. Identificationof a family of muscarinic acetylcholine
receptor genes. Science 1987;237(4814):527–532.
72. Bonner TI. The molecular basis of muscarinic receptor diversity. Trends Neurosci 1989;12(4):148–
151.
73. Wess J. Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 1996;10(1):69–
99.
74. Barnes PJ. The role of anticholinergics in chronic obstructive pulmonary disease. Am J Med
2004;117(Suppl 12A):24S–32S.
75. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-
Roisin R, van Weel C, Zielinski J. Global strategy for the diagnosis, management, and prevention
of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med
2007;176(6):532–555.
76. Tashkin DP. Is a long-acting inhaled bronchodilator the first agent to use in stablechronic obstructive
pulmonary disease? Curr Opin Pulm Med 2005;11(2):121–128.
77. Disse B, Speck GA, Rominger KL, Witek TJ, Jr., Hammer R. Tiotropium (Spiriva): Mechanistical
considerations and clinical profile in obstructive lung disease. Life Sci 1999;64(6–7):457–464.
Medicinal Research Reviews DOI 10.1002/med
886 rGUO ET AL.
78. Disse B, Reichl R, Speck G, Traunecker W, Rominger KL, Hammer R. Ba 679 Br, a novel long-acting
anticholinergic bronchodilator. Life Sci 1993;52(5–6):537–544.
79. Haddad EB, Mak JC, Barnes PJ. Characterization of [3H]Ba 679 BR, a slowly dissociating mus-
carinic antagonist, in human lung: Radioligand binding and autoradiographic mapping. Mol Phar-
macol 1994;45(5):899–907.
80. Casarosa P, Bouyssou T, Germeyer S, Schnapp A, Gantner F, Pieper M. Preclinical evaluation of
long-acting muscarinic antagonists: Comparison of tiotropium and investigational drugs. J Phar-
macol Exp Ther 2009;330(2):660–668.
81. Sykes DA, Dowling MR, Leighton-Davies J, Kent TC, Renard E, Trifilieff A, Charlton SJ. The
influence of receptor kinetics on the onset and duration of action, and the therapeutic index of
NVA237 and tiotropium. J Pharmacol Exp Ther 2012;343(2):520–528.
82. D’Urzo A, Ferguson GT, van Noord JA, Hirata K, Martin C, Horton R, Lu YM, Banerji D,
Overend T. Efficacy and safety of once-daily NVA237 in patients with moderate-to-severe COPD:
The GLOW1 trial. Resp Res 2011;12:156.
83. Tautermann CS, Kiechle T, Seeliger D, Diehl S, Wex E, Banholzer R, Gantner F, Pieper MP,
Casarosa P. Molecular basis for the long duration of action and kinetic selectivity of tiotropium for
the muscarinic M3receptor. J Med Chem 2013;56(21):8746–8756.
84. Kruse AC, Hu J, Pan AC, Arlow DH, Rosenbaum DM, Rosemond E, Green HF, Liu T, Chae PS,
Dror RO, Shaw DE, Weis WI, Wess J, Kobilka BK. Structure and dynamics of the M3muscarinic
acetylcholine receptor. Nature 2012;482(7386):552–556.
85. Smith DA, Jones BC, Walker DK. Design of drugs involving the concepts and theories of drug
metabolism and pharmacokinetics. Med Res Rev 1996;16(3):243–266.
86. Tashkin DP, Celli B, Senn S, Burkhart D, Kesten S, Menjoge S, Decramer M. A 4-year trial of
tiotropium in chronic obstructive pulmonary disease. N Engl J Med 2008;359(15):1543–1554.
87. van Noord JA, Smeets JJ, Custers FL, Korducki L, Cornelissen PJ. Pharmacodynamic steady state
of tiotropium in patients with chronic obstructive pulmonary disease. Eur Respir J 2002;19(4):639–
644.
88. Traynor K. Aclidinium bromide oral inhalation powder approved for COPD. Am J Health Syst
Pharm 2012;69(17):1446.
89. Prat M, Fernandez D, Buil MA, Crespo MI, Casals G, Ferrer M, Tort L, Castro J, Monleon
JM, Gavalda A, Miralpeix M, Ramos I, Domenech T, Vilella D, Anton F, Huerta JM, Espinosa
S, Lopez M, Sentellas S, Gonzalez M, Alberti J, Segarra V, Cardenas A, Beleta J, Ryder H.
Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinol esters as potent and
long-acting muscarinic antagonists with potential for minimal systemic exposure after inhaled
administration: Identification of (3R)-3-{[hydroxy(di-2-thienyl)acetyl]oxy}-1-(3-phenoxypropyl)-
1-azoniabicyclo[2.2.2]octane bromide (aclidinium bromide). J Med Chem 2009;52(16):
5076–5092.
90. Kesten S, Jara M, Wentworth C, Lanes S. Pooled clinical trial analysis of tiotropium safety. Chest
2006;130(6):1695–1703.
91. Suppli Ulrik C. Aclidinium bromide: Clinical benefit in patients with moderate to severe COPD.
Open Respir Med J 2012;6:150–154.
92. Singh D, Magnussen H, Kirsten A, Mindt S, Caracta C, Seoane B, Jarreta D, Garcia Gil E. A
randomised, placebo- and active-controlled dose-finding study of aclidinium bromide administered
twice a day in COPD patients. Pulm Pharmacol Ther 2012;25(3):248–253.
93. Maltais F, Milot J. The potential for aclidinium bromide, a new anticholinergic, in the management
of chronic obstructive pulmonary disease. Ther Adv Respir Dis 2012;6(6):345–361.
94. Gupta V, Singh D. Aclidinium bromide for the treatment of chronic obstructive pulmonary disease.
Expert Rev Respir Med 2012;6(6):581–588.
95. Jones PW, Singh D, Bateman ED, Agusti A, Lamarca R, de Miquel G, Segarra R, Caracta C, Garcia
Gil E. Efficacy and safety of twice-daily aclidinium bromide in COPD patients: The ATTAIN study.
Eur Respir J 2012;40(4):830–836.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r887
96. Glossop PA, Watson CA, Price DA, Bunnage ME, Middleton DS, Wood A, James K, Roberts
D, Strang RS, Yeadon M, Perros-Huguet C, Clarke NP, Trevethick MA, Machin I, Stuart EF,
Evans SM, Harrison AC, Fairman DA, Agoram B, Burrows JL, Feeder N, Fulton CK, Dillon BR,
Entwistle DA, Spence FJ. Inhalation by design: Novel tertiary amine muscarinic M3receptor an-
tagonists with slow off-rate binding kinetics for inhaled once-daily treatment of chronic obstructive
pulmonary disease. J Med Chem 2011;54(19):6888–6904.
97. Haddad EB, Patel H, Keeling JE, Yacoub MH, Barnes PJ, Belvisi MG. Pharmacological charac-
terization of the muscarinic receptor antagonist, glycopyrrolate, in human and guinea-pig airways.
Br J Pharmacol 1999;127(2):413–420.
98. Sykes DA, Dowling MR, Charlton SJ. Exploring the mechanism of agonist efficacy: A relationship
between efficacy and agonist dissociation rate at the muscarinic M3receptor. Mol Pharmacol
2009;76(3):543–551.
99. Hoeren M, Brawek B, Mantovani M, Loffler M, Steffens M, van Velthoven V, Feuerstein TJ. Partial
agonism at the human alpha2A-autoreceptor: Role of binding duration. Naunyn Schmiedebergs Arch
Pharmacol 2008;378(1):17–26.
100. Pantaleo N, Chadwick W, Park SS, Wang L, Zhou Y, Martin B, Maudsley S. The mammalian
tachykinin ligand-receptor system: An emerging target for central neurological disorders. CNS
Neurol Disord Drug Targets 2010;9(5):627–635.
101. Pennefather JN, Lecci A, Candenas ML, Patak E, Pinto FM, Maggi CA. Tachykinins and tachykinin
receptors: A growing family. Life Sci 2004;74(12):1445–1463.
102. Almeida TA, Rojo J, Nieto PM, Pinto FM, Hernandez M, Martin JD, Candenas ML. Tachykinins
and tachykinin receptors: Structure and activity relationships. Curr Med Chem 2004;11(15):2045–
2081.
103. Simonsen KB, Juhl K, Steiniger-Brach B, Nielsen SM. Novel NK3receptor antagonists for the
treatment of schizophrenia and other CNS indications. Curr Opin Drug Discov Dev 2010;13(4):379–
388.
104. Stout SC, Owens MJ, Nemeroff CB. Neurokinin(1) receptor antagonists as potential antidepres-
sants. Annu Rev Pharmacol 2001;41:877–906.
105. Albert JS. Neurokinin antagonists and their potential role in treating depression and other stress
disorders. Exp Opin Investig Drugs 2004;14(10):1421–1433.
106. Lecci A, Maggi CA. Peripheral tachykinin receptors as potential therapeutic targets in visceral
diseases. Exp Opin Ther Targets 2003;7(3):343–362.
107. Quartara L, Altamura M, Evangelista S, Maggi CA. Tachykinin receptor antagonists in clinical
trials. Exp Opin Investig Drugs 2009;18(12):1843–1864.
108. Lindstrom E, von Mentzer B, Pahlman I, Ahlstedt I, Uvebrant A, Kristensson E, Martinsson R,
Noven A, de Verdier J, Vauquelin G. Neurokinin 1 receptor antagonists: Correlation between in
vitro receptor interaction and in vivo efficacy. J Pharmacol Exp Ther 2007;322(3):1286–1293.
109. Duffy RA, Varty GB, Morgan CA, Lachowicz JE. Correlation of neurokinin (NK) 1 receptor
occupancy in gerbil striatum with behavioral effects of NK1antagonists. J Pharmacol Exp Ther
2002;301(2):536–542.
110. Malherbe P, Knoflach F, Hernandez MC, Hoffmann T, Schnider P, Porter RH, Wettstein JG, Bal-
lard TM, Spooren W, Steward L. Characterization of RO4583298 as a novel potent, dual antagonist
with in vivo activity at tachykinin NK1and NK3receptors. Br J Pharmacol 2011;162(4):929–946.
111. Cascieri MA, Ber E, Fong TM, Hale JJ, Tang F, Shiao LL, Mills SG, MacCoss M, Sadowski S, Tota
MR, Strader CD. Characterization of the binding and activity of a high affinity, pseudoirreversible
morpholino tachykinin NK1receptor antagonist. Eur J Pharmacol 1997;325(2–3):253–261.
112. Cascieri MA, Ber E, Fong TM, Sadowski S, Bansal A, Swain C, Seward E, Frances B, Burns D,
Strader CD. Characterization of the binding of a potent, selective, radioiodinated antagonist to the
human neurokinin-1 receptor. Mol Pharmacol 1992;42(3):458–463.
113. Bristow LJ, Young L. Chromodacryorrhea and repetitive hind paw tapping: Models of peripheral
and central tachykinin NK1receptor activation in gerbils. Eur J Pharmacol 1994;253(3):245–252.
Medicinal Research Reviews DOI 10.1002/med
888 rGUO ET AL.
114. Young JR, Eid R, Turner C, DeVita RJ, Kurtz MM, Tsao KL, Chicchi GG, Wheeldon A, Carlson
E, Mills SG. Pyrrolidine-carboxamides and oxadiazoles as potent hNK1antagonists. Bioorg Med
Chem Lett 2007;17(19):5310–5315.
115. Morriello GJ, Chicchi G, Johnson T, Mills SG, Demartino J, Kurtz M, Tsao KL, Zheng S, Tong X,
Carlson E, Townson K, Wheeldon A, Boyce S, Collinson N, Rupniak N, Devita RJ. Fused tricyclic
pyrrolizinones that exhibit pseudo-irreversible blockade of the NK1receptor. Bioorg Med Chem
Lett 2010;20(19):5925–5932.
116. Morriello GJ, DeVita RJ, Mills SG, Young JR, Lin P, Doss G, Chicchi GG, DeMartino J, Kurtz
MM, Tsao K-LC, Carlson E, Townson K, Wheeldon A, Boyce S, Collinson N, Rupniak N, Moore
S. Fused bicyclic pyrrolizinones as new scaffolds for human NK1antagonists. Bioorg Med Chem
2008;16(5):2156–2170.
117. Morriello GJ, Mills SG, Johnson T, Reibarkh M, Chicchi G, DeMartino J, Kurtz M, Davies P, Tsao
KLC, Zheng S, Tong X, Carlson E, Townson K, Tattersall FD, Wheeldon A, Boyce S, Collinson
N, Rupniak N, Moore S, DeVita RJ. Substituted fused bicyclic pyrrolizinones as potent, orally
bioavailable hNK1antagonists. Bioorg Med Chem Lett 2010;20(6):2007–2012.
118. Morriello GJ, Chicchi G, Johnson T, Mills SG, Demartino J, Kurtz M, Tsao KL, Zheng S, Tong X,
Carlson E, Townson K, Wheeldon A, Boyce S, Collinson N, Rupniak N, Devita RJ. Fused tricyclic
pyrrolizinones that exhibit pseudo-irreversible blockade of the NK1receptor. Bioorg Med Chem
Lett 2010;20(19):5925–5932.
119. Di Fabio R, Griffante C, Alvaro G, Pentassuglia G, Pizzi DA, Donati D, Rossi T, Guer-
cio G, Mattioli M, Cimarosti Z, Marchioro C, Provera S, Zonzini L, Montanari D,
Melotto S, Gerrard PA, Trist DG, Ratti E, Corsi M. Discovery process and pharmacologi-
cal characterization of 2-(S)-(4-fluoro-2-methylphenyl)piperazine-1-carboxylic acid [1-(R)-(3,5-bis-
trifluoromethylphenyl)ethyl]methylamide (vestipitant) as a potent, selective, and orally active NK1
receptor antagonist. J Med Chem 2009;52(10):3238–3247.
120. Di Fabio R, Alvaro G, Griffante C, Pizzi DA, Donati D, Mattioli M, Cimarosti Z,
Guercio G, Marchioro C, Provera S, Zonzini L, Montanari D, Melotto S, Gerrard
PA, Trist DG, Ratti E, Corsi M. Discovery and biological characterization of (2R,4S)-
1’-acetyl-N-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}-2-(4-fl uoro-2-methylphenyl)-N-methyl-
4,4’-bipiperidine-1-carboxamide as a new potent and selective neurokinin 1 (NK1) receptor antag-
onist clinical candidate. J Med Chem 2011;54(4):1071–1079.
121. Cirillo R, Astolfi M, Conte B, Lopez G, Parlani M, Terracciano R, Fincham CI, Manzini S.
Pharmacology of the peptidomimetic, MEN 11149, a new potent, selective and orally effective
tachykinin NK1receptor antagonist. Eur J Pharmacol 1998;341(2–3):201–209.
122. Carlsson A, Lindqvist M, Magnusson T. 3,4-Dihydroxyphenylalanine and 5-Hydroxytryptophan
as reserpine antagonists. Nature 1957;180(4596):1200–1200.
123. Le Crom S, Kapsimali M, Barome PO, Vernier P. Dopamine receptors for every species: Gene
duplications and functional diversification in Craniates. J Struct Funct Genomics 2003;3(1–4):
161–176.
124. Carlsson A, Civelli O, Kebabian J, Langer S, Scatton B, Schwartz JC, Sedvall G, Seeman
P, Sokoloff P, Spano P, Van Tol H. Dopamine receptors. Last modified on May 27, 2013.
Accessed on August 29, 2013. IUPHAR database (IUPHAR-DB), http://www.iuphar-db.org/
DATABASE/FamilyMenuForward?familyId=20.
125. Hurley MJ, Jenner P. What hasbeen learnt from study of dopamine receptorsin Parkinson’s disease?
Pharmacol Ther 2006;111(3):715–728.
126. Schneier FR, Liebowitz MR, Abi-Dargham A, Zea-Ponce Y, Lin SH, Laruelle M. Low dopamine
D2receptor binding potential in social phobia. Am J Psychiatry 2000;157(3):457–459.
127. Kienast T, Heinz A. Dopamine and the diseased brain. CNS Neurol Disord Drug Targets
2006;5(1):109–131.
128. Fuxe K, Manger P, Genedani S, Agnati L. In: Riederer P, Reichmann H, Youdim MBH, Gerlach
M, Eds. Parkinson’s Diseases and Related Disorders. Vienna: Springer; 2006. p 71–83.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r889
129. Mihara K, Kondo T, Suzuki A, Yasui-Furukori N, Ono S, Sano A, Koshiro K, Otani K, Kaneko
S. Relationship between functional dopamine D2and D3receptors gene polymorphisms and neu-
roleptic malignant syndrome. Am J Med Genet B 2003;117B(1):57–60.
130. Faraone SV, Khan SA. Candidate gene studies of attention-deficit/hyperactivity disorder. J Clin
Psychiatry 2006;67:13–20.
131. Hummel M, Unterwald EM. D1dopamine receptor: A putative neurochemical and behavioral link
to cocaine action. J Cell Physiol 2002;191(1):17–27.
132. Seeman P, Weinshenker D, Quirion R, Srivastava LK, Bhardwaj SK, Grandy DK, Premont RT,
Sotnikova TD, Boksa P, El-Ghundi M, O’Dowd B F, George SR, Perreault ML, Mannisto PT,
Robinson S, Palmiter RD, Tallerico T. Dopamine supersensitivity correlates with D2High states,
implying many paths to psychosis. Proc Natl Acad Sci USA 2005;102(9):3513–3518.
133. Miyamoto S, Duncan GE, Marx CE, Lieberman JA. Treatments for schizophrenia: A critical review
of pharmacology and mechanisms of action of antipsychotic drugs. Mol Psychiatry 2005;10(1):79–
104.
134. Kapur S, Seeman P. Antipsychotic agents differ in how fast they come off the dopamine D2receptors.
Implications for atypical antipsychotic action. J Psychiatr Neurosci 2000;25(2):161–166.
135. Langlois X, Megens A, Lavreysen H, Atack J, Cik M, te Riele P, Peeters L, Wouters R, Ver-
meire J, Hendrickx H, Macdonald G, De Bruyn M. Pharmacology of JNJ-37822681, a specific
and fast-dissociating D2antagonist for the treatment of schizophrenia. J Pharmacol Exp Ther
2012;342(1):91–105.
136. Schultz W. Multiple dopamine functions at different time courses. Annu Rev Neurosci 2007;30:259–
288.
137. Bartholi G, Haefely W, Jalfre M, Keller HH, Pletsche A. Effects of clozapine on cerebral cate-
cholaminergic neuron systems. Br J Pharmacol 1972;46(4):736–740.
138. Jones C, Kapur S, Remington G, Zipursky RB. Transient D2dopamine receptor occupancy in low
EPS-incidence drugs: PET evidence. Biol Psychiatry 2000;47(8):112s–112s.
139. Carboni L, Negri M, Michielin F, Bertani S, Fratte SD, Oliosi B, Cavanni P. Slow dissociation of
partial agonists from the D2receptor is linked to reduced prolactin release. Int J Neuropsychophar-
macol 2012;15(5):645–656.
140. Kapur S, Seeman P. Does fast dissociation from the dopamine D2receptor explain the action of
atypical antipsychotics?: A new hypothesis. Am J Psychiatry 2001;158(3):360–369.
141. Schmidt ME, de Boer P, Andrews R, Neyens M, Rossenu S, William Falteos D, Mannaert E. D2
receptor occupancy measurement of JNJ-37822681, a novel fast off-rate D2receptor antagonist,
in healthy subjects using positron emission tomography: Single dose versus steady state and dose
selection. Psychopharmacology (Berl) 2012;224(4):549–557.
142. Schmidt ME, Kent JM, Daly E, Janssens L, Van Osselaer N, Husken G, Anghelescu IG, Van
Nueten L. A double-blind, randomized, placebo-controlled study with JNJ-37822681, a novel,
highly selective, fast dissociating D2receptor antagonist in the treatment of acute exacerbation of
schizophrenia. Eur Neuropsychopharmacol 2012;22(10):721–733.
143. Miller DC, Lunn G, Jones P, Sabnis Y, Davies NL, Driscoll P. Investigation of the effect of
molecular properties on the binding kinetics of a ligand to its biological target. MedChemComm
2012;3(4):449–452.
144. Vale W, Spiess J, Rivier C, Rivier J. Characterization of a 41-residue ovine hypothalamic
peptide that stimulates secretion of corticotropin and beta-endorphin. Science 1981;213(4514):
1394–1397.
145. Dautzenberg FM, Hauger RL. The CRF peptide family and their receptors: Yet more partners
discovered. Trends Pharmacol Sci 2002;23(2):71–77.
146. Bale TL, Vale WW. CRF and CRF receptors: Role in stress responsivity and other behaviors. Annu
Rev Pharmacol 2004;44:525–557.
147. Schulz DW, Mansbach RS, Sprouse J, Braselton JP, Collins J, Corman M, Dunaiskis A, Faraci
S, Schmidt AW, Seeger T, Seymour P, Tingley FD, Winston EN, Chen YL, Heym J. CP-154,
Medicinal Research Reviews DOI 10.1002/med
890 rGUO ET AL.
526: A potent and selective nonpeptide antagonist of corticotropin releasing factor receptors. Proc
Natl Acad Sci USA 1996;93(19):10477–10482.
148. Grigoriadis DE. The corticotropin-releasing factor receptor: A novel target for the treatment of
depression and anxiety-related disorders. Expert Opin Ther Targets 2005;9(4):651–684.
149. Holsboer F. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology
2000;23(5):477–501.
150. Chen C. Recent advances in small molecule antagonists of the corticotropin-releasing factor
type-1 receptor-focus on pharmacology and pharmacokinetics. Curr Med Chem 2006;13(11):
1261–1282.
151. Fleck BA, Hoare SR, Pick RR, Bradbury MJ, Grigoriadis DE. Binding kinetics redefine the an-
tagonist pharmacology of the corticotropin-releasing factor type 1 receptor. J Pharmacol Exp Ther
2012;341(2):518–531.
152. Miller DC, Klute W, Brown AD. Discovery of potent, metabolically stable purine CRF-1 antagonists
with differentiated binding kinetic profiles. Bioorg Med Chem Lett 2011;21(20):6108–6111.
153. Ramsey SJ, Attkins NJ, Fish R, van der Graaf PH. Quantitative pharmacological analysis of
antagonist binding kinetics at CRF1receptors in vitro and in vivo. Br J Pharmacol 2011;164(3):992–
1007.
154. Gilligan PJ, Robertson DW, Zaczek R. Corticotropin releasing factor (CRF) receptor modulators:
Progress and opportunities for new therapeutic agents. J Med Chem 2000;43(9):1641–1660.
155. Kehne J, De Lombaert S. Non-peptidic CRF1receptor antagonists for the treatment of anxiety,
depression and stress disorders. Curr Drug Targets CNS Neurol Disord 2002;1(5):467–493.
156. Hollenstein K, Kean J, Bortolato A, Cheng RKY, Dore AS, Jazayeri A, Cooke RM, Weir
M, Marshall FH. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature
2013;499(7459):438–443.
157. Arvanitis AG, Gilligan PJ, Chorvat RJ, Cheeseman RS, Christos TE, Bakthavatchalam R, Beck JP,
Cocuzza AJ, Hobbs FW, Wilde RG, Arnold C, Chidester D, Curry M, He L, Hollis A, Klaczkiewicz
J, Krenitsky PJ, Rescinito JP, Scholfield E, Culp S, De Souza EB, Fitzgerald L, Grigoriadis D, Tam
SW, Wong YN, Huang S, Shen HL. Non-peptide corticotropin-releasing hormone antagonists:
Syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines. J Med Chem
1999;42(5):805–818.
158. Hodge CN, Aldrich PE, Wasserman ZR, Fernandez CH, Nemeth GA, Arvanitis A, Cheeseman
RS, Chorvat RJ, Ciganek E, Christos TE, Gilligan PJ, Krenitsky P, Scholfield E, Strucely P.
Corticotropin-releasing hormone receptor antagonists: Framework design and synthesis guided by
ligand conformational studies. J Med Chem 1999;42(5):819–832.
159. Hann MM. Molecular obesity, potency and other addictions in drug discovery. MedChemComm
2011;2(5):349–355.
160. Decourcelles DC, Leysen JE, Roevens P, Vanbelle H. The serotonin-S2receptor: A receptor-
transducer coupling model to explain insurmountable antagonist effects. Drug Dev Res 1986;8(1–
4):173–178.
161. Smith C, Rahman T, Toohey N, Mazurkiewicz J, Herrick-Davis K, Teitler M. Risperidone irre-
versibly binds to and inactivates the h5-HT7 serotonin receptor. Mol Pharmacol 2006;70(4):1264–
1270.
162. Hosohata Y, Sasaki K, Shen Y, Hattori K, Suzuki J, Nagatomo T. Bopindolol is a slowly dis-
sociating beta1-adrenoceptor antagonist when compared to other beta-blockers. Biol Pharm Bull
1995;18(8):1066–1071.
163. Casarosa P, Kollak I, Kiechle T, Ostermann A, Schnapp A, Kiesling R, Pieper M, Sieger P, Gantner
F. Functional and biochemical rationales for the 24-hour long duration of action of olodaterol. J
Pharmacol Exp Ther 2011;337(3):600–609.
164. Leysen JE, Gommeren W. Drug-receptor dissociation time, new tool for drug research: Receptor
binding affinity and drug-receptor dissociation profiles of serotonin-S2, dopamine-D2, histamine-H1
antagonists, and opiates. Drug Dev Res 1986;8(1–4):119–131.
Medicinal Research Reviews DOI 10.1002/med
DRUG-TARGET RESIDENCE TIME r891
165. Watson C, Jenkinson S, Kazmierski W, Kenakin T. The CCR5 receptor-based mechanism of action
of 873140, a potent allosteric noncompetitive HIV entry inhibitor. Mol Pharmacol 2005;67(4):1268–
1282.
166. Gonsiorek W, Fan X, Hesk D, Fossetta J, Qiu H, Jakway J, Billah M, Dwyer M, Chao J, Deno
G, Taveras A, Lundell DJ, Hipkin RW. Pharmacological characterization of Sch527123, a potent
allosteric CXCR1/CXCR2 antagonist. J Pharmacol Exp Ther 2007;322(2):477–485.
167. Stock N, Volkots D, Stebbins K, Broadhead A, Stearns B, Roppe J, Parr T, Baccei C, Bain G,
Chapman C, Correa L, Darlington J, King C, Lee C, Lorrain DS, Prodanovich P, Santini A,
Evans JF, Hutchinson JH, Prasit P. Sodium [2’-[(cyclopropanecarbonyl-ethyl-amino)-methyl]-4’-(6-
ethoxy-pyridin-3-yl)-6-meth oxy-biphenyl-3-yl]-acetate (AM432): A potent, selective prostaglandin
D2receptor antagonist. Bioorg Med Chem Lett 2011;21(3):1036–1040.
168. Sullivan SK, Hoare SRJ, Fleck BA, Zhu YF, Heise CE, Struthers RS, Crowe PD. Kinetics of
nonpeptide antagonist binding to the human gonadotropin-releasing hormone receptor: Impli-
cations for structure-activity relationships and insurmountable antagonism. Biochem Pharmacol
2006;72(7):838–849.
169. Anthes JC, Gilchrest H, Richard C, Eckel S, Hesk D, West RE, Jr., Williams SM, Greenfeder S,
Billah M, Kreutner W, Egan RE. Biochemical characterization of desloratadine, a potent antagonist
of the human histamine H1receptor. Eur J Pharmacol 2002;449(3):229–237.
170. Smits RA, Lim HD, van der Meer T, Kuhne S, Bessembinder K, Zuiderveld OP, Wijtmans M,
de Esch IJ, Leurs R. Ligand based design of novel histamine H4receptor antagonists; fragment
optimization and analysis of binding kinetics. Bioorg Med Chem Lett 2012;22(1):461–467.
171. Meini S, Bellucci F, Catalani C, Cucchi P, Giolitti A, Santicioli P, Giuliani S. Mul-
tifaceted approach to determine the antagonist molecular mechanism and interaction
of ibodutant ([1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-
ethylcarbamoyl)-cyclopentyl]-amide) at the human tachykinin NK2receptor. J Pharmacol Exp
Ther 2009;329(2):486–495.
172. Malherbe P, Kratzeisen C, Marcuz A, Zenner MT, Nettekoven MH, Ratni H, Wettstein JG, Bissantz
C. Identification of a critical residue in the transmembrane domain 2 of tachykinin neurokinin 3
receptor affecting the dissociation kinetics and antagonism mode of osanetant (SR 142801) and
piperidine-based structures. J Med Chem 2009;52(22):7103–7112.
173. Mould R, Brown J, Marshall FH, Langmead CJ. Binding kinetics differentiates functional antago-
nism of orexin-2 receptor ligands. Br J Pharmacol 2014;171(2):351–363.
174. Ring PC, Seldon PM, Barnes PJ, Giembycz MA. Pharmacological characterization of a receptor
for platelet-activating factor on guinea pig peritoneal macrophages using [3H]apafant, a selective
and competitive platelet-activating factor antagonist: Evidence that the noncompetitive behavior of
apafant in functional studies relates to slow kinetics of dissociation. Mol Pharmacol 1993;43(2):302–
312.
175. Behm DJ, Aiyar NV, Olzinski AR, McAtee JJ, Hilfiker MA, Dodson JW, Dowdell SE, Wang
GZ, Goodman KB, Sehon CA, Harpel MR, Willette RN, Neeb MJ, Leach CA, Douglas SA.
GSK1562590, a slowly dissociating urotensin-II receptor antagonist, exhibits prolonged pharmaco-
dynamic activity ex vivo. Br J Pharmacol 2010;161(1):207–228.
176. Mullins D, Adham N, Hesk D, Wu Y, Kelly J, Huang Y, Guzzi M, Zhang X, McCombie S, Stamford
A, Parker E. Identification and characterization of pseudoirreversible nonpeptide antagonists of the
neuropeptide Y Y5receptor and development of a novel Y5-selective radioligand. Eur J Pharmacol
2008;601(1–3):1–7.
177. Aronne LJ, Tonstad S, Moreno M, Gantz I, Erondu N, Suryawanshi S, Molony C, Sieberts S, Nayee
J, Meehan AG, Shapiro D, Heymsfield SB, Kaufman KD, Amatruda JM. A clinical trial assessing
the safety and efficacy of taranabant, a CB1R inverse agonist, in obese and overweight patients: A
high-dose study. Int J Obes (Lond) 2010;34(5):919–935.
178. Kipnes MS, Hollander P, Fujioka K, Gantz I, Seck T, Erondu N, Shentu Y, Lu K, Suryawanshi
S, Chou M, Johnson-Levonas AO, Heymsfield SB, Shapiro D, Kaufman KD, Amatruda JM. A
Medicinal Research Reviews DOI 10.1002/med
892 rGUO ET AL.
one-year study to assess the safety and efficacy of the CB1R inverse agonist taranabant in overweight
and obese patients with type 2 diabetes. Diabetes Obes Metab 2010;12(6):517–531.
179. http://clinicaltrials.gov/
180. Meltzer HY. Multireceptor atypical antipsychotic drugs. In: Ellenbroek BA, Cools AR, Eds. Atyp-
ical Antipsychotics. Basel: Birkhauser Verlag; 2000. p 191–213.
181. Huirne JA, Lambalk CB. Gonadotropin-releasing-hormone-receptor antagonists. Lancet
2001;358(9295):1793–1803.
182. Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Lissin DV, Monteillet-Agius G, Stewart
PL, Evans CJ, von Zastrow M. mu-Opioid receptor internalization: Opiate drugs have differential
effects on a conserved endocytic mechanism in vitro and in the mammalian brain. Mol Pharmacol
1998;53(3):377–384.
Dong Guo, studied Biopharmaceutical Sciences at Leiden University, the Netherlands. During his
MSc work, he performed traineeships at the Division of Medicinal Chemistry at the University of
Leiden, the Netherlands and at the Division of Neuroscience and Pharmacology at the University
of Copenhagen, Denmark. In January 2010, he started his doctoral project “Drug-target residence
time—a case for the adenosine A1and A2A receptors” under the direction of Prof. Ad P. IJzerman
and Dr. Laura H. Heitman at the Division of Medicinal Chemistry of Leiden University. His areas
of interest include the setup of kinetic assays and investigation of ligand–receptor residence time
at the adenosine A1and A2A receptors.
Julia M. Hillger, studied Biopharmaceutical Sciences at the University of Leiden, the Netherlands.
During her MSc, she performed traineeships at the Division of Medicinal Chemistry at Leiden
University, the Netherlands and at the Center of Biomedical Engineering at the Massachusetts
Institute of Technology, USA. She furthermore performed a literature study on receptor kinetics,
with a focus on neurokinin receptors and slowly dissociating antagonists. In October 2012, she
started her PhD project on “Personalized medicine and label-free technologies” in Prof. IJzerman’s
group at the Division of Medicinal Chemistry of Leiden University. Her main research interest is
in G protein-coupled receptors, in particular genetic influences on drug response and applications
of novel assay technologies.
Ad P. IJzerman, obtained his pharmacy degree from Utrecht University, the Netherlands, in 1980.
He then continued as a PhD student at the Vrije Universiteit of Amsterdam, the Netherlands.
He obtained his PhD degree on the medicinal chemistry of the β-adrenergic receptor in 1985,
after which he was appointed assistant professor of medicinal chemistry at Leiden University in its
Leiden Academic Centre for Drug Research. Since 2000 he holds a full professorship of medicinal
chemistry at the same university. His main research interest is in G protein-coupled receptors and
in novel concepts to intervene with receptor function.
Laura H. Heitman, studied Biopharmaceutical Sciences at the University of Leiden, the Nether-
lands. During her MSc work, she performed traineeships at the Division of Medicinal Chemistry
at the University of Leiden, the Netherlands and at the Division of Pharmaceutical and Biological
Chemistry at the School of Pharmacy in London, UK. In October 2004, she then continued as a
PhD student at the Division of Medicinal Chemistry of Leiden University. She obtained her PhD
degree on “Allosteric modulation of ‘reproductive’ GPCRs” in 2009, after which she was appointed
assistant professor of medicinal chemistry at Leiden University in its Leiden Academic Centre for
Drug Research. Her area of interest is in G protein-coupled receptors, in particular on “receptor
residence time.”
Medicinal Research Reviews DOI 10.1002/med
