The handle
http://hdl.handle.net/1887/138132
holds various files of this Leiden
University dissertation.
Author: Vlachodimou, A.
Title: Targeting the adenosinergic system: Ligand binding kinetics and label-free assays
for the study of SLC29A1 transporter and A2B adenosine receptor
Caffeine is the most commonly consumed psychoactive substance in the world. It is found in a variety of forms, from our daily beverages of coffee, tea, soft and energy drinks, to food, like chocolate and to a multitude of prescription and over-the-counter formulations, including painkillers, cold remedies and weight-loss medications1.
It is naturally sourced from coffee beans (Coffea), kola nuts (Cola), tea (camellia sinensis), and chocolate (Cocoa bean), while its consumption is dated as far back as the Paleolithic period2. The earliest recorded use of caffeine-containing beverages
dates back to the Tang Dynasty of China (618-907 AD), where tea was a popular drink as it was considered to prolong life3, while coffee drinking is dated not earlier
than the middle of the 15th century in the Sufi monasteries of Yemen4. From the Arab
world, coffee drinking spread to Europe and America, leading to its consumption worldwide. Today large amounts of coffee are used in order to reduce fatigue and drowsiness, and to increase mental alertness.
Caffeine administration affects the functioning of a wide range of systems in the human body, including the cardiovascular, respiratory, renal, and nervous system. It is known to mediate its actions via at least three distinct mechanisms, antagonism of adenosine receptors, phosphodiesterase inhibition and release of calcium from intracellular stores5. Concerning coffee-related awakeness and alertness, it is
now well established that caffeine is competing with the endogenous substance adenosine for adenosine receptor binding.
Adenosine exerts its actions via binding to and activating a family of receptors, i.e. the adenosine receptors, while its circulating levels are controlled via a variety of mechanisms and proteins, including membrane transporters. For multiple pathophysiological conditions a distorted adenosinergic tone is involved, hence these receptors and transporters may be potential drug targets. For many years pharmaceutical industry has engaged in the design and synthesis of potential drugs targeting these receptors and transporters. Unfortunately there is either no or a limited number of marketed drugs depending on the target.
For a better understanding of the adenosinergic system, this thesis zooms in at a molecular level to study two subtypes of receptors and transport proteins, i.e. the A2B adenosine receptor and equilibrative nucleoside transporter 1 with a focus on ligand binding kinetics. This introductory chapter provides a background for the research presented, followed by the aim and outline of this thesis.
Cellular regulation of adenosine
Adenosine is a purine ribonucleoside produced endogenously both intracellularly and extracellularly. Extracellular adenosine originates from among others damaged cells6, released via equilibrative transporters, or by ectonucleotidase-mediated
hydrolysis of adenine nucleotides7,8 (Figure 1). Its extracellular concentration varies
greatly, based on the tissue as well as the level of injury or stress experienced9.
triphosphate (ATP) similarly to extracellular hydrolysis of adenine nucleotides or via the transmethylation pathway10 (Figure 1). The S-adenosylmethionine-dependent
transmethylation reaction generates S-adenosylhomocysteine (SAH), which is subsequently hydrolyzed to adenosine and L-homocysteine. This hydrolysis is operational only at low adenosine levels. Once adenosine levels are increased, the equilibrium is shifted and SAH is formed11. The level of intracellular adenosine is
regulated by the metabolism of adenosine to adenosine monophosphate (AMP) via adenosine kinase (AK) or to inosine via adenosine deaminase (ADA) (Figure 1). As a low capacity, high affinity enzyme, AK is regarded as the principal enzyme in regulating intracellular adenosine concentrations under physiological conditions11. So
AK phosphorylates adenosine to AMP, which is further phosphorylated to ADP and ATP (adenosine diphosphate and triphosphate, respectively) by other processes. As a result the intracellular concentration of adenosine is kept low10, which favors the
uptake of extracellular adenosine via equilibrative transporters in order to re-establish equilibrium across the cell membrane12. Of note, ADA becomes more important with
higher adenosine concentrations, such as under ischemic conditions13.
Figure 1: Production, transport and metabolism of adenosine.
ADA: adenosine deaminase, ADO: adenosine, ADP: adenosine diphosphate, AK: adenosine kinase, AMP: adenosine monophosphate, ATP: adenosine triphosphate, cyto-N: cytosolic-nucleotidases, ecto-N: ecto-nucleotidases (including nucleoside triphosphate diphosphohydrolase, 5′-nucleotidase and nucleotide pyrophosphatase/phosphodiesterase), Hcy; Homocysteine, INO: inosine, Met: methionine, SAH: S-adenosylhomocysteine, SAHH: SAH hydrolase, SAM: S-adenosylmethionine. The thickness of arrows indicates the predominant reaction direction.
The next two paragraphs are also covered in review Chapter 2, and are described with some brevity here.
A
2Badenosine receptor (A
2BAR)
The physiological and pathophysiological effects of adenosine are mediated by specific G protein-coupled receptors (GPCRs), the adenosine receptors (ARs). ARs belong to the class A family of GPCRs and they are further divided into four subtypes, namely A1, A2A, A2B and A3 ARs. Each AR subtype shows a distinctive pharmacological profile and tissue distribution. A1 and A3 ARs mainly interact with a Gi protein, and their activation results in inhibition of adenylate cyclase (AC), while activation of A2A and A2B AR has the opposite effect, as they mainly interact with a GS protein. In addition to AC, ARs signal through other pathways, including phospholipase C (PLC), Ca2+ and mitogen-activated protein kinases (MAPKs)9.
A2B is the least studied of the ARs, and since its identification and cloning in 199214, it has been considered as a receptor with low-affinity for adenosine,
hence of less physiological relevance15. However, recent studies have shown that
A2BAR has distinct intracellular signaling pathways and a unique physiological and pathophysiological role. A2BAR has been found to be expressed in numerous tissues and organs, including bowel, bladder, lung, brain, as well as on hematopoietic and mast cells16,17. Interestingly, its expression is affected by diverse signals, such as
inflammation, injury or hypoxia, leading to its upregulation15. Based on the tissue,
its expression levels and the timing of activation or inhibition, A2BAR has been investigated as a potential drug target for many diseases18. Agonists have been
proposed for the treatment of acute lung injury and ischemic heart diseases, while in cases of chronic lung disease, such as asthma, chronic obstructive pulmonary disease and pulmonary fibrosis, as well as in diabetic retinopathy and Alzheimer’s disease antagonists seem to be preferred9,19,20. Moreover, A
2BAR antagonists display
analgesic21 and anti-inflammatory effects, while their use has also been proposed
in cancer (immuno)therapy, as they were found to directly affect the growth and migration of bladder, breast, colon, and prostate cancerous cells22-25.
Equilibrative nucleoside transporter 1 (ENT1)
AR signaling is modulated not only by direct-acting ligands (agonists or antagonists), but also by modulation of the metabolism or cellular uptake of endogenous intra- and extracellular adenosine9. One option for such modulation is
through targeting the nucleoside transporters (NTs), which are responsible for the cellular uptake and efflux of nucleosides, including adenosine. As such they fine-tune many physiological processes, such as the synthesis of nucleic acids and activation of the cAMP signaling pathway. There are two structurally unrelated NT families that belong to the superfamily of solute carriers (SLC)26; the SLC28 concentrative
nucleoside transporter (CNT) and the SLC29 equilibrative nucleoside transporter (ENT) family.
ENT1 is one of the major NTs on plasma membranes and is ubiquitously expressed in the human body27. It is encoded by the SLC29A1 gene and as part of the ENT family
mediates the bidirectional Na+-independent facilitative diffusion of its substrates
down their concentration gradients. Since its identification, ENT1 has been studied as a drug target for both its transportation and inhibition. Many nucleoside analogues used for the treatment of cancer and viral infections are transported by ENT1 as part of their mechanism of action28. In contrast, ENT1 inhibition also offers anti-cancer
possibilities, either by counteracting its overexpression in specific cancer types29,30,
or as add-on cancer therapy for anti-cancer nucleoside drugs transported by CNTs, preventing cellular efflux. Finally, ENT1 has been identified as one of the major if not the most important NT in modulating adenosine levels. As its inhibition leads to increased AR activation, targeting ENT1 offers numerous therapeutic indications related to ARs, including stroke, insomnia and inflammation31,32.
Need for pharmacological intervention
In order to modulate the interaction of adenosine with ENT1 or ARs (for the purposes of this thesis, especially A2BAR) and achieve the desired pharmacological effect, transporter inhibitors and receptor antagonists are of need. Following the discovery of natural products, such as caffeine, theobromine and theophylline, the need for novel, effective and selective pharmaceuticals became indispensable. Hence, the design and synthesis of compounds targeting the adenosinergic system attracted the attention of many medicinal chemists for at least the last thirty years. A considerable number of ENT1 inhibitors have been developed, including two marketed drugs, dilazep and dipyridamole32. However, both drugs are not targeting
ENT1 selectively. Regarding ARs, the list of selective antagonists discovered is longer and in some cases they have been clinically evaluated9. Nevertheless,
there is only one clinically approved AR antagonist; istradefylline, a selective A2AAR antagonist. As a result there is an urge for novel compounds binding to these targets, in addition to novel assays better evaluating the compounds’ in vitro effects, as well as predicting their in vivo outcomes.
Binding kinetics
The problem of compounds showing a promising profile in in vitro studies and failing to do well in clinical trials is not rare in drug discovery programs. Many parameters can lead to the often huge attrition rate in clinical trials, including lack of funding to complete the trial, inability to maintain good manufacturing protocols or to follow the regulatory agencies and organizations, such as FDA and EMEA, guidance, or problems with patient recruitment, enrollment, and retention33. However, the primary
cause for failure in clinical trials is lack of in vivo efficacy34-36, highlighting our inability
to translate in vitro data into successful predictions of efficacy in humans. Therefore, novel strategies that allow for a detailed understanding of a ligand’s mechanism of action are needed.
The traditionally studied parameters of affinity (Ki) and potency (EC50 / IC50) are measured under equilibrium conditions, thus they characterize the thermodynamic drug-target interactions. However, they cannot account for the time-dependent changes in target engagement in an open system like the human body, where drug and target have fluctuating concentrations37. Over the last decade, accumulative data
has indicated that drug-target binding kinetics, as defined by the association (kon) and the dissociation (koff) rate constants, could better account for these fluctuations, rendering them important parameters for improved efficacy (Figure 2)38. Based on
the aforementioned kinetic rate constants, the additional parameters of affinity (KD, defined as koff / kon) and residence time (RT, defined as 1 / koff), a parameter indicating the lifetime of the ligand-target complex, can be determined.
Figure 2: Binding of a ligand to a target as described by its kinetic binding parameters. The
formation of a ligand-target complex is characterized by the association (kon) and dissociation (koff) rate constant of the ligand to and from the target. Residence time (RT) and affinity (KD) can be derived from these rate constants.
The role of RT has been discussed in a number of studies and reviews, which revealed its significance in a number of marketed drugs39. A typical example is
aprepitant, a neurokinin 1 (NK1) receptor antagonist that was found in a retrospective study to have higher in vivo efficacy than other NK1 receptor antagonists with similar thermodynamic affinities40. The enhanced in vivo effect has been attributed to its long
target RT, which results in a longer duration of action. Although RT and koff are now slowly being accepted as important parameters for drug discovery, kon is still largely neglected and understudied compared. It has quite recently, however, been shown to be related to the fast onset of drug action, as well as increased in vivo target occupancy41-43. Indacaterol, a marketed bronchodilator activating β
2-adrenoceptors,
further emphasizes the value of kon. Its fast association to the target correlates well with the rapid onset of action in various experimental models44.
Assays for early stage drug discovery
Drug discovery is a laborious, time-consuming and costly process. Identification of novel molecules and lead optimization are the initial steps of this process, while the proper selection lead compounds plays a crucial role in the following steps. Hence, there is a constant interplay between the fields of medicinal chemistry and in vitro pharmacology, best described as “one goal, many compounds, many assays”. The assays used in in vitro pharmacology can be categorized into two groups: binding assays, evaluating the tenacity (affinity, Ki and KD) and the speed (binding kinetics, kon, koff and RT) that a ligand binds to its target, and functional assays, evaluating the type and the strength of functional effect that a ligand exerts (potency, EC50 / IC50, and efficacy, Emax). For the majority of binding and functional assays, a label attached to the ligand or target is required, while a trend towards novel label-free assays is emerging.
Radioligand-based assays
The advent of radiolabeled ligands in the 1970s facilitated the screening of synthesized small molecules offering a direct way to evaluate the interaction of the compound with the molecular target45,46. Due to their rapid character, technical
ease as well as the need of only a small amount of compound, radioligand-based assays became the gold standard in establishing the binding profile of a drug. In addition, the plethora of available radio-labels, such as 3H (tritium), 125I and 35S,
offered opportunities for optimizing assays based on the system under examination. Furthermore, technological advances in lab equipment and automation, such as harvesters, microplate readers and automated pipetting workstations, dramatically increased the throughput of the assays, hence their prevalence. Currently, radioligand-based binding and functional assays are widely adapted and used in the screening of GPCR and SLC ligands in drug discovery, offering a cost effective way to determine the structure-activity (affinity, kinetics, functional effect) relationship for a series of compounds.
Due to the need of specialized researchers, laboratories, and waste disposal, other, non-radioactive methods were developed, such as bioluminescence- (BRET) and fluorescence-based (FRET) assays. Despite the advantages offered regarding safety, BRET and FRET assays present an important shortcoming; Fluorophores are bulky and as a result they could modify the properties of the ligand or target that they are attached to, and thus potentially generate artifacts and false positive/negative results.
In the current thesis, radioligand binding assays are used for the study of ENT1 transporter and the A2B adenosine receptor. Two radiolabeled molecules, [3H]NBTI
and [3H]PSB 603 are selected based on their high affinity and selectivity for the
targets under investigation. In both cases, tritium is chosen as the radio-label due to its chemically indistinguishable character compared to the unlabeled native ligand.
In the next chapters, several radioligand binding assays are described, serving a distinctive role in the investigation of novel ligands:
- Saturation assay, to determine the radioligand’s equilibrium dissociation constant, KD, and target’s expression/density (Bmax),
- Association and dissociation assay, to quantify the radioligand’s kinetic binding parameters (kon, koff, RT),
- Displacement or competition binding assay, to assess the affinity (Ki) of non-labelled compounds under investigation,
- Competition association assay, to evaluate the kinetic binding parameters of non-labelled compounds under investigation,
- and Wash-out binding assay, to evaluate the wash-resistance of non-labeled compounds under investigation (in relation to their kinetic binding parameters).
Label-free assays
Label-free assays are used as a counterpart of labeled-assays in order to assess both binding and functional effects of the ligands under investigation. Various biosensors have been developed to measure affinity and binding kinetic parameters on isolated proteins or membrane preparations, including but not limited to surface plasmon resonance, surface acoustic wave biosensors and ligand binding assays using mass spectrometry47. Concerning functional responses on whole cells,
label-free assays employ optical, electrical, calorimetric, acoustic, magnetic or other quantifiable signals in order to detect ligand-induced changes in cell adhesion, shape, viability or cell-cell interactions48,49. Best described biosensors measuring
cell morphology changes are the optical- (such as EPIC) and impedance- (such as xCELLigence) based biosensors50,51. Such assays are not measuring a single
signaling pathway, thus yielding an integrated / cumulative response rather than a pathway-biased effect. In addition, the absence of labels and the high sensitivity of measurement allow for the evaluation of the endogenous target function in real-time and under more physiological conditions52, potentially offering a translational bridge
from in vitro to in vivo pharmacology.
Since GPCR signaling is known to result in changes in cell morphology via modulation of the actin cytoskeleton, label-free assays have been widely used to study GPCRs, including some of the ARs53,54. On the other hand, SLC transporters
are not known to exert similar effects on the cytoskeleton, restricting the use of label-free assays to electrophysiological methods, such as patch-clamp and solid supported membrane-based assays55. However, such assays are not suitable for
the study of non-electrogenic membrane transporters, which represent the majority of known SLCs.
developed and validated for the functional study of A2B adenosine receptor and ENT1 transporter:
- A direct label-free assay (Figure 3A), to determine the potency of A2BAR antagonists and signal recovery after washing, supporting the differences in kinetic binding parameters in a functional assay.
- An indirect label-free assay (Figure 3B), to evaluate the potency of ENT1 inhibitors via the concomitant A2BAR signaling and to validate differences in kinetic binding parameters in a functional assay. This is to our knowledge the first case of a biosensor technology applied to study the activity of inhibitors of non-electrogenic membrane transporters56.
Figure 3: Graphic representation of the label-free assays used in this thesis.
A) Direct impedance-based label-free assay assessing the potency of A2BAR antagonists on whole cells. Cells are treated with the antagonist (darkorange triangle) and afterwards with an agonist (lightblue circle). The receptors not occupied by antagonist will be activated by the agonist, resulting in signaling (black arrow). Hence, by increasing the antagonist concentration the receptor signaling will decrease as represented in the graph. Subsequently, the potency of the antagonist can be quantified.
B) Indirect impedance-based label-free assay assessing the potency of ENT1 inhibitors on whole cells. Cell are pre-treated with an ENT1 inhibitor (dark blue triangle) and subsequently with adenosine, an ENT1 substrate / AR agonist (light orange circle). Depending on the inhibitor’s inhibitory potency and concentration, the substrate cannot be translocated inside the cell with the same efficiency. The resulting higher extracellular concentration causes an increased AR activation and signalling (black arrow). Hence, by increasing the ENT1 inhibitor concentration, the extracellular concentration of adenosine (the ENT1 substrate / AR agonist) will be higher, causing an increased AR activation and signalling (black arrow) as represented in the graph. Subsequently, the potency of the inhibitor can be quantified.
OBJECTIVES AND OVERVIEW OF THIS THESIS
Aim
Despite the major efforts and advances in modern drug discovery, many promising drug candidates are withdrawn from clinical trials for reasons related to safety and efficacy. The main reason for failure is the lack of translatability between in vitro and in vivo experiments, resulting in a huge loss of lab animals, time and money. Molecules targeting the adenosinergic system are no exception. As a result a thorough understanding of the mechanism of action at a molecular level is of need, as well as the development of novel physiologically relevant assays. Thus the aim of this thesis was to explore the concept of binding kinetics for A2BAR antagonists and ENT1 inhibitors. Specifically, to provide detailed insights into structural changes influencing the ligand’s binding kinetic profile, we evaluated the role of distinct kinetic profiles in functional assays, and developed novel (kinetic) assays to this end.
Outline
Chapter 2 studies the therapeutic value of ENT1 inhibitors as potentiators of ARs and promising in vitro, in vivo and clinical data are presented as a review. Chapter 3 focuses on the study of A2BAR antagonists. The synthesis of a series of 8-phenylxanthine-based antagonists is described and their affinity is determined. Their binding to the receptor is further evaluated from a kinetic perspective. In addition, an impedance-based label-free assay is developed for the study of A2BAR pharmacology and in particular the link between binding kinetics and pharmacological effect under non-equilibrium conditions. In chapter 4 the need for novel functional assays to study SLCs is addressed. As a result the development, optimization and application of an impedance-based label-free assay, that allows the study of SLC transporters via the concomitant GPCR signaling on whole cells, is reported. For validation purposes, the potency of ENT1 inhibitors were evaluated via ARs signaling. Chapter 5 presents the pharmacological characterization of four reference ENT1 inhibitors. Structure-kinetic relationships (SKR) are drawn in addition to the more classical structure-affinity relationships (SAR) for one of the reference inhibitors and its analogues. Additionally, the direct connection of binding kinetics and functional effect is demonstrated by the use of the label-free assay developed in chapter 4. Chapter 6 discusses the biological evaluation of a novel series of ENT1 inhibitors. Affinity and binding kinetics are determined and followed by extensive SAR and SKR. The kinetic profiles of the inhibitors are evaluated by comparison of their kinetic parameters with in vitro equilibrium parameters. An overall conclusion summarizing the results described in this thesis as well as the forthcoming future opportunities is presented in chapter 7. Hopefully, this thesis will contribute to a better understanding of the molecular pharmacology and medicinal chemistry of A2BAR and ENT1 and provide key in vitro parameters that, when studied in a pharmacokinetics/
pharmacodynamics context, yield an improved in vivo outcome, ultimately resulting in novel safe therapeutics.
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