UNIVERSITY OF PISA
School of Graduate Studies
“Drug sciences and Bioactive Substances
”
PhD THESIS
2011-2013
SSD BIO/10
“Adenosine receptor ligands and Angiogenin in cell
proliferation/differentiation processes”
Candidate:
Supervisor:
Chiara Giacomelli
Prof. Claudia Martini
DIRECTOR OF THE SCHOOL
Prof. Adriano Martinelli
2014
“The content of this thesis is confidential, being protected by the law as arguments secrets. Therefore, the divulgation of the content is sanctioned by articles 325 and 623 of the Italian Criminal Code. "
Pisa, 27/02/2014
Impegno di riservatezza
Il sottoscritto___________________________________________, con qualifica di ________________________presso________________________________________
con riferimento alle informazioni contenute nell’elaborato di tesi di dottorato della Dott.ssa Giacomelli Chiara con titolo: “Adenosine receptor ligands and Angiogenin in cell proliferation/differentiation processes”, in quanto passibili di copertura brevettuale
dichiaro
con la presente di essere stato messo a conoscenza del fatto che tutte le informazioni contenute nella documentazione sopra citata , compresi dati e recapiti riferiti a soggetti fisici o giuridici (*), sono strettamente riservate, essendo presenti argomenti tutelati dalla legge come segreti.
Conseguentemente, consapevole delle sanzioni previste dagli articoli 325 e 623 del Codice penale,
mi impegno
a non divulgare in alcun modo le informazioni riservate e comunque a non utilizzare le informazioni suddette in alcun modo che possa per Voi rivelarsi dannoso, sia direttamente che indirettamente.
Mi impegno altresì a non riprodurre, né integralmente, né in parte la documentazione che mi avete fornito e a restituirVi immediatamente la medesima, a semplice richiesta, senza trattenerne copia alcuna.
Pisa, _____________
In fede
I believe in intuition and inspiration. Imagination is more important than knowledge. For knowledge is limited, whereas imagination embraces the entire world, stimulating progress, giving birth to evolution. It is, strictly speaking, a real factor in scientific research.
CONTENT
CHAPTER 1: Adenosine receptor ligands and Angiogenin in cell
proliferation/differentiation processes ... 1
References ... 2
CHAPTER 2: Allosteric modulation of G protein-coupled receptors. ... 4
2.1 GPCRs ... 4
2.2 Allosteric modulation: opportunities in drugs development... 8
2.2.1 Definition and allosteric receptors models ... 8
2.2.2 Characterization of allosteric modulator. ... 13
2.2.3 Allosteric modulator of GPCRs as drugs ... 14
2.3 Adenosine receptor (ARs) ... 16
2.3.1 Adenosine receptor subtype and function. ... 16
2.4 Adenosine A2B receptor ... 18
2.4.1 Therapeutic potential of A2B adenosine receptor ligands. ... 19
2.4.2 A2BAR agonists. ... 21
2.4.3 A2BARs antagonists. ... 22
2.5 Allosteric modulators of adenosine receptors. ... 24
2.6 Aim of the present work. ... 27
2.7 Materials and Methods ... 28
2.7.1 Investigated Compounds ... 28
2.7.2 Materials ... 28
2.7.3 Human A1 adenosine receptors binding assay ... 28
2.7.4 Human A2A adenosine receptors binding assay ... 28
2.7.5 Human A3 adenosine receptors binding assay ... 28
2.7.6 Equilibrium Binding Assays in Human A2B AR-transfected CHO Cells ... 29
2.7.7 Dissociation Kinetic Binding Assays ... 30
2.7.8 cAMP Level Measurement in Human A1, A2A, A2B, and A3 AR-transfected CHO Cells ... 30
2.7.9 Characterization of Compounds as Allosteric Modulators of the Human A2B AR in Functional Response ... 31
2.7.10 [35S]GTPγS Binding Assays in Human A2B AR-transfected CHO Cells ... 31
2.7.11 Data Analysis ... 32
2.8 Results ... 32
2.8.1 Pharmacological Profile of Compounds 7a,b and 8a as Positive Allosteric Modulators of A2BAR ... 34
2.8.2 Pharmacological Profile of Compounds 8b,c and 9a,b as Negative Allosteric Modulators of A2BAR ... 44
2.9 Discussion ... 48
Reference ... 51
CHAPTER 3: Allosteric modulation of A2BARs promoted mesenchymal stem cell differentiation to osteoblasts and ensured osteoblast survival ... 62
3.1.1 Bone cells ... 63
3.2 Bone remodelling ... 65
3.2.1 The stage of remodelling ... 65
3.3 Alteration of bone remodelling: Osteoporosis ... 66
3.3.1 Osteoporosis therapies ... 68
3.4 From Mesenchymal stem cells (MSCs) to osteoblasts ... 70
3.4.1 Mesenchymal stem cells (MSCs) ... 70
3.4.2 Osteoblastogenesis stages and their regulation ... 72
3.4.3 A2BAR role in MSC differentiation ... 74
3.5 Aim of the present work ... 76
3.6 Matherial and Methods ... 77
3.6.1 Materials ... 77
3.6.2 Cell culture ... 77
3.6.3 Measurement of cyclic AMP levels in human MSCs ... 77
3.6.4 Mineralization assay ... 78
3.6.5 Osteogenic marker expression during MSC differentiation to osteoblasts: real-time RT-PCR analysis ... 79
3.6.6 Cell viability assay (MTS) ... 80
3.6.7 Detection of IL-6 production ... 80
3.7 Results ... 80
3.7.1 Pharmacological profile of A2BAR allosteric modulator, 7b, in MSC ... 81
3.7.2 Effect of 7b, on osteogenic marker expression during osteoblast differentiation from MSCs ... 84
3.7.3 Effect of 7b on MSC-derived osteoblasts mineralization ... 86
3.7.4 Effects of A2B orthosteric and allosteric ligands on osteoblast survival ... 89
3.7.5 Effects of A2B orthosteric and allosteric ligands on IL-6 release ... 89
3.8 Discussion ... 90
References ... 94
CHAPTER 4: Wild-type and recombinant angiogenin differently bind copper(II) ions: biological implication. ... 99
4.1 Angiogenesis ... 99
4.2 Angiogenin ... 101
4.2.1 Ribonucleolytic activity of angiogenin ... 103
4.2.2 Angiogenin binding protein and/or angiogenin receptor ... 105
4.2.3 Angiogenin internalization and nuclear translocation ... 106
4.2.4 Pathological role of angiogenin ... 107
4.3 Copper ... 109
4.3.1 Copper homeostasis ... 110
4.3.2 Copper dyshomeostasis ... 112
4.3.3 Copper-protein complex ... 113
4.4 Aim of the present work ... 113
4.5.2 Protein Expression of r-Ang. ... 115
4.5.3 Protein Expression of 15N 13C r-Ang. ... 115
4.5.4 Site-direct mutagenesis. ... 116
4.5.5 Plasmid transformation in E.Coli BL21. ... 116
4.5.6 Protein Purification. ... 117
4.5.7 Removal of the Met(-1) with AAP. ... 118
4.3.8 Ribonucleolytic Activity. ... 118
4.3.9 Cell Lines and Drug Treatments ... 118
4.3.10 Angiogenin and 45S expression levels ... 119
4.3.11 ERK phosphorylation assay ... 119
4.3.12 Cell viability assay (MTS) ... 120
4.3.13 Tube formation assay ... 120
4.6 Results ... 121
4.6.1 In tube characterization of r-Ang and wt-Ang ... 121
4.6.2 In vitro characterization of r-Ang and wt-Ang ... 127
4.6.3 Copper influence on angiogenin function ... 130
4.7 Discussion ... 144
References ... 147
CHAPTER 5: Future Perspective ... 155
Acknowledgements
I would like to thank Prof. Claudia Martini, Prof. Diego La Mendola and Dr. Maria Letizia Trincavelli for giving me the opportunity to perform this study and for their steady guidance and criticism which have helped shape my attitudes towards research and made this PhD what it is.
Many thanks to Dr. Simona Daniele for all the knowledge and advices that gave me throughout the project. I would also like to thank Dr. Barbara Costa and Dr. Eleonora Da Pozzo for their advice. To all the former and current members of the Laboratory, thank you for the pleasant and friendly working atmosphere.
Thank to Dr. Orjan Hansson for give the possibility to perform protein expression in his laboratory at the Department of Chemistry & Molecular Biology of the University of Göteborg.
I would like to express my gratitude towards my family and friends for the continuous encouragement from near and far and for diverting my attention also to other things.
Thank you to Alessio, first for having time to check this thesis, but above all for being there for me every time, for your trust, for your encouragement during all these years and thank for your love.
Abbreviation
ADA = Adenosine deaminase ALP = Alkaline phosphatase Ang = Angiogenin
ARs = Adenosine receptors ATP = Adenosine-5'-triphosphate BSA = Bovine serum albumin
cAMP = 3'-5'-cyclic adenosine monophosphate CHO = Chinese hamster ovary cells
EC50 = Half maximal effective concentration ECs = Endothelial cells
EL = Extracellular loop
Emax = Maximum possible effect
ERK = Extracellular Signal-Regulated Kinase FK= Forskolin
GDP = Guanosine-5'-diphosphate GFs = Growth factors
GPCR = G-protein coupled receptor GTP = Guanosine-5'-triphosphate
GTPγS = Guanosine 5'-O-[gamma-thio]triphosphate HEK-293 = Human Embryonic Kidney 293 cells HUVECs = Human umbilical vein endothelial cells IC50 = Half maximal inhibitory concentration IL = Intracellular loop
IL-6 = Interleukin-6
MAPK = Mitogen-Activated Protein Kinase MSCs = Mesenchymal stem cells
NAM = Negative allosteric modulator PAM = Positive allosteric modulator Runk2 = Runt-related transcription factor 2 SAM = Silent allosteric modulator
SEM = Standard error of mean
SH-SY5Y = Human neuroblastoma cell line TM = Trans membrane
CHAPTER 1: Adenosine receptor ligands and Angiogenin in cell
proliferation/differentiation processes
Proliferation and differentiation processes have been widely studied during the past decade. The idea that growth is poorly compatible with differentiation has been accepted, and the mechanisms underlying proliferation/differentiation switches are still unclear. Though, diverse proteins and intracellular signals have been emerged as regulator of this switch in several cell types (Dugan 1999; Conti 2001; Teo 2010). The stem cells are the only cell type able to differentiate and proliferate during lifetime. The stem cells are unspecialized cells able to give rise to more specialized cells (differentiation) and/or to renewal themselves (self-renewal/differentiation). Several type of stem cell are present in the adult and are located in the so called “niche”. The niche represents the microenvironment that surrounds stem cells which provides a complex mix of physical and biochemical signals fundamental to define the fate of the cell (Walker 2009) (Fig 1.1).
During the lifetime the proliferation of different cell types is necessary for the growth of the body and consequently to increase tissue mass and volume. In adult tissue, the proliferation is mainly regarded to the tissue repair. Conversely, the differentiation is less significant in growing tissue nevertheless is fundamental in adult one. The ratio of proliferation/differentiation varies depending on the cell-type, however the balance of these processes is fundamental to maintain the tissue homeostasis. An excess of self-renewal may lead to tissue hyperplasia and/or cancer, conversely an excess of differentiation may lead to tissue degeneration and/ or tissue aging. The balance between these processes is tightly regulated by several transcription factor, receptor and signals molecules. The switch from proliferation to differentiation depends on both an inhibition of proliferation and the activation of the differentiation-specific genes that are characteristic of each cell types. However, to date the exact mechanisms for this switch from proliferation to differentiation and viceversa is largely unknown.
Recently the importance of adenosine receptor, and in particular of A2BAR has been pointed out. The A2BAR activation has been involved in the induction of differentiation in different cell type (e.g. mesenchymal stem cells; human lung fibroblasts) (He 2013; Zhong 2005). Moreover the A2BAR has been proved to play a role in proliferation of endothelial cells and coronary artery smooth muscle cells (Feoktistov 2002; Mayer 2011).
Angiogenin is a soluble protein physiologically present in blood plasma regarded as one of the most potent angiogenic factor. Angiogenin has been proved to be essential in
2012). Moreover it is an important regulator of cell proliferation both in physiological condition (Gao 2008; Kishimoto 2005) than in pathological one (Gao 2008;Yuan 2009).
Considering the A2BAR and Angiogenin role in diverse processes of proliferation/differentiation, the aim of the present work was to clarify the molecular mechanism underlying the processes of osteoblastogenesis and angiogenesis, as well as to discover new potential targets for several pathologies. In particular, we focused on the discovery of synthetic and/or physiological modulator of A2BAR and Angiogenin, in order to promote or inhibit their function in different physiological and pathological conditions.
Figure 1.1 Stem cells self-renewal/differentiation maintain the tissue homeostasis. The
Unbalance in favor of one of these processes lead to tissue hyperplasia/cancer or to tissue aging/degeneration (modified from Yamashita 2009).
References
Conti L, Sipione S et al. Shc signaling in differentiating neural progenitor cells. Nat Neurosci 2001; 4: 579–586.
Dugan LL, Kim JS et al. Differential effects of cAMP in neurons and astrocytes. Role of B-raf. J Biol Chem 1999; 274: 25842–25848.
Feoktistov I, Goldstein AE et al. Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation. Circ Res 2002; 90: 531-538.
Gao X and Xu Z. Mechanisms of action of angiogenin. Acta Biochim Biophys Sin 2008; 40: 619-624.
He W, Mazumder A et al. Adenosine regulates bone metabolism via A1, A2A, and A2B receptors in bone marrow cells from normal humans and patients with multiple myeloma. FASEB J 2013; 27: 3446-3454.
Kishimoto K, Liu S et al. Endogenous angiogenin in endothelial cells is a general requirement for cell proliferation and angiogenesis. Oncogene 2005; 24: 445-456.
Mayer P, Hinze AV et al. A2B receptors mediate the induction of early genes and inhibition of arterial smooth muscle cell proliferation via Epac. Cardiovasc Res 2011; 90: 148–156.
Pan SC, Wu LW et al. Angiogenin expression in burn blister fluid: implications for its role in burn wound neovascularization. Wound Repair Regen 2012; 20: 731-739.
Subramanian V and Feng Y. A new role for angiogenin in neurite growth and pathfinding: implications for amyotrophic lateral sclerosis. Hum Mol Genet 2007; 16: 1445-1453.
Teo JL and Kahn M. The Wnt signaling pathway in cellular proliferation and differentiation: A tale of two coactivators. Adv Drug Deliver Rev 2010; 62: 1149–1155.
Walker MR, Patel KK et al. The stem cell niche. J Pathol 2009; 217: 169–180.
Yamashita YM. Regulation of asymmetric stem cell division: spindle orientation and the centrosome. Front Biosci (Landmark Ed) 2009; 14: 3003-3011.
Yuan Y, Wang F et al. Angiogenin is involved in lung adenocarcinoma cell proliferation and angiogenesis. Lung Cancer 2009; 66: 28-36.
Zhong H, Belardinelli L et al. Synergy between A2B Adenosine Receptors and Hypoxia in Activating Human Lung Fibroblasts. Am J Respir Cell Mol Biol 2005; 32: 2–8.
CHAPTER 2: Allosteric modulation of G protein-coupled
receptors.
2.1 GPCRs
G protein-coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and, therefore, a really important target in drug discovery. It has been estimated that more than half of all modern drugs act via GPCRs (Fredriksson 2003). GPCRs share a common seven hydrophobic trans membrane (TM) alpha-helices, with an extracellular amino terminus and an intracellular carboxyl terminus.
Based on the 7 TM sequences similarity, GPCR receptors can be clustered into 5 main families:
• Rhodopsin family (701 members), • Adhesion family (24 members), • Frizzled/Taste family (24 members), • Glutamate family (15 members), • Secretin family (15 members).
The Rhodopsin family has several common characteristics such as NSxxNPxxY motif in TMVII, the DRY motif or D(E)-R-Y(F) at the border between TMIII and IL2, and is further divided in four main groups α, β, γ, and δ. The α-Group of Rhodopsin Receptors (89 members) consists of five main branches: the prostaglandin receptor cluster, amine receptor cluster, opsin receptors cluster, melatonin receptor cluster, and MECA receptor cluster. The MECA receptor cluster consists of the melanocortin receptors (MCRs), endothelial differentiation G-protein coupled receptors (EDGRs), cannabinoid receptors (CNRs), and finally the adenosin receptors (ADORAs) (Fredriksson 2003).
Figure 2.1 Dendrogram of the human G protein–coupled receptor (GPCR) superfamily
with different crystal structures. According to this notation, human GPCRs include the Rhodopsin family, the Secretin and Adhesion families, the Glutamate family, and the Frizzled/TAS2 family. The Rhodopsin family is divided into subgroups: the α-group, the β-group, the γ-group, andthe δ-group, as labeled (Katritch 2013).
GPCRs are characterised by seven transmembrane (TM) a-helices (TM-1 through TM-7) connected by three intracellular loops (IL1, IL2 and IL3) and three extracellular loops (EL1, EL2 and EL3) (Baldwin 1993). GPCRs share the greatest homology within the TM segments. The most variable structures among the family of GPCRs are the carboxyl terminus and the intracellular loop spanning TM5-TM6. The greatest diversity is observed in the amino terminus (Kobilka 2007). A common feature of most GPCRs is a highly conserved disulfide bond between cysteine residue in TM3/EL1 interface and in EL2 (Ballesteros 1995; de Graaf 2008). In recent years arise a great interest in the structure of EL2 that seems to be involved not only in ligand recognition and receptor subtype
selectivity, but beyond that, it plays an important role in receptor activation and ligand efficacy (Avlani 2007; Seibt 2013).
The activation of a GPCR receptor by its endogenous ligand, promotes coupling of the activated receptor to the heterotrimeric G protein complex that consists of an α-,β-, and γ-subunit. This complex undergoes an activation-inactivation cycle consequent to the neurotransmitter-GPCR receptor binding (Fig. 2.2). In the basal state, the βγ-complex and the GDP-α-subunit are associated, and the heterotrimer can bind the activated receptor in an appropriate site. This binding promotes the exchange of GDP for GTP on the G protein α-subunit. The GTP-α-subunit dissociates from the activated receptor as well as from the βγ-complex, and both the α-subunit and the βγ-complex are now free to modulate the activity of several effectors. Signaling is terminated by the hydrolysis of GTP by the GTPase activity, which is inherent to the G protein α-subunit. The resulting GDP-α-subunit reassociates with the βγ- complex to enter in a new cycle (Neer 1995; Cabrera-Vera 2003; Wettschureck 2005).
Figure 2.2 GPCR activation cycle. In the receptor inactive state (top), the α subunit and the
βγ complex of the G protein are associated. Upon agonist binding (right), the receptor undergoes a confirmational change that activates its GEF activity, and it catalyzes the exchange of GDP for GTP on the Gα subunit. GTP-bound Gα and the βγ complex dissociate and activate downstream signaling (bottom). Hydrolysis of GTP to GDP, which may be stimulated by RGS proteins, leads to reassociation of Gα and βγ subunits and termination of G protein signaling (left) (from http://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=305).
GPCRs mediate cellular responses to different extracellular signals ranging from protons, small molecules, such as amine neurotransmitters, hydroxy acids, amino acids,
2008). Moreover, the GPCR regulation and signaling is much more complex than originally envisioned, and includes signaling through G protein independent pathways (e.g. beta-arrestin) (Lefkowitz 2005; Kobilka 2007). Considering that it has been proposed to adopt the term 7 transmembrane, or 7TM, receptors in favor of GPCR (Kobilka 2007).
The great importance of this family of receptors and their physiological role in eukaryotic cells was recently acknowledged by scientific community with the award of the 2012 Nobel Prize in chemistry to Robert J. Lefkowitz and Brian K. Kobilka "for studies of G protein-coupled receptors". This award shows how the study of these receptors has contributed and is still helping the development of new drugs interacting with this family of proteins.
GPCRs are involved in several physiological processes; changes of their expression and function have been related to the pathogenesis of neurological disorders, inflammatory diseases, cancer and metabolic imbalances, highlighting this class of receptors as interesting targets for the discovery and development of new potent and selective drug. Noteworthy, it is estimated that almost 40% of current FDA approved drugs act via GPCRs, of which 26% target class A (rhodopsin-like class) (Kenakin 2005).
For decades, the development of ligands in traditional GPCR drug discovery has focused on targeting the orthosteric binding site of the receptor, which is defined as the site where the endogenous ligand binds to elicit signal transduction. However, it is now well known that the rate of traditional GPCR drug discovery is rapidly in decline (Booth 2004). This is related to the relative intractability of many GPCRs (e.g., peptide receptors) to orthosteric small molecule, as well as the difficulty in selectively targeting orthosteric sites at receptors that display high homology.
Accordingly, in the last decade the interest in the discovery and development of small molecules that target topographically distinct allosteric sites on GPCRs has been aroused (Christopoulos 2002; Christopoulos 2002b; May 2007). Ligands that bind to these allosteric sites are called “allosteric modulators” and offer enormous potential with regards to greater selectivity of action and responsiveness in a spatially and temporally advantageous manner (Keov 2011).
2.2 Allosteric modulation: opportunities in drugs development
2.2.1 Definition and allosteric receptors modelsThe term “allosteric” (from the Greek meaning “other site”) was first used by Monod and Jacob (1961) and subsequently defined by Monod et al. (1963) to describe the ability of ligand to modify the activity of enzymes in either a positive or negative fashion, by the binding to topographically distinct site from the substrate one. Monod et al. (1963) defined these accessory binding sites as “allosteric sites”, in contrast to the substrate-binding (active) site, which was defined as the “orthosteric site”. The allosteric interactions arise when the binding of a ligand to the allosteric site induces a conformational change in the protein and modulates the binding of the substrate to the orthosteric site, and vice versa (Changeux 2012).
Therefore, an allosteric interaction is characterized by: (a) the presence of not overlapping binding sites; (b) the affection of the binding of one ligand to its site to the binding of the second ligand at the other site and vice versa (c) the possibility of an allosteric modulator to control either negative or positive the effects with respect to the binding and/or function of an orthosteric ligand.
The best known example of an allosteric modulator of GPCRs is the G protein itself. GPCRs present at least two binding sites on the same receptor protein, one for the orthosteric ligand and one for the G protein. This represents the simplest model for a positive allosteric interaction (Ehlert 1985; Kenakin 2012). Indeed agonist binding to the orthosteric site results in an alteration of receptor conformation that displays a higher affinity toward the G protein, thus favouring coupling process (Christopoulos 2002b). Conversely the simplest model for a negative allosteric interaction is the binding of GTP to its site on the G protein. This causes a change of G protein structure that is transmitted to the receptor’s conformation as a negatively cooperative effect on agonist binding, thus promoting the uncoupling of the activated G protein from the receptor.
Over the years, a number of more or less complex mathematical models have been developed to describe the behaviour of the affinity and efficacy parameters obtained for a ligand-receptor interaction (Fig. 2.3). In the classical description, (Fig. 2.3a), the affinity of a ligand mathematically is defined as the ratio between the rate of association and dissociation (kon /koff). The first improvement of this model was the original ternary complex model, as described by De Lean et al. (1980) (Fig. 2.3b). The binding of a ligand allows the receptor to form a G protein complex resulting in activation. This represents the simple example of a receptor isomerization mechanism, where the binding of ligand A
promotes a conformation of the receptor that either couples to a G protein or promote an effect in its own (e.g., ion-channels). During his researches, De Lean et al. (1980) also considered the possibility of a closed (cyclic) system operating in equilibrium according to the hypothesis of pre-coupled RG complexes in the absence of bound ligand. However, only in 1989 Costa and Herz demonstrated that GPCR could couple to G protein also in the absence of ligand. This highlighted the possibility of constitutive GPCR activity as well as the necessity to improve the original model described by De Lean et al. (1980) resulting in the development of the two state model (Fig 2.3 c, left) and the cyclic ternary complex model (TCM) (Fig 2.3 c, middle).
Figure 2.3 Pharmacological models of ligand binding. (a) The affinity of a ligand (A) for a
receptor (R) is determined both by its rate of association (kon) and dissociation (koff). (b) Original
ternary complex model (c) Left. The two-state model of receptor activation. Middle. The ternary complex model (TCM). Right. The allosteric ternary complex model (ATCM). (d) The extended ternary complex model (ETC). (e) Left. The cubic ternary complex model (CTC). Right. The allosteric two-state model (ATSM) (Modified from Christopoulos 2002).
Within the two state model of receptor activation (Fig. 2.3c, left), receptors can switch between an inactive (R) and active (R*) state, an equilibrium which is defined by the isomerization constant (L). The efficacy of a ligand is determined by its preference for binding the active over the inactive form of the receptor (α). In parallel, the TCM can
explain the reciprocal effects that GPCR (orthosteric) ligands and G proteins had on each other (De Lean 1980; Ehlert 1985) (Fig. 2.3 c, middle). This represents the simplest probabilistic models that describe the actions of agonists, antagonists, and inverse agonists (Kenakin 1996; Leff 1995; Lefkowitz 1993).
Models combining the two-state model and the TCM have also been developed to describe the effect of an orthosteric ligand and G protein on a receptor that isomerizes between an inactive and an active state: the extended ternary complex model (ETM Fig 2.3 d) (Samama 1993). Although the ETC model improved the original ternary complex, it is thermodynamically incomplete, and Weiss et al. (1996) describe the more complete cubic ternary complex model (CTC, Fig. 2.3 e, left). Parameters within the CTC include the affinity, KA and KG, of the ligand (A) and G protein (G) for the inactive receptor, respectively; the isomerization constant (L), and the thermodynamic coupling factors α, β, γ, and δ. β describe the preference of the G protein for the active over the inactive form of the receptor. The interaction between ligand binding and G protein coupling is described by γ, whereas δ describes the ability of the ternary complex to drive receptor isomerization. The value of α represent the preference of the ligand A for the active over the inactive form of the receptor; if it is greater than one will drive the equilibrium toward the active one; in contrast values lower than one will stabilize the inactive state.
Linkage models such as the ETC and the CTC are useful in that they can incorporate the constitutive activity of GPCRs; however, they are based to the definition of the interacting species, and it represents the major limitation.
The TCM can also be used as a general model to describe any allosteric interaction of two ligands between distinct binding sites on a receptor. This is commonly referred as the “allosteric ternary complex model” (ATCM) that describes the simplest allosteric effect, namely a reciprocal modulation of ligand affinity (Fig. 2.3e, right) (May 2007; Ehlert 1988; Stockton 1983). In this model the interaction is driven by the concentration of each ligand, by the equilibrium dissociation constants KA and KB respectively of the orthosteric ligand (A) and the allosteric ligand (B), and α, the binding cooperativity. This represents the ratio of the dissociation constants of the free receptor to that of the occupied receptor. Values of α greater than 1 denote an allosteric enhancement of affinity for the receptor (positive cooperativity), values of α minor than 1 denote an allosteric reduction of affinity (negative cooperativity); while a value of 1 denotes not effect on binding affinity at equilibrium (neutral cooperativity).
isomerization of the GPCRs and cannot explain the effect of either ligand on the receptor response. Considering the wide spectrum of GPCR conformations, during the last decade, in literature have been describe several ligands that affect orthosteric efficacy in addition to, or independently of, effects on ligand affinity(Litschig 1999; Urwyler 2001; Zahn 2002; Watson 2005; Price 2005) (Fig. 2.4 a). For example, the negative allosteric modulator, CPCCOEt (7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester), does not affect the binding of [3H]glutamate, but it concentration-dependently suppresses maximal response to glutamate in an inositol phosphate accumulation assay (Litschig 1999). Otherwise the allosteric modulator, CGP7930, enhances both the potency and the maximal effect of GABAB receptor agonist (Urwyler 2001), without affecting the binding of the orthosteric antagonist [3H]CGP62349. Even more strikingly, Org27569 was shown to have a complete divergence effects on cannabinoid receptor CB1 agonist binding versus agonist function (Price 2005). This ligand showed a negative allosteric behaviour on CP55940 (CB1 agonist) function, but allosterically enhances [3H]CP 55940 binding.
Figure 2.4 A) Allosteric and orthosteric ligands of G protein-coupled receptors: range of
possible activities. B) The allosteric two-state model (ATSM)which describes allosteric modulator effects on affinity and efficacy (Modified from May 2007).
At the molecular level, divergent effects of allosteric modulators on affinity and efficacy are consistent with the hypothesis that the binding of these compounds biases the conformational equilibria by GPCRs to favour one set of states over another. The extension of the ATCM to accommodate such behaviour was introduced by Hall (2000) as the “allosteric two-state model” (ATSM) (Fig. 2.3e right). This model explicitly incorporates the isomerization of a receptor between active (R*) and inactive (R) states, and introduces additional coupling constants to describe the selective stabilization of these states by
combination of all four R* species (R*, AR*, BR*, and AR*B), as would be the case for ion channel-linked receptors, and that amount of the response is determined by the ratio of all these species (Fig. 2.4b).This represent a limitation of the model that does take into account the interaction with the G-protein, in contrast to the CTC model that quantifies response as the production of activated receptor-G protein species (i.e., R*G, AR*G)
The complex scenario of the allosteric behaviours cannot easily be explained by relatively simple mass-action schemes such as the ATCM and ATSM. Indeed these models can be extended in efforts to accommodate experimental data. To be thermodynamically complete, any model of allosteric interactions should consider the ability of the receptor to isomerize between multiple conformational states and to bind to G-protein. On this basis Christopoulos (1998) describe the “quaternary complex model” (QCM) (Fig. 2.5) that reflects the fact that allosteric modulators of GPCRs present a different behaviours that can extend beyond simple changes on orthosteric ligand binding affinities.
Figure 2.5 The quaternary complex model (QCM) of allosteric interactions at GPCRs; that
take into account the concomitant binding of a orthosteric ligand, A, an allosteric ligand, B, and G-protein, G, on a receptor that can exist in two conformational states (R and R*) (Christopoulos, 2002).
The complexity of all the above model give the idea of the difficulty to discover the allosteric behaviour of a new ligand but their heuristic nature can prove useful for informing the design of additional experiments to deep understand the molecular mechanism and interaction of allosteric modulator.
2.2.2 Characterization of allosteric modulator.
Orthosteric ligands may exhibit a broad range of activities from full agonism to full inverse agonism, a similar range of effects can be observed for allosteric modulators (Fig. 2.4a) (Urwyler 2011). Generally, the allosteric modulator are classified into “negative allosteric modulators” (NAMs) and “positive allosteric modulator” (PAMs). NAMs are non-competitive antagonists, indeed they show insurmountable antagonism. The effect of the modulator cannot be overcome by increasing concentrations of agonist, in contrast to orthosteric competitive antagonists. Instead, the positive allosteric modulators enhance the activity or efficacy of an orthosteric agonist (Bridges 2008).
Ranging from NAMs and PAMs, some compounds may exhibit a mixed mode of action and bind to both sites acting as orthosteric ligands and allosteric modulators. This has, for example, been observed with PAM of A1 adenosine receptors (Baraldi 2007). Allosteric modulators can also enrich active-state GPCR conformations in their own right, expressing the property of agonism in the absence of bound orthosteric ligand. Several examples of allosteric agonist have been reported in recent literature (Neubig 2003, Sachpatzidis 2003; Mitsukawa 2005; Langmead 2006). Furthermore, some allosteric modulators of GPCRs can act as both, allosteric modulators in the presence of an orthosteric ligand and as allosteric agonists in its absence. These are called “ago-allosteric modulators” (Schwartz 2006).
Recently, hybrid ligands also referred to as bitopic, bivalent or dualsteric ligands, have been described. These present a scaffold that binds the orthosteric site connected by a linker moiety to a second scaffold, which binds to the allosteric site (Valant 2008; De Amici 2010; Smith 2011; Smith 2011b). The bitopic ligands present the advantage of a higher selectivity due to the binding of a specific allosteric site; the major disadvantage of these compounds is the large structure that in many cases is correlated to lower drug-like physicochemical properties. Finally, another recent class of allosteric modulator is represented by the silent allosteric modulators (SAMs) that bind to an allosteric site but without showing any modulatory effect (Burford 2011).
Allosteric interaction is characterized by different properties and presents several aspects. One of the most important characteristic is the so call “probe dependence”, indeed the extent and direction of an allosteric interaction can vary with the nature and the structure of the orthosteric ligand used as a probe of receptor function (Kenakin 2005; Valant 2012).
GPCRs couple to several different signaling pathways (e.g. Gs, Gq or Gi; and additionally to β-arrestin translocation). It is well known that orthosteric agonists can exhibit preference for one of several signaling pathways; this is called “functional selectivity” or “biased signaling” (Kenakin 2011). Allosteric modulators may exhibit the same functional selectivity, preferring a particular receptor conformation that activate (or inhibit) selectively a specific signaling pathway (Kenakin 2012; Muller 2012).
2.2.3 Allosteric modulator of GPCRs as drugs
The interest in the research and development of allosteric modulators has been emerged during the last decade (Wang 2009; Burford 2011). This is due to their advantage in comparison to classical (orthosteric) drugs. The actions of drugs that act via the orthosteric site depend in major part on the affinity of the compound for the receptor of interest. Furthermore, to observe a therapeutic effect it must be maintained at a sufficiently high concentration in the receptor compartment. Under these conditions, orthosteric agonists will induce an activated state and the response will generally be unresponsive to fluctuations in the levels of the endogenous ligand. As a consequence, orthosteric ligands may present toxic effects, desensitization, and long-term changes in receptor up/down regulation. Conversely, PAMs show a reduction of all this effects, since allosteric modulators are only active in the presence of the endogenous agonist. First, PAMs action will be site- and event-specific depending on the concentration of the physiological agonist (Muller 2012). Indeed, allosteric modulation can be described as a “fine-tuning” of a response rather than a general induction of a response. Their effects are naturally limited by the temporal release of endogenous agonist, and overdosing can thus be avoided. Second, positive allosteric modulators have a much lower tendency to induce receptor desensitization than orthosteric agonists because of their use-dependent mechanism of action (Urwyler 2011; Muller 2012). Third allosteric modulators present increased target and subtype selectivity, since allosteric sites are less conserved than orthosteric ones.
Despite their advantages, different disadvantages rose during the discovery and development of allosteric GPCR modulators (Raddatz 2007). First of all, the reduced
conservation of allosteric sites, which allows the selectivity for receptor subtype, may also result in significant species difference; furthermore more frequent polymorphisms may be found in the population. This represent a great problem in the development of new drugs considering that rodent models are used for the investigation of drug effects in vivo after the characterization of allosteric effects at recombinant human receptors in vitro. Second, since the effects of allosteric modulators depend on the concentration of endogenous agonist, their effects may be lost with disease progression especially in neurodegenerative disease. Third, from a medicinal chemistry point of view, allosteric GPCR modulators often have “flat,” non-tractable structure-activity relationships (SAR). In addition, only slight structural modifications sometimes result in important changes in pharmacological responses, from positive to negative allosteric modulation, making difficult the rational design of lead compound (Urwyler 2011; Muller 2012).
Allosteric modulators as drugs are well known for ligand-gated ion channels, for example the benzodiazepines diazepam (Valium®), which allosterically enhances the effect of GABA at the GABAA receptor. A relatively new perspective in drug development, however, is the targeting of allosteric binding sites at GPCRs (Christopoulos 2002; De Amici 2010). Hundreds of compounds acting as allosteric modulator towards GPCRs belonging to all the different classes have been reported in literature. However, only recently, allosteric modulators for GPCRs have been approved as drugs (Fig. 2.6): cinacalcet (Mimpara®), a positive allosteric modulator of the calcium-sensing receptor for the treatment of hyperparathyroidism; maraviroc (UK 427,857; Celsentry®), a negative allosteric modulator of the chemokine receptor CXCR5 as a virus entry inhibitor in HIV therapy; and plerixafor (AMD3100), a negative modulator of the chemokine receptor CXCR4 for stem cell mobilization for transplantation (Scholten 2012).
Figure 2.6 The structure of allosteric modulator already approved as drug.
best investigated (van Koppen 2003; Wess 2005). Furthermore, other well investigated family of receptor include the chemokine receptor (Scholten 2012) and the adenosine receptor (Fredholm 2011; Göblyös 2011). Although recently have been emerged projects on other GPCRs such as P2Y receptors, cannabinoid receptor and protease-activated receptor (PAR) (Muller 2012).
2.3 Adenosine receptor (ARs)
The term “Purinergic receptors” is first defined by Burnstock (1976). Later on, they are divided into two families, P1 and P2 (for adenosine and nucleotides, respectively) (Burnstock 1978). The P1 receptors are divided into four receptor subtypes (A1, A2A, A2B and A3) and the P2 receptors are subdivided in two large families (P2X and P2Y receptors). Beside P1 and P2, P0 receptors are recently suggested as a third family of purinergic receptors which are activated by the nucleobase adenine.
Adenosine is a constituent of a wide range of molecules such as oligo- and polymeric nucleotides (RNAs) as well as a constituent of small nucleotides such as ATP, 3'-5'-cyclic adenosine monophosphate (cAMP), nicotinamide adenine dinucleotide (NAD+), flavinadenine-dinucleotide (FAD), S-adenosyl-L-methionine, which play a crucial role in a broad spectrum of physiological processes. Moreover, adenosine itself is an important signaling molecule that is able to directly trigger all the four specific cell membrane G-protein-coupled receptors: adenosine receptors (ARs).
2.3.1 Adenosine receptor subtype and function.
The endogenous purine nucleoside adenosine is ubiquitous in mammalian cell types and modulates a great variety of physiological and pathological functions via the activation of different subtypes of ARs (Klotz 1998). In view of its implication in several biochemical processes there is an increased interest in the understanding of its pharmacology and physiology.
There is a large sequence identity between the human A1 and A3AR (≈ 49%) and the human A2A and A2BARs (≈ 59%); moreover there is a great similarity among the species for the A1, A2A and A2BAR, whereas A3 is more variable in fact for which there is almost a 30% difference at the amino acid level between human and rat (Muller 2011).
ARs signaling has been generally ascribe through inhibition or stimulation of adenylyl cyclase (also known as adenylate cyclase), nevertheless, recent improvements on adenosine receptors pharmacology demonstrate the importance of the activation of other
pathways, such as phospholipase C (PLC), Ca2+ mobilization, mitogen-activated protein kinases (MAPKs) and signal-regulated protein kinase (ERK 1/2) phosphorylation. (Jacobson 2006; Fredholm 2001)
Adenosine is the endogenous ligand of all the AR subtype, under physiological conditions, its intracellular levels arrives up to 100 nM and is able to interact only with the so call “high-affinity” subtype the A1 and A2AAR. In hypoxic, ischaemic or inflammatory conditions, the adenosine intracellular levels can grow to high micromolar concentrations and can activate the so call “low-affinity” A2B and A3AR subtypes (Beukers 2000; Fredholm 2001b). Despite this classification of receptor has been widely used in literature it is important to note that the activity of adenosine among the subtype is strictly related to both receptor number, and the type of response measured (Fredholm 2001b).
Activation of the A1AR and A3AR subtypes inhibits adenylate cyclase activity through activation of pertussis toxin-sensitive G proteins (Gi) and thus leading to a decrease of intracellular cAMP. (van Calker 1979; Londos 1980; Zhou 1992) Both receptors increase the activity of PLC (Tawfik 2005; Rogel 2005; Abbracchio 1995) and calcium mobilization (Fredholm 2001; Fossetta 2003; Shneyvays 2004). Conversely, activation of the A2AAR and A2BAR subtypes increases adenylyl cyclase activity through the coupling to a Gs protein, thus leading to a raise in the intracellular cAMP levels (Fredholm 2001; Peakman 1994). Either activates the PLC pathways through the coupling of Gq/11 proteins, and it seem to mediates many of the important functions of A2BARs (Linden 1999). Each AR subtype has been reported to mediate distinct effect in different tissues through the activation of specific signaling pathways. In cardiac muscle and neurons, for example, A1ARs and A3ARs can activate pertussis toxin-sensitive K+ channels, as well as KATP channels (Tracey 1998; Fredholm 2001). In the heart, A1AR and A2AAR agonist induced preconditioning has been suggested to occur through ERK activation (Reid 2005). In the striatum, the A2AAR subtype mediates its effects predominantly through the coupling to Golf (Kull 2005), which is similar to Gs and also couples to adenylate cyclase. Activation of the A2AAR also induces formation of inositol phosphates to raise intracellular calcium and activate protein kinase C via Gα15 and Gα16 proteins(Offermanns 1995).
ARs are present in different levels among the district of the body; their expression change and activation are at the base of several pathological processes (Fig 2.7). Considering that, in recent years there are a great interest in the research and development of potent and selective adenosine agonist and antagonist. Adenosine itself for a long time
treatment of paroxysmal supraventricular tachycardia due to its activation of A1 receptors, and as a diagnostic for myocardial perfusion imaging (Adenoscan®, Astellas Pharma, Inc.). In addition, adenosine is under inverstigation in several clinical trials for the treatment of inflammation, neuropathic and perioperative pain, and cardioprotection). Despite this, a large number of compounds, acting as agonist or antagonist, are currently under preclinical trials for the treatment of central nervous system disorders (e.g. Parkinson’s disease), inflammatory disease, pain, asthma, kidney failure, ischemic injury and many other disorders related to AR activity (Muller 2011).
Figure 2.7 Some of the potential therapeutic applications for agonist and antagonist that
target A1AR (Black), A2AAR (Red), A2BAR (Green) and A3AR (Blue) (modified from Fredholm
2010).
2.4 Adenosine A
2Breceptor
A2BAR regulates a number of physiological and pathological events that involve several organs and tissues (Fredholm 2011; Feoktistov 1997). It is expresses at high concentrations in caecum, large intestine and urinary bladder, whereas a lower expression has been revealed in lung, blood vessels, eye, and mast cells. Adipose tissue, adrenal gland, brain, kidney, liver, ovary and pituitary gland are thought to have a very low concentration of A2BAR (Baraldi 2009; Taliani 2013).
The A2BAR is high conserved among the species for example the human receptor shares 86–87% amino acid sequence homology with the rat and mouse subtypes (Feoktistov 1997). The A2BAR presents also a high similarity with the A2A receptor,
considerably higher affinity for the human A2A subtype than for the A2BAR. The reason for this and the amino acids involved are yet unknown, mainly due to the lack of crystal structure. Considering that, molecular models combined with mutagenesis data, can provide valuable information about the receptor’s putative binding pocket(s). Homology modelling studies of the A2BAR have been recently reported, based on crystal structures of its closest relative A2A co-crystallized with non-xanthine or xanthine antagonists (Jaakola 2008; Dore 2011; Liu 2012), or with agonists UK432097, NECA and adenosine(Xu 2011; Lebon 2011). However, despite their similarity, the A2A and A2B receptors present several difference. The A2A receptor possesses four disulfide bonds in the extracellular part of the protein whereas the A2B receptor has been found to have only one disulfide bond (Thimm 2013). Furthermore, the longer extracellular loop 2 (EL2) exhibit only 34% of identity, and 46% of similarity respect to the A2AAR subtype (Schiedel 2011). This is fundamental considering that the EL2 participates in ligand recognition and binding as evidenced by crystal structures and that is not well conserved regarding length, amino acid composition, and number of disulfide bonds in many GPCRs (Venkatakrishnan 2013).
Despite these limitations, several potent and selective A2BAR ligands have been identified. Potent and selective agonists have been developed that are currently in preclinical studies for the treatment of atherosclerosis and coronary artery disorders (Rosentreter 2001; Ortore 2010). Furthermore, considering the involvement of A2BAR activation in several physiological and pathological processes, potent and selective A2BAR antagonists are currently being developed as candidates for the treatment of diabetic retinopathy, cancer (Ryzhov 2008; Kalhan 2012), colitis (Kolachala 2005; Kolachala 2008), and asthma (Zablocki 2006; Kalla 2006).
2.4.1 Therapeutic potential of A2B adenosine receptor ligands.
This receptor is the least well characterized among the ARs primarily due to the lack of suitable, high specific ligands (Muller 2011; Kalla 2009). This combine with the fact that the A2BAR is often co-expressed and related with the similar A2AARs in several cell/tissue types led to the idea that A2BAR does not play an equivalent important biological role (Feoktistov 1998). Recently, the interest on A2BAR role increases based on the discovery of its involvement in pathological conditions such as hypoxia and inflammation, in which the adenosine levels increase to micromolar range and the A2BAR is overexpressed (Eltzschig 2003; Kong 2006; Eckle 2007; Hart 2008; Reutershan 2009; Hasko 2009; Aherne 2011).
The physiological role of A2BAR is far from being completely understood, it has been implicated in several processes including glucose homeostasis (Allaman 2003), modulation of arterial blood pressure and heart rate (Lee 2009), angiogenesis induction (Feoktistov 2003; Feoktistov 2004), the growth and development of some tumors (Panjehpour 2005), inflammation (Linden 2006; Zhong 2006) and inflammation- related or thermal pain (Bilkei-Gorzo 2008). The A2B receptor is considered a perfect pharmaco-therapeutical target of promising new compounds.
A2BAR signaling has been identified as potential pathway in the pathogenesis of lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) (Zaynagetdinov 2010). Treatment of asthma with selective A2BAR antagonists has so far been one of the most significant therapeutic approach among AR ligands. Furthermore, A2BAR has been implicated in the development of some cancers. A2B receptors are overexpressed in several solid tumor cells, probably related to the chronically hypoxic conditions in their core that promote A2B gene expression (Hasko 2009). Moreover, the A2B receptor promote the vascularization of the tumors because it enhances the release of vascular endothelial growth factor (VEGF) (Thimm 2013). Thus its inhibition represent a promising approach in the treatment of cancer, indeed Cekic et al. (2009) demostated that blockade of A2B reduced the growth of bladder and breast tumors. A2BAR antagonists are useful therapeutic agents in type 2 diabetes, as these seem to antagonize the adenosine induced hepatic glucose production determining reduction of blood glucose levels (Harada 2001). Inhibition of A2B receptor activation could furthermore be useful in the treatment of other pathological conditions like pain, diarrhoea and cystic fibrosis (Baraldi 2009).
On the other hand, agonists of the A2BAR might be employed for various indications. Ischemic pre-conditioning (IP) is an experimental approach in which exposure to repeated periods of vascular occlusion prior to an extended ischemic event provides high protection from ischemic injury. The administration of A2B agonists resulted in the same protection (Eckle 2007). Therefore A2B receptor activation mediates cardioprotective action, resulting in a reduction in infarction size and dampening of post-ischemic injuries of heart tissue (Aherne 2011). Additionally, an A2B receptor selective agonist could be used in the treatment of atherosclerosis, erectile dysfunctions and septic shock (Baraldi 2009)
2.4.2 A2BAR agonists.
Actually, A2BAR agonists described in literature can be classified into adenosine-like and nonadenosine-adenosine-like ligands. Nucleoside-based agonists are the result of modifying the endogenous ligand, adenosine (Fig 2.8), by substitution at the N6- and C2-positions of the purine heterocycle and/or at the 5´-position of the ribose moiety. Nonadenosine derivatives so far reported are represented by conveniently substituted pyridine-3,5-dicarbonitrile derivatives.
In the screening of adenosine analogues variously modified at the 2-, 5-, 8-, N6 and 5´-positions (or combinations of these) de Zwart et al. (1998) reported for the first time the structure of NECA (5´-N-carboxamidoadenosine, Fig. 2.8). NECA is not selective among the adenosine subtype even if presents high nanomolar activity towards A2BAR. Subsequently to improve the affinity and the selectivity, a series of modification of NECA structure have been proposed, resulting in the discover of compound 2 by Baraldi et al. (2007b).
An improvement in affinity and selectivity for the A2BAR was achieved with the discovery of a new series of 2-thio-4-aryl-3,5-dicyano-6-aminopyrimidine derivatives(Rosentreter 2001). Recently, a new adenosine A2B receptor agonist 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl] acetamide BAY 60-6583 (Rosentreter 2003) (Fig 2.8), has been patented by Bayer HealthCare and used to study the cardioprotective function of A2B receptors (Albrecht 2006). BAY 60-6583 represent the first potent and selective adenosine A2B receptor agonist with an EC50 value ranging in 3–10 nM for the hA2BAR characterized in cAMP functional assay (Eckle 2007).
Figure 2.8 The chemical structures of A2BAR agonists adenosine, NECA, 2 and BAY
2.4.3 A2BARs antagonists.
The A2BAR antagonist based on structure can be divided into two classes of compounds, xanthines and non-xanthine derivatives. Caffeine and theophylline (Fig 2.9) represent the lead compounds of xanthine derivatives, and are considered classic nonselective antagonists for adenosine receptors. Following further structural exploration of the xanthine moiety by several groups, a series of alkylxanthines was developed. In particular, the discovery of 8-phenylxanthines as selective A2BAR antagonists was made. Among these 8-phenylxanthine derivatives, p-cyanoanilide MRS-1754 (Fig 2.9) and its analogue p-acetylphenyl MRS-1706 of Kim et al. (2000; Kim 2002) presented an high selectivity towards A2BAR. In the meantime Hayallah et al. (2002) presented a negatively charged compound PSB-1115 (Fig 2.9), as selective A2B antagonists.
The CV Therapeutics (CVT) chemists started with 8-phenylxanthines structure to discovery a selective, high affinity A2BAR antagonist which presented also a good pharmaceutical properties. Kalla et al. (2006) screened several heterocycles as bioisosteric group for the phenyl at the 8-position of xanthine and discovered that the 8-(pyrazol-4-yl)xanthines display good A2BAR affinity, although presented a low selectivity. Further investigation on this lead structure to improve the selectivity was made and Kalla et al. (2008) reported the 1-propyl-8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)-xanthine CVT-7124 (Fig. 2.9) which displays high A2BAR affinity (6 nM) and very good selectivity. This further supports the Hayallah et al. observation in the 8-phenyl xanthine series of compounds, that the mono-substitution at the N-1 position of the xanthine core enhances the selectivity. Based on this results Borrmann et al. (2009) discovered the 8-[4-[4-(4-Chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine PSB-603 (Fig 2.9) which has excellent affinity and selectivity and a radiolabeled analogue was prepared.
In parallel a series of 9-deazaxanthines (pyrrolo[2,3-d]pyrimidinones) were initially explored by Grahner et al. (1994) as antagonists for the A1 and A2ARs. The structure-activity relationships (SAR) of 9-deazaxanthines are parallel to those of xanthine derivatives and on this basis a vast number of this compounds were synthetized that presented similar affinity and selectivity of the correspondent alkylxanthines (Kalla 2009b).
Figure 2.9 The chemical structures of some xanthines A2BAR antagonists. The affinity
values for each subtypes are reported from Kalla et al. (2009b) and Fredholm et al. (2011).
Regarding the non-xanthine derivatives, a series of 2-aminopyrazines (Vidal 2007), pyrrolopyrimidines (Castelhano 2003) and triazinobenzimidazolones (Taliani 2012) have been reported. The 2-aminopyrazines was first reported by Almirall Prodesfarma, and Vidal et al. (2007) improved the common core arriving at the N-heteroaryl 4´-furyl-4,5´-bipyrimidin-2´-amines, as high affinity and selective A2BAR antagonists. The best compound of this series was the 2´-amino(3-pyridyl) derivative LAS38096 (Fig 2.10) with an affinity of 17 nM and excellent selectivity.
In parallel, OSI Pharmaceuticals have reported a series of 2-phenyl-7-deazaadenines (pyrrolopyrimidines) that display good A2BAR affinity (Stewart 2004). Among this, the lead compound OSIP-339391 demonstrated excellent A2BAR affinity and promising selectivity (Fig 2.10). Furthermore a tritium-labeled analogue ([3 H]OSIP-339391) was synthesized, which displayed a KD value of 0.41±0.06 nM for binding to human A2BAR expressed in HEK-293 cells.
Recently Taliani et al. (2012) reported a new series of triazinobenzimidazolones (Fig. 2.10). Several investigation on different substituents yielded to compound 6 which represent the most potent and selective compound, with an IC50 of 3.10 nM at A2BARs and no affinity at A1, A2A, and A3ARs.
Figure 2.10 The chemical structures of some non-xanthines A2BAR antagonists. The
affinity values for each subtypes are reported from Fredholm et al. (2011) and Taliani et al. (2012).
2.5 Allosteric modulators of adenosine receptors.
Recently, the research of allosteric modulator, instead of an orthosteric compound, has become the focus of widespread efforts in ligand design and pharmacology of the adenosine receptors. To date in literature has been described selective agonist and antagonist ligands for all ARs subtypes (Muller 2011; Fredholm 2011). In contrast, allosteric modulators are well explored only for A1 and A3ARs. Regarding the A2AAR only recently has been reported a ligand able to modulate its function in a selective way (Goblyos 2009; Goblyos 2011). Most of the examples of allosteric modulators of adenosine receptors are positive allosteric modulators (PAMs) (e.g., they increase the affinity, potency, and/or efficacy of the agonist).
Bruns et al. (1990; 1990b) described the first AR allosteric modulators with a benzoylthiophenes structure. Three compounds, (2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)-(2-chloro-phenyl)-methanone (PD71,605); 2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)phenyl]methanone (PD81,723) and (2-amino-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-3-yl)(4-chloro-phenyl)methanone
subsequent studies, and their structure-activity relationship (SAR) as PAMs has been documented (Kourounakis 2000; Nikolakopoulos 2006; Romagnoli 2008). Baraldi et al. (2000), starting by PD71,605 structure as lead compound, described the 2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophene (T-62) (Fig 2.11). It represents also the first allosteric modulator that was synthesized as radiolabeled ligand (Baraldi 2006). Subsequent modification of the benzoylthiophenes core lead Aurelio et al. (2009) to discover the 3,5-di(trifluoromethyl)benzoylthiophene derivative that act as an allo-agonist of the A1AR (Fig 2.11).
Figure 2.11 The chemical structures of A1AR allosteric modulators.
Recently, to improve the knowledge of allosteric binding site, Narlawar et al. (2010) synthesized a bivalent ligands linking both orthosteric (adenosine-like) and allosteric (PD81,723-like) pharmacophores. The N6 -[2-Amino-3-(3,4-dichlorobenzoyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-yl-9-nonyloxy-4-phenyl]adenosine (LUF6258) (Fig. 2.12) with a nine-carbon atom linker between the two pharmacophores demonstrated to reach the binding of both allosteric and orthosteric sites of the receptor; in fact it showed no significant changes in affinity or potency in the presence of PD81,723.
Figure 2.12 The chemical structures of bivalent ortho/allosteric molecules LUF6258.
Amiloride and its analogs have been characterized as first allosteric modulators of the A2AAR(Gao 2000) (Fig 2.13). However, these compounds are not selective for this subtype; in fact they modulate binding at both A1 and A3ARs (Gao 2003). Moreover, they also compete for orthosteric binding at all subtypes. Recently, 1-[4-(9-benzyl-2-phenyl-9H-8-azapurin-6-ylamino)-phenyl]-3-phenyl-urea derivatives have been studied by Giorgi et al. (2008) (Fig 2.13); they were able to allosteric enhancer the A2A receptor both in radiolabeled studied than in functional studied.
Figure 2.13 The chemical structures of A2AAR allosteric modulators.
The first selective allosteric modulator of A3AR, the 4-methoxy-N-(7-methyl-3-(2-pyridinyl)-1-isoquinolinyl)benzamide) (VUF5455), has been described by Gao et al. (2001) (Fig 2.14). It showed to be selective for the agonistic state of the A3AR despite it displayed also modest affinity as orthosteric antagonist. Later, a 2-cyclopentyl-4-phenylamino-1H-imidazo-[4,5-c]quinoline (DU124183) (Fig 2.14), has been reported to selectively enhanc agonist binding and function at A3ARs, similar to VUF5455. However, in contrast to VUF5455, DU124183 increased also the maximum efficacy of adenosine in cAMP functional assay. DU124183 also displayed a moderate affinity (Ki = 820 nM) to hA3AR in radioligand displacement studies (Gao 2002). The optimization of DU124183 at 4-amino and 2-position led to a series of compounds among which
N-(3,4-dichlorophenyl)-2-cyclohexyl-1H-imidazo-[4,5-c]quinolin-4-amine (LUF6000) (Fig 2.14) represented the best one (Göblyös 2006). It enhanced agonist efficacy in a functional assay and decreased the agonist dissociation rate without influencing agonist potency. LUF6000 presented also an activity as orthosteric ligand towards A1ARs. With the aim to reduce this activity Heitman et al. (2009) synthetized a series of compounds among which the N-{2-[(3,4-dichlorophenyl) amino]quinoline-4-yl}cyclohexanecarboxamide (LUF6096) (Fig 2.14) was the best in allosteric enhancement of A3ARs with negligible orthosteric activity on both A1 and A3ARs.
Figure 2.14 The chemical structures of A3AR allosteric modulators.
No allosteric modulators of A2BAR have been described in the literature thus far (Goblyos 2011). Recently, netrin-1, an 85 kDa protein acting as migration and adhesion cue in the developing central nervous system and in a number of non-neural tissues, has been demonstrated to attenuate neutrophil transmigration and experimental colitis by modulating A2BAR signaling (Aherne 2012). However, development of this macromolecule as a drug is hampered by high costs required for its production and lack of oral bioavailability.
2.6 Aim of the present work.
The overall importance of A2BARs in several pathological condition drive the medicinal chemistry researchers in the discovery and development of A2BAR selective ligands with diverse chemical structure. Herein, we described the discovery of a new class of A2BARs with a 3-ketoindoles structure. We first evaluate the activity of these compounds on human A1, A2A, A2B and A3ARs, which unexpectedly led to the identification of three compounds that positively modulate and four compounds that, conversely, negatively modulate the A2BAR.
Later, we described the detailed biological characterization of all these compounds, based on binding and functional assays using CHO cells expressing human A1, A2A, A2B, and A3ARs, revealing that these compounds behaved as positive or negative allosteric
2.7 Materials and Methods
2.7.1 Investigated CompoundsCompounds 7a,b and 8a-c were obtained by simple and high yield synthetic procedures reported previously (Taliani 2013). Compounds 9a,b are commercially available (Bionet). Purity of tested compounds is ≥ 95% (combustion analysis).
2.7.2 Materials
[3H]DPCPX, [3H]NECA, [125I]AB-MECA and [35S]GTPγS were obtained from DuPont-NEN (Boston, MA); [3H]MRS 1754 was purchased from Scopus Research BV (Veenendaal, Netherlands). Adenosine deaminase (ADA) was obtained from Roche Diagnostics S.p.A. (Monza, Italy). BAY 60-6583 was purchased from Tocris bioscience (Bristol, UK). All other reagents were obtained from standard commercial sources and were of the highest commercially available grade. CHO cells stably expressing human A1, A2A, A2B, and A3ARs were kindly supplied by Prof. K. N. Klotz, Wurzburg University, Germany (Klotz 1998).
2.7.3 Human A1 adenosine receptors binding assay
Aliquots of cell membranes (30 µg proteins) were incubated at 25 °C for 180 min in 500 mL of T1 buffer (50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) containing [3H]DPCPX (3 nM) and six different concentrations of the newly synthesized compounds. Non-specific binding was determined in the presence of 50 µM R-PIA (Da Settimo 2004). The dissociation constant (Kd) of [3H]DPCPX in hA1 CHO cell membranes was 3 nM.
2.7.4 Human A2A adenosine receptors binding assay
Aliquots of cell membranes (30 µg) were incubated at 25 °C for 90 min in 500 mL of T1 buffer (50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) in the presence of 30 nM of [3H]NECA and six different concentrations of the newly synthesized compounds. Non-specific binding was determined in the presence of 100 µM R-PIA (Da Settimo 2004). The dissociation constant (Kd) of [3H]NECA in hA2A CHO cell membranes was 30 nM.
2.7.5 Human A3 adenosine receptors binding assay
Aliquots of cell membranes (30 µg) were incubated at 25 °C for 90 min in 100 mL of T buffer (50 mM Tris-HCl, 10 mM MgCl , 1 mM EDTA, 2 units/mL ADA, pH 7.4) in
