Molecular recognition in adenosine receptors

Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All fights reserved.

153

M o l e c u l a r R e c o g n i t i o n in A d e n o s i n e R e c e p t o r s K.A. Jacobson,* A.M. van Rhee, S.M. Siddiqi, X.-d. Ji, Q. Jiang, J. Kim, and H.O. Kim Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, MD 20892 USA. INTRODUCTION Extracellular adenosine acts as a modulator within many physiological systems [1], in general, to compensate for excessive activity of a given organ and to protect it against the detrimental effects of stress. This is a negative feedback loop, by which locally produced adenosine (originating from degradation of either intracellular or extracellular ATP) both reduces the energy demand and increases the oxygen supply. Adenosine is particularly critical m maintaining the homeostasis of essential organs such as the brain, heart, and kidneys, but is also very important for the immune system. In the brain, exogenously administered adenosine agonists have proven exceptionally efficient in neuroprotection [2] (in part, by counteracting the damage caused by excessive glutamate release), and adenosine has been shown to be revolved in pain, cognition, movement, and sleep [3]. Thus, there is a tremendous impetus for the development of therapeutic agents based on selective interactions with adenosine receptor subtypes. Four subtypes of adenosine receptors have been cloned: A 1, A2A, A2B, and A3 [4]. Adenosine agonists, which are almost exclusively derivatives of adenosine, have been sought as potential anti-arrhythmic, anti-lipolytic' (thus anti-diabetic), and cerebroprotective agents (A1), and hypotensive and anti-psychotic agents (A2A). Adenosine antagonists, of which xanthines and a number of fused heterocyclic compounds are representative [1], have been under development as antiasthmatic, anti-depressant, anti-arrhythmic, renal protective, anti-Parldnson's, and cognition enhancing (h~gs. The adenosine receptors are members of the G protein-coupled receptor family, having seven transmembrane helical regions [5]. Site-directed mutagenesis of the A 1 and A2A receptors [6,7] has yielded much insight into structure function relationships. The emerging receptor models [7-9] are approaching a degree of resolution that promises to assist in the design of improved ligands. The discovery of a novel and distinct adenosine receptor subtype, the A 3 receptor [10], has opened new therapeutic vistas m the purmoceptor field. This receptor subtype has a unique SAR (structure activity relationship) profile, tissue distribution, and effector coupling. Reports on A 3 selective agonists have recently appeared [11-13], but the physiological role of A 3 receptors remains to be clarified.

154 In spite of the massive effort to develop selective ligands, a number of agents that initially looked promising did not survive clinical trials [1]. Nevertheless, the interest in adenosine-based therapy has not waned. On the contrary, as our knowledge of the biological effects of adenosine advances, promising, newly envisioned therapeutic applications have become evident. Recently, the use of adenosine agonists in treating stroke has come into focus [2], since, m acute clinical use the interference by some of the previously documented side effects may be tolerable. RECENTLY-DEVELOPED SELECTIVE LIGANDS Highly selective ligands for adenosine A 1 and A2A receptors [1] have been designed both by classical medicinal chemical approaches and by a functionalized congener approach [14]. By the latter approach, a chemically functionalized chain is incorporated at a specific site on a pharmacophore leading to increased flexibility of substitution and enhancement of potency/selectivity via distal interactions at the receptor. In general, for adenosine agonists (Table 1), modification of the N6-position with hydrophobic moieties (such as CHA, N6-cyclohexyladenosine, 9, and CPA, N 6cyclopentyladenosine, 1 0 ) h a s provided selectivity for A1 receptors (and thus cerebroprotective properties). Substitution at the C2-position (such as occurs in CGS 21680, 2-[4- [(2-cm'boxyethyl)phenyl] ethylamino]-5'-N-ethylcarboxamidoadenosine, 11, and its ethylenediamine conjugate, APEC) has resulted in A2A selectivity. A thio- substitution of the 4'-oxygen of 2-chloroadenosine was found to enhance affinity selectively at A2A receptors [23]. Selective antagonists for A 1 receptors (Figure 2) include many 8-aryl and 8-cycloalkyl xanthine derivatives, such as the amine congener XAC, 12, and CPX, 13, which is -500-fold selective for A1 receptors. A thio group may be substituted at the 2-position, but not 6-position, carbonyl group of the xanthine with retention of high affinity. Selectivity for A2A receptors in xanthines has been more difficult to achieve. However, 8-styrylxanthines, such as CSC (8-(3chlorostyryl)caffeine), 14, are A2A selective. The non-xanthine ZM241385, 15, is the most selective A2.,~ antagonist (6800-fold) yet reported [15]. The most recent challenge in the medicinal chemistry of adenosine receptors has been to produce A3 selective agents. A 3 selective antagonists have not yet been reported. One principle of achieving A3 selectivity among adenosine agonists is the combination of optimal substitutions at the N 6- and 5'-positions of adenosine [9,11]. Specifically, among alkyl, cycloalkyl, and aralkyl N 6substituents, a benzyl group is favored, due to its diminished potency at A1 and A2A receptors (Table 1). The A3-selectivity enhancing effects of N6-benzyl

155 Table 1. Affinities (~tM) of adenosine derivatives at rat brain A 1, A2A, and A 3 receptors, arranged in order of decreasing affinity at rat A3 receptors.a

NLN NHR 3

,>

R2

N

!

HO OH

Compound 1. b 2. c 3. d 4. 5. 6. e 7. f 8. g 9. h 10. i 11. J

R1

R2

R3

CH3NHCO C1 3-I-Bz CH3NHCO H 3-I-Bz CH3NHCO H 3-I-4-NH2-Bz CH3NHCO CH3S 3-I-Bz CH3NHCO CH3NH 3-I-Bz C2HsNHCO H Bz CH3NHCO H H C2HsNHCO H H HOCH 2 H cyclohexyl HOCH 2 H cyclopentyl C2HsNHCO NH(CH2)2-pH Ph(CH2)2-

Ki(A1)

Ki(A2A)

0.82 0.054 0.018 2.14 4.89 0.087 0.0836 0.0063 0.0013 0.00059 2.6

0.47 0.056 0.197 3.21 4.12 0.095 0.0668 0.0103 0.514 0.462 0.015

Ki(A3) 0.00033 0.0011 0.0013 0.0023 0.00312 0.0068 k 0.072 k 0.113 k 0.167 k 0.24 k 0.584 k

a. Ki+ S.E.M. determined in radioligand binding assays expressed in ~M, using the following radioligands: A1, [3H]N6-R-phenylisopropyladenosine in rat cortical membranes; A2A, [3H]CGS 21680, 11, binding in rat striatal membranes; A 3, [i25I]AB-MECA, 3, binding, unless noted, in membranes of CHO cells stably transfected with the rat A3-cDNA; b. CI-IB-MECA; c. IBMECA; d. I-AB-MECA; e. Bz-NECA; f. MECA; g. NECA; h. CHA; i. CPA; j. CGS 21680; k. A 3 affinity determined versus [125I]APNEA (N 6aminophenylethyladenosine) binding.

156 modification are additive with the A3-affinity enhancing effects of the 5'uronamido function, as in NECA (adenosine-5'-N-ethyluronamide), 8. The first such hybrid molecule to show A3 selectivity [9] was NG-benzyl-NECA, 6. In a comparison of various 5'-uronamido groups in mono-substituted adenosine derivatives, the 5'-N-methyluronamide, 7 [11], had particularly favorable A3 receptor vs. A1/A2A affinity. Empirical and QSAR studies [11,20] of substituent effects on the N6-benzyl group have shown that substitution at the 3-position with sterically bulky groups, such as the iodo group, is optimal, leading to the development of the highly potent A3 agonist N6-(3-iodobenzyl)-adenosine-5'-Nmethyluronamide (IB-MECA), 2, which is 50-fold selective for A3 vs. either A1 or A2 receptors in vitro [11] and appears to be highly A 3 selective in vivo [21]. [125I]I-AB-MECA, 3, was introduced as a high affinity radioligand for A 3 receptors [21]. 2-Substitution in combination with modifications at N 6 and 5'-positions was found to further enhance A 3 selectivity [12]. Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5'uronamide analogues as agonists at A3 adenosine receptors, were synthesized starting from 1-O-methyl ~-D-ribofuranoside in 10 steps [12]. 2-Chloro-N6-(3 iodobenzyl)-adenosine-5'-N-methyluronamide, 1, which displayed a Ki value of 0.33 nM, was selective for A3 vs. A1 and A2A receptors by 3600- and 2000-fold, respectively. Compound 1 was 66,000-fold selective for A3 receptors vs. the Na +independent adenosine transporter, as indicated in its weak displacement of [3H]S-(4-nitrobenzyl)thioinosine binding at this transporter in rat brain membranes. In a functional assay of rat A3 receptors expressed in CHO cells, it inhibited adenylyl cyclase with an IC50 of 67 nM. 2-Methylthio-N6-(3 iodobenzyl)adenosine-5'-N-methyluronamide, 4, and 2-methylamino-N6-(3 iodobenzyl)-adenosine-5'-N-methyluronamide, 5, were less potent, but nearly as selective for A3 receptors.

DO XANTHINES BIND TO A3 RECEPTORS? The A3 adenosine receptor cloned from rat [10] was shown to be unique among the subtypes in that agonist action is not antagonized by xanthines, such as theophylline. The affinity of certain xanthines, such as BWA522 (Figure 1), 16, is greater in the human and sheep homologues of the A 3 receptor [16,17] than in the rat. Typical Ki values at rat A 3 receptors of roughly 10 .4 M have been obtained for many xanthines that have nearly nanomolar potency at the A1 or A2A subtypes. X A C , 12, for example at 1 pM, has been used in

157 O .LL

Pr\ N

H I~I I

O LI

H ~

O,-~N /

--~N

Pr\

/

oc

I

~

-

I

Pr

Pr

12, XAC

13, CPX

O OH3 CH3.N.,~ _~1~1

C! .o

~

NH.,J,,.-N,,,J~N/~--'<"O'~

CH3 14, CSC

O

Pr\

.LL

15, ZM241385

H

~

0

.~" ~,~=-~o~coo~ c~ .~ _~

O " ~ N ~ -~N

"~"

I @ (0H2)2

S"/~N ~ ~N

,

NH2

CH3 17

16, BWA522

I

Figure 1. Xanthine (12-14,16,17) and non-xanthine (15) adenosine antagonists.

Bu

~,u 0

o~~.~> I

Bu

o~~~> I

H 18

19

]'1'

HO OH

20

~-~ HO OH

Ki (~M) at Rat A 3 Receptors:

143

6.03

0.229

A1 Receptors:

0.50

4.19

37.3

Figure 2. Ribose moiety at the 7-position anchors xanthine in the A 3 binding site.

158 pharmacological experiments in vitro and in rodents [2] for distinguishing A3 receptors from A1 and A2A receptors. In an effort to synthesize A3 antagonists, we have attempted to maximize the affinity of xanthine derivatives at the binding site [18]. One such xanthine is compound 17, which had a K i value at rat A3 receptors of 9.4 ~M. The presence of an anionic group on the xanthine tended to diminish the affinity at A1 and A2A receptors. Thus, compound 17 is 7-fold selective for rat A3 vs. A2A receptors. Molecular modeling of adenosine receptors (supported by mutagenesis) suggests that two histidine residues in the sixth and seventh transmembrane domains (TM6 and TM7) are important for ligand recognition [5-8]. We have proposed [9] that the ribose moiety of adenosine, that is relatively more important for high affinity binding to A3 receptors than at other subtypes, is coordinated to a conserved histidine residue in TM7 (His278 of hA2A, see below). Consequently, we tested the hypothesis that a means of anchoring xanthines in the A 3 binding site is by adding a sugar moiety at the 7-position to form xanthine-7-ribosides (Figure 2). Several members of this class of compounds, the 1,3-dialkylxanthine-7-ribosides, have been synthesized and were previously found by IJzerman and colleagues to bind weakly to A1 receptors [19]. At rat brain A3 receptors, 1,3-dibutylxanthine-7-riboside (DBXR), 19, was found to bind with a Ki value of 6.03 ~M [9],. whereas the parent xanthine, 1,3dibutylxanthine, 18, displayed a Ki value of 143 p~M. Thus, the presence of the ribose moiety enhances affinity of xanthines at rat A3 receptors, while at A 1 receptors the xanthine-7-riboside derivatives are, as a rule, less potent than the parent xanthines. Functionally, 1,3-d~butylxanthine-7-riboside, 19, as a structural hybrid of classical A1/A2A agonist and antagonist molecules, appeared to act as a partial agonist at rat A 3 receptors [9], providing hope that this was a means of designing antagonists. However, upon structural modification that increased the potency and selectivity of the xanthine ribosides at A 3 receptors, full agonism was achieved. Specifically, the structural parallel between adenosine derivatives and the xanthine-7-ribosides was maintained with respect to A 3 receptor affinity. This parallel lead to the design of 1,3-dibutylxanthine-7riboside-5'-N-methylcarboxamide (DBXRM, Figure 2), 20, having a Ki value of 229 nM at A3 receptors with 1G0-fold and >400-fold selectivity for A3 vs. A1 and A2A receptors, respectively. The selectivity of this compound is a result of incorporation of the 5'-methyluronamide group, found to enhance A 3 selectivity in IB-MECA, 2, and optimization of the alkyl chain length at positions 1 and 3. Unlike 1,3-dibutylxanthine-7-riboside, DBXRM acted as a full agonist in the rat A3 receptor-mediated inhibition of adenylyl cyclase. Thus, there was a tendency

159 towards the increase of efficacy as the affinity increased within the same series of compounds.

SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING OF ADENOSINE R E C E P T O R S In addition to chemical probing of the binding sites of adenosine receptors, we have also used molecular biological approaches to determine structure function relationships for human A2A adenosine receptors [7,22]. Amino acid residues were mutated and the constructs expressed in COS-7 cells. Numerous residues in transmembrane domains 3, 5, 6 and 7, individually replaced with alanine and other amino acids, were identified as essential for ligand recognition. An immunological method [8] was used to determine whether the pharmacologically inactive mutant receptors were properly oriented at the cell surface and not simply retained in an intracellular compartment. Thus, an epitope tag was attached near the N-terminus of the receptor to allow detection with an antibody. This extra l 1-amino acid sequence did not interfere with ligand binding or adenylyl cyclase activation by the human A2A receptor. The pharmacological properties of mutant receptors were determined both m radioligand binding experiments and through stimulation of adenylyl cyclase. Specific binding of [3H]CGS 21680, 11, and [3H]XAC, 12, an A2A agonist and antagonist, respectively, was measured. Although A 1 selective in the rat, XAC has greatly enhanced affinity at human A2A receptors, and it is useful as a nonselective radiotracer in this species. High affinity binding of either agonist or antagonist was absent m the following Ala mutants: F182A, H250A, N253A, I274A, H278A and $281A, although they were well expressed in the plasma membrane. The hych'oxy group of $277 is required for high affinity binding of agonists, but not antagonists, since the $277A mutant bound [3H]XAC but not [3H]CGS 21680 similar to the ~dld type receptor. /h~ N181S mutant lost affinity for adenosine agonists substituted at the N 6 or C-2, but not at the C-5' position. The mutant receptors I274A, $277A, H278A showed full stimulation of adenylyl cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were >30-fold less than those at the wild type receptor. A molecular model based on the structure of rhodopsm was developed concurrently to mutagenesis studies in order to visualize the environment of the ligand binding site. We have found that the low resolution structure of rhodopsin serves as a more versatile template for G protein-coupled receptors (GPCRs) than the coordinates of bacteriorhodopsin [7]. The A2A receptor model was composed in steps including: construction and energ~ minimization of each helix individually, composition of the helical bundle based on consideration of

160

161 Figure 3 [upper left]. Vicinity of the bound ligand NECA in the rhodopsin-based molecular model of the human A2A receptor. Docking of the ligand was based on mutagenesis results, e.g. single amino acid substitution of the receptor, followed by energy minimization [7]. A putative pocket consisting of four aromatic residues shown m space f'filing form surrounds the adenine moiety of adenosine. Figure 4 [lower left]. Vicinity of the ribose moiety of NECA to hydrophilic residues of TM7 m the rhodopsin-based molecular model of the human A2A receptor [7]. Mutation to Ala of each of the residues shown in space filling form prevents the high affinity binding of [3H]CGS 21680.

Figure 5. Putative overlay of the agonist NECA, 8, and the antagonist XAC, 12, h~ the human A2A receptor model according to the 'N6/C8' hypothesis. Transmembrane helical regions are represented as ribbon structures. NECA was docked and minimized as above [7]. The amino group of XAC rests near the exofacial side of the receptor.

162 homology among GPCRs and rotation of amphipathic helices such that predominantly hydrophobic faces are pointed towards the lipid bilayer. Only the final step in construction of the model, i.e. docking of a ligand, NECA, took into account the results of mutagenesis. Thus, the receptor portion of the model, based only on computational methods and previous pharmacological structural insights into adenosine receptors [8,9] and other GPCRs, was highly predictive of which residues were potentially facing m the direction of the binding site. Most of the positions at which alanine mutation was not tolerated, were calculated to have site chains pointing into the binding cleft. This helped to define the placement of the ligand in the binding site. The current model takes into consideration only the contribution of the transmembrane helical domains, however a mutagenesis study by Olah et al. [24] has revealed that a portion of the second extracellular loop is also involved m antagonist recognition by A 1 receptors. Some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2A adenosine receptor. Four aromatic side chains in TM6 and TM7 appear to form a cluster in the receptor model that constitutes a putative binding pocket for the adenine moiety (Figure 3). The residues are H250, Y257, and F182 (all found to be essential by mutagenesis) and Y183 (not mutated in our study) [7]. H250 in TM6 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential since the H250F mutant receptor binds radioligand like wild type. On the other hand replacement of H278 in TM7 with other aromatic residues was not tolerated in ligand binding, since hydrophilic groups at this position are required. According to the molecular model N253 is in proximity to form an Hbond with the exocyclic amino group of adenosine, h~ fact, the N253A mutant receptor did not bind either radioligand with high affinity. The xibose moiety appears to span a region of hydrophilic residues located in TM3 and TM7. Figure 4 shows the proposed interaction of the ribose moiety of NECA with hydrophilic residues of TM7. h~ our A2A adenosine receptor model the 5'-NH in NECA is hych'ogen bonded to $277 and H278. We have found that the T88A (TM3) mutant receptor binds antagonists like wild type receptors, but fails to bind agonists with high affinity [22]. In the biogenic amine receptors, an aspartate residue conserved in TM3 is essential as a counterion to the charged ammonium group of the ligand. In the adenosine receptors a Val residue occurs at the position of this Asp, and the essential T88 occurs roughly one helical turn closer to the cytoplasmic side of the helix. In the model, the ri.bose region is in proximity to T88. Certain mutant A2-x receptors have revealed differences in affinity shifts between agonists and antagonists, for example: $281N (agonists become more potent and antagonists less potent) and mutations of T88 (agonists become much less potent). Therefore, a partially overlapping set of amino acid residues

163 in the receptor are proposed to be involved in agonist vs. antagonist binding. We attempted to contrast the agonist and antagonist binding domains in the A2A receptor model. Based on SAR insights, the nitrogens of xanthines and adenosine derivatives, although both purines, certainly do not occupy similar coordinates in the receptor-bound state. At least three models for mapping of agonists onto antagonists have been proposed [25]. Extending this analysis to the entire receptor model clearly favors one of the three hypotheses, i.e. the 'N6/C8 ' model. In this hypothesis, the C-8 substituent of xanthines overlays the N 6 substituent of adenosine analogues. Figure 5 shows the relative orientation when the antagonist XAC is docked into our NECA-occupied receptor model according to this hypothesis, whereas in applying the 'flipped' hypothesis the C8substituent side chain of XAC protrudes into the helical segment (not shown), which is energetically untenable.

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13 Kim HO, Ji XD, Melman N, Olah ME, Stiles GL, Jacobson KA. J. Med. Chem. 1994; 37:4020-4030. 14 Jacobson KA, Ukena D, Padgett W, Kirk KL, Daly JW. Biochem Pharmacol 1987; 36:1697-707. 15 Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PWR, Jones G, Collis MG. Br J Pharmacol 1995; 115:1096-1102. 16 Linden J, Taylor HE, Robeva AS, Tucker AL, Stehle JH, Rivkees SA, Fink JS, Reppert SM. Mol Pharmacol 1993; 44:524-532. 17 Salvatore CA, Jacobson MA, Taylor HE, Linden J, Johnson RG. Proc Natl Acad Sci U S A 1993; 90:10365-10369. 18 Kim HO, Ji XD, Melman N, Olah ME, Stiles GL, Jacobson KA. J Med Chem 1994; 37:3373-3382. 19 IJzerman AP, van der Wenden EM, von Frijtag Drabbe Kiinzel J, MathSt RAA, Danhof M, Borea PA, Varani K. Naunyn Schmiedebergs Arch Pharmacol 1994; 350:638-645. 20 Siddiqi SM, Pearlstein RA, Sanders LH, Jacobson KA. Bioorg Med Chem 1995, 3:1331-1343. 21 Jacobson KA, Nikodijevic O, Shi D, Gallo-Rodriguez C, Olah ME, Stiles GL, Daly JW. FEBS Lett 1993; 336:57-60. 22 Jiang Q, van Rhee AM, Kim J, Yehle S, Wess J, Jacobson IC J Biol Chem 1995, submitted. 23 Siddiqi SM, Jacobson KA, Esker JL, Melman N, Tiwari KN, Secrist JA, Schneller SW, Cristalli G, Johnson CA, IJzerman AP. J Med Chem, 1995; 38:1174-1188. 24 Olah ME, Jacobson KA, Stiles GL, J Biol Chem 1994; 269:24692-24698. 25 van der Wenden EM, Price SL, Apaya RP, IJzerman AP, Soudijn W. J Comp Aided Mol Design 1995; 9:44-54.