Peptidyl Conjugates of Adenosine 5′Carboxylic Acid Synthesized and Evaluated as Ligands for P2 Purinoceptors

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The role of extracellular ATPin vivoand the various cellular responses mediated by P2 purinoceptors have not yet been fully elucidated, in part depending on the lack of subtype-specific high affinity antagonists. Here we describe the synthesis of a

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  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  229,  363–369 (1996) ARTICLE NO . 1811 Peptidyl Conjugates of Adenosine 5  -Carboxylic Acid Synthesizedand Evaluated as Ligands for P2 Purinoceptors Leif Ja¨rlebark,* Mikael Erlandsson,* Asko Uri,* , † Brian F. King,‡ Airat U. Ziganshin,§Charlotta Johansson, Ø and Edith Heilbronn* ,1 *  Department of Neurochemistry and Neurotoxicology, The Arrhenius Laboratories for Natural Sciences, StockholmUniversity, Stockholm, Sweden;  †  Institute of Chemical Physics, Tartu University, Tartu, Estonia;  ‡  Department of  Anatomy and Developmental Biology, University College London, London, United Kingdom;  §  Department of Pharmacology, Kazan Medical University, Kazan, Russia; and   Ø Swedish NMR Centre, Stockholm, Sweden Received October 29, 1996The role of extracellular ATP  in vivo  and the various cellular responses mediated by P2 purinoceptorshave not yet been fully elucidated, in part depending on the lack of subtype-specific high affinity antagonists.Here we describe the synthesis of a new class of compounds, peptidyl derivatives of adenosine 5  -carboxylicacid, among which some have inhibitory effects in certain P2 purinoceptor-carrying biological systems,e.g., glioma and smooth muscle cell lines and isolated smooth muscle tissue preparations from guinea pigvas deferens and urinary bladder.    1996 Academic Press, Inc. Extracellular ATP, endogenously released or exogenously applied to various tissues andcells elicits a variety of P2 purinoceptor-mediated responses, depending upon the tissue. Re-sponses include ATP-induced activation of cation-selective channels (1, 2), activation of phos-phatidyl inositol-specific phospholipase C (PLC) and transient increases in intracellular Ca 2 / levels (3, 4),  e.g.  as previously observed by us in skeletal muscle myotubes (5) and in outerhair cells of the mammalian cochlea (6). P2 purinoceptors (P2Rs) have been subdivided intoat least six subtypes: P 2X , P 2Y , P 2U , P 2T , P 2Z  and P 2D  (subscripts denote pharmacologicallydefined but not yet cloned P2R subtypes, see ref. 8) the characterization was mainly done onthe basis of the relative potencies of their natural agonists (ATP, ADP and UTP) and syntheticnucleotide derivatives. The recent cloning of a number of P 2U , P 2Y  and P 2X  purinoceptorsubtypes(7,8,9,10)nowprovidesthepossibilitytocharacterizethemonamolecularbasis.TheP2X purinoceptor family comprises ion channel receptors (with unique structure) permeable toNa / , K / and Ca 2 / , while P2Y (including P 2U ) purinoceptors are G protein-coupled receptorproteins (GPCRs), with the characteristic motif of seven membrane-spanning helices, oftencoupled to stimulation of PLC and inositol 1,4,5-trisphosphate (IP 3 ) formation, but other secondmessenger pathways may also be activated.Thelackof highaffinitysubtype-specificantagonistsfor P2Rshasdelayedstudiesof receptorfunction  in vivo.  Great efforts have been made to obtain such antagonists (e.g. 11-14), butneitherhighaffinitynor subtype-selectivederivativeshaveyetbeenachieved. Afewantagonistswith moderate affinity exist,  e. g.  aromatic polysulfonic acids, such as Evans blue, Reactiveblue 2, Reactive red, suramin and pyridoxalphosphate-6-azophenyl-2  -4  -disulfonic acid(PPADS) but none of them have the desired subtype specificity.In1994,Uri et al.  introducedanewclassofcompounds(11),peptidederivativesofadenosine5  -carboxylic acid, as ATP analogues for P2R studies. The present work exploits gainedexperience and continues the effort. In the newly synthesized ATP analogues, the triphosphate 1 To whom correspondence should be addressed. Fax:  / 46 (8) 161371.0006-291X/96 $18.00 Copyright    1996 by Academic Press, Inc.All rights of reproduction in any form reserved. 363  Vol. 229, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS chain of ATP is substituted by a small peptide comprising four aspartic (Asp) and glutamic(Glu) acid residues in various combinations. Thus, all the conjugates carry negative charges(at pH 7.4), in a small volume in close proximity to the adenosine (Ado) part of the molecule,thereby conserving the properties of the lead compound from Uri  et al.,  AdoCAsp 4  (thetetraaspartic acid derivative of adenosine 5  -carboxylic acid), a weak antagonist of ATP-induced Ca 2 / -transients in C6 rat glioma cells (11). The biological activity of the new com-pounds was evaluated in P2R-bearing cells and tissue preparations. MATERIALS AND METHODS All compounds were synthesized using standard Fmoc solid phase peptide synthesis (SPPS) techniques [for a fulldescription of the procedure, see Uri  et al.  (11)]. However, in the present work, the procedure for anchoring the firstamino acid to the solid support was a modification of the method of Sieber (15), see below. Synthesized conjugateswere analyzed with respect to their molecular masses on an Applied Biosystems Bio-Ion 20 plasma desorption time-of-flight mass spectrometer and the structures were verified with a Varian Unity 300 MHz NMR instrument.  Anchoring the first amino acid to the resin (modified from ref. 15).  Fmoc-amino acid (288  m mol), 2,6-dichloroben-zoylchloride (288  m mol) and pyridine (475  m mol) were dissolved in dichloromethane. After 30 min under N 2  atmo-sphere, 200 mg of Wang resin (0.72 mmol/g) and N-methylmorpholine (572  m mol) were added to the reaction mixture,followed after 5 minutes by dimethylaminopyridine (40  m mol). The reaction was allowed to proceed for 5 hours.Remaining underivatized hydroxyl groups were capped with acetic anhydride (400  m mol, 30 minutes) and the resinwas washed several times with dimethylformamide. A loading level of 0.43 mmol/g was achieved. The continuedsynthesis of the conjugates has been described previously (11).  Biological testing Culturing C6 glioma cells.  C6rat glioma cells were cultured inHam’s F-10 mediumsupplemented with L-glutamine,gentamycin, newborn calf serum and fetal calf serum as previously described (11). Culturing DDT  1  MF-2 smooth muscle cells.  DDT 1  MF-2 cells (16) were grown in suspension in Dulbecco’s modifiedEagle’s medium (DMEM) containing 4.5 g/l glucose, 5% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillinand 100  m g/ml streptomycin. Cells were subcultured 2-3 times per week and used at a density of approximately 10 5 cells/ml (17).[ Ca 2 / ] i  measurements in C6 glioma cells.  Cells were washed twice in 2 ml Krebs-Ringer-Hepes (KRH) buffer(conc. in mM: NaCl 125; KCl 5; MgSO 4  1.2; KH 2 PO 4  1.2; CaCl 2  2; glucose 6; Hepes 25; pH adjusted to 7.4). Afteraddition of fura-2/AM (2 mM), a cell-permeant Ca 2 / -sensitive fluorescent probe (18), the cells were incubated in thedark for 1 hour. The medium was changed with 2 ml fresh KRH buffer and the cells incubated for another 15 minutesto remove non-hydrolyzed dye. Immediately prior to measurement, the medium was replaced again and the cell culturedish placed in the spectrofluorometer. After base line stabilization, the ligand was rapidly added under mixing, thedish reinserted within 10 seconds. Changes in fluorescence were recorded until base line levels were reached again(3-10 min). For desensitization or inhibition studies, ligands were added sequentially (5 min. interval) without changeof medium. The cytosolic calcium concentration ([Ca 2 / ] i ) was quantified using the Ca 2 / ionophore ionomycin (10 m M) and quenching of the fluorescence by MnCl 2  (20 mM) to determine maximum and minimum values of fura-2fluorescence. Calculations were made according to Grynkiewicz  et al.  (18).[ Ca 2 / ] i  measurements in DDT  1  MF-2.  Cells were washed and resuspended in Hank’s balanced salt solution (HBSS;containing 1.2 mM CaCl 2 , supplemented with 0.1% BSA and 20 mM HEPES, pH 7.4) to a concentration of 10 6 cells/ ml and loaded with fura-2/AM for 40 min at 37  C, then washed twice in HBSS and resuspended. Cells were washedagain prior to measurements, placed in a cuvette (10 6 cells in 2 ml of HBSS) and measurements performed in aHitachi F-2000 spectrofluorimeter. Maximum and minimum fluorescence parameters were obtained by use of TritonX-100 and EGTA respectively, while autofluorescence of the cells was determined before incubation with fura-2. Pharmacological evaluation in smooth muscle preparations.  Relaxant and contractile responses in the followingtissue preparations were measured by standard methods previously described: P 2Y -mediated relaxation of carbachol-contracted rabbit thoracic aorta (with endothelium) and guinea pig taenia coli (19, 20); P 2X -mediated contraction of guinea pig vas deferens and urinary bladder (19, 21). Measurements of the different tissues followed the same principle;strips of dissected muscle were mounted in organ baths filled with Krebs solution [modified Krebs buffer (conc. inmM): NaCl, 133; KCl, 4.7; MgSO 4 , 0.6; NaHCO 3 , 16.3; glucose, 7.7; NaHPO 4 , 1.4; CaCl 2 , 2.5] perfused with O 2  / CO 2  (95%/5%) and maintained at 37  C. Changes in muscle tension after electrical field stimulation or addition of compound were recorded on a Grass polygraph. Pharmacological evaluation using recombinant P2X  1  purinoceptors.  ATP-evoked membrane currents were recordedunder voltage-clamp (Axoclamp 2A) using defolliculated  Xenopus  oocytes (as described in ref. 26) microinjectedwith P2X 1  transcripts (40 nl of 1  m g/  m l). Oocytes were placed in an electrophysiological chamber (0.5 ml) and364  Vol. 229, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSTABLE 1Numbering and Composition of the Peptidyl Derivatives of Adenosine 5  -Carboxylic Acid (AdoCO-X (1-6) )# Pos. #1 2 3 4 5 6R1 Glu Glu Glu Glu — —R2 Glu Glu Glu Asp — —R3 Glu Glu Asp Asp — —R4 Glu Asp Asp Asp — —R5 Glu Asp Glu Asp — —R6 Asp Asp Asp Glu — —R7 Asp Asp Glu Glu — —R8 Asp Glu Glu Glu — —R9 Asp Glu Asp Glu — —R10 Gly Asp Asp Asp Asp —R11 Gly Gly Asp Asp Asp AspR12 ( b Asp) Asp Asp Asp — —R13 ( b Asp) ( b Asp) ( b Asp) Asp — —R14 (D-Asp) Asp Asp Asp — —AdoCAsp 4  was first described in ref. 11.superfused (at 5 ml/min) with an amphibian Ringer solution [containing (in mM): NaCl, 110; KCl, 2.5; HEPES, 5;BaCl 2 , 1.8; adjusted to pH 7.4 and used at 18  C]. Current recordings were carried out as previously described (27).ATP and peptidyl conjugates were added to the superfusate (for final concentrations, see text). ATP was applied for30 s every 20 min. and each concentration of antagonist was applied for 20 min. RESULTS AND DISCUSSION Synthesis and Analysis 14 newly-synthesized conjugates (Table 1), and AdoCAsp 4 , were purified on reversed phase(RP) C 16  HPLC, the correct fractions were collected and lyophilized, yielding white solidswith overall purities of more than 90%, as determined by analytical RP HPLC and  1 H-NMR.PDMS indicated the correct molecular masses. Assigned proton peaks from NMR analyseswere consistent with the proposed structures. Representative data for the compound R1 arelisted below. UV spectroscopy:  l max  (water, pH 7.0)  Å  257 nm, characteristic for adenosinederivatives; massspectroscopy:799(M / H), 821(M / Na), 843(M / 2Na), calculatedmolecularmass  Å  798;  1 H-NMR data for compound R1 (D 2 O; pD was adjusted to 7.0 using NaOD;  d  ,ppm.): H-2 and H-8:  d  Å 8.20 (s)  d  Å 8.14 (s); H-1  :  d  Å 6.17 (d) J Å 6.9; H-2  :  d  Å 4.93 (d / d)J Å 5.1, 6.8; H-3  :  d  Å 4.63 (d / d) J Å 2.5, 4.9; H-4  :  d  Å 4.70 (d) J Å 2.4;  a -glu:  d  Å 4.47 (d / d)J Å 5.0, 9.3 (1H),  d  Å 4.29-4.38 (m) (2H),  d  Å 4.15 (d / d) J Å 4.7, 8.2 (1H);  b / g -glu:  d  Å 1.85-2.38 (m) (16H).  Biological Testing of the Compounds The compounds were tested for agonist and/or antagonist action on fura-2-loaded C6 cells,which express both excitatory P 2Y  and P 2U  purinoceptors (11). The compounds were added ata concentration of 100 m M in all tests, and ATP was used as agonist at an equimolar concentra-tion,  i. e.  100  m M. All compounds, except R1, R2 and R9, reduced (32-58% decrease) ATP-induced increases of cytosolic [Ca 2 / ], none of them acted as agonists.The EC 50  value for ATP-induced [Ca 2 / ] i  increase in DDT 1  MF-2 smooth muscle cells wasapproximately5 m M; subsequently, thisconcentrationwasusedfor measuringinhibitoryeffectsof the compounds on ATP-induced Ca 2 / transients in suspended DDT 1  MF-2 cells. Resultsshow inhibition spanning from 23-29% for AdoCAsp 4 , 27-58% for R12, 20-53% for R13, and 365  Vol. 229, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS FIG. 1.  Concentration-dependent inhibition by AdoCAsp 4  (10–300  m M) and R1 (10–300  m M) of guinea pig vasdeferens smooth muscle (neurogenic) contraction evoked by electrical field stimulation (0.5 ms, 100 V, 4–32 Hz) inthe presence of 0.3  m M atropine and 1  m M phentolamine. 16-47% for R14 of 5  m M ATP-induced Ca 2 / increase after a 5 minute preincubation with 100 m M of either compound.Additionally, AdoCAsp 4 , R1, and R13 were evaluatedas antagonists to thevascular endothe-lium-dependent (P 2Y -mediated) relaxation of rabbit aorta, precontracted with noradrenaline(NA), but were found to be inactive. Continued testing of P 2Y  receptors (in guinea pig taeniacoli), showed the compounds to be very weak agonists,  i. e.  mediating relaxation of carbachol-precontracted smooth muscle. Neither of the above-mentioned compounds, nor R7, R9, R10and R11 antagonized the relaxant effect of ATP, however R1 (at 100  m M) was found topotentiate relaxation evoked by electrical field stimulation.Compounds R7 and R9-11, each at 100  m M, antagonized (10-35% inhibition) transmuralnerve stimulation (0.5 ms, 100 V, 2-16 Hz) of vas deferens, in the presence of the adrenergicantagonist, phentolamine (1  m M). AdoCAsp 4  and R1 both inhibit contraction of vas deferenssmooth muscle and urinary bladder by electrical field stimulation, probably via a P2X 1  subtype(23, 24). Adrenergic and cholinergic components were removed by addition of phentolamineand atropine (0.3  m M) to the organ bath Krebs buffer. AdoCAsp 4  and R1 (300  m M) producedup to 85% and 63% inhibition (frequency-dependent) in vas deferens, respectively (Fig 1),while the effect was weaker in bladder, up to 40% and 30%, respectively (Figs 2a and 2b). a , b -MethyleneATP-evoked contraction was not comparably inhibited, neither was that byATP, NA (vas deferens), ACh (bladder), histamine or KCl. Further experiments investigatedinvolvement of presynaptic P 1 -purinergic adenosine receptors. Results suggest a presynaptic 366  Vol. 229, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS FIG. 2.  Concentration-dependent inhibition by AdoCAsp 4  (10–300  m M) and R1 (10–100  m M) of guinea pigurinary bladder smooth muscle contraction evoked by electrical field stimulation (0.5 ms, 100 V, 1–32 Hz) in thepresence of 0.3  m M atropine and 1  m M phentolamine. action, but no effect on transmitter release, rather, they suggest a nerve-blocking local anaes-thetic-like effect. Consequently, further studies should include analysis of ion channel activity.The activity of AdoCAsp 4  was further analyzed for pre- and postjunctional activities inguineapigurinarybladder, inthepresenceof atropine, ablocker of themuscarinicacetylcholinereceptor. Activation of the nicotinic receptor results in purinergic nerve stimulation, transmitterrelease and contraction of the smooth muscle preparation. The contractile responses to bothcarbachol and nicotine were reduced by 35% at 100  m M AdoCAsp 4  (n Å 2; 40  m M, n Å 1),indicating that AdoCAsp 4  is either a weak antagonist of the postjunctional purinoceptor, or of a prejunctional nicotinic receptor, or has non-specific effects directly on the smooth muscle,compromising its excitability.In  Xenopus  oocytes microinjected with recombinant P2X 1  cRNA, AdoCAsp 4  producedmixed results (Fig 3). The compound was not an agonist at the expressed P2X 1  purinoceptor.In contrast, its use as antagonist of ATP-induced currents yielded interesting results: atlow concentration (0.82  m M), AdoCAsp 4  potentiated ATP-mediated responses while, at8.2 m M, AdoCAsp 4  caused an irreversible antagonism that was accompanied by a significantdecrease in membrane stability and increased membrane conductivity. The increased con-ductance reversed with washout (after 1 hour) but not the responsiveness to ATP. Theseresults suggest that the blocking activity of this compound, also observed in organ prepara-tions of guinea pig bladder and vas deferens, was due to a destabilizing (and depolarizing)membrane effect of the compound. The irreversible blockade of ATP-mediated responses FIG. 3.  ATP-induced inward currents recorded under voltage clamp (V h  Å0 60 mV) from defolliculated  Xenopus oocytes microinjected with P2X 1  purinoceptor cRNA. ATP was superfused for approximately 30 s, every 20 min;AdoCAsp 4  was superfused for 20 min before addition of ATP (in the presence of AdoCAsp 4 ). The washout periodfor AdoCAsp4 was 1 h.367
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