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Biological properties of a new fluorescent biphalin fragment analogue
Life Sciences 70 (2002) 893–897
Biological properties of a new fluorescent biphalin fragment analogue Andrzej W. Lipkowskia,b,c, Aleksandra Misickaa,b,d, Dariusz Kossonb, Piotr Kossonb, Magdalena Lachwa-Fromb, Agnieszka Brodzik-Bienkowskab, Victor J. Hrubya,* a b
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA Medical Research Centre, Polish Academy of Sciences, Warsaw, PL 02106 c Industrial Chemistry Research Institute, Warsaw, PL 01793 d Department of Chemistry, Warsaw University, Warsaw, PL 02093 Received 6 February 2001; accepted 14 August 2001
Abstract Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH←X, where X5Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X5DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both m and d opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Analgesia; Opioid peptides; Fluorescence probe
Introduction The discovery of opioid peptides thirty years ago opened a new chapter in the study of pain formation and modulation. This discovery also created hope for new types of analgesics. However, after years of study, only a few peptide analogues are under study as potential analgesic drugs. Biphalin is one example of an opioid peptide analogue that is under development as an analgesic. Biphalin is a dimeric peptide [(Tyr-D-Ala-Gly-Phe-NH-)2] in which two enkephalin type pharmacophores are connected “head-to-head” by a hydrazide bridge [1]. It * Corresponding author. Tel.: 520-621-6332; fax: 520-621-8407. E-mail address: [email protected] (V.J. Hruby) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 6 7 -9
894
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
has high affinity to all three types (m, d and k) of opioid receptors [2,3]. When administered intravenously, it has antinociception potency comparable to morphine [4]. Administered intrathecally or intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception [4,5]. This in vivo data suggests good permeability of biphalin through the blood-brain barrier. Indeed, a study with [125I-Tyr1]-biphalin gave evidence that biphalin can reach brain and spinal sites to elicit antinociception [6]. All of these promising results have stimulated extensive structure-activity studies of the parent biphalin and its analogues [7–10]. Recent structure-activity study of biphalin shows that the full dimeric sequence is not required for high biological potency. Indeed, elimination of the tripeptide from one “arm” of biphalin does not reduce the biological potency significantly [11]. Lipophilic amino acids [11] or other lipophilic elements [12] could replace the residue phenylalanine. This has led us to the idea of replacing the phenylalanine with a strongly fluorescent moiety. Such a group can allow extensive studies of in vivo metabolism, permeability distribution, and other studies. Fluorescent peptides also are good tools for studies of the mechanisms of peptide-receptor complex formation [e.g. 13, 14]. This paper describes the synthesis and biological properties of a biphalin fragment analogue, AA2016 (3), in which the phenylalanine residue has been replaced with a dansyl (DNS) moiety (Figure 1). Materials and methods Synthesis The synthesis of Na-Boc-Tyr-D-Ala-Gly-Phe-NHNH2 was performed by the method described previously [11]. The Boc-protected tetrapeptide hydrazide was reacted with dansyl chloride in pyridine at room temperature overnight. After evaporation of the pyridine, the Na-Boc-protecting group was removed with trifluoroacetic acid. The product was purified by gel filtration, followed by preparative RP-HPLC. The final product was homogenous by TLC and HPLC analyses, with correct FAB-MS and amino acid analysis. The compound when activated with 254 nm UV light gives a yellow fluorescence. The absorption/emission spectrum is presented on Figure 2. In biological studies AA2016 was used as its triflouroacetate salt.
Fig. 1. Chemical structure of AA2016.
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
895
Fig. 2. Absorption/emission spectrum of AA2016 in water.
Receptor binding Receptor affinity to the m and d receptors was evaluated by competitive displacement of selective radioligands on brain homogenates as previously described [15]. The ligands used were [3H]DAMGO and [3H]Deltorphin II for m and d receptors, respectively. Antinociception assay All experimental procedures used in animal testing followed guidelines on the ethical standards for investigation of experimental pain in animals [16] and were approved by the Animal Research Committee of Medical Research Centre, Polish Academy of Sciences. The antinociceptive activity of peptide, using the tail flick assay was measured in male Wistar rats after intrathecal (i.t.) application. The antinociception has been presented as a percent of the maximal possible effect (%MPE) using 7 sec. as a cut-off time. The details of the protocol used have been published previously [7].
Table 1 Binding affinities of biphalin, its fragment and AA2016 Binding Ki (nM)* Compound 1 (Tyr-D-Ala-Gly-Phe-NH-)2 (Biphalin) 2 Phe-Tyr-D-Ala-Gly-Phe-NH-NH←Phe 3 Phe-Tyr-D-Ala-Gly-Phe-NH-NH←DNS (AA2016)
d
m
2.6** 15** 2.0
1.4** 0.74** 1.1
* The standard errors were less than 20% of the presented values, ** taken from [11].
896
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
Fig. 3. Antinociceptive effect of AA2016 after i.t. application in rats. Confidence limit p ,0.05 between: * particular group and placebo, and 1 between marked groups.
Results and discussion Similar to biphalin, the newly synthesized fragment analogue AA2016 (3) expressed high receptor binding affinity for the d and m receptor types (Table 1). Its affinity profile more closely corresponds to biphalin than the original fragment 2. The high affinity to opioid receptors fully correlated with its antinociceptive activity (Figure 3). Even at a dose of 0.2 nmol, the analogue produces strong (MPE.50%) antinociception similar to that reported for biphalin [6]. The antinociceptive effect is dose dependent. Increasing the dose to 0.5 nmol and over increases both the level and duration of the observed antinociception. An overdose of the compound, up to 1.0 nmol, produces reversible, long lasting antinociception, without any visible signs of side effects, such as rigidity or respiratory depression. In conclusion, we have designed a new peptide opioid analogue of biphalin with high opioid biological activity, similar to the parent biphalin. The presence of a fluorescent group makes this compound a potentially very useful tool for further pharmacokinetic and pharmacodynamic studies in vivo, as well as for detailed macromolecular studies of its interactions with opioid receptors. Acknowledgments We thank The National Institute of Drug Abuse, U.S. Public Health Service Grant DA 06284 and the Medical Research Centre of Polish Academy of Sciences for supporting this work. The views expressed here are those of the authors and do not necessarily reflect those of the USPHS.
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
897
References 1. Lipkowski AW, Konecka AM, Sroczynska I. Double-enkephalins - synthesis, activity on guinea-pig, and analgesic effect. Peptides 1982; 3:697–700. 2. Lipkowski AW, Konecka AM, Sroczynska I, Przewlocki R, Stala L, Tam SW. Bivalent opioid peptide analogues with reduced distances between pharmacophores. Life Sci. 1987; 40:2283–2288. 3. Slaninova J, Appleyard SA, Misicka A, Lipkowski AW, Knapp RJ, Weber SJ, Davis TP, Yamamura HI, Hruby VJ. [125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes. Life Sci. 1998; 62: PL199–204. 4. Silbert BS, Lipkowski AW, Cepeda MS, Szyfelbein SK, Osgood PF, Carr DB. Analgesic activity of a novel bivalent opioid peptide compared to morphine via different routes of administration. Agent Actions 1991; 33: 382–387. 5. Horan PJ, Mattie A, Bilsky EJ, Weber SJ, Davis TP, Yamamura HI, Malatynska E, Appleyard SM, Slaninova J, Misicka A, Lipkowski AW, Hruby VJ, Porreca F. Antinociceptive profile of biphalin, a dimeric enkephalin analogue. J. Pharmacol. Exp. Ther. 1993; 265:1446–1454. 6. Abbruscato TJ, Williams SA, Misicka A, Lipkowski AW, Hruby VJ, Davis TP. Blood-to central nervous system entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives. J. Pharmacol. Exp. Therap. 1996; 276:1049–1057. 7. Misterek K, Maszczynska I, Dorociak A, Gumulka SW, Carr DB, Szyfelbein SK, Lipkowski AW. Spinal co-administration of peptide substance P antagonist increase antinociceptive effect of the opioid peptide biphalin. Life Sci. 1994; 54:939–944. 8. Romanowski M, Zhu X, Ramaswami V, Misicka A, Lipkowski AW, Hruby VJ, O’Brien DF. Interaction of highly potent dimeric enkephalin analog, biphalin, with model membranes. Biochim. Biophys. Acta 1997; 1329:245–258. 9. Misicka A, Lipkowski AW, Horvath R, Davis P, Porreca F, Yamamura HI, Hruby, VJ. Structure-activity relationship of biphalin. The synthesis and biological activities of new analogues with modifications in positions 3 and 4. Life Sci. 1997; 60:1263–1269. 10. Li G, Haq W, Xiang L, Lou BS, Hughes R, Deleon IA, Davis P, Gillespie TJ, Romanowski M, Zhu X, Misicka A, Lipkowski AW, Porreca F, Davis TP, Yamamura HI, O’Brien D, Hruby VJ. Modifications of the 4,49-residues and SAR studies of biphalin, a highly potent opioid receptor active peptide. Bioorg. Med. Chem. Lett. 1998; 8:555–560. 11. Lipkowski AW, Misicka A, Davis P, Stropova D, Janders J, Lachwa M, Porreca F, Yamamura HI, Hruby VJ. Biological activity of fragments and analogues of the potent dimeric opioid peptide, biphalin. Bioorg. Med. Chem. Lett. 1999; 9:2763–2766. 12. Maszczynska I, Lipkowski AW, Carr DB, Kream RM. Alternative forms of interaction of substance P and opioids in nociceptive transmission. Lett. Peptide Sci. 1998; 5:395–398. 13. Turcatti G, Zoffmann S, Lowe JA, Drozda SE, Chassaing G, Schwartz TW, Chollet A. Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands. J. Biol. Chem. 1997; 272:21167–21175. 14. Gaudriault G, Nouel D, Farra CD, Beaudet A, Vincent, JP. Receptor-induced internalization of selective peptide mu and delta opioid ligands. J. Biol. Chem. 1997; 272:2880–2888. 15. Misicka A, Lipkowski AW, Horvath R, Davis P, Kramer TM, Yamamura HI, Hruby VJ. Topographical requirements for delta opioid ligands: common structural features of dermenkephalin and deltorphin. Life Sci. 1992; 51:1025–1032. 16. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 16:109–110.
Biological properties of a new fluorescent biphalin fragment analogue Andrzej W. Lipkowskia,b,c, Aleksandra Misickaa,b,d, Dariusz Kossonb, Piotr Kossonb, Magdalena Lachwa-Fromb, Agnieszka Brodzik-Bienkowskab, Victor J. Hrubya,* a b
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA Medical Research Centre, Polish Academy of Sciences, Warsaw, PL 02106 c Industrial Chemistry Research Institute, Warsaw, PL 01793 d Department of Chemistry, Warsaw University, Warsaw, PL 02093 Received 6 February 2001; accepted 14 August 2001
Abstract Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH←X, where X5Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X5DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both m and d opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Analgesia; Opioid peptides; Fluorescence probe
Introduction The discovery of opioid peptides thirty years ago opened a new chapter in the study of pain formation and modulation. This discovery also created hope for new types of analgesics. However, after years of study, only a few peptide analogues are under study as potential analgesic drugs. Biphalin is one example of an opioid peptide analogue that is under development as an analgesic. Biphalin is a dimeric peptide [(Tyr-D-Ala-Gly-Phe-NH-)2] in which two enkephalin type pharmacophores are connected “head-to-head” by a hydrazide bridge [1]. It * Corresponding author. Tel.: 520-621-6332; fax: 520-621-8407. E-mail address: [email protected] (V.J. Hruby) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 6 7 -9
894
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
has high affinity to all three types (m, d and k) of opioid receptors [2,3]. When administered intravenously, it has antinociception potency comparable to morphine [4]. Administered intrathecally or intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception [4,5]. This in vivo data suggests good permeability of biphalin through the blood-brain barrier. Indeed, a study with [125I-Tyr1]-biphalin gave evidence that biphalin can reach brain and spinal sites to elicit antinociception [6]. All of these promising results have stimulated extensive structure-activity studies of the parent biphalin and its analogues [7–10]. Recent structure-activity study of biphalin shows that the full dimeric sequence is not required for high biological potency. Indeed, elimination of the tripeptide from one “arm” of biphalin does not reduce the biological potency significantly [11]. Lipophilic amino acids [11] or other lipophilic elements [12] could replace the residue phenylalanine. This has led us to the idea of replacing the phenylalanine with a strongly fluorescent moiety. Such a group can allow extensive studies of in vivo metabolism, permeability distribution, and other studies. Fluorescent peptides also are good tools for studies of the mechanisms of peptide-receptor complex formation [e.g. 13, 14]. This paper describes the synthesis and biological properties of a biphalin fragment analogue, AA2016 (3), in which the phenylalanine residue has been replaced with a dansyl (DNS) moiety (Figure 1). Materials and methods Synthesis The synthesis of Na-Boc-Tyr-D-Ala-Gly-Phe-NHNH2 was performed by the method described previously [11]. The Boc-protected tetrapeptide hydrazide was reacted with dansyl chloride in pyridine at room temperature overnight. After evaporation of the pyridine, the Na-Boc-protecting group was removed with trifluoroacetic acid. The product was purified by gel filtration, followed by preparative RP-HPLC. The final product was homogenous by TLC and HPLC analyses, with correct FAB-MS and amino acid analysis. The compound when activated with 254 nm UV light gives a yellow fluorescence. The absorption/emission spectrum is presented on Figure 2. In biological studies AA2016 was used as its triflouroacetate salt.
Fig. 1. Chemical structure of AA2016.
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
895
Fig. 2. Absorption/emission spectrum of AA2016 in water.
Receptor binding Receptor affinity to the m and d receptors was evaluated by competitive displacement of selective radioligands on brain homogenates as previously described [15]. The ligands used were [3H]DAMGO and [3H]Deltorphin II for m and d receptors, respectively. Antinociception assay All experimental procedures used in animal testing followed guidelines on the ethical standards for investigation of experimental pain in animals [16] and were approved by the Animal Research Committee of Medical Research Centre, Polish Academy of Sciences. The antinociceptive activity of peptide, using the tail flick assay was measured in male Wistar rats after intrathecal (i.t.) application. The antinociception has been presented as a percent of the maximal possible effect (%MPE) using 7 sec. as a cut-off time. The details of the protocol used have been published previously [7].
Table 1 Binding affinities of biphalin, its fragment and AA2016 Binding Ki (nM)* Compound 1 (Tyr-D-Ala-Gly-Phe-NH-)2 (Biphalin) 2 Phe-Tyr-D-Ala-Gly-Phe-NH-NH←Phe 3 Phe-Tyr-D-Ala-Gly-Phe-NH-NH←DNS (AA2016)
d
m
2.6** 15** 2.0
1.4** 0.74** 1.1
* The standard errors were less than 20% of the presented values, ** taken from [11].
896
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
Fig. 3. Antinociceptive effect of AA2016 after i.t. application in rats. Confidence limit p ,0.05 between: * particular group and placebo, and 1 between marked groups.
Results and discussion Similar to biphalin, the newly synthesized fragment analogue AA2016 (3) expressed high receptor binding affinity for the d and m receptor types (Table 1). Its affinity profile more closely corresponds to biphalin than the original fragment 2. The high affinity to opioid receptors fully correlated with its antinociceptive activity (Figure 3). Even at a dose of 0.2 nmol, the analogue produces strong (MPE.50%) antinociception similar to that reported for biphalin [6]. The antinociceptive effect is dose dependent. Increasing the dose to 0.5 nmol and over increases both the level and duration of the observed antinociception. An overdose of the compound, up to 1.0 nmol, produces reversible, long lasting antinociception, without any visible signs of side effects, such as rigidity or respiratory depression. In conclusion, we have designed a new peptide opioid analogue of biphalin with high opioid biological activity, similar to the parent biphalin. The presence of a fluorescent group makes this compound a potentially very useful tool for further pharmacokinetic and pharmacodynamic studies in vivo, as well as for detailed macromolecular studies of its interactions with opioid receptors. Acknowledgments We thank The National Institute of Drug Abuse, U.S. Public Health Service Grant DA 06284 and the Medical Research Centre of Polish Academy of Sciences for supporting this work. The views expressed here are those of the authors and do not necessarily reflect those of the USPHS.
A.W. Lipkowski et al. / Life Sciences 70 (2002) 893–897
897
References 1. Lipkowski AW, Konecka AM, Sroczynska I. Double-enkephalins - synthesis, activity on guinea-pig, and analgesic effect. Peptides 1982; 3:697–700. 2. Lipkowski AW, Konecka AM, Sroczynska I, Przewlocki R, Stala L, Tam SW. Bivalent opioid peptide analogues with reduced distances between pharmacophores. Life Sci. 1987; 40:2283–2288. 3. Slaninova J, Appleyard SA, Misicka A, Lipkowski AW, Knapp RJ, Weber SJ, Davis TP, Yamamura HI, Hruby VJ. [125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes. Life Sci. 1998; 62: PL199–204. 4. Silbert BS, Lipkowski AW, Cepeda MS, Szyfelbein SK, Osgood PF, Carr DB. Analgesic activity of a novel bivalent opioid peptide compared to morphine via different routes of administration. Agent Actions 1991; 33: 382–387. 5. Horan PJ, Mattie A, Bilsky EJ, Weber SJ, Davis TP, Yamamura HI, Malatynska E, Appleyard SM, Slaninova J, Misicka A, Lipkowski AW, Hruby VJ, Porreca F. Antinociceptive profile of biphalin, a dimeric enkephalin analogue. J. Pharmacol. Exp. Ther. 1993; 265:1446–1454. 6. Abbruscato TJ, Williams SA, Misicka A, Lipkowski AW, Hruby VJ, Davis TP. Blood-to central nervous system entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives. J. Pharmacol. Exp. Therap. 1996; 276:1049–1057. 7. Misterek K, Maszczynska I, Dorociak A, Gumulka SW, Carr DB, Szyfelbein SK, Lipkowski AW. Spinal co-administration of peptide substance P antagonist increase antinociceptive effect of the opioid peptide biphalin. Life Sci. 1994; 54:939–944. 8. Romanowski M, Zhu X, Ramaswami V, Misicka A, Lipkowski AW, Hruby VJ, O’Brien DF. Interaction of highly potent dimeric enkephalin analog, biphalin, with model membranes. Biochim. Biophys. Acta 1997; 1329:245–258. 9. Misicka A, Lipkowski AW, Horvath R, Davis P, Porreca F, Yamamura HI, Hruby, VJ. Structure-activity relationship of biphalin. The synthesis and biological activities of new analogues with modifications in positions 3 and 4. Life Sci. 1997; 60:1263–1269. 10. Li G, Haq W, Xiang L, Lou BS, Hughes R, Deleon IA, Davis P, Gillespie TJ, Romanowski M, Zhu X, Misicka A, Lipkowski AW, Porreca F, Davis TP, Yamamura HI, O’Brien D, Hruby VJ. Modifications of the 4,49-residues and SAR studies of biphalin, a highly potent opioid receptor active peptide. Bioorg. Med. Chem. Lett. 1998; 8:555–560. 11. Lipkowski AW, Misicka A, Davis P, Stropova D, Janders J, Lachwa M, Porreca F, Yamamura HI, Hruby VJ. Biological activity of fragments and analogues of the potent dimeric opioid peptide, biphalin. Bioorg. Med. Chem. Lett. 1999; 9:2763–2766. 12. Maszczynska I, Lipkowski AW, Carr DB, Kream RM. Alternative forms of interaction of substance P and opioids in nociceptive transmission. Lett. Peptide Sci. 1998; 5:395–398. 13. Turcatti G, Zoffmann S, Lowe JA, Drozda SE, Chassaing G, Schwartz TW, Chollet A. Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands. J. Biol. Chem. 1997; 272:21167–21175. 14. Gaudriault G, Nouel D, Farra CD, Beaudet A, Vincent, JP. Receptor-induced internalization of selective peptide mu and delta opioid ligands. J. Biol. Chem. 1997; 272:2880–2888. 15. Misicka A, Lipkowski AW, Horvath R, Davis P, Kramer TM, Yamamura HI, Hruby VJ. Topographical requirements for delta opioid ligands: common structural features of dermenkephalin and deltorphin. Life Sci. 1992; 51:1025–1032. 16. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 16:109–110.