SEMMELWEIS UNIVERSITY

Doctoral School of Pharmaceutical and Pharmacological Sciences

NATURAL OPIOID PEPTIDES, SYNTHETIC ANALOGS, PEPTIDASES: PHARMACOLOGICAL ANALYSIS AND DRUG DESIGN

by András Z. Rónai

Ph.D. Dissertation

Supervisor: Prof. Dr. Kálmán Magyar, D.Sc. Member of Hungarian Academy of Sciences

Department of Pharmacology and Pharmacotherapy Semmelweis University, Budapest

Budapest, 2004

1

To Sándor Bajusz

2 CONTENTS 1. Introduction 2. List of abbreviations 3. Results and Conclusions 3.1. Chapter 1. Critical overview of the use of isolated organs in opioid research 3.2. Chapter 2. Pharmacological characterisation of enkephalins, b-endorphin and its fragments 3.3. Chapter 3. Development of potent analgesic enkephalin analogs such as (D-Met2,Pro5) -enkephalinamide 3.4. Chapter 4. Characterisation of structural principles for the receptor type-selectivity of enkephalin analogs 3.5. Chapter 5. Detection of a novel dipeptidyl-carboxypeptidase in rodent blood vessel and human adrenomedullary tumors 3.6. Chapter 6. Design and pharmacological characterisation of novel, pure and highly d -opioid receptor type-selective and k-opioid receptor type.selective peptide antagonists: the principle of N-acylation. 3.7. Chapter 7. Present and future: endomorphins, nociceptin. 4. General summary and conclusions 5. Acknowledgement 6. References 7. Patents 8. Appendix.

3 1. Introduction The cornerstone of opioid peptide history was the discovery of enkephalins (Tyr-Gly-Gly- Phe-Met, (Met5) enkephalin and Tyr-Gly-Gly-Phe-Leu, (Leu5)-enkephalin) in porcine brain by Hughes, Kosterlitz and their coworkers in 1975 (53). The history appeared to obey the principle of receptor theory declaring that a receptor should have one or more endogenous ligand(s). The first opioid receptor model has been set forth by Beckett and Casy (11). The direct evidence for the existence of opioid receptors (i.e. the accomplishment of receptor binding assay) has been supplied simultaneously and independently by three teams (97, 142, 151). Soon after the the discovery of enkephalins the heterogeneity of opioid receptors, i.e. the existence of multiple receptor types has also been proved and the main pharmacological properties of receptor types (m, k and d as major ones and also, tentatively, s) have also been documented (33, 77, 81). Since the (Met5)-enkephalin sequence is present in a pituitary hormone, b-lipotropin (LPH(1-91), b-LPH), between sequence positions 61-65, the attention has been focussed on this peptide hormone. b-LPH is the biological source of several natural opioid sequences, each bearing the Tyr-Gly-Gly-Phe-Met motif at the N-terminus (a-, b- and g-endorphins) but not of enkephalins. Of these active sequences, the untriakontapeptide b-endorphin (LPH(61- 91), b-EP) is the most important (15, 44, 76). A third distinct opioid peptide family, based on the N-terminal Tyr-Gly-Gly-Phe-Leu motif, the dynorphin-neo-endorpin group has also been recognized by the late seventies (35, 57). The large molecular-weight precursors of these three opioid peptide families, pre-proopio- melanocortin for endorphins, pre-proenkephalin for enkephalin-related peptides and pre- prodynorphin have duly been identified in the early eighties (23, 56, 87). In the same period (late seventies-early eighties) two entirely novel natural opioid peptide groups have been recognized. One is referred to as the family of Tyr-Pro-peptides; in this group remarkable opioid agonist activity was present if the N-terminal dipeptide motif was followed by aromatic amino acid. Casomorphin-related peptides, hemorphins and cytochrophins fell strictly into this family (16-18) with a-gliadorphin (37) and Tyr-MIF-1/Tyr-W-MIF-1 (26, 59) at the "outskirts" of assembly. The other group, under the heading of "amphibian skin opioid peptides", was heralded by the discovery of dermorphin (27); The characteristic N- terminal motif in this peptide (and the ones to follow) was Tyr-D-Ala/D-Met-Phe--. The former family was "crowned" by the discovery of endomorphins in 1997 (158) whereas the latter gave rise to dermenkephalin and deltorphins (28). The primary structures of all the three

4 major types (i.e. d, m and k) of opioid receptor proteins were deduced between 1992 and 1994 (29, 62; for review see 155). This discovery gave further impetus to the analyses directed at ligand binding, receptor heterogeneity and signal transduction (e.g. 100, 159). My experiments were carried out in two periods, between 1976 and 1984 and 1988 and 2001, with a distinct and painful "silent gap" between 1984 and 1988. There was a chrononological overlap between some of the thematically distinct projects but, occasionally, several issues were re-instated after a dormant period of 8-10 years. Between 1976 and 1981, the work was done at the Institute of Drug Research, in cooperation with preparative biochemists (Groupleader L. Gráf) , peptide synthetic chemists (Groupleader S. Bajusz) and behavioral pharmacologists (Groupleader J.I. Székely). My first task, being the Groupleader of "In vitro pharmacological unit" was to characterize the pharmacological properties of (Met5)-enkephalin, b-endorphin and its fragments (Chapter 1). This entailed of finding a proper assembly of isolated organs and experimental paradigms suitable for differentiating pharmacologically between morphine-like non-peptidic substances and opioid peptides and also between opioid peptides themselves (Chapter 2). The next tasks were to give logistic support to the design of analgesic enkephalin analogs (Chapter 3), to characterize the structural principles in enkephalin analogs responsible for receptor type selection and to find the "thumb rules" of correlating in vitro pharmacological properties with analgesia (Chapter 4). Among the potential factors affecting the in vivo effectiveness of enkephalin analogs, one had to consider their increased stability against biodegradation. Natural enkephalins and some other pro-enkephalin derived natural peptides are rapidly destroyed by various peptidases. While studying these aspects, I became aware of a novel peptidase activity in some rodent and human tissues (Chapter 5); these pursuits took place between 1981-1983 and 1990- 1997,.respectively. It is a perpetual challange in receptor chemistry/pharmacology to design competitive, receptor type/subtype-selective antagonist when the structure and pharmacological profile of natural and synthetic agonist ligand sets are known (Chapter 6). This chapter is an etude of pharmacologist with the additional curiosity that, by lacking a better candidate for the task, the drug design had to be done by myself in the case of d-opioid receptor antagonist peptide analogs and, in part, also in the case of k-opioid receptor antagonist peptides. To relate drug structure to pharmacological activities the knowledge of conformational properties is an important issue. In the case of b-LPH-related peptides the conformational analyses were organised by the biochemist (39, 40), in the case of (D- Met2,Pro5)-enkephalinamide by the synthetic chemist (49) whereas for some other sets of

5 analogs I made the structured proposal for the conformational analysis/modelling (50, 113, 145). Each project gave a conclusive and well-defined result but never the achievement to my real satisfaction. I consider them as "truncated" projects mostly because the lack of proper funding; several sprouts are still open to further cultivation. In this context, I wish to quote the proverbial sentence attributed to Professor W.D.M. Paton: "My task is to invent an original idea and prove that it is worthy of attention. All the other details may be done by others".

2. List of abbreviations Note: all abbreviations are written out in full when they are first mentioned in the text For specifying primary structures of peptedes, either the three-letter or the single-letter conventional amino acid codes are used. Ac= acetyl ACE= angiotensin-converting enzyme, EC 3.4.15.1 All= allyl ART= isolated, perfused rabbit ear artery AT-I=angiotensin-I AT-II= angiotensin-II ß-EP= b-endorphin, porcine, b-lipotropin-(61-91) (unless otherwise indicated, purified from porcine pituitary extract BK= bradykinin BOC or t-BOC= tertiary butoxycarbonyl CD= circular dichroism CNM= isolated, field-stimulated cat nictitating membrane (medial part) CNS= central nervous system DADLE= (D-Ala2,D-Leu5)-enkephalin DAMGO= (D-Ala2,MePhe4,Gly5-ol)-enkephalin DPDP= (D-Pen2,D-Pen5)-enkephalin EDTA= ethylenediamine-tetraacetic acid EKC= ±ethylketocyclazocine For= formyl FT-IR= Fourier-transform infrared GPI= isolated longitudinal muscle strip-Auerbach plexus of guinea-pig ileum

6 HFR= hippuryl-phenylalanyl-arginine HHL= hippuryl-histidyl-leucine HRF= hippuryl-argynyl-phenylalanine HYR= hippuryl-tyrosyl-arginine

IC50= 50% inhibitory concentration icv= intracerbroventricular (third ventricle, lateral horn) injection

Ke= equilibrium dissociation constant LPH= b-lipotropin-(1-91), porcine (unless otherwise indicated, purified from porcine pituitary extract) LVD= rabbit vas deferens ME= (Met5)-enkephalin ME-RF= (Met5)-enkephalin-Arg6,Phe7 MVD= isolated mouse vas deferens NEP= neutral endopeptidase, EC 3.4.24.11 PD-117 302=(±)-trans-N-methyl-N,N-[2-(l-pyrrolidinyl)-cyclohexyl]benzo[b]thiophene-4 -acetamide monohydrochloride Piv= pivaloyl POMC= pro-opiomelanocortin Prop= propionyl RVD= isolated rat vas deferens t-Bu= tertiary butyl TIPP= Tyr-Tic-Phe-Phe

3.Results and Conclusions 3.1. Chapter 1. Critical overview of the use of isolated organs in opioid research The isolated organs used in opioid research are autonomically innervated nerve-smooth muscle preparations. Opioid receptors are confined almost exclusively to neural elements in these organs (for review, see 75). They belong to G protein-couped receptors, characterized by seven transmembrane helical pattern. Owing to the signal transduction properties (opening of K+ channels, inhibition of transmembrane Ca2+-fluxes, less characteristically, increasing intracellular free Ca2+, inhibition of adenylyl cyclase, stimulation of MAPK , see 140) the characteristic action of opioid receptor agonists in isolated organs is to inhibit the nerve

7 stimulation-induced release of neurotransmitters without affecting smooth muscle function directly. Because the electrical excitability of neural elements and of smooth muscle differ considerably, by conveniently chosen field stimulation parameters one can stimulate nerves with good selectivity; of course, one must check the extent of selectivity when introducing an entirely novel preparation. From opioid point of view (and this principle is valid also for any other receptor) an isolated organ is characterized by its receptor type supply and by the proportion of spare receptors (for definition of terms see 88). In spite of the fact that these preparations are used in opioid research since the mid-fifties (most active period beeing the seventies and mid- eighties) the satisfactory characterization of the abovementioned aspects was accomplished only by the late eighties (85). To characterise a novel opioid agonist, the first strategic step is to characterise its dose- dependent inhibitory action in one or more, opioid receptor-containing isolated organ(s). This is done by the construction of dose-response ([E]/[A]) curves. Technically, since the unmanipulated curve is a rectangular hyperbola, the simplest way of convenience is a semilogarithmic plot, and applying a logarithmic regression to the mid-portion of resulting sigmoidal curve. The important parameters of curve are its left-to right position along the abscissa (characteristic of agonist potency), the maximum along the ordinate (characteristic of the extent an agonist is able to generate an effect, effectiveness or, if specified, efficacy) and the slope parameter. Logarithmic regression is of good accuracy to determine the left-to right position (characterised most conveniently by the agonist concentration producing a given inhibitory effect, generally 50%, i.e. the IC50) and the slope. Recently we use curve fitting by the Hill equation (71, 72, 105) which is the most recommended route of parameter acquisition (88Neubig et al., 2003). The next step is to characterise the opioid receptor type whereby the agonist acts. This is done by determining the quantitative aspects of interaction between a competitive receptor antagonist of known receptor type selectivity profile and the agonist in question. The characteristic parameter calculated for the antagonist is the equilibrium dissociation constant, Ke or its negative logarithm, pA2 (4, 70, 75, 88). Ke is inversely whereas pA2 directly related to the antagonist affinity and, in case of antagonists with a certain receptor type selectivity, each antagonist-receptor type pair can be characterised by a given antagonist affinity range. From the beginning, I used naltrexone (106) or, occasionally, naloxone, for this purpose; both opioid antagonists have an approximately 20-fold affinity preference for the m-opioid receptors (where, by giving a liberal width, the Ke of naltrexone is

8 characterised by the 0.2-0.8 nM range) as compared to d or k. With the availability of d- opioid receptor type-selective antagonists naltrindole and naltriben (98, 99) and our own peptide antagonists, the BOC-Tyr-Pro-Gly-Phe-Leu-Thr-peptides (113-115, 125-127, see Chapter 6) and the k-opioid receptor antagonist nor-binaltorphimine (98) I used also these compounds to characterize opioid agonist receptor type preferences and receptor type sets in isolated organs (46, 80). Besides charcterizing these two agonist properties (i.e. potency and receptor type preference) one can also characterize the property of an agonist referred to as full or partial agonism (2). For characterising a novel antagonist candidate the same parameters of agonist-antagonist interaction should be determined but, in this case, one should use agonists of known selectivity for one or the other receptor type. As a m-opioid receptor preferrimg/selective agonist I used first normorphine then (D-Ala2,MePhe4,Gly5-ol)-enkephalin (DAMGO). The d- opioid receptor agonists used were (Met5)-enkephalin first, followed by (D-Ala2,D-Leu5)- enkephalin (DADLE), (D-Pen2,D-Pen5)-enkephalin (DPDP), occasionally deltorphin-II (46, 80, 92-94, 113-115, 125-127). The k-opioid receptor agonists were the mixed k/m agonist benzomorphan, ±ethylketocyclazocine (EKC)(80, 113-115, 122, 125-127), later the pure k agonist benzothiophene-acetamide, PD-117 302 (80, 126). From transmitter point of view GPI is "cholinergic", all vasa deferentia (mouse, rat, rabbit) and also rabbit ear artery and cat nictitating membrane are "noradrenergic", with purinergic (e.g. ATP) and peptidergic (e.g. neuropeptide Y, NPY) co-transmission (58, 61, 79; for review see 75). Longitudinal muscle strip-Auerbach plexus of guinea-pig ileum I used the method devised originally by Paton and Vizi (94) without any major modification (e.g. 105, 118). The reason why this preparation is still a standard one in opioid testing battery stems from early opioid history. When relating the agonist potency of a number of natural, semisynthetic and synthetic opiates in GPI to the experimental and clinical analgesic potency, a good correlation has been reported (67, 69)(except for pro-drugs, of course). During my postgraduate training in Professor F.F. Foldes' laboratory (Monefiore Hospital & Albert Einstein College, N.Y., 1974-1975), I had the opportunity of testing besides conventional opiates a number of novel morphinone-, morphinan- and benzomorphan- derivatives being currently tested in a clinical setting, and the correlation for our data set was also significant (117, Rónai and Foldes, unpublished).

9 The opioid receptor type supply of GPI is characterized by the presence of m- and k-opioid receptors with high receptor reserve (i.e. high"spare" receptor contingent) and the absence of functional d receptors (77, 86, for review see 75). Mouse vas deferens The preparation has been devised at the Aberdeen' team (48) and they used it to advantage in their efforts which eventually led to the discovery of enkephalins (52, 53). However, the original procedure had a number of cumbersome elements which were disadvantageous for the routine use of preparation in pharmacological research, especially in the analyses of natural opioid peptides. They prepared the vasa pairwise, connected by a tissue rich in enzymes and biologiacally activce substances such as prostanoids (prostate, seminal vesicle pieces). To avoid these disadvantages, I modified the method at several points (118). I prepared a single vas, carefully cleaned by "stripping". This modification necessitated the application of new parameters in field stimulation in order to obtain steady contractile responses. I used paired rectangular pulses with 100 ms pulse distance (and 1 ms pulse width with supramaximal voltage drop) and repeated this cycle by 10 s. Using a single pulse, paired pulses or even a short train with three pulses (with identical individual pulse parameters) yielded similar sensitivity of preparation to the inhibitory effect of opioid agonists. Presently, the majority of pharmacologists use this or a similar procedure for the experimentation with MVD. The special value of MVD in the early periods of opioid peptide research was the presence of a receptor type preferentially responsive to (Met5)-enkephalin; this type, after the organ, has been designated as d (77). The Aberdeen'team, as an authirity on the issue, overemphasized the dominance of d, and rather neglected the contribution of m- and k-opioid recceptor types in the action of agonists in this preparation. As a matter of fact, the proportion of these receptor types has turned out to be highly strain-dependent in this organ (14, 150) and also the spare receptor contingents for the respective types was different (85). The strains used by my teams (mainly CFLP, occasionally OFA and recently, NMRI) could be characterized by d receptors with high receptor reserve and fewer m- and k-opioid receptors (2, 80) By using routinely the MVD besides the GPI in characterising the opioid properties of novel enkephalin analogs supplemented by determining the antagonism by naltrexone, I could detect the changes in the opioid character of synthetic substances as compared to the parent natural peptide enkephalin (9, 10, 105-109, 131, 149).

10 Rat vas deferens (RVD) The organ is not responsive to the agonist effects of morphine and related opiates but has been described to contain an opioid receptor set snsitive to the agonism by b-endorphin (73, 137, 157). This distinctive profile prompted Wüster and his coworkers (157) to propose the existence of a novel receptor type in this preparatrion, designated as e.. However, some benzomorphans (34) and also morphine (110) still retain affinity to these receptors as attested by their opioid antagonist action in RVD. Since, according to these reports, RVD had the potential of offering novel information about the properties of opioid peptides I have also analysed the effect of b-endorphin, several enkephalin analogs and non-peptide opioids in this preparation (110, 111). I could confirm the prominent, naltrexone-reversible agonism by b-EP. When correlating the order of agonist potencies of some 16 enkephalin analogs found in RVD with the same order in GPI and MVD, a significant correlation was found between RVD and GPI but not between RVD and MVD but the absolute potencies in RVD were considerably lower in RVD than either in GPI or MVD (111). The affinity of naltrexone determined against b-EP and a potent analgesic enkephalin analog, (D-Met2,Pro5)-enkephalinamide (8, 146, see Chapter 3) was found similar to but not quite identical with the affinity range characteristic of a m-opioid rceptorial interaction (110, 111). Besides naltrexone, morphine was also a competitive antagonist of the enkephalin analog (110). In the early postnatal period (between days 12-30) a significant change takes place in the opioid mechanisms of RVD (110,111). There is an indication that the maturation of neural mechanisms is completed in this phase (110) and the CNS opioid receptors in rats also undergo a change in this period. The change is characterized in RVD by a gradual reduction of agonist efficacy without a sinilar change in antagonist affinty. As a result, by the end normorphine (a weak agonist at day 12) becomes an opioid antagonist and (D-Met2,Pro5)- enkephalinamide a partial agonist whereas b-EP still retains the character of a full agonist (110, 111). At present, the majority of experts accept the opinion that RVD contains dominantly a m- opioid receptor-like receptor type with very low receptor reserve (144, for review see 75) and deny the existence of special receptors for b-EP. I should add that the endogenous opioid b- EP must have a high intrisic effeicacy at m-opioid receptors. The preparation has not been enlisted in our routine opioid testing battery,

11 Rabbit vas deferens (LVD) Oka and his coworkers have reported the presence of predominantly k-opioid receptor type (90) in LVD. With minor methodological changes, I used the preparation for testing the actions of EKC and PD- 117 302 as reference agonists , the effect of (Met5)- enkephalin.Arg6,Phe7 (ME-RF) a proenkephalin-derived endogenous opioid agonist (5), and also an alleged ligand prototype of a novel k-opioid receptor subtype (156) and for the characterisation of one of our novel, k-opioid receptor-selective peptide antagonists (80, 92, 93, 129). Cat nictitating membrane This autonomically innervated "noradrenergic" isolated organ has long been known as one responsive to the presynaptic inhibitory action of opiate agonists (152). Because it held the promise of investigating the interaction between opioid and dopaminergic mechanisms (54), I sought to characterise its opioid receptor supply with particular attention to the action of enkephalin analogs (105, 106). From determining the parameters of agonism for normorphine, b-EP and a number of enkephalin analogs and also the parameters of antagonism by naltrexone, besides the dominant m-like opioid receptors, the presence of d-like receptors could also be substantiated. Because the preparation technically is not an easy one and has unfavourable diffusion properties and offered no advantages as compared to the other opioid receptor-containing isaolated organs, it was excluded from further experimentation. Rabbit ear artery (ART) The preparation has been reported insensitive to the inhibitory acton of morphine but responsive to the presynaptic inhibitiry effect of (Met5)-emkephalin (63). However, thge antagonism of this presynaptic inhibition by opioid receptor antagonists has not been investigated, therefore, the the "opioid" designation for these receptors could not be declared. By modifying the original method for the preparation of ART (23) and also the parameters of experimentation (102, 103, 105, 107, 116, 122, 129, 130) the organ was rendered highly suitable for the reproducible testing of opioid agonist actions and presynaptic inhibition by other receptorial agents. I could confirm the absence of m-like opioid receptors in ART (107); apart from this, the opioid receptor supply seemed to resemble to the one found in MVD (105, 129). It means the presence of dominantly d-like opioid receptors besides the later demonstrated k-like opioid receptor type (80, 122, 129).

12 q From the point of view of my experimentation, the preparation became prominently important because it was the one where I first detected the novel dipeptidyl- carboxypeptidase enzyme activity converting (Met5)-enkephain-Arg6,Phe7 to (Met5)- enkephalin (83, 116, 129, 130, see Chapter 5).

Summary and conclusions q I conducted exploratory analyses in six, opioid receptor-containing isolated organs, in field-stimulated longitudinal muscle strip-Auerbach plexus of guinea-pig ileum (GPI), mouse (MVD), rat (RVD) and rabbit (LVD) vas deferens, cat nictitating membrane medial part (CNM) and perfused rabbit ear artery (ART). q Significant, innovative methodological changes were made in the case of MVD and ART. For the other preparations the originally described methods were installed with only minor modifications q The consensus opioid receptor types, defined in the same period (1976- 1977) when my exploratory experiments were performed (1976-1978) were µ, d and ?. To characterise the opioid receptor supply of isolated organs I used normorphine then DAMGO as µ-opioid receptor type preferring/selective agonists, (Met5)-enkephalin then DADLE, DPDP and deltorphin II as d-receptor preferring/selective agonists and EKC and PD-117 302 as ?/µ- and ?-receptor agonists, respectively. Of the competitive opioid antagonists initially the moderately µ receptor type- preferring naltrexone, later type-selective antagonists were used. q For the routine analyses of novel enkephalin analogs and antagonist candidates I used the MVD where all the three major opioid receptor types are present. For comparative purpose, I used regularly also the GPI, characterised by µ and ? receptor supply. q I have demonstrated the presence of d and ? opioid receptors in ART. Otherwise, this preparation was used mainly to prove the existence of a novel peptidase (Chapter 5).

13 q I have studied the postnatal development of opioid receptors in RVD. I have confirmed the potent agonism by ß-endorphin in this preparation. I found the receptors similar to but not quite identical with the µ type. The postnatal changes are characterised by a gradual loss of mechanism(s) responsible for the efficacy component of agonist action. q I have used LVD (characterised by dominantly ? receptors) occasionally and decided to exclude CNM from the testing battery. q For further, detailed analyses one may select a preparation with a single/dominant receptor type (RVD for m-like, LVD for k, and hamster vas deferens (82)for d. If partial agonism at a given receptor type is the issue to be investigated, high or low receptor reserve may also be a matter of consideration; alternatively, partial receptor pool inactivation strategy can also be used for this purpose (2).

3.2. Chapter 2. Pharmacological characterisation of enkephalins,

b- endorphin and its fragments Initially I have used just two opioid receptor-containing isolated organs for testing the opioid activities of natural opioid peptides. One of them, the field-stimulated longitudinal muscle strip-Auerbach plexus of guinea-pig ileum (GPI, 95) has long been used for the in vitro analysis of morphine and semisynthetic-synthetic opiates (67, 69). The other, the field- stimulated mouse vas deferens (MVD, 48) played a pivotal role in the discovery of enkephalins (52, 53). The neural elements of these organs are supplied by (mostly) presynaptic, inhibitory opioid receptors, therefore, opioid agonists are characterised by an inhibitory effect in these organs, specifically antagonised by opioid receptor antagonists (for technical details see Chapter 2). Synthetic (Met5)-enkephalin and the synthetic derivative (Nle5)-enkephalin (similar to natural (Leu5)-enkephalin) are 15-30 times more potent inhibitors in MVD than in GPI, the

50% inhibitory concentrations (IC50) being in the 4-20 nanomolar (nM) range in the former whereas in the 100-400 nM range in the latter (7, 107, 118). Intact porcine b-LPH is ineffective in GPI up to the concentration of 2x10-6 M while the complete tryptic digest of

14 peptide exerts an inhibitory action reversible by the opioid antagonist naloxone (43). Considering the cleaving site-specificty of trypsine, LPH-(61-69) could be suspected as the fragment responsible for the opioid agonist activity, where the first five amino acids are identical with the (Met5)-enkephalin sequence; indeed, the fragment was duly indentified in the digest by peptide analytical methods. No other fragments present in the digest had any opioid agonist activity (Rónai and Berzétei, unpublished). I compared systematically the in vitro opioid activities of synthetic (Met5)-enkephalin, LPH-(61-69) fragment, LPH-(61-79)-and LPH-(61-91)-peptide (b-endorphin) (118). Using MVD and GPI as assay systems, LPH-(61-69) fragment and LPH-(61-79)-peptide appeared to follow the pattern characteristic of (Met5)-enkephalin, i.e. they were more potent agonists in MVD than in GPI whereas b-EP was approximately equipotent in GPI and MVD (Fig. 1, 105, 107, 118). By studying the quantitative aspects of opioid peptide antagonism by naltrexone (for technical details see Chapter 2), it appeared that in GPI (Met5)-enkephalin and b-EP acted mainly or exclusively on a similar receptor type as the opiate normorphine whereas in MVD the receptors for normorphine and the opioid peptides were different (105, 106). In the meantime the field-stimulated rat vas deferens (RVD) has been reported to contain specific opioid receptors (tentatively designated as e type) for b-EP (73, 137, 157). I have also found b-EP as a prominently effective agonist in this preparation at all stages of postnatal development (109-111 see also Chapter 2). ....The precursor of b-LPH (designated later as POMC) is the natural, endogenous source of b-EP (15, 76) and, possibly, of LPH-(61-79)-peptide (42, 43) but not of (Met5)-enkephalin or LPH-(61-69) fragment. When incubating purified prorcine b-LPH with crude porcine pituitary homogenate,at pH 8.0, there was a time-dependent generation of opioid activity (38, 39, 41, 42). Biochemical analysis of products gave evidence for the cleavage of peptide bond between residues 60-61, 77-78 and 79-80. For the appearance of opioid activity, the cleavage of 60-61 bond was necessary. By comparing the opioid activities of product mixtures in GPI and MVD the presence of active peptides shorter than b-EP can be concluded. By applying bacitracin, an inhibitor of multiple peptidases but not interfering with the generation of actve opioid sequences, this spectrum of products was not altered but the absolute amount of opioid activity was increased significantly. Therefore, it could be concluded that 1) the liberation of N-terminal Tyr-Gly-Gly-Phe-Met motif in a C-terminally extended form is the key step in generating actve opioid sequences, 2) the production of active sequences and their

15 Fig. 1. The opioid agonist potencies of (Met5)-enkephalin, b-endorphin and its shorter fragments (LPH-(61-69) and -(61-79)) in field-stimulated longitudinal muscle strip of guinea -pig ileum and mouse vas deferens.

1000

100 (nM) 50

10 Log IC

1 60 65 70 75 80 85 90 95 Sequence position (61 - X) Legend: On the ordinate the logarithm of 50 % inhibitory concentrations are given, on the abscissa the sequence positions in porcine b-lipotropin. LPH-(61-65)= (Met5)-enkephalin, LPH-(61-91)= b-endorphin. Symbols: open circles: MVD; open triangles: GPI. Points represent the mean, vertical lines the S.E.M. of 4-100 independent determinations. Calculations were applied to data sets obtained in 1976-1977. biodegradation takes place within the same time frame and 3) among the active opioid products peptide(s) shorter than b-EP is/are predominant. Having at hand a set of peptides with potent opioid agonist activities in vitro we-as well as several other teams- were interested as to their analgesic activity in vivo in the conventional analgesic assays. Dealing with peptides, their penetration through the blood-brain barrier could be expected to be poor; therefore their direct application to the liquor space had to be sought. I devised a rapid method for the injection of substances into the lateral horn of the third ventricle (intracerebroventricular, icv injection) in non-anesthetised rats and mice to faciliate the technical output of Behavioral Pharmacological Unit headed by J.I. Székely

16 (132). By using essentially the same injection coordinates in rats as had been described in anesthetised animals by Axelrod and his coworkers (89) a syringe fixed into a Hamilton dispenser, supplied with a needle with custom-cut size served as the technical tool. In the light of many subsequent misinterpretations I wish to put on record that from pharmacological point of view injection of substances into the liquor space is not a preferable route and should be dictated only by necessity; a very judicious interpretation of results should be exercised. Highly purified b-LPH produced naloxone-reversible analgesia in the rat tail-flick test when injected icv (132). Since intact b-LPH was devoid of opioid activity (I tested each batch in vitro before in vivo use) I concluded that an active opioid fragment (most probably b-EP, see below) had to be liberated before its action. Given by the same route (Met5)-enkephalin was practically ineffective as analgesic in the same test in the same species, i.e. rat (7, 39, 44). LPH-(61-79) was marginally effective whereas b-EP induced potent, long-lasting opioid analgesia (39, 44, 146-148). The ineffectiveness of shorter endorphin fragments and enkephalin on the one hand and the high analgesic potency of b-endorphin on the other hand could be attributed to the differences in the opioid receptor type selection , the species and the route of administration (108, 112, see also Chapter 4) rather than to the higher metabolic stability of b-EP (38, 39, 42).

Summary and conclusions q Intact porcine b-lipotropin had no opioid agonist effect but its complete tryptic digest contained fragment(s) with opioid agonist activity in isolated organs. Enzymes present in pituitary membranes also generated opioid fragments from LPH. q Only the fragments having Tyr61 at their N terminus were effective agonists 5 q (Met )-enkephalin (LPH-(61-65)), LPH-(61-69) and LPH-(61-79) were 16-28 times more potent agonists in MVD than in GPI whereas b- endorphin (LPH-(61-91)) was equipotent in the two preparations. q In GPI all LPH fragments acted at the same or similar receptor type where the opiate normorphine whereas the peptides and normorphine acted at different receptor types in MVD.

17 q b-endorphin was a potent opioid analgesic in the rat tail flick test when given intracerebroventricularly whereas the shorter fragments were practically devoid of analgesic activity. The difference can be explained primarily by the different pattern of agonism at different opioid receptor types. q b-LPH produced naloxone-reversible analgesia in the same test given by the same route. Since intact LPH is inactive as opioid agonist, an active fragment (most probably b-endorphin) had to be generated before the action.

3.3. Chapter 3. Development of potent analgesic enkephalin analogs such as (D-Met2,Pro 5)-enkephalinamide The design of enkephalin analogs to be discussed below was done entirely by Sándor Bajusz at the Institute of Drug Research. Owing to his originality and professionalism not only the in vitro testing but also the in vivo analgesic assays served only as ancillary factors in the impressively rapid way to success. Whatever I did learn about the principles of peptide analog design I learnt it from him. After the discovery of enkephalins, all over the world the peptide chemist/pharmacologist teams embarked on the task of developing potent analgesic enkephalin analogs followed , in the beginning, similar speculative patterns. By looking at the structure of parent natural opioid peptide pair, i.e. Tyr-Gly-Gly-Phe-Met/Leu, one may stipulate the following premises: q the pair of glycines, having no side chains, are likely to serve as spacers between the two aromatic residues, Tyr1 and Phe4 q the glycine pair is also permissive for the formation of "turn"-based conformational states q considering the phenyletlylamine like moiety present both in morphine and tyrosine (although the issue of overlapping has been debated repeatedly) and also the presence of two benzene rings both in some potent analgesic synthetic opiates (e.g. fentanyl- derivatives) and enkephalins, one can assume that Tyr1 and Phe4 are crucial for receptorial interaction in enkephalins (see e.g. 30, 51) q the amino acid in postion 5 should have a hydrophobic side chain but otherwise it might be a site for certain variability

18 q natural enkephalins could be assumed to be good substrates of a number of peptidases in the organism as indeed they are (for review see 36). Accordingly, the first steps in design strategy are as follows q introduce D amino acids with aliphatic or aromatic side chains into poistion 2 or 3 or both. The correct substituent(s) would promote a favorable conformational state, protect from inactivation by aminopeptidase(s) and may "offer" the side chain for additional receptor interaction q find a proper substituent in position 5 For synthetic convenience, the first issue was decided by using (Nle5)-enkephalin as parent peptide and GPI as the in vitro assay (7). D-Ala in position 2 was preferred to D-Phe and improved in vitro agonist potency considerably; D-Ala was not accepted in position 3 or simultaneously in positions 2 and 3. These findings outlined a strict logic for further synthetic work. The unique approach to the second issue, devised by S. Bajusz was the introduction of proline into position 5. This substituent is more hydrophobic than either Met or Leu, provides a special conformational environment and renders the C-terminus accessible only to a very limited set of peptidases. At this stage, introducing also MVD and the antagonism by naltrexone in the in vitro analyses (106) I have found that introduction of proline in itself did not improve opioid potency as compared to the parent peptide (Nle5)-enkephalin either in GPI (7, 105, 107) or in MVD (107, 108, 131) but altered entirely the character of opioid activity (105, 106, 108, 109, 112, 131, for details see Chapter 4). This latter tendency became more pronounced by C-terminal amidation. Since (D-Ala2,Pro5)-enkephalin was the first analog with analgesic effect comparable to that of morphine in the rat tail-flick test given by intracerebroventricular route (6, 7) and C-terminal amidation further increased in vivo analgesic potency, this line of design was pursued. The "hit" compound, the highly potent analgesic (rat tail flick, icv route) (D-Met2,Pro5)-enkephalinamide (HU patent GO-1350/76; Belgian patent No. 858453; US patent 4465625/84; 8, 106, 107, 108, 109, 112, 131) emerged from the first ten analog attempts. It was 8-20 times more potent agonist than normorphine in all isolated organs where normorphine was an effective agonist (105, 107-109,112, 131, 149). Its receptor type selection got close to that of normorphine but the enkephalin analog still retained some capability of interacting with the receptor type preferred by (Met5)-enkephalin

(i.e. the d type) as it was shown by the intermediate Ke against it in MVD (106, 109, 112). It was approximately 80 times more potent analgesic than morphine in the rat tail flick test when

19 given by icv route and it was practically equipotent with the opiate parenterally (8, 149). The analgesia was competitively antagonised by naloxone both in rats and mice (146). For at least five years (D-Met2,Pro5)-enkephalinamide was one of the best-selling enkephalin analogs on the peptide reagent market; the only competitor was the analog developed at Sandoz, FK-33 824 ( (D-Ala2,MePhe4,Met5-ol)-enkephalin, (134). In spite of the early expectations on the contary, enkephalin analog-based analgesics held no realistic chance to become therapeutically useful agents. We promoted our peptide to phase I clinical trial (31)(where all the research associates who deserved the "healthy" status voluntered for serving as subjects), but the project was reasonably terminated by 1981-1982.

Summary and conclusions q At the Institute of Drug Research in 1976-1977 we initiated a project aimed at developing potent analgesic enkephalin analogs q I gave a logistic support to drug design by S. Bajusz by testing the enkephalin analogs in GPI and MVD q Enkephalin analogs with enhanced in vitro agonist potency could be produced by modifying the parent YGGFM natural sequence at position 2 (by aliphatic D amino acid substituents) and at the C-terminus 2 5 q The "hit compound", (D-Met ,Pro )-enkephalinamide was 80 times more potent analgesic than morphine in the rat tail-flick test when given intracerebroventricularly q It was 8-20 times more potent agonist than normorphine in all isolated organs where the opiate was effective. q The receptor type preference of agonism by the enkephalin analog got closer to the opiate normorphine but some weak agonism at the receptor type where the parent natural peptide (Met5)-enkephalin acted was still present

20 3.4. Chapter 4. Characterisation of structural principles for the receptor type-selectivity of enkephalin analogs The conclusions were drawn from the pharmacological profile of enkephalin analogs bearing hydrophobic, aliphatic D-amino acid substituents (D-Ala-, -Met-, -Nle) in postion 2 and aliphatic, hydrophobic D-or L-amino acid or Pro in postion 5, in the latter case both with free and amidated C-terminus. Furthermore, tetrapeptide-amide analogs were also synthetised 2 2 in the (D-Met /D-Nle )-series (i.e. Tyr-D-Met/D-Nle-Gy-Phe-NH2), where the pharmacological analyses gave valuable informations about the principles of receptor type- selective agonism ( 9, 108, 131; HU patent 178 004 (GO-1448/1979), 1985)). I wish to make it clear even at this stage that, owing to the technical approach, the discussed issue is not the selectivity of binding to one or the other receptor type but the selectivity of agonism. The substituent in position 2 is not a primary determinant of receptor type selection, the decisive structural features are found at the C-terminus (108, 131). At the d-opioid receptors, potent and selective agonism is produced by analogs bearing a hydrophobic, aliphatic D- or L- amino acid in position 5 with a free carboxylic function. Changing the carboxylic function in Nle for a more acidic but also bulkier sulfonic acid or phosphonic acid moiety (10; HU patent 181 013 (155/80), 1987) the pattern is similar for L-pentane-sulfonic/phosphonic acid (NleS/NleP, respectively) containing analogs (112). Replacement of C-terminal aliphatic amino acid for proline-still with a free carboxylic function- reduces agonist potency at d-opioid receptors (105, 108, 131). However, as it was shown by the naltrexone antagonism of (D-Ala2,Nle5)-/(D-Ala2,Pro5)-enkephalin pair in MVD (105) the presence of proline may still retain the capability of analog to interact with the d- opioid receptor type. The key factor in m-opioid receptor selective/preferring agonism is C- terminal amidation (105, 108, 131) or abolition of carboxylic function by other means (e.g. by substituting with an -ol function (68, 134). Since the tetrapeptide-amide analogs are at least as potent agonists at m-opioid receptors as the ones containing even a favourable substituent in position 5 (131), one must assume that the d-opioid receptor area interacting with the fifth residue is missing, inaccessible or entirely different in the m-opioid receptor type. Molar ellipticity data calculated from the CD spectra of a number of enkephalin analogs (145) suggested that the reduction in molecular flexibility (i.e. a higher tendency to exist in an ordered conformation) is an advantage for m-receptorial agonism, but no such a correlation exists for d-receptorial action.

21 A further task was to find structural the principles in the enkephalin analogs responsible for the analgesic effectiveness in vivo. There are two sides of this coin: the drug side and the analgesic assay including species and route of administration. The drug side includes receptor type selection, agonist potency at the respective receptor types, susceptibility to biodegradation and penetration properties. The last issue is more or less common for the najority of natural peptides and synthetic analogs and can be characterised by poor bioavailability by oral administration and poor penetration through blood.brain barrier. Therefore, when testing the analgesic effect of opioid peptides, the strategy of central injection routes was followed, either into the liquor space (third ventricle, "intracerbroventricular", fourth ventricle, "intracisternal" or spinal cord, "intrathecal") or by site-directed microinjections into neural tissue. The majority of analgesic assays employed determination of pain perception, which can be influenced by opioids at peripheral, spinal and supraspinal sites. The two species used most frequently for these assays were rat and mouse. At the beginning, working with natural opioid peptides ((Met5)-enkephalin, b-endorphin and its shorter opioid fragments, see Chapter 1), we have found that the only effective analgesic was b-EP in the rat tail-flick test given by icv route (44, 146-148); empirically, we set the upper dose limit around 100 nmol/rat. One the one hand, it was true that b-EP was considerably more resistant against enzymic degradation against a number of peptidases as compared to the shorter fragments (38, 39, 42) including the aminopeptdases which play a leading role in the in vivo inactivation of enkephalins and endorphins (for review see 36). On the other hand, the opioid receptor type selection profile of b-EP in vitro was at least as conspicuously different from the others (105, 107, 108, 110, 111, 118). The majority of reports stating the presence of substantial analgesic activity of enkephalins and shorter endorphin fragments in the same test given by similar route came from groups where the assay was performed in the mouse (e.g. 20). Thus, even at that time, we suspected but could not prove that supraspinal opioid analgesia is mediated differently in rats and mice, respectively. When relating the analgesic potency of of enkephalin, shorter endorphin fragments and a limited set of enkephalin annalogs in the rat tail flick test by icv route and their in vitro opioid profile (GPI, MVD) I have found that all the endorphin fragments and enkephalin analogs sharing the in vitro character of (Met5)-enkephalin (in terms of receptor type-selection of agonism) were conspicuously devoid of significant analgesic effect (108). The majority of enkephain analogs contained D-amino acid in position 2, therefore, they could be assumed to

22 be resistant against aminopeptidases. Apart from this, no close correlation could be substantiated between biodegradation and analgesic potency of enkephalin analogs (6). When we had several enkephalin analogs at hand which shared the in vitro opioid receptor selection profile of (Met5)-enkephalin but their C-terminal modification (aliphatic D-amino acid, L- aminopentane-sulfonic or phosphonic acid) rendered them, at least partly, resistant to caboxypeptidase action as well (10) I had the opportunity to go further into the correlation issue (112). There was -still is- a consensus, that in supraspinal opioid analgesia m receptor agonism plays a decisive role both in rats and mice as well as in humans. Thus, one may stipulate that the analgesic potency of enzyme-resistant enkephalin analogs given by icv route should be proportional to their in vitro agonist potencies at m-opioid receptors. It was not the case when the analgesic assay was the rat tail flick and the administration route intracerebroventricular (112). As an example, (D-Ala2,Nles5)enkephalin and (D-Met2,Nles5)- enkephalin had agonist potencies at GPI m-opioid receptors (IC50= 23.4 and 18.6 nM, resp, 2 5 (20) practically identical with that of (D-Met ,Pro )-enkephalinamide (IC50=19.7 nM (109).However, the former two analogs were 40 times more potent at MVD d-receptors (MVD/GPI potency ratios being 41.8 and 38.0, resp) whereas the agonism by the latter at d- opioid receptors was very minor indeed (MVD/GPI potency ratio being 0.85 (105, 108, 109)). The analgesic potency of (D-Met2,Pro5)-enkephalinamide could be characterised in the rat tail flick test by an icv ED50 of 0.04 nmol/rat (8, 149) whereas the corresponding values for the other two analogs were 178 nmol/rat and 89 nmol/rat, respectively (10, 112). ...I concluded that d-opioid receptor-selective agonism interfered with the expression of m- opioid receptor mediated analgesia in the rat tail-flick test when the analogs were given by icv route (112). The sophistcated wording was purposeful. The phenomenon could be attributed to negative allosteric interaction at the level of receptor molecules in the same membrane, opposing net effects on the same neuron or opposing influences through different neuronal populations (possibly at different brain sites). Negative allosteric modulation can be ruled out because soon afterwards a positive allosteric modulatory interaction between m- and d- opioid receptor ligands has been reported (135).

Summary and conclusions Using the presently standing opioid receptor type specifications (i.e. d, µ and ?), the conclusions to be drawn will concern the structural principles

23 determining the µ or d receptor type-selection of agonism in vitro and the properties affecting supraspinal analgesia in the rat. Thus, q Introduction of aliphatic, hydrophobic D amino acid (D-Ala, D-Met or D-Nle) into position 2 of parent enkephalin structure (YGGFM/Nle) enhances in vitro agonist potency but is not a determining factor in receptor type selection. q The structural factors determining the µ or d receptor selection of agonism are found at the C-terminus of enkephalin analogs. q An aliphatic, hydrophobic amino acid (Met, Leu, Nle, Ile etc.) with free carboxylic function in position 5 is the prerequisite of d receptor- selective agonism. C-terminal amidation, particularly with Pro in position 5, deletion of fifth amino acid with amidated residual tetrapeptide will result in µ receptor selective/preferring agonism. q The tendency of an enkephalin analog to assume an ordered structure (i.e. a tendency for conformational rigidity) may promote agonism at µ- opioid receptors; no such a correlation exists regarding d receptor agonism. q Accepting -reasonably- that in the case of non-peptide opiates supraspinal analgesia fairly correlates with agonist potency at µ-opioid receptors, I realised that in the case of enkephalin analogs this correlation is altered by the presence of d receptor agonism. In the rat tail-flick test, giving the analogs intracerebroventricularly, the presence of preferential d receptor agonism does not permit the analgesic manifestation of agonism -even potent agonism- at µ-opioid receptors.

3.5. Chapter 5. Detection of a novel dipeptidyl-carboxypeptidase in rodent blood vessel and human adrenomedullary tumors The purportedly novel dipeptidyl-carboxypeptidase was detected in the course of experiments aimed at the in vitro characterisation of the opioid properties of a proenkephalin-

24 derived heptapeptide, Tyr-Gly-Gly-Phe-Met-Arg-Phe (YGGFMRF, (Met5)-enkephalin- Arg6,Phe7, ME-AP (47, 122, 129, 130).

Fig 2. The cleavage sites in (Met5)-enkephalin and (Met5)-enkephalin-

Arg6,Phe7.(scheme after 36, 143, 154)

DAP ¯ ß ACE Tyr--Gly--Gly--Phe--Met Ý Ý APs NEP XXX ß ß ß Tyr--Gly--Gly--Phe--Met--Arg--Phe DAP • • ACE Ý

Legend: Abbreviations: ACE= angiotensin converting enzyme (EC 3.4.15.1); APs= aminopeptidases (leucine-aminopeptidase, aminopeptidase M); DAP= dipeptidyl- aminopeptidase ("Enkephalinase B"); NEP = neutral endopeptidase (EC 3.4.24.11, "Enkephalinase A" (138); XXX= the novel dipeptidyl-carboxypeptidase. Thickness of arrows and letter sizes indicate relative importances in biotransformation.

The C-terminal heptapeptide of precursor (23) appears regularly among the endogenous products of processing particularly in the brain and may serve as an endogenous opioid receptor agonist with distinct biological profile on its own right (5, 156).There are several tissue peptidases which can hydrolyse the heptapeptide by attacking at different peptide bonds; the comparative biodegradation profile of (Met5)-enkephalin and the heptapeptide is shown in Fig.2.

25 In isolated organ preparations (GPI, MVD, RVD, ART) ME-AP followed the agonist activity pattern characteristic of (Met5)-enkephalin when no peptidase inhibitors were added to the assay media (122, 129). In rabbit ear artery ME-AP was equipotent with (Met5)- enkephalin in the absence of enzyme inhibitors whereas high concentration (10-5 M) of ACE inhibitor captopril caused a tenfold rightward shift in the concentration-response curve of heptapeptide (116, 129, 130). In other isolated organs captopril caused either no change or potentiation (47, 130). Thus, the most reasonable assumption was that in ART the heptapeptide was converted rapidly and almost completely to (Met5) enkephalin by a captopril-inhibited enzyme (129); indeed, the conversion could be demonstrated also by high- voltage paper electrophoresis. Since i) the conconversion was not inhibited by a lower concentration (10-6M) of captopril and ii) the preferential location of ACE in blood vessels is the luminal membrane of endothelium while both the administration of peptides and their effect took place in the vicinity of adventitia (the terminal arborisation of supplying nerves is mostly confined to the subadventitial layer of musculature) I presumed that the enzyme was not identical with ACE. I re-opened the issue some six years later. Although cleavage at the -Met5-Arg6- bond was not likely to be due to the action of neutral endopeptidase, I did not entirely discount NEP or a similar enzyme. The initial test system was the same (i.e. ART) but I added ME-AP-amide, bradykinin and angiotensin I to the potential substrates. Angiotensin I and II (AT-I and II) as well as bradykinin exerted a powerful presynaptic inhibitory effect in ART through a prostanoid-mediated pathway (102, 103) whereas the action of ME-AP, as expected, was independent of prostanoid generation. The results are shown in Fig. 3. which is a re-drawn and extended version of the figure published in 1995 (116).

26 Fig.3. The presynaptic inhibitory effect of (Met5)-enkephalin-Arg6,Phe7 (A), its amide (B), (Met5)-enkephalin (C), bradykinin (D) and angiotensin I (E) in the absence (?) and in the presence (?) of captopril in perfused, neurally stimulated rabbit ear artery in vitro.

27 Legend: The concentration of captopril used from "A to E" was 10-5 M. In panel "A" the effect of (Met5)-enkephalin-Arg6,Phe7 is shown also in the presence of 10-6 M (? ) and 10-4 M (?) captopril. In panel "E" the effect of angiotensin-II without captopril (¦ ) and the action of angiotensin-I in the presence of 3x10-8 M (Sar1,Val5,Ala8)-angiotensin-II (Ñ) is shown.

It is apparent that q The ME-APÞME conversion was inhibited by high but not low concentration of captopril q ME-AP-amide was not a substrate of the enzyme 5 q There was only a very modest interaction of captopril with (Met )-enkephalin q There was no functional indication of significant, captopril-sensitive AT-IÞAT-II conversion q The effect of bradykinin was potentiated significantly by captopril Furthermore, ME-AP conversion was not affected by 10-6 M of NEP inhibitor thiorphan and was unaltered by endothel removal (116). Based on these informations, on the analogy of well-known synthetic substrate of ACE, hippuryl-histidyl-leucine (Hip-His-Leu, HHL, (91) I have asked the chemists to synthetise Hip-Arg-Phe and Hip-Phe-Arg (see bradykinin C- terminal dipeptide), respectively. Using HHL and HRF substrates the presence of both ACE and the purportedly novel peptidase could be demonstrated in the vessel wall also by biochemical means (83). Furthermore, because i) the enzyme could be assumed to be located to neural elements in ART and ii) these neurons are postganglionic sympathetic neurons and iii) the adrenal medulla, which can be considered as a modified postganglionic neuronal assembly is the richest source of proenkephain, I hypothetised that the adrenal medulla must be a rich source also of the novel enzyme. Indeed,using human adrenal tumors as potential enzyme source"our" enzyme as well as ACE was found in pheochromocytoma but not in adrenocortical tumors (124). Both enzyme activities were present in particulate but not in soluble fraction. ACE could be inhibited by low whereas "our" enzyme only by high concentration of captopril. I was trying to find grant support to isolate this enzyme, with no success. Since the procedure in itself may not be a technically difficult one, I did not want to advertise further this easy opportunity. Therefore, I delayed the publication of some enzyme kinetic analyses, which will be given below. In these series I used a single enzyme-rich human pheochromocytoma speciemen (No. 125/3), and besides HHL and HRF I included also

28 the synthetic substrate corresoponding to the C-terminal dipeptide of human atrial natriuretic peptide, i.e. HRY. Because the enzyme reaction was linear between 0.05 mg and 0.3 mg protein enzyme source and 15 min -120 min reaction time, I could use relatively lower enzyme concentration and shorter incubation period as compared to the previous series.

Furthermore, P2 fraction prepared from speciemen No. 125/3 could be taken up by the puffer without the addition of any detergent. According to the Lineweaver-Burk plot (Fig.4.) the enzymes present in pheochromocytoma membranes hydrolyse HRF and HRY substrates at a higher rate as compared to HHL. The Henri-Michaelis Menten parameters (calculated from two independent experiments, each sample run in duplicate) are shown in Table 1.

Fig. 4. The Lineweaver-Burk plot of hydrolysis of Hip-His-Leu, Hip-Arg-Tyr and Hip-Arg-Phe substrates by enzymes present in P2 fraction prepared from human pheochromocytoma speciemen No. 125/3

1/V x108

4 HHL

3

2 HRY 1 HRF

0 -10 -5 0 5 10

x103 1/S

29 Table. 1. The Henri-Michaelis-Menten parameters of substrate

hydrolysis by P2 fraction of human pheochromocytoma speciemen No. 125/3.

Substrate Km Vmax -1 -1 (mM) (mol x (mg prot) x min ) Hip-His-Leu 1.31 3.72x10-8 Hip-Arg-Phe 1.24 9.24x10-8 Hip-Arg-Tyr 0.86 7.04x10-8

At 10-6 M captopril inhibited both HHL and HRF hydrolysis in a non-competitive manner, the inhibition against the former being significantly stronger (Fig 5, upper panel). At 10-7 M captopril and below the inhibition was competitive in the case of HHL-hydrolysing enzyme and there was no inhibitory interaction with HRF hydrolysing enzyme activity (Fig. 5, lower -8 panel. For the competitive captopril? HHL interaction a Ki of 1.69x10 M (1.58 and 1.80 x10-8 M, resp.) could be calculated for the inhibitor. The effect of various enzyme inhibitors on the hydrolysis of 10-3M HRF substrate is shown in Table 2.

Table 2. The effect of enzyme inhibitors on the hydrolysis of -3 10 M HRF substrate by P2 fraction prepared from human pheochromocytoma speciemen No. 125/3.

Inhibitor Concentration Inhibition (M) (%) EDTA 10-3 97.3 N-phosphoryl-Arg-Phe 10-6 0 10-5 0 Thiorphan 10-6 9.1

30 Fig. 5. The Lineweaver-Burk plot of substrate hydrolysis in the absence and presence of captopril

Legend: Hydrolysis in the absence of captopril (? ) and in the presence of 10-7 M (¦ ) or 10-6 M (? ) captopril (upper panel) and 10-7 M (? ) or 3 x 10-8 M (¦ ) captopril (lower panel)

Summary and conclusions I detected a purportedly novel dideptidyl carboxypeptidase in neural elements of rabbit ear artery and also in human adrenomedullary tumor. 5 7 7 q Its substrates should have an -RF (as in (Met )-enkephalin-Arg ,Phe ), -- RY (as in atrial natriuretic peptide) or--FR (as in bradykinin) at the C- terminus q It is a metallopeptidase because it is inhibited by EDTA

31 q Its active centrum is likely to differ from the "thermolysin-like" enzymes because it was not inhibited by N-phosphoryl-Arg-Phe q It is different from angiotensin converting enzyme because it is inhibited (noncompetitively) only by high but not low concentrations of captopril q It is different from neutral endopeptidase because the cleavage site specificity of NEP is different and the enzyme was not inhibited by the NEP inhibitor thiorphan q It is different from brain dipeptidyl-carboxypeptidase "PDP-3" (24) because it does not cleave HHL, only weakly inhibited by captopril and not at all by thiorphan

3.6. Chapter 6. Design and pharmacological characterisation of novel, pure and highly d-opioid receptor type-selective and k-opioid receptor type- selective peptide antagonists: the principle of N-acylation. In the enkephalin series, N-monoallyl substitution yielded partial agonists (96); for producing antagonists, N,N-diallyl-derivatives had to be synthetised (78). The synthetic procedure may not be an easy one because in spite of repeated efforts, such derivatives could not be synthesized at the beginining of projects. Looking for a possible equivalent led to the introduction of a novel structural principle, N-terminal acylation. Peptide chemists routinely use N-t-butoxycarbonyl moiety to "protect" the N-terminus, therefore, the protected peptide was available for testing. Tertiary butyl is bulkier than monoallyl; however, N-acylation would not permit the protonation of nitrogen, which, according to the standing textbook axioms, is a prerequisite of opioid activity. I hoped that it be true for agonism and not for antagonism as well. Wishful thinking is occasionally gratified; so it was this time. The design of d-opioid receptor selective peptide antagonists Stimulated by the revival of "address and message" theory (98, 139) I have set the design game as follows. The N-terminal Tyr-Pro motif was found regularly in several, µ-opioid receptor-preferring natural ligands such as morphiceptin (21), hemorphin, cytochrophin (16, 17) and Tyr-MIF-1/Tyr-W-MIF-1 (26,59). I wanted to explore if by the addition of C- terminal d-opioid "receptor-addressing" sequences the receptor type selection could be altered. These sequences were -Phe-Leu (taken from (Leu5)-enkephalin), -Phe-Leu-Thr (32)

32 and -Phe-Leu-Thr(OtBu) (12). In a model pair, the additive was used with and without a Gly3 spacer and with an eye on the possible conformational consequences, an analog with Tyr-Gly- Pro- N-terminal sequence was also synthesised (HU patent 2551/91 (23571), 1991; (113-115, 124-128) The opioid properties of resulting compounds are shown in Table 3.

Table 3. The opioid properties of C-terminally extended Tyr-Pro- -peptides in isolated organs

Peptide Antagonism Agonism

Ke (µM) IC50 (µ M) d µ ? Tyr-Pro-Phe-Leu 11 28 N.T. >1,000 NtBOC------>100 >100 N.T. >100 Tyr-Pro-Gly-Phe-Leu 15 84 N.T. >100 NtBOC------0.60 79 N.T. >100 Tyr-Gly-Pro-Phe-Leu 27 N.T. N.T. 32 NtBOC------28 N.T. N.T. >100 Tyr-Pro-Gly-Phe-Leu-Thr 19 >10 N.T. 11 NtBOC------0.55/0.08* 200-300* >100* >100 N,N-diallyl------0.33 >30 N.T. >10 NtBOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu) 0.03-0.04* 200-370* >200 >200

Notes to Table 3. The Ke values were determined against agonists of good pharmacological selectivity ((Met5)-enkephalin for d, normorphine for µ and EKC for ?/µ) or against highly receptor type-selective agonists (DAMGO for µ, DADLE, DPDPE or deltorphin-II for d and PD-117 302 for ?); the latter experimental type was marked by asterisk. In general, the data were obtained in MVD but for key compounds also in GPI (µ, ?). Abbreviations: tBOC= tertiary butoxycarbonyl; tBu= tertiary butyl. N.T.: not tested.

It is apparent that substantial d-opioid receptor antagonism appeared first in the N- terminally acylated Tyr-Pro-Gly-Phe-Leu peptide, and both the affinity and the selectivity was further improved by -Thr6 or -Thr(OtBu)6 addition. The competitive type of antagonism was shown by Schild-analysis (115, 126, 128). The antagonism was pure in the sense that

33 none of the effective antagonists had any appreciable opioid agonist activity even at very high concentrations. . The N-terminus equally accepted both acyl (BOC) and alkyl (diallyl) moiety . The best analog had the same affinity to d-opioid receptors as naloxone and, in contrast to the opiate antagonist, the peptide had a 5,000-10,000-fold d over µ (or ?) receptor type selectivity (128). The naltrexone-related naltrindole (99) has much higher affinity but it is inferior in the extent of type-selectivity (approx. 100-fold, 46, 80, , 121,126). TIPP (Tyr-Tic- Phe-Phe) (136) has slightly higher affinity and matching selectivity whereas 2',6'dimethyltyrosyl-tetrahydro-isoquinolinic acid (19) is clearly superior in affinity and the selectivity is at least matching. Tyr-Pro-Phe- and Tyr-Pro-Gly-Phe sequences are so-called turn-inducing ones but predispose for different types of turns. On the other hand, Tyr-Gly-Pro- is known as a "turn- braking" sequence. Fortunately, a group of excellent conformational analysts was also interested in turns induced by the Tyr-Pro- motif.

Table 4. The preferred conformations of some Tyr-Pro-peptides based on their CD and FT-IR spectra taken in nonpolar solution Peptide1 Preferred conformation Antagonist effect BOC-YPFL short a-helical or open ß-turn NO or ?-turn BOC-YGPFL NO single preferred NO conformational state BOC-YPGFL Type II ß-turn YES BOC-YPGFLT Type II ß-turn YES Footnote: 1 single-letter amino acid codes are used

From the circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectra of peptides taken in nonpolar or nonpolar + polar solution, the conformations proposed for the didactically key compounds and their pharmacological effects are shown in Table 4 (from 50, 113-115, 126). It is apparent that a type II ß-turn is a prerequisite of antagonist property. This

34 type of folded conformation brings the aromatic moieties (Tyr1 and Phe4, resp.) into close spatial vicinity whereas these two residues exist in an extended position in BOC-YPFL (Fig. 6).

Figure 6. Molecular dynamic simulation of the preferredconformation of BOC-YPGFL (from the courtesy of A. Perczel, unpublished) and BOC -YPFL (taken from (50) in nonpolar solution

Note: The position of H-bonds are shown in dotted line

Conclusions q By proper C-terminal extension(s) and N-substituent(s) pure, compeptitive d-opioid receptor antagonists with good affinity and very high type-selectivity can be produced in the Tyr-Pro-series q Besides the sequence, the novelty of prominent importance is the N-acyl substituent, which does not permit N-protonation t q Since N,N-diallyl- or N BOC-substituted active sequence yield matching pharmacological effects, the protonation state of nitrogen appears to be indifferent for the receptorial interaction by these structures

35 q A type II ß-turn conformation is a prerequisite of pharmacological activity, possibly due to providing a proper steric relationship between the aromatic moieties and also for the placement of C-terminal, type-specific address

The design of ?-opioid receptor-selective peptide antagonists While attending the European Peptide Symposium in 1980, S. Bajusz became aware of a presentation about the recently identified venom sequence of Philippine cobra (55). With the aim of developing peptide antagonist analogs at the skeletal muscle nicotinic acetylcholine receptors, he choose the Tyr-Lys-Lys-Trp-Trp pentapeptide (located between sequence positions 24 - 28 of venom) as the starting point of the synthetic project. Neither this sequence nor its C-terminally amidated derivative had the desired effect. On the other hand, since it contained tyrosine at the N-terminus and in two amino acid distance in the peptide backbone a pair of bulky, hydrophobic aromatic residues (Trp4,5), opioid receptorial action seemed possible. Although a basic L-amino acid pair in positions 2,3 is a disadvantage for ideal opioid receptorial interaction (86), the same basic residues were attractive in the light of abundance of basic amino acids in the structure of recently discovered brain opioid peptides, dynorphins and neo-endorphins (35, 57). My primary aim was to obtain, in the long run, antagonists rather than agonists. Since the reluctance of synthetic chemists to produce N,N- diallyl derivatives appeared to be a standing aversion, in fact, this was the first project where the idea of N-acylation has been introduced (for the reasoning see the previous section). In spite of the fact that within the first tested pair the indication of desired pharmacological effect was present (see the first two peptides in Table 5), the finding and its novelty were not enough to obtain the approval for project modification at the Institute of Drug Research. The project was re-started some ten years later at the Research Group of Peptide Chemistry, HAS-

Eötvös University. I have set the line of design first. Soon after the start, a bright young chemist, G. Orosz has joined the team, and the design became an amusing joint venture. Since the early eighties the preference of dynorphins and neo-endorphins for the k-opioid receptors

36 became a common knowledge (21), we set the aim for producing antagonists selective for this receptor type (92-94, 125). The pharmacological testing was performed in isolated organs,

MVD and GPI; the results for the didactically important peptides are shown in Table 5.

Table 5. The opioid properties of peptides designed from the

Tyr-Lys-Lys-Trp-Trp-NH2 starting sequence

Peptide Antagonism (Ke, mM) Agonism k m d (IC50, µM)

H2N-Tyr-Lys-Lys-Trp-Trp-NH2 N.T. >100 N.T. >100 NtBOC------18.6 0.84 >100 >100 N-Piv ------25.9 8.32 N.T. >100 N-Prop------16.1 @29 N.T. >30 N-Ac ------2.61 23.4 N.T. >50 N-Form------20.7 >100 N.T. >100 N-All ------40.7 N.T. N.T. >100

N,N-All2------2.61 22.5 N.T. >100 t N BOC-Tyr-Arg-Lys-Trp-Trp-NH2 @270 1.76 N.T. >30 t N BOC-Tyr-Lys-Lys-Trp-Pro-NH2 @50 >100 >10 >100 t N BOC-Phe-Lys-Lys-Trp-Trp-NH2 2.31 1.31 N.T. >50 t N BOC-Tyr-D-Nle-Lys-Trp-Trp-NH2 >10 0.24 >10 >10 ------Phe------>100 27.7 108 >100

t N BOC-Tyr-Lys-Trp-Trp-NH2 5.39 117.3 >30 >100 ------Arg------2.50 @40 @16 >30 N-Ac------@40 @60 N.T. >30

t N BOC-Tyr-D-Lys-Trp-Trp-NH2 1.20 0.74 7.05 >10

37 Footnotes The antagonism was tested against EKC (k/µ), normorphine or DAMGO (m) and (Met5)- enkephalin or DADLE (d). Abbreviations: tBOC= tertiary butoxycarbonyl, Piv= pivaloyl; Pro= propionyl; Ac= acetyl; For= formyl; All= allyl. performed in isolated organs, MVD and GPI; the results for the didactically important peptides are shown in Table 5. Since there was no such a conceptual background in this case as for the design of d-opioid receptor antagonists, the structure set is far from optimisation. The minimal primary aim, i.e. the development of a definitely k-opioid receptor-selective antagonist(s) of substantial absolute affinity (meaning a Ke in the lower micromolar range) starting from the µ-opioid t receptor-preferring peptide N BOC-Tyr-Lys-Lys-Trp-Trp-NH2 (which had only a poor affinty for k-opioid receptor type), has been attained. Thus, I consider as „hits” N-Ac- and N,N- t diallyl-Tyr-Lys-Lys-Trp-Trp-NH2 of pentapeptide series and N BOC-Tyr-Lys-Trp-Trp-NH2 and -Tyr-Arg-Trp-Trp-NH2 of tetrapeptide series. The former tetrapeptide competitively antagonised the effect of EKC in rabbit vas deferens (an isolated organ with ?-opioid receptor dominance) with a Ke of 3.57 µM (93). All the „hits” had Ke values in the lower- and mid- micromolar range and 8-20-fold ? over µ selectivity ratios. Although it follows from the previous section/chapters as well, I mention that the structural characteristics of the present series do not favor d-opioid receptorial interaction; a noticeable exception was NtBOC-Tyr-D-

Lys-Trp-Trp-NH2. The guidelines for future structure optimisation can be summarised as follows: Apart from the Tyr1/Phe1, three domains in the present ligand structures appear to be crucial for selective ? receptorial interaction. These are the N-terminal N-substituent, the side chain of amino acid in postion 2 and the C-terminal bulky aromatic, hydrophobic residues. The result of alterations at a given domain depends a great deal on the actual structure in the rest of the molecule. Thus q Similarly to the d-opioid receptor antagonist structures discussed in in the previous section, int he present series the protonation state of terminal N is indifferent for receptorial interaction q The receptor pocket where the N-substituent is bound is likely to have a rather rigorous geometry because the the „acceptance” of substituent is strongly affected by the rest of the molecule (compare e.g.the Ac/BOC pairs in the penta- and tetrapeptide series)

38 q A basic function in the side chain in position 2 is a prerequisite of k-opioid receptorial action. D-amino acid is an advantage for µ-opioid receptorial effect. q From the available structure-activity relationships the necessity of tryptoptophane pair at the C-terminus seems likely for m-receptors, cannot be firmly established as yet for k- receptors. Summary and conclusions q By following the address and message theory, by extending the Tyr-Pro- N-terminal motif by d-receptor address sequences (-Leu-Phe, -Leu-Phe- Thr or -Leu-Phe-Thr(OtBu) and using proper N-substituents (NtBOC- or N,N-diallyl), pure, competitive d-opioid receptor antagonists could be produced with good affinity and almost absolute receptor type-selectivity t q Since N,N-diallyl- or N BOC-substituted active sequence yield matching pharmacological effects, the protonation state of nitrogen appears to be indifferent for the receptorial interaction by these structures t q The most prominent antagonist, N BOC-Tyr-Pro-Gly-Phe-Leu- t Thr(O Bu) had a Ke of 30-40 nM at d-opioid receptors and more than 5,000-fold d over µ or d over ? selectivity ratio. q A type II ß-turn conformation is a prerequisite of pharmacological activity, possibly due to providing a proper steric relationship between the aromatic moieties and also for the placement of C-terminal, type- specific address q Starting from the Tyr-Lys-Lys-Trp-Trp sequence found in snake venom, pure, competitive ?-opioid receptor antagonists with moderate affinity and fair receptor type-selectivity could be produced by proper structural modifications with particular reference to the N-substituent. q The "hit" compounds were N-acetyl- and N,N-diallyl-Tyr-Lys-Lys-Trp- t Trp-NH2 of pentapeptide series and N BOC-Tyr-Lys-Trp-Trp-NH2 and

-Tyr-Arg-Trp-Trp-NH2 of tetrapeptide series. They had Ke values at ?-

39 opioid receptors in the lower or mid-micromolar range and 8-20-fold ? over µ selectivity ratios. q These series of experiments have set the guidelines for future structure optimisation. q In both structures the most significant novelty was the N-acylation principle which does not permit nitrogen protonation. In both structures, the protonation state of N appeared to be indifferent for receptorial interaction.

3.7. Chapter 7. Present and future: endomorphins, nociceptin, central autonomic regulation. In the late nineties, a young biologist, Éva Makó, "inherited" from Professor G. Ádám of Eötvös University prepared her PhD thesis in my laboratory. Among other topics, her tasks were to perform a detailed in vitro pharmacodynamic analysis with the d-opioid receptor antagonist peptide NtBOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu), and to characterize the d- and ?- opioid receptor supply of four isolated organs (80). She received her degree by 2001. While she was staying at the laboratory, Zadina et al. has reported on a novel, µ-opioid receptor agonist brain peptide pair, Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2, designated as endomorphin-1 and -2, respectively (158). We joined the research of endomorphins immediately (133) and tested several novel endomorphin analogs since then (65, 66, 153). The partial agonist properties of endomorphins was a particularly interesting issue (3, 74, 141), therefore, we sought techniques for the analysis of partial agonism in isolated organs (2, 104). Mahmoud Al-Khrasani has prepared his PhD thesis in my laboratory, mostly from the experiments with endomorphins; he received his degree by June 2004. Presently, we are trying to find a solution on the still misterious puzzle of the biosynthetic route of endomorphin production. We extended our attention to another novel, opioid-related endogenous peptide, nociceptin (13, 45, 64, 84, 101). There will be a shift also in the technical field we are planning to work with. We will work mostly with isolated brain slices (1, 60, 123)., cell cultures and lymphocyte preparations. Of the CNS functions, autonomic integrative mechanisms will receive distinguished attention (119, 120, 123).

40 4 General summary and conclusions The actual results were presented in six sections; the general summary and conclusions are given below by the sections

Chapter 1. Critical overview of the use of isolated organs in opioid research q I conducted exploratory analyses in six, opioid receptor-containing isolated organs, in field-stimulated longitudinal muscle strip-Auerbach plexus of guinea-pig ileum (GPI), mouse (MVD), rat (RVD) and rabbit (LVD) vas deferens, cat nictitating membrane medial part (CNM) and perfused rabbit ear artery (ART). q Significant, innovative methodological changes were made in the case of MVD and ART. For the other preparations the originally described methods were installed with only minor modifications q The consensus opioid receptor types, defined in the same period (1976- 1977) when my exploratory experiments were performed (1976-1978) were µ, d and ?. To characterise the opioid receptor supply of isolated organs I used normorphine then DAMGO as µ-opioid receptor type preferring/selective agonists, (Met5)-enkephalin then DADLE, DPDP and deltorphin II as d-receptor preferring/selective agonists and EKC and PD-117 302 as ?/µ- and ?-receptor agonists, respectively. Of the competitive opioid antagonists initially the moderately µ receptor type- preferring naltrexone, later type-selective antagonists were used. q For the routine analyses of novel enkephalin analogs and antagonist candidates I used the MVD where all the three major opioid receptor types are present. For comparative purpose, I used regularly also the GPI, characterised by µ and ? receptor supply.

41 q I have demonstrated the presence of d and ? opioid receptors in ART. Otherwise, this preparation was used mainly to prove the existence of a novel peptidase (Chapter 5). q I have studied the postnatal development of opioid receptors in RVD. I have confirmed the potent agonism by ß-endorphin in this preparation. I found the receptors similar to but not quite identical with the µ type. The postnatal changes are characterised by a gradual loss of mechanism(s) responsible for the efficacy component of agonist action. q I have used LVD (characterised by dominantly ? receptors) occasionallyand decided to exclude CNM from the testing battery.

Chapter 2. Pharmacological characterisation of enkephalins, b- endorphin and its fragments q Intact porcine b-lipotropin had no opioid agonist effect but its complete tryptic digest contained fragment(s) with opioid agonist activity in isolated organs. Enzymes present in pituitary membranes also generated opioid fragments from LPH. 61 q Only the fragments having Tyr at their N terminus were effective agonists 5 q (Met )-enkephalin (LPH-(61-65)), LPH-(61-69) and LPH-(61-79) were 16-28 times more potent agonists in MVD than in GPI whereas b- endorphin (LPH-(61-91)) was equipotent in the two preparations. q In GPI all LPH fragments acted at the same or similar receptor type where the opiate normorphine whereas the peptides and normorphine acted at different receptor types in MVD. q b-endorphin was a potent opioid analgesic in the rat tail flick test when given intracerebroventricularly whereas the shorter fragments were practically devoid of analgesic activity. The difference can be explained

42 primarily by the different pattern of agonism at different opioid receptor types. q b-LPH produced naloxone-reversible analgesia in the same test given by the same route. Since intact LPH is inactive as opioid agonist, an active fragment (most probably b-endorphin) had to be generated before the action.

Chapter 3. Development of potent analgesic enkephalin analogs such as (D-Met2,Pro 5)-enkephalinamide q At the Institute of Drug Research in 1976-1977 we initiated a project aimed at developing potent analgesic enkephalin analogs q I gave a logistic support to drug design by S. Bajusz by testing the enkephalin analogs in GPI and MVD q Enkephalin analogs with enhanced in vitro agonist potency could be produced by modifying the parent YGGFM natural sequence at position 2 (by aliphatic, hydrophobic D amino acid substituents) and at the C- terminus 2 5 q The "hit compound", (D-Met ,Pro )-enkephalinamide was 80 times more potent analgesic than morphine in the rat tail-flick test when given intracerebroventricularly q It was 8-20 times more potent agonist than normorphine in all isolated organs where the opiate was effective. q The receptor type preference of agonism by the enkephalin analog got closer to the opiate normorphine but some weak agonism at the receptor type where the parent natural peptide (Met5)-enkephalin acted was still present

43 Chapter 4. Characterisation of structural principles for the receptor type-selectivity of enkephalin analogs Using the presently standing opioid receptor type specifications (i.e. d, µ and ?), the conclusions to be drawn will concern the structural principles determining the µ or d receptor type-selection of agonism in vitro and the properties affecting supraspinal analgesia in the rat. Thus, q Introduction of aliphatic, hydrophobic D amino acid (D-Ala, D-Met or D-Nle) into position 2 of parent enkephalin structure (YGGFM/Nle) enhances in vitro agonist potency but is not a determining factor in receptor type selection. q The structural factors determining the µ or d receptor selection of agonism are found at the C-terminus of enkephalin analogs. q An aliphatic, hydrophobic amino acid (Met, Leu, Nle, Ile etc.) with free carboxylic function in position 5 is the prerequisite of d receptor- selective agonism. C-terminal amidation, particularly with Pro in position 5, deletion of fifth amino acid with amidated residual tetrapeptide will result in µ receptor selective/preferring agonism. q The tendency of an enkephalin analog to assume an ordered structure (i.e. a tendency for conformational rigidity) may promote agonism at µ- opioid receptors; no such a correlation exists regarding d receptor agonism. q Accepting -reasonably- that in the case of non-peptide opiates supraspinal analgesia fairly correlates with agonist potency at µ-opioid receptors, I realised that in the case of enkephalin analogs this correlation is altered by the presence of d receptor agonism. In the rat tail-flick test, giving the analogs intracerebroventricularly, the presence of preferential d receptor agonism does not permit the analgesic manifestation of agonism -even potent agonism- at µ-opioid receptors.

44 Chapter 5. Detection of a novel dipeptidyl-carboxypeptidase in rodent blood vessel and human adrenomedullary tumors I detected a purportedly novel dideptidyl carboxypeptidase in neural elements of rabbit ear artery and also in human adrenomedullary tumor. 5 7 7 q Its substrates should have an -RF (as in (Met )-enkephalin-Arg ,Phe ), -- RY (as in atrial natriuretic peptide) or--FR (as in bradykinin) at the C- terminus q It is a metallopeptidase because it is inhibited by EDTA q Its active centrum is likely to differ from the "thermolysin-like" enzymes because it was not inhibited by N-phosphoryl-Arg-Phe q It is different from angiotensin converting enzyme because it is inhibited (noncompetitively) only by high but not low concentrations of captopril q It is different from neutral endopeptidase because the cleavage site specificity of NEP is different and the enzyme was not inhibited by the NEP inhibitor thiorphan q It is different from brain dipeptidyl-carboxypeptidase "PDP-3" (24) because it does not cleave HHL, only weakly inhibited by captopril and not at all by thiorphan

Chapter 6. Design and pharmacological characterisation of novel, pure and highly d-opioid receptor type-selective and k-opioid receptor type- selective peptide antagonists: the principle of N-acylation. q By following the address and message theory, by extending the Tyr-Pro- N-terminal motif by d-receptor address sequences (-Leu-Phe, -Leu-Phe- Thr or -Leu-Phe-Thr(OtBu) and using proper N-substituents (NtBOC- or N,N-diallyl), pure, competitive d-opioid receptor antagonists could be produced with good affinity and almost absolute receptor type-selectivity

45 t q Since N,N-diallyl- or N BOC-substituted active sequence yield matching pharmacological effects, the protonation state of nitrogen appears to be indifferent for the receptorial interaction by these structures t q The most prominent antagonist, N BOC-Tyr-Pro-Gly-Phe-Leu- t Thr(O Bu) had a Ke of 30-40 nM at d-opioid receptors and more than 5,000-fold d over µ or d over ? selectivity ratio. q A type II ß-turn conformation is a prerequisite of pharmacological activity, possibly due to providing a proper steric relationship between the aromatic moieties and also for the placement of C-terminal, type- specific address q Starting from the Tyr-Lys-Lys-Trp-Trp sequence found in snake venom, pure, competitive ?-opioid receptor antagonists with moderate affinity and fair receptor type-selectivity could be produced by proper structural modifications with particular reference to the N-substituent. q The "hit" compounds were N-acetyl- and N,N-diallyl-Tyr-Lys-Lys-Trp- t Trp-NH2 of pentapeptide series and N BOC-Tyr-Lys-Trp-Trp-NH2 and

-Tyr-Arg-Trp-Trp-NH2 of tetrapeptide series. They had Ke values at ?- opioid receptors in the lower or mid-micromolar range and 8-20-fold ? over µ selectivity ratios. q These series of experiments have set the guidelines for future structure optimisation. q In both structures the most significant novelty was the N-acylation principle which does not permit nitrogen protonation. In both structures, the protonation state of N appeared to be indifferent for receptorial interaction.

46 5. Acknowledgement I wish to express my gratitude to Professor Kálmán Magyar, D.Sc., Member of Hungarian Academy of Sciences for honouring me by the acceptance of supervisorship over the thesis.

I am indebted also to Professor Klára Gyires, D.Sc., who, by applying a tactful but persistent pressure induced me to accomplish the thesis.

Most of all, I wish to thank my friend, Professor Sándor Bajusz, D.Sc., to whom this work was dedicated, everything he gave me. It was a pleasure to work with him, learn from him. He is one of the bests I have ever met in the profession. He is always bristling with original ideas, good-humoured and honest; a rare assembly of qualities nowadays.

The list of coworkers at different Institutions I have worked with/at is given below. Apologies to those -not by neglect but by possible skip of memory- who are not mentioned.

Department of Pharmacology and Pharmacotherapy, Semmelweis University Abbas, Mamode Yusuf Al-Khrasani, Mahmoud Balogh Jeno Elor, Guy Fürst Zsuzsanna Gábriel Lilla Gulyás Antal Gyires Klára Hadházi Pál

47 Illés Péter Juhász Krisztina Kató Erzsébet Makó Éva Péter Sámuel Somogyi György Szalai Judit Vizi E. Szilveszter Wachtl Istvánné

Department of Pharmacodynamy and Isotope Department, Semmelweis University Lengyel József Magyar Kálmán Nagy Tibor Tarcali József

1st Department of Anatomy, Semmelweis University Fehér Erzsébet Palkovits Miklós

2nd Department of Internal Medicine, Semmelweis University Adlesz Vilmos Rácz Kálmán

Montefiore Hospital and Medical Center & Albert Einstein College, N.Y. Amaki, Yoshikiyo Fishman, Jack Foldes, Francis F.

48 Hahn, Elliot F. Norton, Baiba

The Rockefeller Institute, N.Y. Ceccarelli, Bruno Gorio, Alfredo Hurlbut, Paul Mauro, Alexis

Catholic University, Nijmegen Crul, Jan F. Kleinhans, Wim vonWesel, Henny

Institute for Drug Research, Budapest Bajusz Sándor Berzétei Ilona Borsi József Dunai-Kovács Zsuzsanna Gráf László Háry Anikó Jaszlits László Kenessey Ágnes Kurgyis József Miglécz Erzsébet Mohai Zsuzsa Patthy András Székely József I. Tardos László

49 Tarnawa István Turán András

Institute of Experimental Medicine, Hungarian Academy of Sciences Barna István Fekete Márton I.K. Gacsal Éva Hársing László G. Kanyicska Béla Major Zsuzsa Mergl Zsuzsa Seregi András Serfozo Péter

Biological Research Center of Hungarian Academy of Sciences, Szeged Benyhe Sándor Borsodi Anna Kramer Mihály Soós József Tóth Géza Wollemann Mária

Central Research Institute of Chemistry, Hungarian Academy of Sciences Kardos Julianna Simonyi Miklós

50 Research Group of Peptide Chemistry, Hungarian Academy of Sciences/Eötvös University Botyánszki János Hepp József Hollósi Miklós Kalmár Bernadett Kocsis László Magyar Anna Majer Zsuzsa Medzihradszky Kálmán Medzihradszky-Schweiger Hedvig Orosz György Perczel András Valtinyi Lidia

Department of Comparative Physiology, Eötvös University Ádám György Juhász Gábor Kékesi Katalin Kirilly Zsuzsanna

6. References 1. Al-Khrasani, M., Elor, G., Abbas, M.Y., Rónai, A.Z.. The effect of endomorphins on the release of 3H-norepinephrine from rat nucleus tractus solitarii slices. Regulatory Peptides 111, 97-101, 2003. 2. Al-Khrasani, M., Orosz, G., Kocsis, L., Farkas, V., Magyar, A., Lengyel, I., Benyhe, S., Borsodi, A., Rónai A.Z. Receptor constants for endomorphin-1 and endomorphin 1-ol indicate differences in efficacy and receptor occupancy. Eur.J.Pharmacol .421, 61-67, 2001.

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122. Rónai A.Z., Hársing L.G., Berzétei I.P. Bajusz S., Vizi E.S. [Met5]-enkephalin -Arg6-Phe7 acts on vascular opiate receptors. European J. Pharmacol. 79, 337-338, 1982. 123. Rónai A.Z., Kató E., Al-Khrasani M., Hajdú M., Müllner K., Elor G., Gyires K., Fürst S., Palkovits M. Age and monosodium glutamate treatment cause changes in the stimulation-induced [3H]-norepinephrine release from rat nucleus tractus solitarii -dorsal vagal nucleus slices. Life Sci. 74, 1573-1580, 2004. 124. Rónai A.Z., Lengyel J., Nagy T., Orosz G., Adlesz V., Rácz K., Magyar K. Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu- cleaving dipeptidyl-carboxypeptidases in human adrenal tumors. Neuropeptides 31, 585-588, 1997b. 125. Rónai A.Z., Magyar A., Botyánszki J., Orosz G., Blahunka A., Medzihradszky K. Two families of novel, receptor-type selective antagonists: what do they tell us? Regulatory Peptides, Suppl. I., S57-58, 1994. 126. Rónai A.Z., Magyar A., Medzihradszky K. Kinetic parameters of antagonism by the delta opioid receptor selective peptide antagonist Boc-Tyr -Pro-Gly-Phe-Leu-Thr against selective and non-selective agonists in the mouse vas deferens. Neuropeptides 25, 127-129, 1993b. 127. Rónai A.Z., Magyar A., Orosz G., Blahunka A., Kátay E., Mak M., Majer-Decker Zs., Sohar P., Hollósi M. Receptor-related conclusions drawn from the actions of type -selective opioid antagonists and novel Tyr-D-Pro-derivatives. Med. Sci. Monitor 1, Suppl. I, 19-20, 1995b. 128. Rónai A.Z., Magyar A., Orosz G., Borsodi A., Benyhe S., Tóth G., Makó É., Kátay E., Babka E., Medzihradszky K. The opioid antagonist properties of highly d -receptor selective BOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu) peptide and its Phe1 and Mel1 analogues. Arch. int. Pharmacodyn. 330, 361-369, 1995c.

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7. Patents Bajusz S., Rónai A., Székely J., Gráf L., Mohai L. Eljárás morfin-szeru hatással rendelkezo pentapeptidek és származékaik eloállitására. HU GO-1350/76; Pentapeptides and process 2 5 for their preparation (covering (D-Met ,Pro NH2)-enkephalin). Belgian patent No. 858453. US patent 4465625/1984. Bajusz S., Rónai A., Székely J. Eljárás új enkefalin-származékok eloállitására. HU 178 004(GO-1448/1979), 1985. Bajusz S., Rónai A., Székely J. Eljárás új enkefalin analógok eloállitására. HU 181 013 (155/80), 1987.

65 Rónai A., Botyánszki J., Hepp J., Medzihradszky K., Borsodi A. Eljárás uj opioid receptorantagonista penta- és hexapeptidszármazékok eloállitására. HU 2551/91 (23571), 1991.

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