Research topics in the medicinal chemistry and molecular pharmacology of opioid peptides—present and future

Life Sciences, Vol. 39, pp. 1825-1843 Printed in the U.S.A.

Pergamon Journals

MINIREVIEW RESEARCH TOPICS IN THE MEDICINAL CHEMISTRY AND MOLECULAR PHARMACOLOGY OF OPIOID PEPTIDES--PRESENT AND FUTURE*

Rao S. Rapaka, Ph.D.

National I n s t i t u t e on Drug Abuse Rockville, Maryland 20857

i.

Introduction

2. Research Topics A. Biosynthesis and Processing of Precursors B. ~ / a - S p e c i f i c i t y in Enkephalin Analogs C. Conformational R e s t r i c t i o n in Analog Design D. Retro-lnverso Analogs E. Amphiphilic Secondary Structures F. I n h i b i t o r s f o r Enkephalin-Degrading Enzjanes G. Conformational Aspects of Opioid Peptides H. Lipids in Drug-Receptor Interaction I. Analysis of Opioid Peptides J. Opioi d Receptor Ty!oes K. Miscellaneous Aspects 3. Peptide Analogs in Drug Design-Advantages 4. Future Goals *This minireview is intended to discuss some of the important research topics only and is not intended as an exhaustive review. I t is impossible to r e f e r to a l l the p e r t i n e n t reviews and s c i e n t i f i c papers. A limited number of references only are given and interested readers may obtain other related references from the c i t e d a r t i c l e s , NIDA Research Monographs 69 &70 and, The Peptides: Analysis, Synthesis and Biology, Vol. 6, Opioid Peptides: Biology, Chemistry and Genetics. 0024-3205/86 $3.00 + .00

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Introduction

The conquest of pain has been long sought by monks, medical p r a c t i t i o n e r s , quacks, and laymen a l i k e , and each of them has attempted to achieve t h e i r goal in t h e i r own way. Opium has been used f o r centuries for i t s analgetic effects by the ancient cultures. In 1806, Serturner discovered the analgetic e f f e c t s of morphine. However, the side effects of the opiates, such as addiction l i a b i l i t y and r e s p i r a t o r y depression, were soon realized. This f u r t h e r motivated research for other compounds, e i t h e r from n a t u r a l l y occurring plant drugs or synthetic sources. A number of useful compounds--such as meperidine, methadone, d-propoxyphene, nalorphine, phenazocine, etc.--were synthesized. However, the goal to discover an ideal analgetic without the undesirable side effects has not been achieved. In the meantime, advances in the neurosciences, endocrinology, protein synthesis, molecular biology, and other sciences have resulted in a better understanding of brain function and i t s modulation. The characterization of opiate receptors ( I - 3 ) and l a t e r the i s o l a t i o n and structure determination of the enkephalins (4) have i g n i t e d an explosive i n t e r e s t in the understanding of the role of the endogenous |igands in general, and t h e i r role in brain function in p a r t i c u l a r . Subsequent developments in recombinant DNA technology added an important tool to research on precursors of the endogenous ligands. The opiate receptor--the s i t e where the opiates i n t e r a c t to produce the b i o l o g i c effect--was soon to be called the opioid receptor, as the endogenous ligands are peptides. A number of other opioid peptides were soon discovered. Following t h e i r discovery, several hundred synthetic analogs were made and tested. Extensive pharmacological testing and studies on binding at the receptor s i t e showed that the opioid receptors are heterogenous (5) and that at least four types of receptors e x i s t (5,6). As information accumulated on opioid peptide families and t h e i r functions, i t seemed that i t would be useful to discuss the pertinent questions on the medicinal chemistry and molecular pharmacology of opioid peptides. Research on opioids is progressing in several d i r e c t i o n s . Some of the important topics that have a t t r a c t e d a great deal of attention by researchers are: biosynthesis, processing of precursors, endogenous antagonists, s p e c i f i c i t y in drug design, conformational r e s t r i c t i o n in analog design, i n h i b i t o r s of enkephalin-degrading enzymes as analgetic drugs, conformational features of peptides, r e l a t i o n s h i p between ligand conformation and receptor s e l e c t i v i t y , r e t r o - i n v e r s o analogs, amphiphilic helices, receptor types and t h e i r s t r u c t u r e , drug-receptor i n t e r a c t i o n , role of l i p i d s in the i n t e r a c t i o n , and a n a l y t i c a l methodology. Some of these aspects are reviewed in a preliminary fashion below. For related information, see reviews (7-13). Research Topics A. Biosynthesis and Processing of Precursors Biosynthesis of opioid peptides is being a c t i v e l y investigated ( f o r reviews see 14-16). A number of t r a n s l a t i o n a l and p o s t t r a n s l a t i o n a l events are involved in the generation of active peptides, and these processes occur in a well-defined order. Processing of the opioid peptides appears to be t i s s u e - s p e c i f i c and d i f f e r e n t sets of processing enzymes seem to be responsible f o r the t i s s u e - s p e c i f i c processing of the neuroendocrine peptides. Pairs of basic amino acids serve as signals for the processing

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enzymes. The enzymes appear to be trypsin-like and, quite often, carboxypeptidase-like a c t i v i t y is also invo]ved (17,18). Other posttranslational modifications include phosphory~ation, acetylation, sulfation, methylation, glycosylation, amidation, etc. Processing of precursor peptides to bioactive peptides is largely explored using synthetic peptides (18,19). In addition to the "dibasic residues," X-Arg-Pro- appears to be another processing signal as postulated byMatsuo (20), or referred to as the single arginine cleavage (21). This is exemplified by the generation of metorphamide (21), or adrenorphin (20) from proenkephalin (corresponding to the peptide segment encompassing residues 210-217 of the precursor). Another example of single arginine cleavage is generation of dynorphin(l-8) from prodynorphin (corresponding to the peptide segment encompassing residues 209-216 of the precursor). Single arginine cleavage was also postulated for the processing of dynorphin B-29 to d~1orphin B(l-13) at the Thr-Arg bond (22). Here i t is to be noted that Arg is followed by Ser (not Pro}. Another interesting processing event is amidation. Mechanism of C-terminal amidation was investigated by Bradbury et al. (23). C-terminal amidation involves the action of a specific enzyme and glycine is a mandatory amino acid in the C-terminal position. The amide does not arise from ammonia or by direct amidation or transamidation. The mechanism involves removal of hydrogen from the C-terminal glycine and spontaneous hydrolysis of the i~ino linkage. Amidation is well known for neurohypophyseal peptides, oxytocin and vasopressin (24}, and, more and more amidated opioid peptides are being isolated. Metorphamide or adrenorphin Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH 2 is an amidated peptide (21). Dermorphin, Tyr-D-Ala-Gly-Phe-Tyr-Pro-SerNH2, is another amidated peptide and is isolated from the skin of the South American frog of the phyllomedusa species (25). Recently, another amidated peptide, amidorphin, was isolated from bovine adrenal medulla (26}. A very significant aspect in the isolation of this peptide is that i t was logically deduced to be a processed product of the known bovine proenkephalin A sequence, as i t has the necessary processing signals including a terminal glycine essential for amidation and the other significant aspect of the paper by Seizinger et al. (26) is that the expected product was f i r s t synthesized to establish the presence of the proposed endogenous product. An important aspect of amidation is its effect on receptor s e l e c t i v i t y . This is reflected in the decreased a-receptor a f f i n i t y of Met5-enkephalinamide as compared to Met5-enkephalin. However, amidation of Val-8, as in metorphamide, causes an increased a f f i n i t y at the N-receptor site with an insignificant change at the u- and a-sites (21). Li (27) and colleagues have determined the primary sequence for B-endorphins from various species. They a l l consist of 31 amino acids with Tyr-Gly-Gly-Phe-Met at the N-terminal sequence. Although minor differences are noted in the primary sequence for B-endorphins isolated from various species as compared to the human, i t appears the amino acid sequence in general is highly conserved. These various B-endorphins isolated from several species, due to minor changes in the primary sequence, way be considered as the naturally occurring analogs of the Bh-endorphi'n. Biological a c t i v i t y of some of these endorphins was evaluated and i t was found that the ratio of binding a c t i v i t y to analgetic potency varied for these endorphins. The discrepancies between receptor a f f i n i t y in v i t r o and analgesic potency i n v i v o may reflect differences in "efficacy" between analogs. However, other factors such as different transport, absorption and degradation properties, which play an important role in the in vivo assay, are more l i k e l y to be the cause of these discrepancies.

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In addition, smaller fragments of B-endorphin appear to be present in the p i t u i t a r y glands of several animals. In fact, B-endorphin (I-27) was isolated from porcine tissue (28) and was later synthesized (29). This naturally occurring Bh-enderphin (I-27) i n h i b i t s the analgetic properties of Bh-endorphin. Other synthetic fragments of Bh-endorphin (I-28), and (I-26) also i n h i b i t analgesia induced by Bh-endorphin (30). These observations have led Li and colleagues to formulate the following hypothesis, " I n h i b i t i o n of a peptide hormone by a naturally occurring segment of the same hormone may be a general phenomenon in b i o l o g i c a l l y active pepti des." B. u / a - S p e c i f i c i t y in Enkephalin Analogs After the realization that the opioid receptor is heterogenous and that there are at least three types of receptors (~, a, and K) and possibly a fourth type (~), several groups have attempted to understand the features essential for receptor s e l e c t i v i t y (31-42). Several thousand analogs of enkephalins were synthesized and receptor s p e c i f i c i t y was investigated (31, 38, 43-49 and references therein). Following rational design, a-receptor specific enkephalin analogs, such as H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH (DSLET) and H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH (DTLET), were developed (46,50). To summarize b r i e f l y , the principal characteristics for a-receptor binding a f f i n i t y for noncyclic (excIuding the conformationally r e s t r i c t e d analogs) enkephalin analogs are (45-47): a) b) c)

d)

hydrophilic side chain in position 2 (51), aromatic residue in position 4 of the peptide sequence (46), a C-terminal COOH group on the f i f t h amino acid, as modifications such as reduction to alcohol (50-54), amidation (55), e s t e r i f i c a t i o n (56-58) or removal (59) generally decrease the a-receptor selectivity. A conformationa] role was proposed for terminal COOH group (of the f i f t h residue) in the recognition of the a-receptor (46). lengthening of the enkephalin peptide chain by Thr 6 residue; for example, the hexapeptide H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH, where GIy 2 is replaced b~v D-Ser (as in a), and the pentapeptide sequence is extended by Thr D, is a highly selective a-agonist (51). Similarly H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH is another a-selective agonist (50).

Characteristics of high s p e c i f i c i t y for ~-binding sites are (45-47, 60), a) b) c) d)

a hydrophobic D-residue in the second position, shortening of the enkephalin sequence to the tetrapeptide by removal of the f i f t h amino acid residue (46), removal of the terminal carboxyl group (59), replacement of Phe4 residue with l i p o p h i l i c alkyl chain (46) and stereo-orientation of the Phe4 residue (61-62).

Although these modifications produced s i g n i f i c a n t l y improved receptor-specific ligands, alternate approaches were also attempted. C. Conformational Restriction in Analog Design One of the synthetic approaches that has resulted in the development of highly receptor-selective ligands is conformational r e s t r i c t i o n or constriction. For an excellent minireview on conformational r e s t r i c t i o n in peptides via amino acid side chains, see Hruby (63). Linear peptides are highly f l e x i b l e and e x i s t in a conformational equilibrium. Moreover, the

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conformational states may be influenced by the solvent and the surrounding environment. Hence, i f one of the various conformers is the most active form, then "dilution of the active forms" can be avoided by reducing the number of inactive forms. Formation of cyc]ic peptides is one of the main modes. Someother structural modifications leading to conformational restriction include N-methylation of the aMide NH, rep]acement of the C:H (to C~-CH3), formation of disulfide bpnds and other cyclizations via side-chains. These conformationally{restrained analogs might have increased agonist or antagonist properties due :to enhanced conformationa] integrity, thus possessing increased s p e c i f i c i t y and biological a c t i v i t y . These analogs may have increased enzymatic s t a b i l i t y , as well. This technique has already been u t i l i z e d for developing analogs with desired properties in the oxytocin and bradykinin series. In the opioid peptides, a number of conformationally restricted analogs were synthesized for the enkephalin peptides. As the i n t e g r i t y of the Tyr residue has to be maintained at position I and as manipulations are permissible at positions 2 and 5, most of these analogs were modified at positions 2 and 5. One of the earliest syntheses was by Schiller and col]eagues using D-~, w-diaminobutyric acid as the second residue and affecting the cyclization of w-amino group to the C-terminal carboxyl group (64-66). Two of the cyclic analogs involving the m-amino group to the C-terminal carboxyl group are H-Tyr-c[-NY-D-A2bu-Gly-Phe-Leu] and H-Tyr-c-[-NY-D-Lys-GIyPhe-Leu-]. Studies with these analogs, and their linear analogs, and a number of other modified analogs revealed that the cyclic analogs and their linear counterparts may involve a common mode of binding in their interaction with identical receptor subsites (67,68). General]y, cyclic peptides exhibited preference for u-receptors over a-receptors and were biologically more active, whereas the corresponding open chain analogs did not show similar preference or enhanced a c t i v i t y . Similarly, other cyclic peptides were synthesized u t i l i z i n g cyclizations between the side chains of Lys and Orn, with those of Asp and Glu (69). An example of such a side-chain cyclized product is H-Tyr-D-Orn-Phe-Asp-NH2 (70). This compound is the hlghest u-receptor selectlve cyclic analog syntheslzed to date and has an IC50 (MVD)/ICso (GPI) ratio of 107 (68-70). I t is comparable in its u-receptor s e l e c t i v i t y to DAGO (59). Another type of side-chain to side-chain cyclization was attempted using Cys or Pen residues in the 2 and 5 positions (71, 72). Using this technique, Hruby and colleagues (72-77, and references therein) synthesized a series of analogs. Among these analogs, [D-Pen2, CysS]-enkephalin, [D-Pen2, Penb]-enkephalin and [D-PenL,D-Penb]-enkephalin are some of the very highly selective a-receptor selective compounds . [D-Pen2,D-Pen5]Enkephalin demonstrates the beneficial effects of further conformational restriction of gem-dimethyl groups of the Pen moiety. Another example of u t i l i z a t i o n of conformational restriction in analog design is the synthesis of a somatostatin analog, D-Phe-CLy_s-Tyr-D-Trp-Lys-Thr-P~n-Thr-NH2, a very potent, ,-opioid receptor selectiv-e c~o-u-n-d--(77, TS]. .... Other types of s t e r i c a l l y constrained enkephalins are those that contain e-amino isobutyric acid and cyclopentane-l-carboxylic acid residues (79). Another mode of conformational restriction is the synthesis of peptides incorporating ~,B-dehydroamino acids (referred to as the dehydro- or A-amino acids) or the cyclopropyl amino acids (v-amino acids). These amino acid substitutions confer unique chemical and stereochemical properties, as they effect both chemical r e a c t i v i t y and conformation. For reviews see Noda et al. (80) and Stammer (81,82). A substitution at the second position, i . e . , a-Ala2-enkephalin f a c i l i t a t e d interaction at the u-receptor site (due to

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enhanced l i p o p h i l i c i t y ) , whereas s u b s t i t u t i o n at the 3-position in enkephalin may have f a c i l i t a t e d the i n t e r a c t i o n at the a-receptor s i t e . Studies on a-Phe4-enkephalins indicated that the Z-compound (phenyl ring and C=O are trans) s t e r i c a l l y favors i n t e r a c t i o n with the a-receptor s i t e (83). An additional advantage of s u b s t i t u t i o n with amino acids is that peptide linkages with ~, B-dehydro-amino acid residues are highly r e s i s t a n t to enzymatic degradation at the C-terminal side and completely r e s i s t a n t at the N-terminal side (84). Enkephalin analogs with cyclopropyl amino acid residues seem to he r e s i s t a n t to enzymatic degradation as well (82). To date, conformational r e s t r i c t i o n appears to provide the most successful approach in the design of synthetic analogs with high receptor selectivities. Another o r i g i n a l approach for improving the s e l e c t i v i t y of receptor binding, although less explored, is formation of dimeric peptides (45,84,85). Dimeric penta- and tetrapeptide analogs of enkephalins were shown to e x h i b i t enhanced a f f i n i t y and s e l e c t i v i t y f o r the a-receptor, although the results are less drama t i c . D. Retro-lnverso Analogs The b i o l o g i c a l a c t i v i t i e s of peptides generally depend on both primary and secondary s t r u c t u r e . For some c y c l i c peptides i t was shown that retro-isomers--where the direction of the amide bonds linking the residues is reversed retain b i o l o g i c a | a c t i v i t y . In these cyclic peptides, i t may be assumed that biological a c t i v i t y results from the three-dimensional topology and not from the peptide backbone. The concept of retro-peptides has also been extended to linear peptides (86). To obtain closeness in three-dimensional s t r u c t u r e , a f u r t h e r modification of c h i r a l i t y at each chiral center was also investigated. These modified analogs are named " r e t r o - i n v e r s o - p e p t i d e s . " Several p a r t i a l l y modified retro-inverso-peptides were synthesized for a number of b i o l o g i c a l l y active peptides. These modifications helped in assessing the r e l a t i v e importance of side-c~ain versus backbone structure in conferring biological a c t i v i t y . An additional reason for synthesis of these analogs is the expected resistance to biodegradation. This concept was applied in developing analogs of enkephalins (87), enkephalinase i n h i b i t o r s such as retrotniorphan (88), and dermorphi n (89). E. Amphiphilic Secondary Structures Prediction of formation of certain types of secondary structures from the amino acid sequences is very helpful in the rational design of b i o l o g i c a l l y active peptides. The amphiphilic environment of the aqueous solution and l i p i d b i l a y e r of the membrane could impose a secondary structure on t~e peptide. I f the secondary structure formation is i n i t i a t e d by the membrane, i t is l i k e l y to be characterized by the amphiphilic d i s t r i b u t i o n of the individual amino acid side chains. Amphiphilic helical structures have been hypothesized to be important in determining the b i o l o g i c a l a c t i v i t y of a number of peptides. This approach was successfully u t i l i z e d in the design of B-endorphin analogs (90-93). For a review on amphiphilic secondary structures, see Kaiser and Kezdy (94). F. I n h i b i t o r s f o r Enkephalin Degrading Enzymes One of the main problems in the bioassay of the peptides and in t h e i r use as analgetics has been t h e i r ease of biodegradation (for reviews see 19, 38, 43, 45, 88). Enzymes involved in the metabolism of opioid peptides, as well as

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their isolation, assay, and s p e c i f i c i t y are described by Marks et al. (19 and references therein). Enkephalins are easily metabolized; Tyr1-Gly 2 bond is cleaved by aminopeptidases, Gly3-Gly 4 bond by enkephalinase and Phe4-LeuS(Met5) by carboxypeptidases. Although extensive research has been carried out on these enzymes, the s p e c i f i c i t y of enzymes and the precise mechanisms of action are s t i l l not established. In addition to the specific cleavage at a particular amide bond, sequential processing has also been proposed as an alternate mechanism of action (17,]8,95). In order to prevent the degradation of the enkephalins, a variety of approaches have been u t i l i z e d such as replacement of D-amino acid for L-amino acid, N-methylation of the amide linkages, replacement of amide linkages, or substitution with unusual amino acids (dehydroamino acids, etc.). A closely related but more innovative approach is the rational design of inhiDitors for the enkephalin degrading-enzymes (88). These compounds prolong the duration of action of the endogenously released enkephalins and, hence, may be devoid of the major side effects of narcotic analgetics. A number of enkephalinase inhibitors are derived from Phe-Leu bearing carboxy] groups (to chelate Zn required for this metalloenzyme) and are of the general formula: R-CH( CH2@-CONH-CH[CH2CH(CH3 ) 2] COOHwith R=-COOH,-CH2COOH, -NH-CH2-COOH and -NH-CH2-CH2-COOH. Highly potent enKephalinase inhibitors were also obtained from L-Phe-B-Ala derivatives. Another series of enkephalinase inhibitors was obtained from compounds that contained a thiol group, R-CH-(CH2~)CONH-CHECH2CH-(CH3)2]-COOH, where R=-CH2SH and -SH-CH2-CO. Although these are excellent enkephalinase inhibitors, they are also angiotensin converting enzyme (ACE)inhibitors. To enhance the s p e c i f i c i t y , the "retro" compound HS-CH2CH(CH2@) NH-CO-CH2-COOH, retrothiorphan, was developed which, while retaining enkephalinase inhibitors property, exhibited s i g n i f i c a n t l y less ACE inhibitor property. In summary, this type of rational of design has provided researchers with a number of inhibitors, such as thiorphan, retrothiorphan, and kelatorphan. The clinical use of these compounds, however, is not promising due to the limited b i o a v a i l a b i l i t y of some. Developmentof synthetic analogs that are o r a l l y active, or have good b i o a v a i ] a b i l i t y may be of therapeutic use, and this is expected to be achieved in the near future. G. Conformational Aspects of Opioid Peptides Conformational aspects of opioid peptides have been examined extensively (for a review on enkephalins, see 96). Most of the work has been focused on enkephalins, on morphiceptins, and to some extent on endorphins. The presence of a B-turn in enkephalins was proposed, from X-ray crystallographic data and solution studies (for reviews see 68,96,97). The number of hydrogen bonds (2 or 3), and the type of B-turn (type I or II or I' or I I ' ) a r e not yet agreed upon universally and much more work is warranted in this area. Fortunately, several physical methods are available to study conformational aspects of peptides (for recent reviews see 98-I02) and i t is anticipated that a much more clearer understanding of conformation-biological a c t i v i t y will soon be forth-coming. Yamashiro and Li (34) reviewed the s t r u c t u r e - a c t i v i t y features of endorphins and for a general review on dynorphin peptides see Goldstein (33). Some of the other opioid peptides of interest are dermorphin and dynorphin. Few conformational studies are reported on dynorphin in the literature (103-I06). From circular dichroism studies on dynorphin(1-13), Maroun and Mattice concluded that dynorphin does not have an ordered structure in water (103). However, addition of a detergent such, as dodecyl sulfate, enhances helical content to some. extent. From fluorescence studies on the b i o l o g i c a l l y active Trp4-analogs of dynorphin(1-13), Schiller observed that

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Tyrl-Trp 4 are at least 15 A° apart (unlike the enkephalin Tyrl-Trp ~ analog, which appears to have a folded conformation)and the predominant conformation is where the N-terminal tetrapeptide is almost completely extended. In a d d i t i o n , S c h i l l e r proposedrthat these conformational differences between dynorphin and Leu~-enkephalin may play a role in preferences for various opioid receptor subclasses.

To date, few conformational studies have been done on dermorphin or related peptides (107-111). Salvadori et al. (107) suggested the presence of a linear extended structure from NMR studies and, due to lack of significant dependence on PMR parameters over a concentration range, the results indicated a monomeric peptide. Arlandini et al. (108) using various spectroscopic techniques, suggested that dermorphin may preferentially exist in an extended, f l e x i b l e conformation and that an intermolecularly stabilized form exists in DMSO. Recently, from FT-IR, laser Raman, CD, conformational energy calculations, and molecular modeling studies, i t was proposed that dermorphin contains two B-turns--a type I I I B-turn between residues one and four and a type II B-turn between residue five and the terminal amide group. This conformation with two B-turns is highly folded and mimics the "morphine-type" conformation; and due to this conformation, i t may act as a potent ~-receptor agonist. This highly folded conformation and the hydrophobic environment may explain its s t a b i l i t y as well (109). In a recent study by Toma et al. ( i l l ) a satisfactory agreement was obtained between the preferred solution conformations and the calculated conformations. The solution conformations was determined 'H-nmr in dimethyl sulfoxide and the calculated conformations was deduced using conformational energy calculations using semiempirical partitioned energy function methods. Studies were also extended to the L-Ala~-dermorphin analog~ Th~ results indicated the predominance of type I B-turn around Pro°-SeW and an extended structure in the sequence of Phe3-Gly4-Tyr5. Although earlier conformational studies mainly addressed the correlation of structure between opioid peptides and r i g i d opiates, the discovery of multiple opioid receptor and the realization that different opioid receptor types may have differing conformational requirements (64-66, 69) considerably influenced the direction of research in this area . Conformational analysis using theoretical methods has been extensively reviewed byGorin et al. ( l l 2 ) and Schiller (96). In addition, computer-assisted drug design (i13) and multidisciplinary approaches (114) have also played a significant role in the understanding of conformational features. Recently, a computational procedure for determining the energetically favorable binding sites on macromolecules has been reported (I15) and this may be of help in drug design. For reviews on theoretical methods for the analysis of peptide conformations and related information see Zimmerman (I00) and Hagler ( l O l ) . The role of conformation and i t s r e l a t i o n to b i o l o g i c a l a c t i v i t y is not f u l l y established. The peptide may e x i s t in a number of conformations and may continuously i n t e r c o n v e r t , e s p e c i a l l y in the case of small peptides such as the enkephalins. I t is a question of whether a b i o a c t i v e conformation preexists, such as in the hydroxylation of protocollagen by proline hydroxylase (116) or is adopted (such as the "zipper"-type model, 117). I t is conceivable that the opioid receptors have conformational requirements f o r i n t e r a c t i o n which d i f f e r depending on the receptor type (64-66, 69) and several groups have proposed a r e l a t i o n s h i p between conformation and r e c e p t o r - s e l e c t i v i t y (118-127). A folded B-turn conformation, s i m i l a r to morphine, was proposed to be the bioactive conformation f o r morphine-like or u-receptor a c t i v i t y by Smith and G r i f f i n (121). On the contrary, Camerman et a l . (122) proposed for ~-receptor binding that an extended conformation is the preferred conformation. Doi et a l . (123) found that Boc-Tyr-Gly-Gly-

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(4-bromo)Phe-Met-OH is composed of a n t i p a r a l l e l s-sheets, that this structure is s t a b i l i z e d by intermolecular hydrogen bonds, and that the Boc-peptide has considerable analgetic a c t i v i t y (although less than the z w i t t e r i o n i c peptide). From these observations, they proposed that the dimeric extended conformation may be involved in binding to the a-receptor. This proposal is supported by the finding that dimeric enkephalins e x h i b i t enhanced binding to a-sit~s (84). _Later, the same group (124) determined the crystal structures of Leub- and Metb-enkephalins and t h e i r (4-bromo)-Phe4-analogs. From these studies they found that Leub-enkephalins have type I ' s-turn conformation and Met5-analogs possess an extended dimeric conformation. They proposed that dimeric extended forms i n t e r a c t with the a-receptor, while the B-turn conformer interacts with the ~-receptor s i t e s . Recently, Renugopalakrishnan et al. (125), from t h e i r conformational studies using FT-IR and laser Raman spectroscopies, proposed that B-turn containing enkephalins i n t e r a c t with a-receptor sites and B-sheet containing peptides i n t e r a c t with u-receptor sites. Future studies might help in determining the conformation-receptor s e l e c t i v i t y in the opioid peptides. H. Lipids in Drug-Receptor Interaction So f a r , the ligand-receptor i n t e r a c t i o n has been discussed as i f these were the only components involved. The s i t u a t i o n is much more complex. I n t e r a c t i o n between the opioid peptides and l i p i d bilayers might be a very important event in the molecular mechanisms of ~)iological a c t i v i t y of these peptides (106, 128, 129). "The l i p i d phase of the membrane acts as a matrix for the receptors and is essentia] for the f u n c t i o n a l i t y and b i o l o g i c a l arrangement of the receptor proteins" (130). This does not mean that lipids are the peptide receptors, but rather that they are a means to capture peptides ( e i t h e r by hydrophobic or e l e c t r o s t a t i c interactions or both) and Facilitate interaction with the receptor (131). Gysin and Schwyzer investigated the head group and structure specific interaction of dynorphin and enkephalin liposomes, a-Agonists interacted more with phosphatidylserine (PS) and a mixture of lecithin and phosphotidic acid (PC/PA), than with cerbroside sulfate (CS). ~-Agonists interacted more strongly with CS liposomes than with PS and PCIPA, and~-agonist dynorphin(1-13) interacted more strongly and more equally with all the systems. This makes i t possible to distinguish ligand types by l i p i d head group specificity. Several other groups studied the interaction of lipids and detergents with hormonal peptides (132), methionine enkephalin (133), and dynorphin(1-13) (103). The significance of these investigations has yet to be explored by further research. I. Analysis of Opioid Peptides An area of growing interest and need is the quantitative analysis of opioid peptides. Although RIA methods are largely being used at present, instrumental methods such as HPLC and LC/MS that are accurate, specific, sensitive, and reproducible are required (for reviews see 134-139). For additional information on LC/MS methodology in biomaterials, see (140-142). These methods are essential to provide specific analysis of low levels of endogenous peptides (active components, precursors, and metabolites) and to understand pathological states mediated through either lower or higher levels of these peptides. These methods should be available in conjunction with immunoassay methods to determine the levels of synthetic peptides i f they are used as drugs. These methods should also be available for the pharmacokinetic evaluation of the various bioactive moieties in biofluids.

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O. Opioid Receptor Types Current research on opioid receptors has been reviewed extensively (32, 35-37, 143-149). Sub-types have already been identified for some receptors (149). In the near future, the receptor types will be mapped and the subtle differences involved in their structure and organization will be better understood. At present, i t may be speculated that an endogenous ligands might exist for ~-, 6- and K-receptors. In a recent paper, Oka et al reported that morphine could be found in the bovine CNS and in the skin of the toad and other animals (150). This raises the question of whether there exists an endogenous peptide ligand for the ~-receptor and whether i t is peptidic in nature. A PCP receptor has been identified (151) and evidence suggests that the endogenous ligand is present and is peptidic in nature (152, 153). This will be an active area of research in drug abuse in the near future. K. Miscellaneous Aspects of Opioid Peptides One of the significant aspects in the opioid research is the development of peptide antagonists such as the N-cyclopropylmethyl, N-allyl and the N-diallyl enkephalin analogs (154-159). Synthesis of opioid peptide analogs as photoaffinity agents for the characterization of opioid receptor types is another important area of research. To date, several photoaffinity reagents have been synthesized and their interaction with the receptors studied (160-167). Much more work s t i l l needs to be done in these areas. Clinical significance of opioid peptides in humans is currently an active area of research ( for reviews see 168,169). Attempts are under way to develop c l i n i c a l l y useful drugs and evaluate them (170,171). One example is the use of FK 33-824, to detoxify hard-core heroin abusers (172). For reviews or additional information on peptides in the treatment of opiate addiction see Bhargava (173,174), Ritzmann et al (175) and Chipkin et al. (176). 3. Peptide Analogs in Dru9 Design-Advantages Extensive research has been carried out on naturally occurring and synthetic opiates in order to obtain more useful analgetic agents. ,Opioid peptides offer more promise than the opiates in drug development because: I. These compounds are endogenous and, on metabolic degradation (unlike the opiates), break down to amino acids. Hence, the metabolites are nontoxic and do not cause kidney and liver damage (unlike the opiates). 2. Since the peptides are made up of several subunits, the amino acid residues, a large number of analogs can be synthesized from a few basic building blocks. Moreover, simple modifications, such as substitution of unnatural amino acids, s t e r i c a I l y constrained amino acids, or other modifications, may be attempted to develop analogs with a desired biological a c t i v i t y . 3. The peptide analogs (unlike the opiates) are f l e x i b l e conformationally and, are ideal molecules to investigate relationships between 3-D structure and biological a c t i v i t y . They may be cyclized or subjected to other synthetic modifications, so that analogs of varying and controlled f l e x i b i l i t y can be synthesized and studied.

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4. As these molecules are substantially polar, so]ution studies can be performed with different solvents and ions (calcium, sodium, etc.) to better understand their influence on peptide conformations. 5. Technology for the synthesis of peptides is readily available and solid-phase peptide synthesis is already automated to a considerable extent so that even complex molecules can be synthesized in large quantities. Even the ultimate goal of synthesizing pro-enkephalin (about 250 residues) and pro-dynorphin to provide scientists with adequate material for studies on processing mechanisms seems possible (177). Recently, synthesis of polypeptides by recombinant DNA methods has become a powerful tool in the armamentarium of the peptide/protein chemist (178-183). 6. To date, the compounds that are highly selective for a-receptor have been opioid peptides. 7. Due to the unique character of some of the peptide drugs, i . e . t h e i r i n a b i l i t y to cross placental b a r r i e r (due to placental enzymatic deactivation of the peptides), these drugs have a d i s t i n c t advantage for being used as analgetics in pregnant females (170, 171). They are d e f i n i t e l y superior to meperidine in this respect. 4. Future Goals At present, research in opioid peptides is progressing at a rapid rate. h i g h l i g h t some of the important aspects:

To

Analgetic peptides are to be designed that show minimal drug dependence potential. The peptides of potential i n t e r e s t dre enkephalins, endorphins, dynorphins, dermorphins, morphiceptins, and related opioid peptides. Some of the goals are: a) the development of peptide analogs that can be administered o r a l l y , that have long duration of action, and that have diminished potential for abuse; and b) synthesis of i n h i b i t o r s of enkephalin-degrading enzymes as potential analgetics. Extensive SAR studies on the opioid peptides may be of great value in the rational design of these analogs. I t is f u r t h e r a n t i c i p a t e d that SAR studies on opioid peptides might r e s u l t in the development of antagonists and mixed agonist-antagonists that are c l i n i c a l l y useful in the treatment of opiate addiction. Several hundred analogs of the opioid peptides have been evaluated to aid in delineating the essential features of the ~-and a-receptors. The synthesis of even more s p e c i f i c analogs f o r a l l types of receptors can be anticipated. The receptor types are well d i f f e r e n t i a t e d on the basis of binding studies and/or pharmacological studies. However, considerable information on sites and functions is s t i l l lacking. P a r t i c u l a r l y , the component peptides of the d i f f e r e n t receptors must be isolated and characterized, and receptors or receptor models must be synthesized or reaggregated to understand mechanism(s) of action. I t may be possible to study the conformation of the receptor in response to environment-mediated conformational changes ( i . e . , l i p i d s , ions, solvents, ionic strength, p o l a r i t y , e t c . ) and the conformation of the drug-receptor complex i t s e l f . A number of conformational studies have been conducted on opioid peptides, although most were performed on enkephalin peptides since they are r e l a t i v e l y small pentapeptides and are e a s i l y accessible. Conformational studies using t h e o r e t i c a l , s o l i d - s t a t e and solution methods have to be extended to a l l the

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p e r t i n e n t opioid peptides and t h e i r analogs. The effects of the surrounding environment (solvent, pH, ionic e f f e c t s , e t c . ) m u s t be examined; and, f i n a l l y , the receptor-bound conformation of the drug must be determined. Advanced methods such as X-ray crystallography, FT-IR, laser Raman, nuclear magnetic resonance, C.D., and computer-assisted drug design w i l l play an increasing role in these studies. Research in the opioid peptides is progressing at a rapid rate. In the near future, we may look forward to s t i l l more e x c i t i n g d~iscoveries, and there is room for optimism to believe that t h e r a p e u t i c a l l y useful peptide analgetics may soon be developed. ACKNOWLEDGMENTS: The author expresses his gratitude to Dr. H. W. K o s t e r l i t z , U n i v e r s i t y of Aberdeen, Aberdeen, Scotland f o r the review of the manuscript and for his valuable suggestions. References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15.

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