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Future Med Chem. Author manuscript; available in PMC 2012 December 1.

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Published in final edited form as:
Future Med Chem. 2012 February ; 4(2): 205–226. doi:10.4155/fmc.11.195.

Opioid glycopeptide analgesics derived from endogenous
enkephalins and endorphins
Yingxue Li1, Mark R Lefever1, Dhanasekaran Muthu1, Jean M Bidlack2, Edward J Bilsky3,
and Robin Polt*,1
1Department of Chemistry & Biochemistry, BIO5, The University of Arizona, Tucson, AZ 85721,
USA
2Department

of Pharmacology, University of Rochester Medical Center, 601 Elmwood Avenue,
Rochester, NY 14642, USA
3Department

of Pharmacology, University of New England College of Osteopathic Medicine, 11
Hill Beach Road, Biddeford, ME 04005, USA

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Abstract
Over the past two decades, potent and selective analgesics have been developed from endogenous
opioid peptides. Glycosylation provides an important means of modulating interaction with
biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the
resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows
penetration of cellular barriers that block efficient drug distribution, including the blood–brain
barrier. Extremely potent and selective opiate agonists have been developed from endogenous
peptides, some of which show great promise as drug candidates.

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The success rate of CNS drug development is lower than that of other therapeutic areas [1].
There are multiple reasons for this dearth of new drugs, including the sheer complexity of
the brain and its neuropathologies; a propensity for CNS drugs to cause CNS-mediated side
effects that limit dosing and compliance; a lack of validated biomarkers to inform whether a
given neurotherapeutic agent is engaging the target in sufficient concentrations to modulate
the CNS target; and the presence of the blood–brain barrier (BBB), across which CNS
agents need to penetrate. Among these challenges, the BBB is considered to be most
problematic for peptide or protein-based therapies [2–4]. However, peptides and proteins do
offer distinct advantages for developing efficacious and well-tolerated treatments for CNS
diseases, such as chronic pain, Alzheimer's disease and Parkinson's disease. These
advantages include intrinsic affinity and selectivity of the peptide for native receptors and
the metabolism of the peptide into smaller fragments and amino acids (versus the variety of
active metabolites seen with small molecules). In addition, peptides and proteins can bind to
multiple sites across a receptor protein, offering greater opportunity to fine tune the
receptor–effector response [5,6]. Historical challenges for peptide and protein drug
development are being addressed, including modifications that increase stability, techniques
that increase yields and lower total synthetic costs, and technology to improve tissue
targeting, including access to the CNS. A number of monographs have been written since

© 2012 Future Science Ltd
*
Author for correspondence: Tel.: +1 520 621 6322 Fax: +1 520 621 8407 polt@u.arizona.edu.
Financial & competing interests disclosure
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or
financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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the beginning of this century that describe formulations and articulate solutions of increasing
sophistication to address the problems of peptide-based drugs [7–9].

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Opioid receptors
The classical opioid receptors [10] are divided into three subtypes, the μ receptor (MOR or
MOP), the δ receptor (DOR or DOP) and the κ receptor (KOR or KOP). Another receptor,
the nociceptin or orphanin receptor (NOP or ORL1), is widely distributed in the CNS, and is
clearly related to the opioid receptors in terms of its molecular biology but is generally not
regarded as an opioid receptor as it does not respond to classical opioid agonists or
antagonists. IUPHAR recommends use of the terminology MOP, DOP, KOP and NOP,
replacing the older recommendation for OP1 and OP2, for example [201]. All of the opioid
receptors have been cloned from various species, including mouse, rat and human. Opioid
receptors are GPCRs that consist of highly homologous seven-transmembrane helical
domains, and are linked with extracellular peptide loops of very limited size (Figure 1)
[11,12].

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The MOP receptor [13] is distributed presynaptically in various brain regions, including the
limbic structures, the brainstem (i.e., the periaqueductal grey area) and in the superficial
dorsal horn of the spinal cord. It was initially characterized in functional smooth muscle
preparations of the guinea pig ileum (GPI). This receptor remains the principal target of
opioid analgesics currently in clinical use, with the μ referring to morphine. The MOP
receptor is widely distributed in other areas of the brain, as well as non-CNS tissues, most
relevantly in the immune system, and the cardiovascular and gastrointestinal systems.
Pharmacological activation of MOP not only modifies the transmission and perception of
nociceptive stimuli, but also a host of other effects, including reduced respiratory drive in
response to increased levels of CO2, opioid-induced bowel dysfunction, abuse liability and
pruritus (itching). Repeated or prolonged MOP activation results in adaptations that manifest
as tolerance and physical dependence, further complicating management of chronic pain
sufferers and patients with substance abuse disorders. Mixed opioid agonists/ antagonists
and partial MOP agonists have been used to limit some of the opioid side effects with a
mixed degree of success. Clearly, opioid pharmacology is a complex issue [14].

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The DOP receptor [15] was originally characterized using the mouse vas deferens smooth
muscle tissue preparation (mouse vas deferens [MVD] assay) [16]. The receptor is widely
distributed anatomically, being found in the same general anatomical areas as MOP. DOP
also contributes to a variety of physical and emotional effects, including initiation of
movement [17], regulation of pain and reward circuitry, as well as other complex CNS
behaviors (mood/ affect and anxiety). The issues of cellular co-localization of DOP and
MOP, as well as the formation of functional ‘hetero dimers’ continue to be the subject of
considerable interest [18–20]. Despite the progress that has been made in the localization of
opioid receptors [21], the precise localization and neuronal and cellular pathways through
which these three receptor types work remains incompletely understood [22,23]. Although
DOP receptors contribute to analgesia, euphoria and physical dependence, DOP agonists
may be able to produce broad spectrum analgesic efficacy with reduced propensity to
produce classic opioid side effects [24].
The KOP receptor, named after the κ-agonist ketocyclazocine, is not as well studied as MOP
and DOP, although it has attracted interest from pharmaceutical companies, as either a
stand-alone drug [25,26] or in conjunction with agonism of other opioid receptors [27].
Stimulation of CNS KOP receptors is generally associated with dysphoria and
psychomimetic effects, along with limited analgesic efficacy, especially in men [28].
Peripherally active KOP agonists produce antinociception in animal models of pain [29] and

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are being explored as targets for analgesia in several chronic pain states [18,30,31]. KOP
antagonists are also being investigated as treatments for addiction and depression [32].

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The diversity of the endogenous neuropep-tides and their receptors provide many
opportunities for drug discovery [33]. If there were a straightforward methodology for
converting endogenous neuropeptides into useful CNS drugs, then a new and potentially
sea-changing pharmacopeia would be available for the treatment of CNS disorders.

Endogenous opioid peptide agonists
Since the discovery of the two endogenous pentapeptides, Met-enkephalin and Leuenkephalin in the 1970s, perhaps as many as 300 endogenous neuropeptides have been
identified in widespread locations throughout the CNS Table 1. Endogenous opioid peptides
and their receptors undergo modulation in response to various physiological conditions, such
as inflammation, tissue injury, pain and other stressors [34].

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The three classes of endogenous opioid peptides (enkephalins, endorphins or dynorphins)
are typically assigned to the three types of opioid receptors (DOP, MOP and KOP,
respectively). This approach is misleading since the absolute selectivity of each peptide is
limited, and it neglects the fact that there are many cleavage variants of the neuropeptides,
splice variants for the receptors and, likely, variations in their glycoforms. The endogenous
peptides are not orthogonal and neuropeptide receptors might, for example, just as easily be
thought of as ‘metorphamide receptors’ or ‘enkephalin receptors’ [35]. Moreover, αendorphin and γ-endorphin have been found to be inactive at sites that are sensitive to βendorphin [36] and other sites have been found that are sensitive to γ-endorphin, which have
been referred to as ‘non-opioid’ in nature [37,38].

Schwyzer's membrane compartment concepts
Schwyzer articulated critical roles for the membrane in peptide–receptor binding events
(Figure 2) [39]. Although his ‘membrane compartment theory’ may have overstated the
influence of the membrane in differentiating receptor selectivity (μ vs δ vs κ), it is clear that
the membrane environment does play critical roles in pre-organizing the peptide
conformation prior to binding, as well as the peptide–receptor binding event itself [40]. We
view binding as a three-step process:

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■ Adsorption of the peptide ligand to the membrane. This promotes receptor binding by
reducing a 3D search for the receptor to a faster 2D search. Surface-assisted ‘reductionof-dimensionality’ calculations, performed by Polya in 1921, were examined by Max
Delbrück in which he quantitatively demonstrated the viability of this theory [41,42];
■ Conformational changes in the peptide induced by the asymmetric environment of the
membrane. Amphipathicity of the peptide is believed to reorganize the peptide–
membrane aggregates into minimal energy states [43,44];
■ The binding event itself. GPCR binding and activation is a complex, multifaceted
phenomenon. It is, however, beyond the scope of this review.

The neurovascular unit & the BBB
In order to enter the brain and CNS, pharmaceutical agents must first penetrate the BBB
(Figure 3). There are several strategies available to make a peptide metabolically stable but
the transport of a peptide across BBB is still remains a major hurdle in developing CNS
drugs [45]. Contrary to the belief that small molecules with molecular weights under 400
readily cross the BBB, it has been observed that almost 98% of small molecules do not
readily cross the BBB. Further, evidence has accumulated that peptides can penetrate the
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CNS by several different mechanisms [46–49]. These observations clearly indicate molecule
size is not the primary factor, but rather the overall physiochemical characteristics of the
molecule is critical for BBB transport. Since the BBB is composed of endothelial
membranes, many research groups focus on designing peptides with increased lipid
solubility. Simply increasing the lipid solubility of a drug molecule may have undesirable
effects, such as decreasing solubility and bioavailability, and increasing plasma protein
binding.

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The BBB is composed of endothelial membranes that function as a continuous lipid barrier
that protects the brain from toxic substances by preventing their entrance from bloodside to
CNS. The BBB is also an enzymatic barrier that poses additional challenges in developing
peptide/protein-based CNS drugs. Further, it has been viewed recently as a regulatory
interface between the CNS and circulation with nutritional, homeostatic and communication
functions. Understanding of the principles and physiology of BBB has improved a great deal
in the past decade. Moreover, evidence is accumulating that many peptides and proteins
cross the BBB in amounts sufficient to affect CNS function. It is now clear that the BBB is
not an absolute physical barrier but a regulatory tool that controls the delivery of the
substances to the CNS. Strategies based on this principle are proving to be very successful
[50]. Other strategies using ‘molecular umbrellas’ [51], ‘Trojan horses’ [52] and BBB
‘shuttles’ [53] have been proposed by various groups. We have successfully applied
glycosylation as a strategy to improve the BBB penetration, as well as the stability and
systemic availability of enkephalins and the larger endorphin-like peptides.

Glycopeptide synthesis
Initially, O-linked glycopeptides were considered to be exotic substances, and were difficult
to evaluate as drugs simply because the synthetic methods required to produce them in
tangible amounts were lacking. In the last 40 years this situation has changed, however,
glycopeptides are still significantly more difficult to produce than simple peptides, even if
the requisite Fmoc–amino acid glycosides are commercially available, and particularly so if
the glycosides require synthesis [54,55]. Enzymatic approaches have been incorporated into
chemical methods for serine and threonine glycosides [56]. The ideal methodology should
produce high yields of pure diastereomers, typically β-anomers for the highest stability.
Many classical methods produce high yields of the desired anomers, but require the
production of labile glycosyl donors or very reactive (e.g., unstable) promoters [57].

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Production of O-linked glucosides and lactosides of enkephalins and endorphins requires the
corresponding acetate-protected glycosides of Fmoc–serine or Fmoc–threonine. The
benzophenone Schiff base appeared to be a very good protection for amino groups of serine
and threo-nine for glycoside formation [58]. O-glycosides of these amino acids with
different monosaccharides, aminosugars and deoxysugars were obtained with excellent yield
and very high stereoselectivity [59]. Glycoside peracetates have been used as building
blocks for solid-phase glycopeptide synthesis, and significant improvements have been
made in the production of the glycoside building blocks [60]. An even more efficient and
direct approach to these precursors has now been developed that proceeds directly from
Fmoc–serine or Fmoc–threonine as glycosyl acceptors, and either β-D-glucose per-acetate or
β-lactose peracetate in the presence of ‘minimally competent’ Lewis acids, such as
indium(III)bromide (Figure 4) [61].
The glycopeptides can be synthesized manually based on established solid-phase Nfluorenylmethoxycarbonyl methods (Fmoc chemistry) (Figure 5). The side chain-protected
amino acids used by our research group were Fmoc–Lys(Boc)-OH, Fmoc–Glu(OtBu)-OH,
Fmoc–Asn(Trt)-OH, Fmoc–D-Thr(But)-OH, and Fmoc–Tyr(But)-OH. For support we have

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In an effort to enhance CNS bioavailability. Best results (e. and the N-terminal Boc group. using mechanization. was naive and incorrect. Glycoside placement proved critical for affinity as determined by radioligand binding studies. NIH-PA Author Manuscript Concurrently. Glycopeptide analgesics based on enkephalins Our initial attempts at improving the CNS bioavailability of opioid peptides came from collaborations between the Chemistry and Pharmacology Departments at the University of Arizona led by Victor Hruby [62]. The crude glycopeptides were precipitated in cold diethylether. 250 × 55 mm or equivalent) was used.. In 1983 this group successfully synthesized and characterized a series of cyclic penicillamine containing enkephalin analogues (e. with substitution typically ranging from 0.1% TFA to obtain glycopeptides of greater than 97% purity.g. we synthesized a series of enkephalin analogues (Tables 2–4).Li et al. F3CCOOH:Et3Si H:H2O:PhOCH3:CH2Cl2 (8:0. Table 2 highlights some of the initial enkephalin glycopeptides that were synthesized [67].5:0. the enkephalin glycosides did penetrate the BBB very effectively and produced potent and longlasting antinociception in mice after intravenous (iv. Glycosylation sites close to the N-terminus resulted in reduced affinity for both DOP and MOP. and that we would observe antinociceptive activity following systemic administration.2– 0.. in retrospect. we tested an alternative hypothesis.4. BBB penetration could be increased. which also removed the side chain protection. which may be regarded as an extremely hindered case of amino acid coupling. which survived the hydrazine treatment but was removed by TFA. with an acetonitrile–water (CH3CNH2O) gradient containing 0. Coupling of the Fmoc–amino acids was achieved using manual coupling methods with or without microwave heating or. Homogeneity of the pure glycopeptides was confirmed by analytical reverse-phase HPLC and MS. One hypothesis was that by increasing the lipophilicity of an already quite lipophilic DPDPE. with extended reaction times or heating only for coupling of the glycosidic residue and the residue following.) or intraperitoneal (ip.g.0]-undecane in DMF for 10 min with argon bubbling as agitation. A superior method used Boc protection for the last amino acid. NIH-PA Author Manuscript The Fmoc group was removed from the N-terminus of the growing glycopeptide chain using a mixture of 3% piperidine and 2% diaza-1. available in PMC 2012 December 1. Extension of the Future Med Chem. A preparative scale C18 column (Phenomenex® 250 × 22 mm. It was predicted that CNS delivery of the enkephalin molecule across the BBB would be increased. typically. Manual coupling reactions and critical couplings performed during mechanized coupling were typically monitored using Kaiser's ninhydrin test. Author manuscript. which was confirmed in an in vitro BBB model that used bovine brain microvessel endothelial cells [64]. Incorporation of an unnatural amino acid (D-penicillamine) and the cyclic constraint into the peptide enhanced both its stability and DOP selectivity. as determined by GPI and MVD assays. While the Glut-1 hypothesis eventually proved to be incorrect.8 meq/g.5:0. The acetyl protecting groups of the glycosides and the Nterminal Fmoc could be removed with 80% hydrazine hydrate (H2NNH2•H2O) in CH3OH with argon agitation 3X for approximately 2 h. if that method is employed. Page 5 NIH-PA Author Manuscript used Rink amide MBHA 1% DVB resin. whereby attachment of a glucose molecule to the modified enkephalin peptide would make the overall ligand a substrate for the Glut-1 transporter [65]. The synthetic glycopeptides were cleaved from the Rink resin with a ‘TFA cocktail’. .) injection [66]. which. redissolved in H2O and then lyophilized prior to reverse-phase HPLC. and efficacy. highest purities of the crude glycopeptides) were obtained by coupling well below the resin capacity and then capping the excess capacity with acetic anyhydride (Ac2O). Use of NMP as a solvent rather than the more polar DMF also aided these couplings.3-bicyclo[5. DPDPE) that had higher affinity and selectivity for DOP [63].05:1).

The addition of a glucoside to a serine in the sixth position of the peptide resulted in retention of modest selectivity for DOP over MOP in functional MVD/ GPI tissue assays and in receptorbinding studies [69]. Tyr-D-Thr-Gly-Phe-Leu-Thr) were produced. ip.Li et al. 9 and 10.and time-related antinociception following ip. . with the βmelibioside being the most potent of the three. injection into mice. In addition. It should be noted that we stayed with the linear enkephalin parent peptide as it had roughly equal affinity for DOP and MOP receptors. When compared with morphine.c. However. glycopeptide 12 was significantly more potent following systemic routes of administration (iv.or tri-saccharides provided any additional benefit to pharmacokinetic and pharmacodynamic properties. several other modifications were made to explore the geometry of the attachment point (D vs L amino acid) of the glycoside and to see if the more sterically hindered threonine attachment differed from serine in its effects on activity. and if bis. Both glycopeptides 3 and 4 produced dose. potency. potency. a number of glycopeptides were synthesized to determine if the type of monosaccharide altered the transport characteristics and systemic potency of the lead peptide pharmacophore. The three disaccharides (β-lactose. In situ BBB studies in rats also indicated.1 nmol A50 values) following intra cerebroventricular (i. that the glycopeptide penetrated the BBB more effectively than its unglycosylated peptide counterpart 9 [70].. the β-xylose was approximately two-times more potent than β-glucose or α-mannose following iv. despite the increase in MW and increased water solubility. and displayed a slight preference (~tenfold) for functional activity in the mouse MVD assay versus GPI assay [68]. we synthesized additional glycopeptides that incorporated a trisaccharide (β-maltotriose) to see if additional size/ bulk of the carbohydrate moiety would lead to further increases in iv. The geometry of the glucoside attachment did not impact functional potency/efficacy in the in vitro or in vivo assays (L-Ser vs D-Ser or L-Thr vs D-Thr). The experimen iv. NIH-PA Author Manuscript In an effort to further explore the structure-activity of glycosylation [71]. and sc.v.or tris-monosaccharides were viable alternative strategies for improving BBB transport and systemic potency [72]. Both the parent peptide (9) and glycosylated analogue (12) were extremely potent in the mouse tail-flick assay following intra cerebroventricular injection.). to produce any antinociception in the mouse 55°C tail-flick. The primary obstacles for better characterization of these glycopeptides were the somewhat tedious synthesis of the cyclic disulfides and the relatively low potency of the compounds (~30 mg/kg A50 values). For the monosaccharides. β-glucoside 3 and 4 (Table 2) were tested in mice for their ability to produce CNS-mediated antinociception after systemic administration. available in PMC 2012 December 1. retained high affinity for both DOP and MOP. The experimental data Future Med Chem. This was important in assessing potential effects of glycosylation on preferential biasing for DOP or MOP. with some compounds retaining moderate DOP selectivity while others had approximately equal affinity for DOP and MOP. glycopeptide 12 resulted in lower levels of physical dependence as indicated by naloxone-precipitated withdrawal. The parent unglycosylated peptides. Both compounds were extremely potent (<0. The initial Roques-based linear peptides tested had either an L-Ser or L-Thr added to the sixth position of the peptide. Page 6 NIH-PA Author Manuscript modified enkephalin peptide at the C-terminus allowed for glycosylation while preserving opioid receptor affinity. β-maltose and β-melibiose) were all more potent than the best monosaccharide tested. administration. whereas the unglycosylated control peptides did not produce any measurable effects. relatively weak binding to KOP.) administration but required very large doses iv. NIH-PA Author Manuscript Larger quantities of a linear enkephalin glycoside based on Roques's so-called ‘deltaenkephalin’ or DTLET (YtGFLT. Based on these results. Author manuscript. and were compared with the unglycosylated peptide control 1. Two glycopeptides. if di.

possibly due to enhanced DOP signaling under inflammatory conditions. Glycopeptide 17 produced potent reversal of the tactile allodynia and thermal hyperalgesia post-ligation. the peripherally selective antagonist naloxone methiodide did not alter the agonist actions of the compound. 17 was compared with gabapentin in a rat spinal nerve ligation model of neuropathic pain. Subtype selective DOP (naltrindole) and MOP (β-FNA) antagonists each partially blocked the antinociceptive actions of 17 and.. NIH-PA Author Manuscript Based on this modest library of glycopeptides. Finally. While not the most potent of the disaccha-rides. Page 7 NIH-PA Author Manuscript indicated a modest fall off in binding affinity and potency in the in vitro and in vivo functional assays. although. 17 had greater potency than morphine. This effect becomes pronounced at near maximal and supramaximal antinociceptive doses. UNPUBLISHED DATA].Li et al. The KOP-selective antagonist nor-BNI was without effect. As expected. 17 was almost equal to morphine in terms of potency. The mixed MOP/ Future Med Chem.. when combined. we chose the β-lactoside (17) as the lead molecule to pursue more advanced in vivo characterization. Serum and brain stability of the glycopeptides also increased with these substitutions. In an in situ model of BBB transport the disaccharide proved to be the most readily transported with the trisaccharide having a reduced RBr value (though still superior to the unglycosylated control).v. available in PMC 2012 December 1. Based on these findings. indicating greater water solubility (parent peptide < monosaccharide < disaccharide < trisccharide). The general antagonist naloxone completely blocked the actions of 17 in the 55°C tail-flick assay in mice. The first assay used was a postsurgical incision model of the hind paw [73]. NIH-PA Author Manuscript As a lead molecule.and tris-monosaccharides) to more fully explore the structure–activity relationship. Collectively. they completely eliminated the agonist actions. One of the initial screens for side effect was to inject increasing doses of 17 or morphine and collect locomotor data in an automated open field assay. In all cases. the additional glycosyl bulk reduced potency following i. 17 was also tested in several rat models of pain to determine how broad an antinociceptive spectrum the compound might have. and the one compound tested iv. acetic acid and carrageenan assays following systemic administration (all of these pain assays have an inflammatory component). We confirmed this profile in vivo by pre-treating mice with various opioid antagonists. Morphine and other MOP agonists stimulate forward locomotion in imprinting control region mice. 17 produced full efficacy in the GTPγS assay with a modest selectivity for DOP over MOP. The disaccharide 17 produced potent antinociception in the formic acid. the data indicate that a mixed DOP/MOP agonist has a broad spectrum of antinociceptive effects in acute and chronic pain models. was significantly less potent than the original glycopeptide (β-glucoside and L-serine attachment). We also extended the hexa-peptide to include one to two additional Ser or Thr attachment points with β-glucose (bis. . Author manuscript. the compound was much easier and less costly to synthesize compared with melibiose. Morphine and 17 both produced dose-related reversal of the tactile allodynia associated with the injury [D GIUVELIS ET AL. This profile was similar to what was observed in the functional MVD and GPI tissue assays. including ones that have inflammatory and/or neuropathic pain components. Additional studies confirmed aspects of the in vivo studies [67]. On a μmol/kg basis. whereas gabapentin only reversed tactile allodynia at the doses examined [D GIUVELIS ET AL. UNPUBLISHED DATA]. We extended the in vivo work by adding additional pain assays to assess efficacy. In contrast. Larger carbohydrates reduced octanol:saline partitioning (logD value). including being highly water soluble (>50 mg/ml). Similar results were seen in a subchronic inflammatory pain model induced by complete Freund's adjuvant.c. administration. in this case. Glycopeptide 17 also had some additional desirable characteristics. we advanced 17 into a more complete characterization of its antinociceptive efficacy and side-effect profile.

we used a common paradigm involving twice-daily injections of the approximate A90 doses of the agonist (or vehicle) for 3 days. potency ratios to produce antinociception for 17 versus morphine (more formal pharmacokinetic measures are currently being conducted). Equivalent doses of 17 (in terms of analgesia) resulted in a significantly reduced rightward shift (<fivefold). one of the other gross observable differences between 17 and morphine is a lack of Straub tail and muscular rigidity with 17. on the other hand. The former may have been due to the apparent higher concentrations of glycopeptide 17 in the peripheral circulation compared with CNS. requires significant occupation of the MOP receptors at sites both responsible for antinociception and tolerance/dependence. overwhelming the MOP populations in the enteric nervous system. In both cases.v. On the morning of day 4. The level of physical dependence/severity of withdrawal was consistently lower with the 17 exposure compared with equivalent exposures of morphine [74]. full dose–response curves were constructed for each compound in the agonist. The working hypothesis for explaining these results is that the antinociceptive effects of the mixed DOP/MOP compound synergize at the cellular or network level. available in PMC 2012 December 1. To further characterize the side-effect profiles of the glycopeptide. We had predicted that the DOP/MOP profile would have a reduced effect on these parameters compared with equivalent doses of morphine. Repeated doses of morphine resulted in an approximately 13-fold rightward shift in the A50 value indicating substantial development of antinociceptive tolerance. Interestingly.c. NIH-PA Author Manuscript For assessment of physical dependence liability. We quantified this effect in dose–response curves versus antinociception with both morphine and the mixed agonist 17. for example). thus. Morphine and glycopeptide 17 have similar durations of action and AUC values. we were interested in evaluating the tolerance and physical dependence liability of the compound relative to morphine. making the comparisons more straightforward. NIH-PA Author Manuscript Based on the mixed DOP/MOP profile of 17. Glycopeptide 17 also inhibited upper-gastrointestinal transit and suppressed respiratory response to elevations in CO2 on the minute ventilation parameter. These values were estimated from the i. We further investigated the initial inhibition of locomotor activity by pretreating mice with naloxone methiodide or nor-BNI.and vehicle-treated animals. This was not the case. . This indicated that stimulation of peripheral opioid receptors can produce a transient decrease in exploratory locomotor behavior and there may be a modest κ-agonist effect of the compound in the CNS that contributes to reduced stimulation of locomotor activity but does not contribute to the antinociceptive effects in the 55°C tail-flick assay. Page 8 NIH-PA Author Manuscript DOP agonist 17 produced an initial and transient decrease in forward locomotion that was replaced by a very mild stimulation of activity at later time points. Both pretreatments attenuated the effects of 17 on locomotion and completely eliminated both effects when the two opioid antagonists were administered simultaneously. A predominantly MOP-selective agonist. The respiratory depression observations indicate that a slight Future Med Chem. two commonly used assays for assessing MOP effects were used (gastrointestinal transit and respiratory depression). versus iv. injection of the general opioid antagonist naloxone was used to precipitate withdrawal and several indices of withdrawal were recorded (vertical jumps and paw tremors.Li et al. we used both an acute (single high-dose administration of agonist) and chronic (twice-daily injections for 3 days) dependence protocol. whereas it took much higher doses of 17 to produce the muscular rigidity and Straub tail compared with its antinociceptive effects. For tolerance studies. Author manuscript. whereas the processes that drive tolerance and/or physical dependence are additive or subadditive. including the formation of heterodimers with the glycopeptide (17) versus the small molecule (morphine) that lead to activation of different signaling pathways. The potential of morphine to produce both effects overlapped. Other explanations are possible.

The reinforcing effects of 17 were also evaluated in rhesus monkeys [75].based MOP agonists To further explore and exploit the biousian hypothesis [84]. In the conditioned place preference studies. This was based not only on the extensive characterization we had done with 17. (<1 nmol A50 value) (KITSOS ET AL. Under the conditions examined 17 did not support selfadministration in rhesus across a series of doses/concentrations.89]. In addition. and the larger peptide-based deltorphin II analogues [79–82]. including glycopeptide 25. such as BW373U86 and SNC80. In rat self-administration studies 17 maintained significantly lower numbers of infusions than the more MOP selective agonists morphine and fentanyl. available in PMC 2012 December 1. the level of locomotor stimulation with 17 was markedly reduced compared with morphine. . although the results are more difficult to interpret due to species differences with respect to pharmacokinetics. The stimulation of forward locomotion is generally interpreted as an activation of mesolimbic dopamine systems and an indicator of abuse liability. Page 9 preference for DOP over MOP is not sufficient to differentiate from a MOP selective agonist. MANUSCRIPT IN PREPARATION].Li et al.v. Glycopeptide 25 is also systemically bioavailable following iv. drug self administration in rodents. MAUSCRIPT IN PREPARATION). and p. DAMGO . NIH-PA Author Manuscript A series of glycosylated deltorphin analogues were synthesized that retained their high affinity. NIH-PA Author Manuscript Additional studies were conducted with 17 with respect to its abuse liability. Their pharmacology was assessed in vitro and in vivo [88. the prior work with 17 (mixed DOP/ MOP agonist) indicated to us that greater DOP selectivity might be needed in order to differentiate a lead candidate from currently available MOP analgesics.86] was used as a lipophilic peptide message [87]. This may be due to differences in ligand/receptor biasing or interactions with unique homo. These preliminary data suggest that we the efficacy of the δ-agonist 25 is at least equal to the mixed μ/δ-agonist 17 with a side-effect profile on gastrointestinal transit that is superior to morphine and 17. and additional moieties added to provide a water soluble address to produce a series of MOP-selective ligands. dosing. selectivity and efficacy at the DOP. It was believed that the exploration of the biousian hypothesis [84] within the context of a pure MOP agonist would simplify interpretation of the results (Figure 6). As mentioned previously. These experiments suggest that the reinforcing effects of 17 are less than the prototypical MOP agonists morphine and fentanyl. We are currently conducting further assessments of efficacy and side effects of glycopeptide 25 and comparing them to morphine and to 17. NIH-PA Author Manuscript Current studies with DOP-selective peptides With respect to our analgesic drug-development efforts.o. the classical μ-agonist (Ki: 0. (Table 4) This compound exhibits a low nM EC50 value in the GTPγS assay and is very potent when injected i. The binding and antinociceptive Future Med Chem. whereas the later are effective. the cumulative latency to delivery of the first three infusions of 17 (at the peak of the dose– effect curve) was significantly longer than morphine and fentanyl [STEVENSON ET AL. and the further improvement in side-effect profiles [76–78].c. The former are ineffective in acute nociceptive assays that have high stimulus intensities. Through an in vitro screening process. morphine produced a significant place preference whereas antinociceptive equivalent doses of 17 did not (similar to vehicle). We have also been interested in exploiting potential differences between the small-molecule DOP agonists. but also the literature demonstrating involvement of DOP receptors in neuropathic and other chronic pain states. Author manuscript.53 nM) DAMGO [85.or hetero-dimers of the DOP [83]. Our group has also conducted preliminary studies using conditioned place preference and iv. although the oral dose requires a proprietary coformulation technology developed by Unigene. two to three lead compounds emerged.

a plot of the BBB transport or antinociceptive A50 values versus the membrane affinity produced a Ushaped or V-shaped curve (Figure 6). focusing their pioneering efforts on endogenous opioid hormones of mammalian origin including β-endorphin [90] and other seemingly diverse peptides from arthropods such as bee venom. Critically. irrespective of the identity of the glycoside attached to the peptide. ■ The peptide remains in aqueous solution. available in PMC 2012 December 1. Binding was determined in Chinese hamster ovary cell membranes as before.Li et al. It is in understanding the similarities and differences in exactly how these two peptides interact with membranes that will provide a rationale and opportunity for improving peptide drug delivery and effective design of CNS drugs. which effectively determines the amount of time the glycopeptide spends on the endothelial membrane of the BBB. and that are likely to be extremely addictive. especially when it is not interacting with the phospholipid membrane [84].c. Affinity for the membrane is still required for effective binding and activation of the GPCR but a certain amount of membrane hopping is required for effective drug transport. These larger glycopeptides Future Med Chem. effectively removing it from solution. is to balance the degree of glycosylation. which in turn depends on the biousian character of the drugs [84]. In fact.v. both β-endorphin and melittin interact strongly with biological membranes. One may consider two extremes that result in poor delivery of a peptide drug:. Antinociception (A50 values) was determined after i. Unpublished studies in mice showed that disaccharide 29 produced behaviors (Straub tail and hyperlocomotion) suggestive of ‘narcotic intoxication’ at very low doses and had an extreme addiction liability as indicated by naloxone precipitated withdrawal studies. While μ-agonists. as well as other membranes that the glycopep-tide is likely to encounter. Page 10 NIH-PA Author Manuscript effects of 26–29 are shown in Table 5 with values for morphine sulfate and DAMGO included for comparison. this biousian approach also preserves a degree of hydrophilicity for the overall peptide. effectively preventing it from binding to biological membranes. administration using the mouse 55°C tail-flick assay. there does not seem to be much appetite for adding drugs to the pharmacopeia with side-effect profiles worse than morphine. no defined structure is observed in aqueous media (Figure 8a) [92]. the analgesic potencies of the glycopeptides are largely determined by their ability to penetrate the BBB by transcytosis [67]. which represent the simplest model for a biological membrane. Author manuscript. Since the binding affinities and receptor preferences of the μ-selective DAMGO derivatives are similar. could provide some useful clinical features that that morphine and other μ-selective anal-gesics do not possess. Kaiser's pioneering studies on the structure of β-endorphin NIH-PA Author Manuscript The late Emil Kaiser led a group of researchers at the Rockefeller Institute (USA) in studies of the structure and function of naturally occurring peptides. Thus. Our group has incorporated a design strategy for BBB penetration that exploits amphipathic α-helices that can interact strongly with cellular membranes to enhance endocytotic events. . binding and GPCR activation. or iv. In contrast. such as peptide 26 or glycopeptide 27. the goal in producing glycopeptides that are capable of effective CNS delivery. the longer glycopeptides related to β-endorphin tend to adopt amphipathic helical structures in SDS micelles as well as phospholipid bicelles (Figure 8b) [93]. ■ The peptide binds tightly to biological membrane. Similarly. or melittin (Figure 7)[91]. NIH-PA Author Manuscript Thus. All of our glycosylated enkephalins display well-defined secondary structures in sodium dodecyl sulfate (SDS) micelles.

Each series of helices was prepared in three different glycosylation states. Peptide 41 was not soluble in H2O.c. the α-helical region is similar in both cases [99]. Page 11 NIH-PA Author Manuscript displayed an ensemble of random coil conformations in aqueous solvent despite they are 17 residues in length. one peptide series with unglycosylated L-serine (S°).94]. and in crystals [97.c. we focused on the importance of the glycoside in the context of the short enkephalin-based glycopeptides to enhance membrane hopping and BBB penetration.101]. A~G. Note that helicity was essentially absent in aqueous media (Table 7). Two amino acid residues (position 8 or 12) were substituted with one of three amino acids.v. but tend to fold into largely α-helical conformations in the presence of organic solvents [95] and lipid micelles [96]. and in the A50 values obtained after i. Many. was not soluble in water.103].Li et al. and SDS/H2O (Figure 10) [100. or strongly helix-preferring α-aminoisobutyric acid (Aib or B) to produce seven different peptides of decreasing helix stability (B~B. 41. and subjected to the 55°C tail-flick test in mice. All 21 compounds were characterized in terms of δ/μ/κ-binding. and a series of disaccharide glycopeptides bearing β-O-lactose (Lact) on L-serine (S**). This biousian behavior (i. there are large variations in the A50 Future Med Chem. More importantly although. satisfying both the GPCR and the membrane binding requirements. helix-preferring Lalanine (Ala or A). and the data (not shown) is only preliminary. potency in vivo. We think that the flexibility of the linker (proline vs GABA vs DAVA) may play an important role as well.v. Inooka and coworkers have been able to demonstrate that micelle-bound conformation of a peptide ligand is closely related to the actual receptor-bound conformation. However. Although there were only modest variations in the binding selectivity. or even a deleterious effect on iv. The most helical compound. the glycosylation could have no effect (32 vs 33). H2O/F3CCH2OH (data not shown here).. It is evident from these observations that amphipathicity of the glycopeptide (not simply hydrophilic or hydrophobic nature alone) is essential for membrane transport. . and intermediate levels of helicity for the others in relatively smooth. administration (Tables 9–11). B~A. both after i. Biousian behavior has also been observed for other endogenous GPCR peptide ligands [84. G~A and G~G). as evidenced by iv. helix-weakening glycine (Gly or G). bolus injection is currently being studied. The degree of helicity was conveniently expressed as%helicity per residue [102. administration (bypassing the BBB). as evidenced by peptide 35 compared with glycopeptide (Table 6) [35]. if not all peptide ligands that activate GPCRs lack a well-defined structure in aqueous buffer. A~A. stepwise fashion (Figure 11). G~G disfavoring helical conformations. Glycopeptide opioids based on endorphins NIH-PA Author Manuscript Initially. available in PMC 2012 December 1. that is. the intrinsic stability of the membrane bound helix can decisively affect drug transport. random coil state in aqueous environment and highly folded state in membrane environment) appears essential for the effective transport of these larger peptides across the BBB [84]. A~B. and by circular dichroism in H2O. although not monotonic. but readily dissolved in the presence of SDS. (Table 8) with B~B strongly favoring helical conformations.e. Administration with iv. In fact. in the presence of SDS micelles there was a clear trend. potency (32 vs 34).98]. a series of monosaccharides bearing a single β-O-D-glucose (Glc) on L-serine (S*). The much larger endorphin-based peptides did not always require glycosylation in order to achieve effective membrane hopping rates to produce effective drug transport (Figure 9). These closely related seven peptides and 14 glycopeptides were characterized by high field (600 MHz) NMR in the presence of SDS and D2O/ H2O. NIH-PA Author Manuscript To determine the effect of helix stability a series of opioid C-terminal amide peptide 16mers 41–47 were prepared using seven different helical segments with a minimal perturbation of the structure. Author manuscript.

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Side-effect profiles of the analgesics can be manipulated by altering the agonist features of the enkephalins or the endorphin message segments. The address domain can be altered in order to optimize the biological transport rates of the endorphin-based glycopeptides based on the endogenous endorphins. the enkephalins and endorphins.Li et al. We are optimistic that interest in glycopeptide drugs derived from neuropeptides will increase. The effects of the endogenous peptide ligands are local in nature due to their poor transport properties. play numerous roles in the CNS. δ and κ receptors. μ. Page 18 Executive summary NIH-PA Author Manuscript ■ The three opiate receptors. The endogenous peptide neurotransmitters are highly amphipathic. spending the bulk of their independent existence after cleavage from larger precursor proteins and prior to release stored in vesicles contained within the presynaptic membrane. We have dubbed this effect ‘biousian behavior’ in which the glycopeptide may exist either in a relatively constrained membrane bound state. ■ The larger endorphins have a message–linker–address peptide format. such as morphine. . A flexible linker domain allows the receptor binding requirements and the membrane binding requirements to be met simultaneously. available in PMC 2012 December 1. NIH-PA Author Manuscript ■ Glycopeptide-based drugs have been developed that exert analgesic effects in mice far in excess of the classical narcotics. Principal among these roles is the modulation of acute and chronic pain states. ■ Glycosylation of the relatively short enkephalins allows them to leave the membrane environment to engage in ‘membrane hopping’. Future Med Chem. We expect that the applications will not be limited to opiate agonists and that a large number of the 250 plus endogenous neuropeptides can be converted into glycopeptide drugs capable of penetrating the blood–brain barrier. or various combinations. such as μ/κ receptors and mixed μ/δ-agonists. NIH-PA Author Manuscript ■ The biousian behavior permits the glycopeptides and related serine phosphates to penetrate the blood–brain barrier. ■ Peptide sequences can be varied to produce glycopeptide drugs that favor any of the three opiate receptors. Demerol® and Oxycontin®. The key is to add a water soluble moiety to the peptide in such a way that it enhances water solubility without interfering with its interaction with the membrane. or an aqueous state as a ‘random coil’ ensemble. The degree of glycosylation can be adjusted to optimize the biological transport rates of the enkephalin-based glycopeptides to provide drugs based on the endogenous enkephalins. Upon release they float in the postsynaptic membrane where they bind and activate opiate receptors. The address exists as an amphipathic helix and is largely responsible for binding to the membrane. probably via transcytosis. and that all major pharmaceutical companies will establish research programs in the area. The message exists as a turn structure and is largely responsible for receptor binding and activation. Author manuscript. and their endogenous peptide neurotransmitters.

Opioid receptors The μ-opioid receptor G-protein-coupled receptor derived from bovine rhodopsin by homology modeling [12]. . NIH-PA Author Manuscript Future Med Chem. available in PMC 2012 December 1. Page 19 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1. Author manuscript.Li et al.

Author manuscript. Studies show that active (folded) conformations are favored in the membrane and inactive (random coil) conformations are favored in the absence of a membrane. available in PMC 2012 December 1. . Page 20 NIH-PA Author Manuscript Figure 2. Incorporation of glycosides. Membrane hopping NIH-PA Author Manuscript Endogenous opioids associate with the membranes (kon >> koff) and bind to one or more of the opioid receptors via a membrane-bound conformation (Fisher's lock and key). NIH-PA Author Manuscript Future Med Chem. can shift the kon/koff equilibrium to facilitate ‘membrane hopping’. represented by the 270° arc.Li et al.

Page 21 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3. Author manuscript. The neurovascular unit The neurovascular unit forms the blood–brain barrier that prevents the passage of most peptides and other polar substances from the capillaries into the brain. NU: Neutrophil. TJ: Tight junction. P: Pericyte.Li et al. EC: Endothelial cell. . available in PMC 2012 December 1. AE: Astrocyte endprocess. NIH-PA Author Manuscript Future Med Chem. BL: Basolateral membrane.

. Minimally competent Lewis acids as glycosidation promotors Lewis acids such as InBr3 can dissociate (lower pathway) from the displaced acetate to form acetic acid and regenerate the Lewis acid catalyst. Page 22 NIH-PA Author Manuscript Figure 4. require a full equivalent of the Lewis acid. Stronger Lewis acids remain associated with the acetate (upper pathway) to produce a Brønsted acid and.Li et al. Author manuscript. NIH-PA Author Manuscript NIH-PA Author Manuscript Future Med Chem. generally. available in PMC 2012 December 1.

Treatment with hydrazine hydrate (H2NNH2•H2O) in methanol (CH3OH) was required to remove the acetates from the glycoside moiety prior to cleavage from the Rink resin.Li et al. Page 23 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 5. which is cleaved with the TFA cocktail. . Glycopeptide assembly MBHA-functionalized Rink polystyrene resin was used to provide the C-terminal amides upon cleavage after classical Fmoc construction of the glycopeptides. A Boc-protected amino acid may be used for the final amino acid (O-tBu-Tyr). available in PMC 2012 December 1. Author manuscript. NIH-PA Author Manuscript Future Med Chem.

where Awater = the Connolly surface area of the hydrophilic moiety (Å2) and Alipid = the Connolly surface area of the rest of the lipophilic peptide message segment YaG(N-MeF). Author manuscript. and A50 values derived from mouse intravenous tail-flick data are plotted on the Y-axis. NIH-PA Author Manuscript Future Med Chem. The amphipathicity values were calculated using the formula A = e–Awater/Alipid. Page 24 NIH-PA Author Manuscript Figure 6. . as predicted by the biousian hypothesis [84].Li et al. available in PMC 2012 December 1. Both analyses produce a U-shape or V-shape. Antinociception studies indicate a U-shaped or V-shaped curve when the A50 potency values are correlated with predicted amphipathicity NIH-PA Author Manuscript The hydrodynamic values (glucose units) or Connolly-derived amphipathicity values are plotted along the X-axes.

NIH-PA Author Manuscript Future Med Chem. Representations of the amphipathic helical region in β-endorphin NIH-PA Author Manuscript (A) Human β-endorphin [12–28] represented both as an α-helical net projection (left) and as a π-helical net projection (right) [90]. (B) Human β-endorphin [12–30] represented as an axial projection of a π-helix [91]. Author manuscript.Li et al. The lipophilic (hydrophobic) residues are circled. Page 25 NIH-PA Author Manuscript Figure 7. . available in PMC 2012 December 1.

Micelle-bound structures of glycopeptide analogues determined by NMR related to (A) enkephalins and (B) endorphins [92. Page 26 NIH-PA Author Manuscript Figure 8.Li et al. available in PMC 2012 December 1. NIH-PA Author Manuscript NIH-PA Author Manuscript Future Med Chem. Author manuscript. .93].

NIH-PA Author Manuscript Future Med Chem. available in PMC 2012 December 1. Page 27 NIH-PA Author Manuscript Figure 9.Li et al. Author manuscript. . Biousian behavior in a helix context NIH-PA Author Manuscript Modulation of amphipathic helix stability should modulate interactions with biological membranes and ‘searching’ for the receptor.

Author manuscript. Page 28 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 10. Circular dichroism to measure helicity Modulation of the amphipathic helix stability can be directly measured by examining the circular dichroism behavior in (A) sodium dodecylsulfate. . and measuring the elipticity at 222 nm. (B) trifluoroethanol and (C) water. Future Med Chem. available in PMC 2012 December 1.Li et al.

NIH-PA Author Manuscript NIH-PA Author Manuscript Future Med Chem.Li et al. Page 29 NIH-PA Author Manuscript Figure 11. Author manuscript. Helicity in sodium dodecyl sulfate as a function of peptide address sequence and glycosylation state. available in PMC 2012 December 1. .

Their common names. Peptide Sequence Subtype Leu-enkephalin YGGFL δ receptor/μ receptor Met-enkephalin YGGFM μ receptor/δ receptor Metorphamide YGGFMRRV-NH2 δ receptor/μ receptor Peptide E YGGFMRRVGRPEWWMDYQKRYGGFL25 μ receptor/κ receptor β-endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE31 μ receptor/δ receptor γ-endorphin YGGFMTSEKSQTPLVTL17 μ receptor/unknown α-endorphin YGGFMTSEKSQTPLVT16 μ receptor/unknown Dynorphin A YGGFLRRIRPKLKWDNQ17 κ receptor (μ receptor) Dynorphin B YGGFLRRQFKVVT13 κ receptor (μ receptor. Author manuscript. sequences and suggested receptor binding activities are shown. available in PMC 2012 December 1. δ receptor) β-neoendorphin YGGFLRKYP κ receptor (μ receptor. . δ receptor) FGGFTGARKSARKLANQ ORL1 Endomorphin-1 YPWF-NH2 μ receptor Endomorphin-2 YPFF-NH2 μ receptor Dermorphin YaFGYPS-NH2 μ receptor Deltorphin A YmFHLMD δ receptor Deltorphin C YaFDVVG-NH2 δ receptor Enkephalins Endorphins Dynorphins NIH-PA Author Manuscript Nociceptin/orphanin FQ Nociceptin Endomorphins Dermal peptides NIH-PA Author Manuscript † A few of the opioid peptides isolated from the mammalian CNS and from the skin of amphibians are illustrated. Page 30 Table 1 † NIH-PA Author Manuscript Endogenous opioid peptides . Weaker binding activities are in parentheses. Future Med Chem. δ receptor) α-neoendorphin YGGFLRKYPK κ receptor (μ receptor. δ receptor) Dynorphin1–8 YGGFLRRQ8 κ receptor (μ receptor.Li et al.

† GPI: Guinea pig ileum.5 MVD 40. Author manuscript. available in PMC 2012 December 1.1–6.3 19 48.000 60 110 3700 26 GPI Study of a series of cyclic disulfide glycosides of enkephalin showed that placement of the glycoside was critical to maintain opioid activity and that δ receptor selectivity was reduced in this series of compounds. Page 31 Future Med Chem. 9.9 3 6.000 45–53 42 7700 30–43 μ receptor IC50 (nM) 560 13 24 520 5. .NIH-PA Author Manuscript † 26–46 85 32 10 48 4 5 6 7 8 9 53. NIH-PA Author Manuscript Table 2 Li et al.4 δ receptor 3900 Structure 2 1 Compound NIH-PA Author Manuscript Cyclic disulfides related to DPDPE . MVD: Mouse vas deferens.

08 0.017 65.038 0.022 0.4 24.040 0.0 ND ND 16.79 ± 0.3 ND ND 34.5 11 12 13 14 15 16 31.7 11.8 54. Author manuscript.84 Intravenous (μmol/kg) ND: Not determined. 46. Glycopeptide monosaccharides based on DOP-preferring enkephalin sequences developed by BP Roques.4 ± 0.0 7.035 0.7 1.NIH-PA Author Manuscript Future Med Chem.068 2384 Intracerebroventricular (nmol) 22.12 μ receptor Ki (nM) ND ND ND ND 49 ± 4.2 23. NIH-PA Author Manuscript Table 3 Li et al.8 15.0 ± 1.8 11.71 10 290 ± 38 δ receptor 4. 9.033 0.0 ± 2.45 32.2 2.8 297. available in PMC 2012 December 1.0 16.092 0. DtGFLS (DTLES) and DsGFLT (DSLET).4 9.3 κ receptor 0.4 7.4 ± 0.0 ± 1.8 39.6 46.023 0. Page 32 .2 12.1 ± 41 Structure 9 Morphine Compound A50 NIH-PA Author Manuscript Linear glycopeptide amides as δ receptor/μ receptor opioid agonists.

8 4.16 3.63 54.33 7.00 ± 0.34 19 20 21 22 23 24 25 1100 ± 13 3.1 30% at 32.12 41.034 0.0 ± 0.12 μ receptor Ki (nM) 0.20 ± 1.20 7.380 0. available in PMC 2012 December 1.0 12.9 6.062 ND 0.0 ± 0.0 ± 5.70 Structure 17 Morphine Compound Linear glycopeptide amides as μ receptor/δ receptor opioid agonists. Glycopeptide disaccharides.NIH-PA Author Manuscript NIH-PA Author Manuscript Future Med Chem.22 3% at 10.9 ± 0.86 25.7 ± 0. Author manuscript.9 ± 0.0 ND 3.55 9.19 8. Page 33 .018 2384 Intracerebroventricular (nmol) 310 ± 66 130 ± 6. 6. and phosphates based on δ receptor-preferring enkephalin sequences developed by BP Roques.0 ± 1.0 ± 0.93 ND 56.3 κ receptor A50 5.7 30.84 Intravenous (μmol/kg) ND: Not determined.8 10.73 14.79 ± 0.3 ± 0.48 18 290 ± 38 δ receptor 9.3 ND 42.65 0.093 0.5 ± 0.82 ND 2.34 0.0 μM 0. 13. DtGFLS (DTLES) and DsGFLT (DSLET).9 5.0 mg/kg 140.2 ND ND ND 31. NIH-PA Author Manuscript Table 4 Li et al.061 0.

3.12 μ receptor 26 290 ± 38 δ receptor Ki (nM) 990 ± 35 Structure DAMGO Morphine Compound NIH-PA Author Manuscript Glycopeptides based on DAMGO.02 600 ± 44 0. A50 33 42 31% at 10 μM 2.6 1400 ± 180 27 28 29 30 31 Future Med Chem.14 730 ± 66 54% at 10 μM 1600 ± 129 55 ± 4.84 Intravenous (μmol/kg) NIH-PA Author Manuscript Table 5 Li et al.88 7.20 1.05 0.0 19 2.27 0.3 κ receptor Glycopeptide disaccharides and phosphates based on the μ receptor-preferring sequences.15 0.0 ± 1.8 350 ± 51 270 ± 2.5 30% at 38.72 0.3 12.73 4.5 160 ± 10 190 ± 9.30 ± 0.2 ± 0.8 ± 0.1 mg/kg 1.79 ± 0.16 1.68 ± 0. Page 34 .34 0.0 30 2384 Intracerebroventricular (nmol) 570 ± 9.NIH-PA Author Manuscript 1.3 270 ± 9.006 0.56 ± 0.66 ± 0.0 2. Author manuscript.30 ± 0. 1. available in PMC 2012 December 1.

NIH-PA Author Manuscript Table 6 Li et al. † DAVA: Delta amino valeric acid.11 0.32 Intravenous (μmol/kg) For endorphin analogues 32. the glycosylation state showed only minor effects on binding and intravenous potency.36 0. GABA: Gamma amino butyric acid.3 2.7 2.2 2.143 0. Page 35 Future Med Chem. S* = Glc. S** = Lact..57 Intracerebroventricular (nmol) A50 1.2 29 39 16 12.0 3.6 δ receptor 37 0.3 μ receptor Ki (nM) 64 11 11 49 56 38 5. presumably by affecting transport. 33 and 34.41 1.064 0.6 9.6 2.12 2.046 0. 38) provides the best delivery.06 0.162 0. S = OH.165 0.8 κ receptor 0.g.29 (>> 10) 1. available in PMC 2012 December 1.3 4.NIH-PA Author Manuscript † YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL 32 33 34 35 36 37 38 39 40 DAVA GABA GABA GABA GABA GABA P P P Link NLBEKALKSL-NH2 NLBEKALKS**L-NH2 NLBEKALKS*L-NH2 NLBEKALKSL-NH2 S*L-NH2 SL-NH2 NLBEKALKS**L-NH2 NLBEKALKS*L-NH2 NLBEKALKSL-NH2 Helical address sequence 32 4.97 4. P: Proline.28 1.3 31 16 11 8.059 0. The addition of a carbohydrate (35 vs 36) clearly shows a dramatic effect on intravenous potency.13 0. and combining the two features into the address region (e.58 0. . Message sequence Compound NIH-PA Author Manuscript The amphipathic helix address . Author manuscript.

0 0. Author manuscript.0 7.1 1.7 0.1 NLAEKBLKSo/*/**L-NH2 NLAEKALKSo/*/**L-NH2 NLAEKGLKSo/*/**L-NH2 NLGEKALKSo/*/**L-NH2 NLGEKGLKSo/*/**L-NH2 2 Not soluble S° = OH 0. all of these compounds were highly water soluble.5 4.8 0.5 S* = Glc 0.7 7.0 0. † Aib: α-aminoidobutyric acid.3 1.1 8.0 0. NIH-PA Author Manuscript Table 7 Li et al.9 7.4 7. Page 36 Future Med Chem. P: Proline.7 S** = Lact Helicity per residue in H2O buffer (%) NLBEKALKSo/*/**L-NH2 NLBEKBLKSo/*/**L-NH Helical address sequence Variation in the intrinsic helicity was achieved with minimal impact on other properties by altering only two amino acid residues with B (Aib). A (Ala) or G (Gly).2 8. . Except for the extremely helical peptides without glycosylation. and showed only random coil behavior in aqueous solution. Water solubility could be affected by altering the glycosylation state of the S.0 0. YtFGL YtFGL 41 42 Message sequence Series NIH-PA Author Manuscript Intrinsic helix stability in H2O .NIH-PA Author Manuscript † YtFGL YtFGL YtFGL YtFGL YtFGL 43 44 45 46 47 P P P P P P P Link 12.0 6. S: Serine.2 7. available in PMC 2012 December 1.

8 S** = Lact Helix amphipath stability was greatly altered in the presence of sodium dodecyl sulfate by altering the two amino acid residues with B (Aib). Page 37 Future Med Chem. available in PMC 2012 December 1.3 31.5 54.0 NLAEKBLKSo/*/**L-NH2 NLAEKALKSo/*/**L-NH2 NLAEKGLKSo/*/**L-NH2 NLGEKALKSo/*/**L-NH2 NLGEKGLKSo/*/**L-NH2 6.5 24.7 35.6 35.9 56.4 37.8 4. NIH-PA Author Manuscript Table 8 Li et al. S: Serine.6 35.8 33.7 S* = Glc 2.5 13.7 40.8 2 So = OH Helicity per residue in sodium dodecyl sulfate micelles (%) NLBEKALKSo/*/**L-NH2 NLBEKBLKSo/*/**L-NH Helical address sequence Aib: α-aminoidobutyric acid.† NIH-PA Author Manuscript NIH-PA Author Manuscript YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL 41 42 43 44 45 46 47 P P P P P P P Link 58. Author manuscript.4 14. P: Proline. † 70 60.8 33. Message sequence Compound Amphipathic helix stability in the presence of micelles . .7 44. A (Ala) or G (Gly). Water solubility and the degree of helix formation (%-helix/residue) was also affected by altering the glycosylation state of the S.0 11.

79 0. Page 38 Future Med Chem. S: Serine. S* = Glc.6 16 δ receptor 14 21 17 2.66 Intracerebroventricular (nmol) A50 Binding and intracerebroventricular antinociception as a function of helix stability: unglycosylated peptides. . S** = Lact.57 1.55 0.5 28 2.8 10 2. Message sequence Compound P P P P P P P Link NLGEKGLKSL-NH2 NLGEKALKSL-NH2 NLAEKGLKSL-NH2 NLAEKALKSL-NH2 NLAEKBLKSL-NH2 NLBEKALKSL-NH2 NLBEKBLKSL-NH2 Helical address sequence 19 14 15 3.NIH-PA Author Manuscript NIH-PA Author Manuscript YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL 41* 42*/32 43* 44* 45* 46* 47* P: Proline.92 0. NIH-PA Author Manuscript Table 9 Li et al.3 8.96 0.5 9.7 μ receptor Ki (nM) 17 23 49 5.7 9. available in PMC 2012 December 1. Author manuscript. S = OH.18 0.8 27 κ receptor 1.

54 1.3 7.5 2.47 1. NIH-PA Author Manuscript Table 10 Li et al.44 1.3 9.9 κ receptor 2.7 6. S: Serine. .12 2. S = OH.1 5.9 14 3.3 8.6 11 δ receptor 3. available in PMC 2012 December 1.4 11 6.9 2.2 6. S** = Lact.13 Intracerebroventricular (nmol) A50 Binding and intracerebroventricular antinociception as a function of helix stability: glucopeptides.1 2.2 6. Message sequence Compound P P P P P P P Link NLGEKGLKS*L-NH2 NLGEKALKS*L-NH2 NLAEKGLKS*L-NH2 NLAEKALKS*L-NH2 NLAEKBLKS*L-NH2 NLBEKALKS*L-NH2 NLBEKBLKS*L-NH2 Helical address sequence 7. S* = Glc.9 12 2.1 μ receptor Ki (nM) 4.NIH-PA Author Manuscript NIH-PA Author Manuscript YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL 41* 42*/33 43* 44* 45* 46* 47* P: Proline.6 6. Page 39 Future Med Chem. Author manuscript.58 1.46 0.

1 1.11 0.7 2.5 13 4.2 μ receptor Ki (nM) 2.3 3. S** = Lact.5 3.2 1. S* = Glc. .23 0.50 0.21 0. intracerebroventricular and intravenous antinociception as a function of helix stability: lactosides > 40 8.3 Intravenous (μmol/kg) A50 NIH-PA Author Manuscript Table 11 Li et al.6 6.2 1.1 4. available in PMC 2012 December 1.5 5.4 4.7 4.57 Intracerebroventricular (nmol) A50 Binding. Author manuscript.2 8. S = OH.76 0. Page 40 Future Med Chem.97 <1 3. Message sequence Compound P P P P P P P Link NLGEKGLKS**L-NH2 NLGEKALKS**L-NH2 NLAEKGLKS**L-NH2 NLAEKALKS**L-NH2 NLAEKBLKS**L-NH2 NLBEKALKS**L-NH2 NLBEKBLKS**L-NH2 Helical address sequence 4.9 δ receptor 1.6 14 12 5.5 κ receptor 0.14 0.7 2.6 4. S: Serine.NIH-PA Author Manuscript NIH-PA Author Manuscript YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL YtFGL 41** 42**/34 43** 44** 45** 46** 47** P: Proline.4 0.