Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities.

Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.

Effect of anchoring 4-anilidopiperidines to opioid peptides.

We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.

Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities.

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr(1)-DAla(2)-Gly(3)-Phe(4)-Gly(5)-Trp(6)-O-[3',5'-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers.

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4' positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-L-phenylalanine residues at 4 and 4' positions. Binding values are: Kµ(i)=0.51 nM and Kδ(i)=12.8 nM for compound 9, Kµ(i)=0.09 nM and Kδ(i)=0.11 nM for analogue 10.

Design and synthesis of trivalent ligands targeting opioid, cholecystokinin, and melanocortin receptors for the treatment of pain.

It has been known that co-administration of morphine with either cholecystokinin (CCK) receptor or melanocortin (MC) receptor antagonists enhance morphine's analgesic efficacy by reducing serious side effects such as tolerance and addiction. Considering these synergistic effects, we have designed trivalent ligands in which all three different pharmacophores for opioid, CCK, and MC receptors are combined in such a way as to conserve their own topographical pharmacophore structures. These ligands, excluding the cyclic compound, were synthesized by solid phase synthesis using Rink-amide resin under microwave assistance in very high yields. These trivalent ligands bind to their respective receptors well demonstrating that the topographical pharmacophore structures for the three receptors were retained for receptor binding. Ligand 10 was a lead compound to show the best biological activities at all three receptors.

Synthesis and evaluation of new endomorphin analogues modified at the Pro(2) residue.

Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Delta(3)Pro), azetidine-3-carboxylic acid (3Aze) and dehydro-alanine (DeltaAla) have been used to prepare [Delta(3)Pro(2)]EM-2 (1), [Aze(2)]EM-1 (2), [Aze(2)]EM-2 (3), [3Aze(2)]EM-1 (4), [3Aze(2)]EM-2 (5) and [DeltaAla(2)]EM-2 (6). Binding assays and functional bioactivities for mu- and delta-receptors are reported. The highest affinity, bioactivity and selectivity are shown by peptides 2 and 3 containing the Aze residue.

The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety.

In order to improve metabolic stability, a ring structure with a cystine moiety was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)Bzl]), which is a lead compound of our developing bifunctional peptide possessing opioid agonist and NK1 antagonist activities. TY038 (Tyr-cyclo[D-Cys-Gly-Phe-Met-Pro-D-Cys]-Trp-NH-[3',5'-(CF(3))(2)Bzl]) was found as a highly selective delta opioid agonist over mu receptor in conventional tissue-based assays, together with an effective NK1 antagonist activity and good metabolic stability with more than 24h half life in rat plasma.

A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists.

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Structure-activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors.

Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[d-Cys-Gly-Trp-Cys]-Asp-Phe-NH(2)) showed potent binding and agonist activities at delta and mu opioid receptors but weak binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands.

Synthesis of stable and potent delta/mu opioid peptides: analogues of H-Tyr-c[D-Cys-Gly-Phe-D-Cys]-OH by ring-closing metathesis.

Ring-closing metathesis has emerged as a powerful tool in organic synthesis for generating cyclic structures via C-C double bond formation. Recently, it has been successfully used in peptide chemistry for obtaining cyclic molecules bridged through an olefin unit in place of the usual disulfide bond. Here, we describe this approach for obtaining cyclic olefin bridged analogues of H-Tyr-c[D-Cys-Gly-Phe-Cys]-OH. The synthesis of the new ligands was performed using the second generation Grubbs' catalyst. The resulting cis-8 (cDADAE) and trans-9 (tDADAE) were fully characterized and tested at delta, mu, and kappa opioid receptors. Also the linear precursor 13 (lDADAE) and the hydrogenated derivative 11 (rDADAE) also were tested. All the cyclic products containing a olefinic bond are slightly selective but highly active and potent for the delta and mu opioid receptors. Activity toward the kappa opioid receptors was absent or very low.

Partial retro-inverso, retro, and inverso modifications of hydrazide linked bifunctional peptides for opioid and cholecystokinin (CCK) receptors.

Partially modified retro-inverso, retro, and inverso isomers of hydrazide linked bifunctional peptides were designed, synthesized, and evaluated for bioactivities at delta/mu opioid receptors and CCK-1/CCK-2 receptors. All modifications of the CCK pharmacophore moiety affected bioactivities for the CCK-1 and CCK-2 receptors (up to 180-fold increase in the binding affinity with higher selectivity) and for the delta and mu opioid receptors. The results indicate that the opioid and CCK pharmacophores in one molecule interact with each other to induce topographical changes for both pharmacophores.

Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for delta/mu opioid receptor agonists and neurokinin-1 receptor antagonists.

A series of bifunctional peptides that act as agonists for delta and mu opioid receptors with delta selectivity and as antagonist for neurokinin-1 (NK1) receptors were designed and synthesized for potential application as analgesics in various pain states. The peptides were characterized using radioligand binding assays and functional assays using cell membrane and animal tissue. Optimization was performed on the fifth residue which serves as an address moiety for both receptor recognitions. It had critical effects on both activities at delta/mu opioid receptors and NK1 receptors. Among the synthesized peptides, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF3) 2 (5) and H-Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 (7) had excellent agonist activity for both delta opioid and mu opioid receptors and excellent antagonist activity for NK1 receptors. These results indicate that the rational design of multifunctional ligands with opioid agonist and neurokinin-1 antagonist activities can be accomplished and may provide a new tool for treatment of chronic and several pain states.

Development of novel enkephalin analogues that have enhanced opioid activities at both mu and delta opioid receptors.

Enkephalin analogues with a 4-anilidopiperidine scaffold have been designed and synthesized to achieve therapeutic benefit for the treatment of pain due to mixed mu and delta opioid agonist activities. Ligand 16, in which a Dmt-substituted enkephalin-like structure was linked to the N-phenyl-N-piperidin-4-yl propionamide moiety, showed very high binding affinities (0.4 nM) at mu and delta receptors with an increased hydrophobicity (aLogP = 2.96). This novel lead compound was found to have very potent agonist activities in MVD (1.8 nM) and GPI (8.5 nM) assays.

Structure-activity relationships of bifunctional peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors.

Cholecystokinin (CCK) has been identified as a pronociceptive endogenous peptide which also possesses antiopioid actions. CCK may be upregulated in conditions of chronic pain or during sustained morphine administration resulting in attenuation of opioid-mediated pain relief. These complex interactions between opioids and endogenous CCK receptor systems have suggested the need for a new paradigm in drug design for some states of chronic pain. In these circumstances the rational design of potential drugs for the treatment of these conditions must be based on one ligand for multiple targets. We have designed a single peptide which can interact with delta and mu opioid receptors as agonists and with CCK receptors as antagonists. The ligands were designed based on a model of overlapping pharmacophores of opioid and CCK peptide ligands, which incorporates opioid pharmacophores at the N-terminal and CCK tetrapeptide pharmacophores at the C-terminal of the designed ligands. We measured binding and activities of our bifunctional peptides at opioid and CCK receptors. Compound 11 (Tyr-d-Ala-Gly-d-Trp-NMeNle-Asp-Phe-NH(2)) demonstrated opioid agonist properties at delta and mu receptors (IC(50) = 63 +/- 27 nM and 150 +/- 65 nM, respectively in MVD and GPI tissue assays) and high binding affinity at CCK-1 and CCK-2 receptors (K(i) = 320 and 1.5 nM, respectively). Compound 9 (Tyr-d-Nle-Gly-Trp-Nle-Asp-Phe-NH(2)) displayed potent agonist activity at delta and mu receptors (IC(50) = 23 +/-10 nM and 210 +/- 52 nM, respectively in MVD and GPI tissue assays), with a balanced binding affinity for CCK-1 and CCK-2 receptors (K(i) = 9.6 and 15 nM, respectively). These results provide evidence supporting the concept that opioid and CCK receptors have overlapping pharmacophores required for binding affinity and biological activity and that designing overlapping pharmacophores of two peptides into a single peptide is a valid drug design approach.

Design and synthesis of novel hydrazide-linked bifunctional peptides as delta/mu opioid receptor agonists and CCK-1/CCK-2 receptor antagonists.

A series of hydrazide-linked bifunctional peptides designed to act as agonists for delta/mu opioid receptors and antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors and in functional assays. SAR studies in the CCK region examined the structural requirements for the side chain groups at positions 1', 2', and 4' and for the N-terminal protecting group, which are related to interactions not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities (0.1-10 nM) for both delta and mu opioid receptors generally showed lower binding affinities (micromolar range) at CCK-1 and CCK-2 receptors, but were potent CCK receptor antagonists in the GPI/LMMP assay (up to Ke = 6.5 nM). The results indicate that it is reasonable to design chimeric bifunctional peptide ligands for different G-protein coupled receptors in a single molecule.

4-{N-[2-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-3-phenylpropionyl]anilino}-1-phenethylpiperidinium chloride methanol disolvate.

The structure of the title compound, a fentanyl derivative with formula C(36)H(36)N(3)O(3) (+)·Cl(-)·2CH(3)OH, crystallizes as a racemic mixture. The organic cation has an extended conformation and the structure displays O-H⋯O, O-H⋯Cl and N-H⋯Cl hydrogen bonding.

An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons.

We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect (vehicle), and delivered as repeated daily bolus doses (0.5 microg to 4 microg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala(2), Glu(4)]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.