Biphalin: The Foundation of Bivalent Ligands.

Seldom in medicinal chemistry does one ligand present the ability to study two separate phenomena in a pharmacological process. The discovery of biphalin with other homodimeric ligands has given scientists a tool that not only explores how to increase the efficacy of the ligand, but also explore the possible interactions of hetero and homo dimerization of the receptors themselves. As a straight ligand, biphalin has allowed scientists to increase efficacy by direct modification of the residues to affect the message-address interactions with receptors. This led to the exploration of ligand linkers to increase efficacy and it was this modification of the linkers led to discoveries that suggested dimerization of receptor system occurs as a secondary modulation of signal transduction. Even more recently, exploration of the advances in linkers through the discovery of bitopicity seems to modulate the actual receptors to increase the binding and signal transdcution of the ligand. This is accomplished by possible slight conformational changes in the receptors before binding of the ligand located at the end of the linker. These advances were made by the work of the late Prof. Andrzej W. Lipkowski. This review gives the foundation of biphalin and in turn celebrates the contributions of Prof. Lipkowski made in this area.

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.

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.

Development of potent µ and δ opioid agonists with high lipophilicity.

An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-ylpropionamide moiety at the C-terminal was performed and has resulted in highly potent ligands at µ and δ opioid receptors. In general, ligands with the substitution of D-Nle(2) and halogenation of the aromatic ring of Phe(4) showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay.

Use of plasmon waveguide resonance (PWR) spectroscopy for examining binding, signaling and lipid domain partitioning of membrane proteins.

AIMS: Due to their anisotropic properties and other factors, it has been difficult to determine the conformational and dynamic properties of integral membrane proteins such as G-protein coupled receptors (GPCRs), growth factor receptors, ion channels, etc. in response to ligands and subsequent signaling. Herein a novel methodology is presented that allows such studies to be performed while maintaining the receptors in a membrane environment. MAIN METHOD: Plasmon waveguide resonance (PWR) spectroscopy is a relatively new biophysical method which allows one to directly observe structural and dynamic changes which occur on interaction of GPCRs (and other integral membrane proteins) with ligands and signaling molecules. The delta opioid receptor (DOR) and its ligands serve as an excellent model system to illustrate the new insights into GPCR signaling that can be obtained by this method. KEY FINDINGS: Among our key findings are: 1) it is possible to obtain the following information directly and without any need for labels (radioactive, fluorescent, etc.): binding affinities, and the ability to distinguish between agonists, antagonists, inverse agonist, and partial agonists without a need for second messenger analysis; 2) it is possible to determine directly, again without a need for labels, G-protein binding to variously occupied or unoccupied DORs, and to determine which alpha-subtype is involved in allowing structurally different agonist ligands to have differential effects; 3) GTPgammaS binding can be examined directly; and 4) binding of the DOR with different ligands leads to differential segregation of the ligand-receptor complex into lipid rafts. SIGNIFICANCE: The implications of these discoveries suggest a need to modify our current views of GPCR-ligand interactions and signaling.

New paradigms and tools in drug design for pain and addiction.

New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at mu and delta opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the delta opioid receptor.

Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.

The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-d-penicillamine, 5-d-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D(1) = 2.0(+/-1.2) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 10(-8) cm(2)/V.s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.

A novel 3-step enantioselective synthesis of pyrenylalanine with subsequent incorporation into opioid, CCK, and melanotropin ligands.

Pyrene possesses unique spectroscopic properties such as a high quantum yield, a long half-life in the excited state, and the ability to form excimers when in proximity to each other in the excited state. These properties allow pyrenylalanine, which is a pyrene moiety incorporated into an amino acid, to be used as a fluorescent probe in peptides and proteins. The common route for the synthesis of pyrenylalanine involves 5 steps, with subsequent separation of the two isomers by recrystallization. This paper reports a novel 3-step asymmetric synthesis of pyrenylalanine with high enantioselectivity, good yields, and facile isomer purification. After synthesis, pyrenylalanine was incorporated into a series of opioid, CCK, and melanotropin peptide ligands in order to study the effects of aromaticity, lipophilicity, and steric properties on their potency and efficacy at their corresponding biological receptors. The change in binding and efficacy of the labeled ligands as compared to the unlabeled ligands demonstrates the possible role of lipophilicity/aromaticity in the binding and signal transduction of the ligand-receptor interaction.

Different structural states of the proteolipid membrane are produced by ligand binding to the human delta-opioid receptor as shown by plasmon-waveguide resonance spectroscopy.

Understanding structure-function relationships and mechanisms of signal transduction in G-protein-coupled receptors (GPCRs) is becoming increasingly important, both as a fundamental problem in membrane biology and as a consequence of their central role as pharmacological targets. Their integral membrane nature and rather low natural abundance present many challenging problems. Using a recently developed technique, plasmon-waveguide resonance (PWR) spectroscopy, we investigated the structural changes accompanying the binding of ligands to the human delta-opioid receptor (hDOR) immobilized in a solid-supported lipid bilayer. This highly sensitive technique can directly monitor changes in mass density, conformation, and orientation occurring in such thin proteolipid films. Without requiring labeling protocols, PWR allows the direct determination of binding constants in a system very close to the receptor's natural environment. In the present study, conformational changes of a proteolipid membrane containing the hDOR were investigated upon binding of a variety of peptide and nonpeptide agonists, partial agonists, antagonists, and inverse agonists. Distinctly different structural states of the membrane were observed upon binding of each of these classes of ligands, reflecting different receptor conformational states, and the formation of each state was characterized by different kinetic properties. Binding constants, obtained by quantifying the extent of conformational change as a function of the amount of ligand bound, were in good agreement with published values determined by radiolabeling methods. The results provide new insights into ligand-induced GPCR functioning and illustrate a powerful new protocol for drug development.

Plasmon-waveguide resonance spectroscopy: a new tool for investigating signal transduction by G-protein coupled receptors.

Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned delta-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems.

Agonist-specific down-regulation of the human delta-opioid receptor.

Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.