Richard S. Agnes

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors.

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.

Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors.

Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male Sprague-Dawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80% of these cells co-express both receptors. Agonist-directed cell lesion in the RVM with the cytotoxin, saporin, using either CCK-saporin to target CCK receptor expressing cells, or dermorphin-saporin to target MOR expressing cells, resulted in concomitant loss of CCK2 and MOR expressing cells, did not alter the basal sensory thresholds but abolished the hyperalgesia induced by microinjection of CCK into the RVM. The findings suggest that these CCK2-MOR co-expressing RVM neurons facilitate pain and can be directly activated by CCK input to the RVM. Furthermore, lesion of these RVM neurons did not affect the initial development of neuropathic pain in the hind paw upon injury to the sciatic nerve, but the abnormal pain states were short lived such that by about day 9 the sensory thresholds had reverted to pre-injury baselines despite the existing neuropathy. These data support our hypothesis and identify CCK2-MOR co-expressing neurons in the RVM as potential therapeutic targets for neuropathic pain.

Suborganelle Sensing of Mitochondrial cAMP-Dependent Protein Kinase Activity

A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments (outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor developed in this study was used to reveal the relative matrix/intermembrane space/outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria.

Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain

Disease states such as neuropathic pain offer special challenges in drug design due to the system changes which accompany these diseases. In this manuscript we provide an example of a new approach to drug design in which we have modified a potent and selective peptide ligand for the CCK-2 receptor to a peptide which has potent agonist binding affinity and bioactivity at delta and mu opioid receptors, and simultaneous antagonist activity at CCK receptors. De novo design based on the concept of overlapping pharmacophores was a central hypothesis of this design, and led to compounds such as H-Tyr-DPhe-Gly-DTrp-NMeNle-Asp-Phe-NH(2) (i.e., RSA 601) which have the designed properties.

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 μ and σ 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 δ opioid receptor.

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.

Opioid and melanocortin receptors: do they have overlapping pharmacophores?

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the δ opioid receptor, 2,6‐dimethyltyrosine (Dmt)‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid (Tic), and a small molecule for the MC receptor, Tic‐DPhe(p‐Cl)‐piperidin‐4‐yl‐N‐phenyl‐propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the δ opioid receptor (Ki = 0.38 nM in binding assay, EC50 = 0.48 nM in GTP‐γ‐S binding assay, IC50 = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC50 = 2.3 μM) at the MC‐3 receptor rather than at the MC‐5 receptor. A study of the structure‐activity relationships demonstrated that the residues in positions 2, 3, and the C‐terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.

Design of peptide agonists

Publisher Summary This chapter describes a systematic approach for designing potent and selective agonists for GPCRs. Sufficient and detailed experiments are provided in the chapter as a model to synthesize the peptide agonists successfully. The design of peptides resulting in potent and selective agonist activities is difficult because of several factors, but success can be normally obtained with a systematic approach. This approach requires highly interdisciplinary considerations that combine knowledge gathered from structure–biological activity relationships, conformational analysis, computer-assisted calculations and molecular design, biophysical analysis of structures (spectroscopy, crystallography, etc.), and in vivo and in vitro assays. In addition, new asymmetric syntheses and other synthetic methodologies are often important in preparing the designed compounds. Once a lead compound (whether from nature or from a peptide library prepared by combinatorial chemistry) has been defined based on the desired agonist activity, it is often desirable to determine the minimal structural requirements for bioactivity.

Designing Peptide Drugs/Ligands for Pathological States. A New Paradigm for Design of Bioactive Peptide Hormones and Neurotransmitters

G-protein coupled receptors and other membrane bound receptors are the target for over 45% of all current drugs. They are important targets because the ligands and receptors modulate or control most biological functions necessary for survival in man, such as fear-flight, feeding behavior, response to stress, response to pain, cardiovascular function, anxiety, learning, sexual behavior, etc. Most of the ligands are polypeptides. The present paradigm for drug discovery and development in this area is to identify the gene/cell/tissue/protein associated with the biological activity and develop a binding assay and functional (2nd messenger) assay. If no endogenous “ligand” is known, assays with “libraries” of “natural products,” peptides and other organic compounds are used. The receptors and the assays chosen utilize receptors, cells, tissues and animals that are “normal.” However, there is growing evidence that the ligands and their receptors/acceptors behave differently in normal and pathological states. Especially in the central nervous system (CNS), many drugs that are effective in normal states become ineffective or less effective in pathological disease states. Evaluation of this problem suggests that a new paradigm is needed.

Design and Synthesis of Bifunctional Peptides: Antagonists at CCKA/CCKB Receptors and Agonists at μ/δ Opioid Receptors

Endogenous CCK has been demonstrated to exhibit anti-opioid effects, particularly in the spinal cord [1], We have hypothesized that the development of novel ligands which possess properties as antagonists at CCK receptors, and as agonists at opioid receptors within the same molecule would present a significant therapeutic advantage in the treatment of pain states refractory to commonly employed opioids such as morphine. Our research has been aimed at the development of ligands that could interact simultaneously and potently with opioid receptors as agonists and with CCK receptors as antagonists.