Cris M. Olsen

Activities of mixed NOP and μ‐opioid receptor ligands

Compounds that activate both NOP and μ‐opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds.

Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): ligand-based analysis of structural factors influencing intrinsic activity at NOP.

The recently discovered fourth member of the opioid receptor family, the nociceptin receptor (NOP) and its endogenous ligand, the heptadecaptide nociceptin, are involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small-molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents a brief overview of the several recently reported NOP ligands, classified as NOP agonists and antagonists, with an emphasis on the analysis of the structural features that may be important for modulating the agonist/antagonist profile (intrinsic activity) of these ligands. Structure-activity relationships in our own series of dihydroindolinone-based NOP ligands and those of the various reported ligands indicate that the lipophilic substituent on the common basic nitrogen present in all NOP ligands plays a role in determining the agonist/antagonist profile of the NOP ligand. This analysis provides a basis for the rational drug design of NOP ligands of desired intrinsic activity and provides a framework for developing pharmacophore models for high affinity binding and intrinsic activity at the NOP receptor. Since NOP agonists and antagonists both have therapeutic value, rational approaches for obtaining both within a high-affinity binding class of compounds are very useful for designing potent and selective NOP ligands with the desired profile of intrinsic efficacy.

Comparison of the Antinociceptive and Antirewarding Profiles of Novel Bifunctional Nociceptin Receptor/μ-Opioid Receptor Ligands: Implications for Therapeutic Applications

The nociceptin receptor (NOPr), a member of the opioid receptor family, is a target for the treatment of pain and drug abuse. Nociceptin/orphanin FQ (N/OFQ), the endogenous peptide for NOPr, not only modulates opioid antinociception, but also blocks the rewarding effects of several abused drugs, such as morphine, cocaine, and amphetamine. We hypothesized that NOPr agonists, with bifunctional activity at the μ-opioid receptor (MOPr), may function as nonaddicting analgesics or as drug abuse medications. Bifunctional small-molecule NOPr agonists possessing different selectivities and efficacies at MOPr were evaluated in an acute thermal antinociception assay, and for their ability to induce conditioned place preference (CPP) and their effect on morphine-induced CPP. 1-(1-Cyclooctylpiperidin-4-yl)-indolin-2-one) (SR14150), a high-affinity NOPr partial agonist, with low MOPr affinity and efficacy, produced analgesia that was naloxone-reversible. SR14150 did not induce CPP alone, nor did it attenuate morphine-induced CPP. 3-Ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), which has high affinity for both NOPr and MOPr, full agonist activity at NOPr, and partial agonist activity at MOPr, was also a potent analgesic and produced CPP alone, but also modestly attenuated morphine CPP. 1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidinl-4-yl)-indolin-2-one (SR16835), a NOPr full agonist and low-affinity MOPr partial agonist, was not antinociceptive, did not produce CPP alone, but attenuated morphine CPP. Our results suggest that NOPr full-agonist activity is required to modulate opioid-induced reward, whereas a bifunctional NOPr/MOPr partial agonist profile may be suitable as a nonaddicting analgesic. The opioid-modulating effects of the NOPr ligands may be used effectively to produce better medications for treatment of drug abuse and pain.

Characterization of opiates, neuroleptics, and synthetic analogs at ORL1 and opioid receptors

Nociceptin/orphanin FQ (N/OFQ) was recently identified as the endogenous ligand for the opioid-receptor like (ORL1) receptor. Although the ORL1 receptor shows sequence homology with the opioid receptors, the nociceptin/ORL1 ligand-receptor system has very distinct pharmacological actions compared to the opioid receptor system. Recently, several small-molecule ORLI receptor ligands were reported by pharmaceutical companies. Most of these ligands had close structural similarities with known neuroleptics and opiates. In this study, we screened several available neuroleptics and opiates for their binding affinity and functional activity at ORL1 and the opioid receptors. We also synthesized several analogs of known opiates with modified piperidine N-substituents in order to characterize the ORL1 receptor ligand binding pocket. Substitution with the large, lipophilic cyclooctylmethyl moiety increased ORL1 receptor affinity and decreased mu receptor affinity and efficacy in the fentanyl series of ligands but had a different effect in the oripavine class of opiate ligands. Our results indicate that opiates and neuroleptics may be good starting points for ORL1 receptor ligand design, and the selectivity may be modulated by appropriate structural modifications.

SR 16435 [1-(1-(Bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed Nociceptin/Orphanin FQ/μ-Opioid Receptor Partial Agonist: Analgesic and Rewarding Properties in Mice

We identified a novel nociceptin/orphanin FQ (NOP)/μ-opioid receptor agonist, SR 16435 [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], with high binding affinity and partial agonist activity at both receptors. It was hypothesized that SR 16435 would produce antinociception and yet, unlike morphine, would have diminished rewarding properties and tolerance development. Antinociception was assessed in mice using the tail-flick assay, whereas behavioral and rewarding effects were assessed using the place conditioning (PC) paradigm. PC was established by pairing drug injections with a distinct compartment. Behavioral effects were measured after acute and repeated drug administration, and the test for PC was carried out 24 h after four drug- and vehicle-pairing sessions. SR 16435 produced an increase in tail-flick latency, but SR 16435-induced antinociception was lower than that observed with morphine. Given that naloxone blocked SR 16435-induced antinociception, it is highly likely that this effect was mediated by μ-opioid receptors. Compared with morphine, chronic SR 16435 treatment resulted in reduced development of tolerance to its antinociceptive effects. SR 16435-induced conditioned place preference (CPP) was evident, an effect that was probably mediated via μ-opioid receptors, as it was reversed by coadministration of naloxone. NOP agonist activity was also present, given that SR 16435 decreased global activity, and this effect was partially reversed with the selective NOP antagonist, SR 16430 [1-(cyclooctylmethyl)-4-(3-(trifluoromethyl)phenyl)piperidin-4-ol]. Naloxone, however, also reversed the SR 16435-induced decrease in activity, indicating that both opioid and NOP receptors mediate this behavior. In summary, the mixed NOP/μ-opioid partial agonist SR 16435 exhibited both NOP and μ-opioid receptor-mediated behaviors.

Novel α3β4 Nicotinic Acetylcholine Receptor-Selective Ligands. Discovery, Structure−Activity Studies, and Pharmacological Evaluation

Antagonist activity at the α3β4 nicotinic acetylcholine receptor (nAChR) is thought to contribute to the antiaddictive properties of several compounds. However, truly selective ligands for the α3β4 nAChR have not been available. We report the discovery and SAR of a novel class of compounds that bind to the α3β4 nAChR and have no measurable affinity for the α4β2 or α7 subtype. In functional assays the lead compound antagonized epibatidine-induced Ca(2+) flux in α3β4-transfected cells in a noncompetitive manner.

Isolation and characterization of a new atrial peptide-degrading enzyme from bovine kidney

An endopeptidase isolated from bovine kidney displays high affinity and selectivity for the Ser-Phe bond located in the C-terminal region of atrial peptides. Enzymatic activity converts APIII and APII to the less active peptide API. This peptidase is inhibited by both metal chelators and sulfhydryl-reactive agents, suggesting both a tightly bound metal and a cysteine residue are important for enzymatic activity. This enzyme may be important for the processing and/or degradation of atrial peptides.

Activity of new NOP receptor ligands in a rat peripheral mononeuropathy model: Potentiation of morphine anti-allodynic activity by NOP receptor antagonists

The effect of new NOP receptor agonists and antagonists in the rat chronic constriction injury model was investigated. Intraperitoneally administered NOP receptor agonist SR14150 and antagonists SR16430 and SR14148, had no effect on mechanical allodynia when given alone. The nonselective NOP/mu-opioid receptor agonist SR16435, however, produced an anti-allodynic response, similar to morphine and reversible by naloxone. Notably, co-administration of the NOP receptor antagonists potentiated the anti-allodynic activity of both morphine and SR16435. Increased levels of the NOP receptor are implicated in the reduced efficacy of morphine in neuropathic pain. Our results suggest the utility of NOP receptor antagonists for potentiating opioid efficacy in chronic pain.

Structure of a bovine thrombin-hirudin51-65 complex determined by a combination of molecular replacement and graphics. Incorporation of known structural information in molecular replacement.

Crystals of the bovine thrombin-hirudins(51-65) complex have space group P6(1)22 with cell constants a = 116.4, and c = 200.6 A and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 A. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is epsilon-thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement.

Structure-Function and Refolding Studies of the Thrombin-Specific Inhibitor Hirudin

We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.