The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system (CNS) and in the periphery. Positive allosteric modulators (PAMs) of the α7 increase the response to an agonist and are divided into two types depending on whether they also decrease desensitization of the receptor (type II) or not (type I). Therefore, this study aims to investigate whether the enhancement of endogenous α7 nAChR function will result in a beneficial effect in nociceptive, inflammatory and chronic neuropathic pain models. While NS1738 and PNU-120596 were not active to reduce acute thermal pain, measured by hot-plate and tail-flick tests, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. Our results with selective (MEK) inhibitor U0126 argues for an important role of extracellular signal-regulated kinase (ERK1/2) pathways activation in PNU-120596’s antinociceptive effects in formalin-induced pain. The α7 antagonist MLA, via intrathecal and intraplantar administration, reversed PNU-120596’s effects, confirming PNU-120596’s action through central and peripheral α7 nAChRs. Tolerance to PNU-120596 was not developed after chronic treatment of the drug. Furthermore, mixtures of PNU-120596 and choline, an endogenous α7 nAChR agonist, synergistically reduced formalin-induced pain, while interactions of non-antinociceptive doses of PNU-120596 and PHA-543613, a selective α7 nAChR agonist, or nicotine resulted in antinociception. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, -hypomotility and –antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via α7 nAChRs. In the carrageenan inflammatory test both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the chronic neuropathic pain (CCI) model, PNU-120596 had long-lasting (up to 6 hrs), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Subcutaneous and intrathecal administration of MLA reversed PNU-120596’s effects, suggesting the involvement of α7 nAChRs. Finally, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results show a fundamental in vivo difference between type I and II α7 nAChR PAMs, and demonstrate type II’s potential for the treatment of chronic inflammatory pain.

des-Formylflustrabromine (dFBr), isolated from the marine organism Flustra foliacea, is the first selective, positive allosteric modulator (PAM) of α4β2 nicotinic acetylcholine receptors that potentiates the action of the neurotransmitter acetylcholine (ACh). Most agonists for this receptor population are not selective and can activate other nACh receptors. A selective PAM, which activates α4β2 nACh receptors only in the presence of ACh, might find application in the treatment of of various neurological diseases such as Alzheimer’s disease or autism. dFBr was examined and found to produce a biphasic dose-response curve over a wide concentration range (i.e., potentiation at low concentration, but inhibition of the ACh-induced response at high concentrations). Our goal was to examine various structural features of dFBr required for potentiation; a secondary goal was to examine the same for inhibition. To understand the structural requirements of dFBr, a systematic ‘deconstruction reconstruction and elaboration’ approach (see p. 48) was employed to determine the contribution of various structural components of dFBr to its activity at α4β2 nACh receptors. Novel compounds were synthesized and characterized. Human α4β2 nACh receptors were expressed in Xenopus oocytes and the actions of dFBr and its analogs were measured using a two-electrode voltage clamp technique. Dose-response curves were obtained for the compounds in the absence and presence of 100 μM ACh. Structural features of dFBr optimal and/or required for PAM action at 42 nACh receptors were identified. A novel reconstructed analog with all the essential features for PAM action was synthesized and submitted for biological testing. Elaborated analogs of dFBr further helped in identification of various structural features important for PAM action and the inhibition of action of ACh. The ‘deconstruction reconstruction and elaboration’ approach (see p.48) identified important structural features of dFBr that modify its actions as a PAM or an antagonist (NAM? or channel blocker?) at α4β2 nACh receptors. This information should be useful for the subsequent design of novel analogs to evaluate their potential for the treatment of neurological disorders associated with ACh.

No description available.

Schizophrenia is a chronic psychotic illness affecting ~21 million people globally. Currently available antipsychotic agents act through a dopamine D2 receptor mechanism, and produce extrapyramidal or metabolic side effects. Hence, there is a need for novel targets and agents. The mGlu2/5-HT2A receptor heteromer has been implicated in the action of antipsychotic agents, and represents a novel and attractive therapeutic target for the treatment of schizophrenia. A long-term goal of this project is to synthesize bivalent ligands where a 5-HT2A receptor antagonist is tethered to an mGlu2 PAM via a linker. The goals of the investigation were to study the SAR of risperidone (an atypical antipsychotic agent) at 5-HT2A receptors using a “deconstruction-reconstruction-elaboration” approach to determine the minimal structural features of risperidone that contribute to its 5-HT2A receptor affinity and antagonism, and to determine where on the “minimized risperidone” structure an mGlu2 PAM can be introduced. Additional goals included studying the binding modes of various mGlu2 PAMs and identifying where on an mGlu2 PAM a risperidone “partial” structure could be introduced. Biological studies of deconstructed/elaborated analogs of risperidone suggest that the entire structure of risperidone is not necessary for 5-HT2A receptor affinity and antagonism, and that a fluoro group contributes to 5-HT2A binding. 6-Fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole that has only half the structural features of risperidone retains 5-HT2A receptor affinity and antagonist activity, and represents the “minimized risperidone” structure with the piperidine nitrogen atom representing a potential linker site for eventual construction of bivalent ligands. Molecular modeling studies at 5-HT2A receptors suggest that risperidone and its analogs have more than one binding mode. Modeling studies to evaluate binding modes of various PAMs at mGlu2 receptors, coupled with known SAR information, were used to identify a PAM (JNJ-40411813), and the pyridone nitrogen atom of JNJ-40411813 as a potential linker site. Additionally, potential synthetic routes for JNJ-40411813 were explored that might be of value in the synthesis of bivalent ligands. Based on the structural features of 6-fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole, a new pharmacophore for 5-HT2A receptor antagonists, consisting of one aromatic region, a basic protonated amine and hydrogen bond acceptors, has been proposed.