Date of Award

5-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Medicinal Chemistry and Molecular Pharmacology

Committee Chair

Richard M. van Rijn

Committee Member 1

Val J. Watts

Committee Member 2

Jean-Christophe Rochet

Committee Member 3

Nana Gletsu Miller

Abstract

Chronic pain is a debilitating disorder associated with various diseases such as arthritis and diabetic neuropathy and is also commonly observed during withdrawal from chronic use of opioids or alcohol. Mu opioid receptor (MOR) agonists have become a mainstay of pain therapy for thousands of years. While opioids are effective for acute pain, they are not useful for chronic pain and carry several adverse effects. This poor control of chronic pain urges a need for developing novel targets for chronic pain relief. Here we have examined different promising targets including delta opioid receptor (DOR), DOR-MOR heteromers and adenylyl cyclase type I (AC1) which have all been shown to be associated with chronic pain processing. Our studies are providing important insights into mechanisms and efficacy of these targets for development of drugs for chronic pain with fewer side effects. In chapter 2, our aim is to identify the DOR as a possible target for treatment of alcohol withdrawal-induced mechanical allodynia (AWiMA). One of the hallmarks of chronic pain is mechanical allodynia (pain responses to normally innocuous stimuli). Mechanical allodynia is also a component of the alcohol withdrawal syndrome, consisting of several separate symptoms including anxiety and depression, that can facilitate alcohol relapse. The currently available drug treatments for alcoholism rarely combat the complexities of the withdrawal syndrome. Yet, the mechanisms underlying alcohol withdrawal symptoms specifically allodynia are still not well understood, hampering development of novel medications that could treat the alcohol withdrawal syndrome and reduce relapse. Here we propose that the DOR could be a novel target for AWiMA therapy, particularly considering that DOR agonists also reduce alcohol consumption and attenuate anxiety and depression in alcohol-withdrawn mice. To study AWiMA, adult male wild-type and transgenic DOR knockout C57/BL6 mice were exposed to alcohol either by a voluntary drinking or oral gavage exposure. The DOR-selective agonist TAN-67 and antagonist naltrindole were used to examine the involvement of the DOR in AWiMA, which was measured using a von Frey nociception test. We found that AWiMA was exacerbated and prolonged in DOR knockout mice as well as by pharmacological blockade of DORs compared to control mice, indicating a protective role of the DOR in the establishment of AWiMA. However, analgesia induced by TAN-67 was attenuated during withdrawal in alcohol-gavaged mice, suggesting that DORs appear to be desensitized once mice reside in a state of severe alcohol withdrawal. While this data supports the DOR as a drug target for prevention but not treatment of AWiMA, it is important to consider that DOR agonists alleviate other components of the alcohol withdrawal syndrome and decrease the risk of relapse. In chapter 3, our aim is to identify a novel interaction site of the DOR-MOR heteromers using a multipronged approach. Evidence suggests that MORs can interact with DORs to form DOR-MOR heteromers which display unique functions relative to MOR or DOR monomer, and may contribute to side effects of long-term mu opioid therapy for chronic pain. Our goal is therefore to develop drug-like compounds that disrupt the heteromers to lessen the side effects and increase the effectiveness of mu opioids. Yet, a lack of tools to selectively investigate the role of DOR-MOR heteromers in preclinical and clinical models is stifling our ability to target the heteromers with drugs. To aid in the development of such tools, it is important to comprehensively understand interactions between MORs and DORs; i.e., determining which amino acids in the heteromer interface play major roles in the DOR-MOR formation. Biochemical studies and insights gained from the crystal structures of the MOR and DOR reveal that their transmembrane domains five and six may be essential for the DOR-MOR interaction. We then constructed mutants spanning these domains of the DOR to probe for destabilization of the DOR-MOR heteromers. We used a multi-pronged approach including heteromer-selective calcium signaling assay, bimolecular fluorescence complementation, co-immunoprecipitation and computational model to investigate the impact of mutations on the stability and function of the heteromers. , Val5.68 and Arg5.69 We confirmed that amino acid positions Ser5.67 located in the intracellular loop, which had been previously proposed by other labs, were crucial in stabilizing the heteromers. Intriguingly, we identified novel amino acid positions Tyr5.33, Trp5.34, Ile6.53 and Asp6.62 of DOR to be necessary for the heteromer formation. These discrete amino acids are located on the extracellular regions, thereby providing a promising new target for heteromer disruption. These findings will move us closer to our goal to develop small molecule protein-protein interaction inhibitors that can be used with opioid analgesics to prevent or delay adverse effects of chronic opioid therapy. In chapter 4, our aim is to study the effectiveness of AC1 inhibition in a model of (opioid resistant) inflammatory pain. Arthritis which occurs in 20% of US adults is a major source of chronic inflammatory pain. Although opioids are commonly prescribed for acute pain, they are not recommended for use in treating arthritic pain. Recent studies have suggested that AC1 may be a new target of chronic pain. This is supported by evidence showing a significant decrease in chronic pain responses in AC1 knockout mice. Moreover, AC1 is the enzyme that synthesizes cAMP which functions as a second messenger in opioid receptor signaling. Chronic opioid use can lead to so-called cAMP superactivation which has been suggested to contribute to opioid-induced abnormal pain sensitivity and tolerance. We thus hypothesize that inhibition of AC1 will reduce (opioid resistant) pain associated with chronic inflammation. Our studies have demonstrated that a recently identified AC1 inhibitor ST034307 is able to provide analgesia in a Complete Freunds Adjuvant model of acute inflammatory pain and may reduce withdrawal signs observed in opioid-dependent mice.

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