Discovery of Novel, Potent and Selective Glycine Receptor Modulators from Southern Australian Sponges

Abstract

Consisting of five subunits (!1-!4 and "), glycine receptors (GlyRs) orchestrate inhibitory neurotransmission in the central nervous system (CNS). Mutation and/or dysfunction of different GlyR subunits are associated with CNS diseases such as spasticity and movement disorders (GlyR!1), hyperekplexia (GlyR !1, GlyR!2 and GlyR"), epilepsy (GlyR!2 and GlyR!3) and inflammatory pain (GlyR!1 and GlyR!3). In particular, GlyR!3 has recently emerged as the target of choice for the treatment of chronic inflammatory pain due to its discrete distribution at the lamina II dorsal horn spinal cord, the pain terminal. Unfortunately, while known GlyR modulators lack specificity for GlyRs, that render them undrugable, little is known about GlyR modulators from marine natural products (MNPs), despite the important roles of MNPs in drug discovery programs.Together with the biomedical potential of MNPs and the unmet medical needs in treatment of CNS diseases, recent advances in spectroscopic and ion channel technologies strongly suggest the importance of MNPs in the discovery of novel, potent and specific GlyR modulators.Consequently, chapter 1 discusses the various potential medical benefits of GlyRs and/or MNPs in the search for a cure for CNS diseases, with emphasis on chronic inflammatory pain. They include:• Structural diversity and pharmacological aspect of GlyRs;• Possible uses of GlyR modulators in therapy;• The importance of advanced technologies in discovering anti-inflammatory pain drugs from marine resource;• MNPs as a prolific source of bioactive molecules; and• The role of MNPs in the discovery of new pain relief drugs.Chapter 2 prioritizes marine extracts as our first step in identifying MNPs as potential GlyR modulators. Taking advantage of the yellow fluorescence protein (YFP) assay, we screened nBuOH extracts of >2,500 Australian and Antarctic marine invertebrates against GlyR!1, GlyR!2 and GlyR!3 and compared the images for control and tested extracts. This screening yielded 27 active extracts (3 strong, 19 moderate and 5 weak antagonist extracts). Along with this screening, liquid chromatography mass spectrometry (LCMS) analysis established 7 priority specimens described in the following three chapters. and (7E,12Z,20Z,18S)-variabilin (3.53) compounds from three Irciniidae sponges. The isomers 3.51 and 3.52 were successfully isolated and assigned as a single pure compound without derivatisation.Importantly, by means of liquid chromatography-solid phase extraction nuclear magnetic resonance (LC-SPE-NMR), we managed to isolate and elucidate the structure of minor metabolites (3.45-3.50). were unique in that they all featured an unusual functionality, a glycinyl lactam and in case of ircinialactam D, a dihydroxy diacid moiety. Using automated patch clamp electrophysiology (APCE), we evaluated 3.45-3.53 against !1 and !3 GlyRs and identified 3.46 and 3.47 as strong and specific GlyR!1 potentiators with EC 50 values of 1.0 µM and 0.5 µM respectively and 3.53 as a potent and selective GlyR!3 antagonist (IC 50 ...