Consisting of five subunits (α1-α4 and β, 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 (GlyRα1), hyperekplexia (GlyR α1, GlyRα2 and GlyRβ), epilepsy (GlyRα2 and GlyRα3) and inflammatory pain (GlyRα1 and GlyRα3). In particular, GlyRα3 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 n- BuOH extracts of >2,500 Australian and Antarctic marine invertebrates against GlyRα1, GlyRα2 and GlyRα3 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.
Chapter 3 describes 6 new (ircinialactam A (3.45), 8-hydroxyircinialactam A (3.46), hydroxyircinialactam B (3.47), ircinialactam C (3.48), ent-ircinialactam C (3.49) & ircinialactam D (3.50) and 3 known (12E,20Z,18S)-8-hydroxyvariabilin (3.51), (7E,12E,20Z,18S)-variabilin (3.52) 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). Ircinialactams (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 α1 and α3 GlyRs and identified 3.46 and 3.47 as strong and specific GlyRα1 potentiators with EC50 values of 1.0 μM and 0.5 μM respectively and 3.53 as a potent and selective GlyRα3 antagonist (IC50 = 7.0 μM).
Chapter 4 discloses 5 novel cyclic sesterterpenes (-)-ircinianinlactam A (4.11), (-)-oxo-ircinianin lactam A sulfate (4.12), (-)-oxo-ircinianin A (4.13), (-)-oxo-ircinianin lactam A (4.14), and (-)- ircinianin lactone (4.15), two known co-metabolites (-)-ircinianin (4.1) & (-)-ircinianin sulfate (4.2) and one synthetic derivative, ircinianin acetate (4.16). Ircinianins possessed rare functional groups such as the glycinyl lactam in 4.11, 4.12 and 4.14 and a modified tetronic acid in 4.13 and 4.14 with the latter moiety being hitherto unknown in natural products. Importantly, APCE analysis showed 4.2 as a strong GlyRα3 antagonist (IC50 = 3.2 μM), 4.14 as a specific GlyRα1 potentiator and 4.11 as a potent and selective GlyRα3 potentiator (EC50 = 8.2 μM). (-)-Ircinianinlactam A (4.11) was the first compound to exert such activity against GlyRα3 subunit, the emerging pain target.
Chapter 5 discusses ianthellalactam A (5.66) and B (5.67) (new), ethyldictyondendrilin (5.68) (artefact), aplysinopsin (5.1), 8E-3'-deimino-oxoaplysinopsin (5.3), 8Z-3'-deimino-oxoaplysinopsin (5.4), dihydroaplysinopsin (5.10) and tubastrindole B (5.34) (known) from Ianthella cf. flabelliformis. Ianthellalactam A and B were the first examples of linear sesquiterpene glycinyl lactams. APCE analysis revealed 5.3 and 5.4 as modest GlyRα3 antagonists (IC50 = 67.0 μM) and 5.34 as a strong GlyRα1 potentiator (1.0 μM) but a modest antagonist at high concentration (IC50 = 25.9 μM). One pot synthesis produced two strong GlyRα1 antagonists, 8E-3'-deimino-4'-demethyl- 3'-oxoaplysinopsin (5.70) and 8Z-3'-deimino-4'-demethyl-3'- oxoaplysinopsin (5.71), with IC50 values of 8.8 μM.
Finally, chapter 6 describes 30 compounds discovered during this study. Apart from their status (new or known, natural products and/or synthetics), this chapter provides their physicochemical characteristics and bioactivity against GlyRs. Moreover, through a strength-weakness-opportunity- threat (SWOT) analysis, this chapter discusses the possibility of taking the lead compounds (3.46, 3.47, 4.11 & 4.14) to the pipeline or clinical trials.