Ircinialactams: Subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae

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Abstract

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure–activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against α1 and α3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.

Graphical abstract

Screening an extract library of marine invertebrates yielded new glycinyl lactam sesterterpenes with potent and subunit selective modulatory properties against α1 and α3 glycine receptor (GlyR) chloride channels isoforms.

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Introduction

Glycine-gated chloride channel receptors (GlyRs) play a pivotal role in orchestrating inhibitory neurotransmission in the central nervous system.1 Although they are generally known to be concentrated at the post-synaptic densities of neurons in the spinal cord, brainstem and retina, GlyRs are also located pre-synaptically at several synapses.2, 3, 4 GlyRs are members of the Cys-loop ion channel receptor family and comprise a family of five subunits, α1–α4 and β. Functional GlyRs can be formed either as pentameric α subunit homomers or as αβ subunit heteromers.1, 5 The α1–α4 subunits exhibit differential central nervous system distribution patterns that are particularly evident in the superficial dorsal horn of the spinal cord,6 and the retina.7, 8, 9 On the other hand, the β subunit is broadly distributed throughout the brain. The physiological consequences of the differential α subunit distribution patterns are difficult to establish, as there are currently few pharmacological probes that can selectively inhibit specific GlyR isoforms.10, 11 Although several synthetic cannabinoid agonists (e.g., HU-210, HU-308 and WIN-55,212-2) effect a strong pharmacological discrimination between α1-containing and α3-containing GlyRs,12 these ligands are generally unsuitable for physiological or behavioural studies due to non-specific actions at cannabinoid receptors CB1 and CB2. As such, small molecule subunit selective modulators of GlyRs would have significant value as pharmacological tools.

A further reason for identifying novel GlyR subunit-specific ligands is to identify therapeutic lead compounds for muscle relaxation, inflammatory pain, immunomodulation and epilepsy. As α1β GlyRs mediate inhibitory neurotransmission onto spinal motor neurons,1 increasing α1β GlyR activity dampens motor neuron activity and hence reduces muscle contractility. Although generally sparsely distributed, the α3 subunit is strongly expressed in glycinergic synapses on nociceptive sensory neurons in the superficial layers of the spinal cord dorsal horn.6 A variety of evidence13 indicates that inflammatory pain sensitisation is caused by a prostaglandin E2-mediated down-regulation of α3-mediated glycinergic inhibitory currents in nociceptive neurons. This could produce ‘disinhibition’ of nociceptive projection neurons, resulting in the increased transmission of pain impulses to the brain, thereby explaining inflammatory pain sensitisation. Agents that restore (i.e., potentiate) glycinergic currents could therefore have potential as analgesics for chronic inflammatory pain. Although α1 is also expressed in nociceptive neurons, the α3 GlyR is considered a more promising therapeutic target as its sparse distribution outside the dorsal horn implies a reduced risk of side-effects.11, 13, 14

Functional GlyRs are also present in a variety of immune cells, including macrophages and leucocytes, where they are thought to mediate the anti-inflammatory effects of glycine.15, 16 Thus, the systemic administration of enhancing agents specific for α1- or α2-containing GlyRs could limit the damage inflicted by the inflammatory immune response on essential biological molecules, cells and organs. Finally, RNA-edited high-affinity transcripts of α2 and α3 GlyRs are upregulated in human temporal lobe epilepsy and there is evidence for a causative relationship between this upregulation and the condition.17, 18 Thus, antagonists selective for high-affinity α2- or α3-containing GlyRs may be useful for treating this chronic and debilitating neurological disorder. Considering all this information, it would be potentially useful to identify novel compounds with potent and specific potentiating or inhibitory effects on any individual GlyR isoform.

The aim of this study was to identify novel GlyR subunit-specific modulators from marine natural products with potential either as therapeutic leads or as pharmacological probes for unravelling glycinergic mechanisms in the central nervous system and immune system.

Section snippets

Fluorescence-based screening

A collection of >2500 southern Australian and Antarctic marine invertebrates and algae were initially screened against recombinantly expressed α1 and α3 GlyRs using a yellow fluorescent protein (YFP)-based anion influx assay as described in Section 5. While the fluorescence assay employed was robust, some problems associated with autofluorescence of the extracts were encountered. These problems were largely overcome by using cell selection masks during quantitative microscopy to only measure

Discussion

Analogs 3, 5 and 6 all modulated GlyRs in the sub-micromolar to low-micromolar range and exhibited differential effects on α1 and α3 GlyRs. Analog 3 exhibited the strongest subunit-specificity. At a 10 μM concentration, 3 strongly potentiated α1 GlyRs, had little effect on α2 GlyRs and potently inhibited α3. This is the first α3 specific inhibitor yet identified. Until now, the only compounds known to exhibit strong subunit-specific inhibition of GlyRs were the synthetic cannabinoids (HU-210,

Conclusion

In this investigation, we successfully challenged a library of >2500 marine extracts to focus attention on three geographically distinct sponges of the Family Irciniidae that in turn yielded an array of sesterterpene tetronic acids, examples of which were shown to be potent subunit-selective GlyR modulators. The glycinyl lactam pharmacophore identified during this investigation may inspire the development of new pharmacological tools capable of probing the distribution and function of GlyR

Equipment

Chiroptical measurements ([α]D) were obtained on a JASCO P-1010 polarimeter in a 100 × 2 mm cell. UV spectra were acquired from a Cary 50 UV–vis spectrophotometer. NMR spectra were obtained on a Bruker Avance DRX600 spectrometer equipped with a 3 mm HPLC–SEI 1H–13C/D Z-GRD flow probe in the solvents indicated and referenced to residual 1H signals in the deuterated solvents. HPLC–DAD–SPE was performed using an Agilent 1100 Series Separations Module equipped with a Bruker Diode Array Detector coupled

Acknowledgements

We thank L. Goudie (Museum Victoria) for taxonomic analysis and S. Nevin for help with the Patchliner. W.B. was supported by an Australian Development Scholarship and J.W.L. was supported by a fellowship from the National Health and Medical Research Council of Australia. We also acknowledge the support of the Australian Research Council Special Research Centre for Functional and Applied Genomics, the Institute for Molecular Bioscience and The University of Queensland.

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