Discovery and characterisation of novel analgesic neurotoxins, hCav2.2 channel inhibitors, from cone snail and spider venoms

Abstract

Cav2.2 is a voltage-gated calcium channel isoform expressed in nerve terminals of excitable cells and dendrites, where it initiates neurotransmitter release and controls nociception. Cav2.2 is particularly important in controlling signalling in nociceptive pathways such as the ventral and dorsal horn of the spinal cord, dorsal root ganglion (DRG) neurons, and along the dendrites and at presynaptic terminals, where it contributes critically to neurotransmitter release. Although different Cav channel types are expressed in nociceptive pathways. Cav2.2 is up-regulated at the spinal cord during chronic pain and nerve injury states, along with the auxiliary a2d subunit. The Cav2.2 auxiliary subunits a2d1 and a2d2 are the targets of the gabapentinoid drugs (gabapentin and pregabalin), which are currently marketed for use in neuropathic pain treatment. Morphine, a drug used for decades for the treatment of severe pain inhibits Cav2.2 indirectly at the spinal cord level. Ziconotide (Prialtr) is a direct Cav2.2 blocker that produces efficient analgesia, despite side effect problems. Therefore, Cav2.2 inhibition is a validated analgesic strategy, and Cav2.2 inhibitors have therapeutic application in the treatment of neuropathic pain. Venomous animals, such as snakes, scorpions, cone snails and spiders, are rich sources of remarkably potent and selective Cav2.2 inhibitors. There are aproximately 100 different venom components per species of cone snails, leading to an estimate of 50,000 different pharmacologically active components, present in venoms of all living cone snails. Spiders are the most successful venomous animals with an estimated 100,000 extant species. The vast majority of spiders employ a lethal venom cocktail to rapidly subdue their prey. Some spider species produce venom containing g1000 unique peptides. The main aim of this thesis was to discover and pharmacologically characterize new Cav2.2 channel inhibitors from the venoms of cone snails and spiders to help finding alternative drug leads for the treatment of chronic severe pain. Initially we established human cell-based miniature high throughput screening assays. These assays allowed accelerated identification and characterization of novel Cav2.2 channel inhibitor peptides extracted from large venom libraries. o-Conotoxins MoVIA and MoVIB were discovered from a worm hunter cone snail. MoVIA given intrathecally was a potent analgesic in a rat model of induced neuropathic pain. The first Chapter of this thesis gives a general introduction, describes the pathophysiology of pain as and the role of Cav2.2 in pain pathways. Chapter 1 also introduces venom toxins as tools to isolate Cav2.2 channels and as drug leads to threat pain. Parts of Chapter 1 have been published in Toxins, 2013. The pdf version has been attached as Appendix 2 at the end of the thesis. Parts of Chapter 2 have been published in PLoS ONE, 2013 and a pdf version has been attached as Appendix 2. Chapter 2 describes the establishment of robust functional calcium fluorescent assays and binding assays to identify and characterize Cav channel inhibitors. Using these assays the pharmacology of a range of o-conotoxins were compared and importantly, new hCav2.2 inhibitors were discovered and pharmacologically characterised. Additionaly the influence of auxiliary subunits in the pharmacology of o-conotoxins was investigated and is described in Chapter 2. In Chapter 3 it is described the discovery and characterization of two novel o-conotoxins named MoVIA and MoVIB. These toxins were isolated from the venom of the cone snail Conus moncuri, a worm hunting cone snail. Worm huntings are ancestral cone snails. MoVIA and MoVIB were highly selective Cav2.2 channel inhibitors in rat DRG cells, the human SH-SY5Y cells and fish brain. Before this thesis it was thought o-contoxins were only part of fish hunting cone snail venoms and this component of the venom was needed to produce the motor cabal effect needed for the snails to paralyse the prey (fishes), which would otherwise move too quickly. MoVIA and MoVIB were chemically synthesised and the structure-function relashionship and family tree were studied. Interestingly, in vivo studies proved analgesic potential of toxins MoVIA and MoVIB in rats. Therefore these toxins have the potential to be used as lead tools for treatment of neuropathic pain in humans. Chapter 4 reports the discovery and functional characterisation of Cd1a, a new Cav2.2 channel inhibitor toxin isolated from the venom of Ceratogyrus darlingi, a tarantula African spider. Toxins from related spiders are potent Kv and Nav channels. Cd1a pharmacology was investigated at hNav channels using assays established in house and confirmed activity at hCav2.2, but not at other hCav channel subtypes endogenously expressed in SH-SY5Y cells. Interestingly, Cd1a inhibited a range of hNav channel subtypes with high potency, suggesting this toxin is more a Nav than a Cav channel inhibitor. The results described in this thesis will provide new pharmacological tools for the discovery and characterization of Cav inhibitors and may lead to the development of better analgesic options for the treatment of severe pain conditions.