By Yeji Hong
Chronic pain is a frequent condition that affects approximately 20% of the world population. It is recognised as pain that persists longer than normal healing time, recurring for more than 3~6 months (Treede et al., 2015). Chronic visceral pain forms a major component of several gastrointestinal and bladder disorders including irritable bowel syndrome (IBS). The complexity of the disease makes treatment difficult (Saha, 2014) – current available drugs fail to provide effective analgesic effects and have dose-limiting side effects.
Visceral organs are innervated by a network of sensory afferent neurons that express a variety ion channels and receptors that detect mechanical and chemical stimuli. A key mechanism in development of chronic visceral pain is the hypersensitivity of the visceral afferents to physiological stimuli (Grundy et al., 2018). Dysfunction of voltage-gated ion channels in visceral afferents, specifically the voltage-gated sodium channels (Nav) and T-type voltage-gated calcium channel (Cav3) subtypes have been identified in the development and maintenance of chronic visceral pain. Therefore, targeting specific combinations of sodium or calcium channels may be a useful strategy for treating chronic visceral pain.
Interestingly, many venom peptides have shown to modulate the activity of voltage gated ion channels, including Nav and Cav3 channels (Klint, 2012). In particular, peptides from arachnid venoms are amongst the most potent modulators of the therapeutically relevant Nav and Cav3 subtypes. However, selectivity of the analgesics is critical to avoid the modulation of other sodium channels including Nav 1.4, Nav 1.5, and Nav 1.6 which underlie muscle, heart and myelinated motor neuron action potentials. Several spider venom peptides have been discovered that inhibit pain related Nav channels such as Nav1.1 and Nav 1.7 yet avoid Nav1.4, 1.5, and 1.8 (Cardoso&Lewis, 2017). However, it has been found to be more challenging to achieve selectivity against Nav1.6, as it has similarities to Nav1.7 in the regions where these peptides bind.
A recent study conducted at University of Queensland screened 28 different spider venoms with the purpose of identifying spider-venom peptides with high selectivity and potency for the therapeutically relevant ion channels (Cardoso et al., 2020). Through screening venoms against both Nav1.6 and Nav1.7, 10 tarantula venoms with a preference for Nav1.7 inhibition were identified, which at a concentration of 250ug dried venom/mL, each of them nearly fully inhibited hNav1.7 (human Nav1.7) but induced weak or no inhibition of hNav1.6.
A previously uncharacterised venom identified during screening was from the tarantula Theraposa apophysis, one of the largest spiders in the world with a leg span of up to 30cm. It was found that this venom was able to inhibit 100% and 70% of hNav1.7 activity at concentrations of 250 and 25 ug/mL respectively and had little effect on hNav1.6 at these concentrations. Reversed phase HLPC fractionation of the T. apophysis venom revealed peaks of two poorly separated peptides. After reduction and alkylation, these peptides were distinguishable and named as TRTX-Tap1a (Tap1a) and TRTX-Tap2a (Tap2a) peptides. Upon modelling the 3D structures of Tap1a and Tap2a, it was presumed that the dissimilarity in sequences of the two peptides could account for their different potency and selectivity for Nav and Cav3 channels. The next step of the study was generating recombinant Tap1a (rTap1a) and Tap2a (rTap2a) using an E. coli periplasmic expression system. The effects of rTap1a and rTap2a on hCav3.1, hCav3.2, and hCav3.3 were examined; the study revealed that both peptides preferentially inhibited the Cav3.1 subtype. It was also found that rTp1a inhibited Cav3.2 whereas rTap2 did not, whilst both peptides weakly inhibited Cav3.3 at high concentrations. Overall, these experiments revealed that rTap1a and rTpa2a behaved as gating modifiers, as indicated by the hyperpolarising shifts in the voltage-dependence of activation and steady-state inactivation for hNav1.1, hVav1.6, and hNav1.7. It was also found that rTap1a-induced a hyperpolarizing shift in the voltage-dependence of activation of Cav3.1 and steady-state inactivation of Cav3.2.
To elucidate the relative expression of Cav3.2 in cell bodies that innervate the colon and bladder, quantitative reverse-transcription polymerase chain was carried out on cell bodies of the pelvic and splanchnic nerve which are located in the lumbar-sacral and thoracic-lumbar dorsal root ganglia. It was found that there was abundant expression of Cav3.2 and weaker expression of both Cav3.1 and Cav3.3 channels.
Next, it was investigated whether rTap1a had the ability to regulate bladder mechanosensitivity in afferent nerve fibres innervating the bladder of healthy mice. It was found that administration of 10uM of rTap1a into the urinary bladder significantly reduced neuronal firing induced by bladder distension, indicating the rTap1a is able to directly modulate bladder afferent excitability. Additionally, the researchers identified that rTap1a reduces the evoked visceral pain in their mouse model of IBS and it also has the ability to reduce action potential firing in colonic pain pathways to reduce mechanical sensitivity of colonic nociceptors.
Other previous studies have shown that inhibition of Nav1.1 alone or combined inhibition of Nav1.1 and Nav1.3 reduces colonic nociceptor firing, whilst combined inhibition of Nav1.1 and Nav1.6 reduces colonic and afferent responses to visceral pain behaviour. It has also been shown that colon-innervating neurons in mice with chronic visceral hypersensitivity had enhanced responses to Nav1.1 activation, whilst Nav1.1 inhibitors relieved chronic visceral pain in a mice with IBS, indicating that Nav1.1 participates in chronic visceral pain. Furthermore, previous studies have indicated that Cav3 channel blockers reduce hypersensitivity in mice, supporting the role of Cav3.2 channels in chronic visceral pain transmission in IBS (Francois et al., 2013). Overall, these previous studies consolidated that inhibition of Nav and Cav3.2 channels through the administration of inhibitors can provide visceral pain relief.
These previous studies support the study conducted at University of Queensland, whose results show that rTap1a administration can provide visceral pain relief in IBS mice through inhibition of Nav and Cav3.2 channels, nearly ablating neuronal mechanosensitive in the afferent fibres that innervate the colon and bladder. Tap1a is a potent peptide drug that could potentially relieve pain in IBS patients, where there are currently no effective treatments for. Their data suggests that Tap1a, derived from the T. apophysis spider, can target both Nav and Cav3 channels to provide analgesia through inhibition of the ion channels. Although its mechanism of action remains elusive, the benefit achieved by its administration in IBS mice shows promise of its therapeutic potential for treating chronic visceral pain.
References:
Treeded RD, Rief W, Barke A, Aziz Q, Bennett MI, Benoliel R, et al. (2015) A classification of chronic pain for ICD-11. Pain Journal. 156(6): 1003–1007. Available from: doi: 10.1097/j.pain.0000000000000160
Saha L. (2014) Irritable bowel syndrome: Pathogenesis, diagnosis, treatment, and evidence-based medicine. World J Gastroenterol. 14; 20(22): 6759–6773. Available from: doi: 10.3748/wjg.v20.i22.6759
Grundy L, Caldwell A, Brierley SM. (2018) Mechanisms underlying overactive bladder and interstitial cystitis/painful bladder syndrome. Front Neurosci. 12: 931. Available from: doi: 10.3389/fnins.2018.00931
Klint JK, Senff S, Rupasinghe DB, Er SY, Herzig V, Nicholson GM, King GF. (2012) Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads. Toxicon. 60: 478–91. Available from: doi: 10.1016/j.toxicon.2012.04.337
Cardoso FC, Lewis RJ. (2017) Sodium channels and pain: from toxins to therapies. Br J Pharmacol. 175:2138–57. Available from: doi: 10.1111/bph.13962
Cardoso FC, Castro J, Grundy L, et al. (2020)A spider-venom peptide with multitarget activity on sodium and calcium channels alleviates chronic visceral pain in a model of irritable bowel syndrome.Pain. Available from: doi:10.1097/j.pain.000000000000204
Francois A, Kerckhove N, Meleine M, Alloui A, Barrere C, Gelot A, et al. (2013) State-dependent properties of a new T-type calcium channel blocker enhance CaV3.2 selectivity and support analgesic effects. Pain.154:283–93. Available from: doi: 10.1016/j.pain.2012.10.023
Imperial Bioscience Review 