ProTx I

Chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse effect experienced by cancer patients receiving treatment with paclitaxel. The voltage-gated sodium channel 1.7 (Nav1.7) plays an important role in multiple preclinical models of neuropathic pain and in inherited human pain phenotypes, and its gene expression is increased in dorsal root ganglia (DRGs) of paclitaxel-treated rats. Hence, the potential of change in the expression and function of Nav1.7 protein in DRGs from male rats with paclitaxel-related CIPN and from male and female humans with cancer-related neuropathic pain was tested here. Double immunofluorescence in CIPN rats showed that Nav1.7 was upregulated in small DRG neuron somata, especially those also expressing calcitonin gene-related peptide (CGRP), and in central processes of these cells in the superficial spinal dorsal horn. Whole-cell patch-clamp recordings in rat DRG neurons revealed that paclitaxel induced an enhancement of ProTx II (a selective Nav1.7 channel blocker)-sensitive sodium currents. Bath-applied ProTx II suppressed spontaneous action potentials in DRG neurons occurring in rats with CIPN, while intrathecal injection of ProTx II significantly attenuated behavioral signs of CIPN. Complementarily, DRG neurons isolated from segments where patients had a history of neuropathic pain also showed electrophysiological and immunofluorescence results indicating an increased expression of Nav1.7 associated with spontaneous activity. Nav1.7 was also colocalized in human cells expressing transient receptor potential vanilloid 1 and CGRP. Furthermore, ProTx II decreased firing frequency in human DRGs with spontaneous action potentials. This study suggests that Nav1.7 may provide a potential new target for the treatment of neuropathic pain, including chemotherapy (paclitaxel)-induced neuropathic pain.SIGNIFICANCE STATEMENTThis work demonstrates that the expression and function of the voltage-gated sodium channel Nav1.7 are increased in a preclinical model of chemotherapy-induced peripheral neuropathy (CIPN), the most common treatment-limiting side effect of all the most common anticancer therapies. This is key as gain-of-function mutations in human Nav1.7 recapitulate both the distribution and pain percept as shown by CIPN patients. This work also shows that Nav1.7 is increased in human DRG neurons only in dermatomes where patients are experiencing acquired neuropathic pain symptoms. This work therefore has major translational impact, indicating an important novel therapeutic avenue for neuropathic pain as a class.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (NaV) channel 1.7 (NaV1.7), which has been identified as a primary pain target. However, in developing NaV1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other NaVsubtypes that are critical for survival. Since spider venoms are an excellent source of NaVchannel modulators, we screened a panel of spider venoms to identify selective NaV1.7 inhibitors. This led to identification of two novel NaVmodulating venom peptides (β/μ-theraphotoxin-Pe1a and β/μ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (β/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the NaV1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and NaVchannel subtype selectivity. Several analogues had improved potency against NaV1.7, and altered specificity against other NaVchannels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of NaV1.7.

Background and purpose:Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Nav1.7 voltage-dependent Na+channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Navchannel targets.Experimental approach:We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high-throughput automated patch-clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Nav-expressing cells in pull-down and cell distribution studies.Key results:Two of the produced ProTx II analogues, Biot-ProTx II and ATTO488-ProTx II, best emulate the pharmacological properties of unlabelled ProTx II, whereas other analogues remain high affinity blockers of Nav1.7. The biotinylated version of ProTx II efficiently works for the pull-down of several Navisoforms tested in a concentration-dependent manner, whereas the fluorescent ATTO488-ProTx II specifically labels the Nav1.7 channel over other Navisoforms tested in various experimental conditions.Conclusions and implications:The properties of these ProTx II analogues as tools for Navchannel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also be useful in the design of new drug screening strategies.