Reversal of peripheral neuropathic pain by the small-molecule natural

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Reversal of peripheral neuropathic pain by the smallmolecule natural product physalin F via block of CaV2.3 (Rtype) and CaV2.2 (N-type) voltage-gated calcium channels Zhiming Shan, Song Cai, Jie Yu, Zhongjun Zhang, Tissiana Gabriela Menna Vallecillo, Maria Jin Serafini, Ann Mary Thomas, Nancy Yen Ngan Pham, Shreya Sai Bellampalli, Aubin Moutal, Yuan Zhou, Guo-Bo Xu, Ya-Ming Xu, Shizhen Luo, Marcel Patek, John M. Streicher, A. A. Leslie Gunatilaka, and Rajesh Khanna ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00166 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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ACS Chemical Neuroscience

Reversal of peripheral neuropathic pain by the small-molecule natural product physalin F via block of CaV2.3 (R-type) and CaV2.2 (N-type) voltage-gated calcium channels

Zhiming Shana,b,†,1, Song Cai†,1, Jie Yu†,c, Zhongjun Zhanga, Tissiana Gabriela menna Vallecillo†, Maria Jin Serafini†, Ann Mary Thomas†, Nancy Yen Ngan Pham†, Shreya Sai Bellampalli†, Aubin Moutal†, Yuan Zhou†,d, Guo-Bo Xu¶, Ya-Ming Xu¶, Shizhen Luo†, Marcel Patek#, John M. Streicher†,£, A. A. Leslie Gunatilaka¶, and Rajesh Khanna†,£,¥,*

aDepartment

of Anesthesiology, Shenzhen People's Hospital & Second Clinical Medical College of Jinan

University, Shenzhen 518020, P.R. China; bDepartment of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, P.R. China; Department of Pharmacology† and £Neuroscience Graduate Interdisciplinary Program, College of Medicine; ¶Natural Products Center, School of Natural Resources & the Environment, College of Agriculture & Life Sciences, The University of Arizona; ¥The Center for Innovation in Brain Sciences, The University of Arizona Health Sciences, Tucson, Arizona 85724, USA; cCollege

of Basic Medical Science, Zhejiang Chinese Medical University, Hangzhou 310058, P.R. China; dThe

First Hospital of Jilin University, 71 Xinmin Street, Changchun 130021, P. R. China; and BrightRock Path Consulting, LLC, Tucson, Arizona.

1co-first

authors

*To whom correspondence should be addressed: Dr. Rajesh Khanna, Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ 85724, USA Office phone: (520) 626-4281; Fax: (520) 626-2204; Email: [email protected]

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Abstract No universally efficacious therapy exists for chronic pain, a disease affecting one-fifth of the global population. An overreliance in the prescription of opioids for chronic pain despite their poor ability to improve function has led to a national opioid crisis. In 2018, the NIH launched a Helping to End Addiction Long-term plan to spur discovery and validation of novel targets and mechanisms to develop alternative non-addictive treatment options. Phytochemicals with medicinal properties have long been used for various treatments worldwide. The natural product physalin F, isolated from the Physalis acutifolia (family: Solanaceae) herb, demonstrated antinociceptive effects in models of inflammatory pain, consistent with earlier reports of its anti-inflammatory and immunomodulatory activities. However, the target of action of physalin F remained unknown. Here, using whole-cell and slice electrophysiology, competition binding assays, and experimental models of neuropathic pain, we uncovered a molecular target for physalin F’s antinociceptive actions. We found that physalin F: (i) blocks CaV2.3 (R-type) and CaV2.2 (N-type) voltage-gated calcium channels in dorsal root ganglion (DRG) neurons; (ii) does not affect CaV3 (T-type) voltage-gated calcium channels or voltage-gated sodium or potassium channels; (iii) does not bind G-protein coupled opioid receptors; (iv) inhibits the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in spinal cord slices; and (v) reverses tactile hypersensitivity in models of paclitaxel-induced peripheral neuropathy and spinal nerve ligation. Identifying CaV2.2 as a molecular target of physalin F may spur its use as a tool for mechanistic studies and position it as a structural template for future synthetic compounds.

Keywords: Natural products; physalin F; N-type voltage-gated calcium channels; non-opioid; neuropathic pain


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INTRODUCTION Chronic pain is a growing global affliction for which no universally efficacious therapy exists. In recent years, there has been an increase in the use of prescription and non-prescription opioids for chronic pain despite their poor ability to improve function.1 This overreliance has contributed to an alarming epidemic of opioid overdose deaths and addictions2. The heterologous mechanisms underlying pain phenotypes further implies that a “one-size-fits-all” approach will not be viable.3 In calling for speedy scientific solutions to the national opioid public health crisis, the National Institutes of Health kickstarted initiatives to spur discovery and validation of novel therapeutic targets to facilitate the development of non-opioid based pain therapeutics. Along with a reframing of prevention strategies (e.g. abuse-deterrent formulations) of opioids and stricter prescribing practices and prescription-monitoring programs4, new drugs are urgently needed. In the quest for new analgesics, natural products and their metabolites remain interesting therapeutic resources. The large biodiversity of substances isolated from medicinal plants, particularly in Asia and South America, offers an assortment of novel structural templates for drug discovery. Despite the power of synthetic chemistry which can be deployed to take advantage of these new chemical entities, natural products themselves remain of high interest. A challenge, however, in the clinical development of natural products is identifying their mechanism of action and target(s). For example, physalin F, a steroidal derivative isolated from Physalis acutifolia (family: Solanaceae), was demonstrated to have antinociceptive properties in models of acute and inflammatory pain.5 Apart from suppression of the pro-inflammatory tumor necrosis factor-alpha production, the mechanisms by which physalin F elicited pain relief remained unknown. Most reported studies with natural products have described their mechanisms of action to involve either pro‐ and antioxidant mechanisms, a decrease in pro‐inflammatory mediators, regulation of dopaminergic, cannabinoid, and opioid pathways, or targeting of ion channels.6 In this study, we researched the target of physalin F’s antinociceptive activity. Commonly used in some tropical countries in the world, physalin F was isolated and described from the ethanol extract of the whole plant of Physalis angulata L.7 Physalin F has been reported to (i) inhibit growth of human leukemia cells7; (ii) inhibit activation of macrophages, thus reducing pro-inflammatory cytokine ACS Paragon Plus Environment


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production and lipopolysaccharide-induced lethality8; (iii) inhibit inflammation observed in a collagen-induced arthritis model9; (iv) induce apoptosis of cells via a reactive oxygen species-mediated mitochondrial pathway and suppress activation of NF-κB in cancer cells10; and (vi) induce apoptosis of peripheral blood mononuclear cells, decreasing the proliferation and cytokine production evoked by infection of Human T-lymphotropic virus.11 In addition to these immunosuppressive effects, Villarreal and colleagues recently reported that physalin F, at multiple doses, inhibited writhing behaviors induced by acetic-acid injections as well as both the early (nociceptive pain) and late (inflammatory pain) phases of the formalin test.5 Physalin F was also antinociceptive in the tail-flick assay, which is considered principally spinal as it can be observed even in animals with spinal cord transection that cut descending pathways,12 as well as in the hot-plate assay, which is considered to be a supraspinal integrated response. The authors of this study suggested a yet to be identified central site of action of physalin F. Our studies are focused on identifying non-opioid based antinociceptive compounds (synthetic and natural products). We recently reported that Betulinic acid, a bioactive fraction of the desert plant Hyptis emoryi, was antinociceptive in pre-clinical neuropathic pain models via targeting of N-type (CaV2.2) and Ttype (CaV3.x) voltage-gated calcium channels.13 In another study, we identified two plant natural products: hardwickiic acid – isolated from Salvia wagneriana ,13 and hautriwaic acid – isolated from Eremocarpus setigerus (Euphorbiaceae),14 both of which inhibited voltage-gated sodium channels. These natural products reversed pain behaviors in experimental models of HIV-induced- and chemotherapy-induced neuropathies.14 In contrast, the results presented in this study identify both R-type (CaV2.3) and N-type (CaV2.2) voltage-gated calcium channels as a pharmacological target of physalin F. Expression of CaV2.3 has been reported in regions of the central nervous system as well as in the somatosensory neurons of the peripheral ganglia14, thus linking them to pain pathways. Notably, genetic (CaV2.3 knockout mice) have reduced pain perception15, 16 and pharmacological antagonism of CaV2.3 channel with the tarantula toxin SNX-482, yields analgesia in animal models of neuropathic pain17. The therapeutic importance of pursuing CaV2.2 has been established with CaV2.2-blocking conotoxins for allodynia18 and the dysregulated pain behaviors of CaV2.2 knock-out mice.19, 20


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Physalin F reversed tactile hypersensitivity in rodent models of chemotherapy-induced neuropathy and spinal nerve ligation. Identifying CaV2.3/CaV2.2 as molecular targets of physalin F may promote its use as a tool for mechanistic work and position this seco-steroid as a structural template for development of future synthetic compounds.

RESULTS AND DISCUSSION Physalin F inhibits depolarization-evoked calcium influx in rat dorsal root ganglia neurons Physalin F (C28H30O10, CHEMBL1215434; molecular weight: 526.538 g/mol; Figure 1A) is a secosteroid with reported immunomodulatory and anti-inflammatory actions.11 Since Physalin F was recently reported to have antinociceptive activity5, here we set out to investigate its possible mechanism of action. We focused our efforts on ion channels as they are highly expressed on afferent pain fibers and mediate the transmission and processing of pain signals.21-23 To assess physalin F’s potential in modulating voltage-gated calcium channel function, we used a 90 mM potassium chloride (KCl) stimulus to depolarize the membrane of dorsal root ganglia (DRG) neurons in culture so as to allow opening of voltage-gated calcium channels, thus permitting measurements of evoked calcium influx. Neurons were treated overnight with either 0.1% DMSO (n=276) or a 1µM concentration of physalin F (n=381). DRG sensory neurons treated with physalin F exhibited a ~44% decrease in depolarization-evoked calcium influx in comparison with control (DMSO)-treated neurons (*p0.05, Student’s t-test) (Figure 1C). These results imply that physalin F affects voltage-gated calcium channels, but not sodium channels, in sensory neurons.



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Total calcium current (ICa) in DRG sensory neurons is reduced by Physalin F treatment Since rat DRGs express a heterogenous mix of low voltage-activated (LVA; i.e., T-type (CaV3 family)) and high-voltage activated (HVA; i.e., L-type (CaV1 family), N-type (CaV2.2) , P/Q-type (CaV2.1) , and Rtype (CaV2.3)) calcium channels, we next set out to map the identity of the channel(s) affected by physalin F using voltage-clamp electrophysiology. First, total (LVA and HVA) Ca2+ currents were measured from rat DRG neurons. To do so, we used a current-voltage protocol, holding rat DRG sensory neurons at −60 mV with depolarization steps (200ms-step) from −70 mV to +60 mV in 10mV increments (Figure 2A). At the holding potential used (-60 mV), T-type channels are inactivated and therefore the total calcium conductance measured is mostly carried by high-voltage-activated calcium channels. Typical traces in Figure 2A show a family of calcium currents recorded from DRG neurons treated with control (0.1% DMSO) (n=16) or a 1µM concentration of physalin F overnight (n=19). When compared with the control (0.1% DMSO), physalin F inhibited total calcium current density with a ~42% decrease in peak current density (Figure 2B, C). The data was normalized according to cell capacitance in order to account for the heterogeneity of DRG neuronal populations. To rule out changes in channel gating as the cause of the inhibitory properties of physalin F on total Ca2+ currents, we investigated the effect of physalin F on the biophysical properties of activation and inactivation of the DRG calcium currents as described in the Methods. After converting the current values to conductance (G), the conductance-voltage relationship was fitted with a Boltzmann equation, and the G value for each neuron was normalized to the maximal value (Gmax) derived from the fit. The G/Gmax-voltage relationship is presented in Figure 2D and demonstrates that the voltage-dependence for Gmax was similar between the two conditions. The Boltzmann fitting factors, similar half-maximal activation (V0.5) and slope (k), for the single fits to the data are shown in Figure 2D and are summarized in Table 1; these results show that there were no differences between the treatment conditions. Similarly, steady-state inactivation (protocol as shown in Figure 2E) properties of ICa were unchanged between the conditions (see Figure 2E and Table 1).

Physalin F does not affect T-type calcium currents in DRG sensory neurons


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To delineate the specific type of calcium channel affected, we began by assessing the possible effect of physalin F on T-type currents. To separate T-type currents, we use conditions and protocols described by Todorovic and colleagues.25 Currents were evoked from a holding voltage of −90 mV and stepping, for 200 msstep depolarizations, to potentials between −70 and +60 mV in increments of 10 mV (Figure 3A). Representative traces in Figure 3A show the family of T-type Ca2+ currents recorded from DRG neurons treated with control (0.1% DMSO) (n=18) or a 1 µM concentration of physalin F (n=15). Analyses of these recordings, summarized in the current-voltage (I-V) relations, show that, compared with controls (open blacksymbols), physalin F (filled red symbols) failed to reduce amplitudes of T-type Ca2+ currents at all potentials tested (Figure 3B) or at the peak (at −10 mV) (Figure 3C). Physalin F had no significant effect on macroscopic current inactivation kinetics (Figure 3D) or time-dependent (10 to 90% rise time) activation (Figure 3E). Physalin F did not cause alterations of channel gating, as shown by similar V0.5 values of the Ttype channels in the absence or presence of physalin F (Figure 3F and Table 1). Likewise, physalin F did not alter voltage-dependent kinetics of channel inactivation (Figure 3G and Table 1). Deactivating tail currents for both treatment conditions were measured by fit with a single exponential function: y = A1 × e(-x/ 𝜏1)+ y0, where A1 is the amplitude, 𝜏1 is the decay constant, and y0 is the offset. The resulting 𝜏 values (Figure 3H), exhibited no differences between both conditions. As T-type Ca2+ channels can recover from inactivation when the neuronal membrane of DRGs is undergoing long hyperpolarizations, which is permissive for possible remodeling of the cells’ firing properties. Therefore, we also studied the effects of physalin F on this property of recovery from inactivation. For this we used a two-pulse protocol with a variable duration between the pulses at −90 mV (Figure 3I) after a 500-ms inactivating pulse (holding voltage = −90 mV; test voltage = −30 mV). Figure 3I shows that in the presence of physalin F (filled red circles), T-type Ca2+ currents recover fully to 100% of the T-type Ca2+ current amplitudes of control values (Figure 3I, open black circles). Thus, the above investigations show no effect of physalin F on T-type Ca2+ current and T-type Ca2+ channel gating properties.

Physalin F reduces N-type calcium currents in DRG sensory neurons



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We next examined if HVA channels were affected by physalin F. We began by studying N-type (CaV2.2) channels. Blockers of the other specific HVA subtypes were employed to consider only the contribution of N-type to the total ICa. A representative recording from a DMSO- or physalin F-treated neuron is illustrated in Figure 4A (voltage protocol shown above the traces). The current-voltage relations for the N-type ICa (normalized to cell capacitance) obtained from 0.1% DMSO (n=16) and 1µM physalin F (n=14) treated sensory neurons are summarized in Figure 4B. When compared with the vehicle, physalin F treated sensory neurons had lower total calcium current density with a ~32% decrease in peak current density (Figure 4B, C). Thus, a majority of the total ICa blocked by physalin F is via N-type channels. We then investigated the consequence of physalin F on the biophysical properties of voltage-contingent activation and inactivation of N-type calcium channels. Data is shown with representative Boltzmann fits for either DMSO or physalin F treatment (Figure 4D, E). Steady-state inactivation or inactivation properties of calcium currents remained unchanged after treatment with either condition. The V0.5 and k, for the single fits are shown in Figure 4D, E and are summarized in Table 1; these results showed that physalin F induced a left shift in the V0.5 activation but had no effect on inactivation properties of CaV2.2 sensory neurons (Table 1).

Physalin F inhibits N-type calcium currents in DRG sensory neurons under both hyperpolarized and depolarized conditions To further understand the interaction of physalin F with CaV2.2 as the basis for its potential antinociceptive activity, we used voltage-clamp electrophysiology to measure CaV2.2 currents in cells posttreatment with either DMSO or a 1µM concentration of physalin F in DRG neurons in either hyperpolarized (data not shown) or depolarized states (data not shown). In both hyperpolarized and depolarized states, treatment with physalin F reduced peak N-type calcium currents measured at +10mV. These results are parallel to the observations reported for ω-conotoxin (tradename Prialt),26 which is also a state independent blocker of N-type calcium channels with a nearly equal efficacy at both holding potentials. In contrast, N-triazole oxindole (TROX-1), a calcium channel antagonist, was reported to preferentially inhibit CaV2.2 currents in rat DRGs under depolarized conditions.27 ACS Paragon Plus Environment

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Physalin F does not affect P/Q-type calcium currents in DRG sensory neurons Having established that physalin F blocks N-type calcium channels, we set out to determine the selectivity by testing the other HVA channels. We isolated P/Q-type (CaV2.1) channels using blockers/toxins to the other subtypes (Figure 5A: gray box). A representative recording from a DMSO- or physalin F-treated neuron is illustrated in Figure 5A (voltage protocol shown above the traces). The current-voltage relations for the P/Q-type ICa (normalized to cell capacitance) obtained from 0.1% DMSO (n=11) and 1µM physalin F (n=11) treated sensory neurons are summarized in Figure 5B. We observed no block by phsalin F of P/Q-type currents (Figure 5B, C). We then investigated the consequence of physalin F on the biophysical properties of voltage-contingent activation and inactivation of P/Q-type calcium channels. Data is shown with representative Boltzmann fits for either DMSO or physalin F treatment (Figure 5D, E). Steady-state inactivation or inactivation properties of calcium currents remained unchanged after treatment with either condition. The V0.5 and k, for the single fits are shown in Figure 5D, E and are summarized in Table 1; these results showed that physalin F did not affect gating properties of CaV2.1 in sensory neurons (Table 1).

Physalin F blocks R-type calcium currents in DRG sensory neurons Next, we isolated R-type (CaV2.3) channels using blockers/toxins to the other subtypes (Figure 6A: gray box). A representative recording from a DMSO- or physalin F-treated neuron is illustrated in Figure 6A (voltage protocol shown above the traces). The current-voltage relations for the N-type ICa (normalized to cell capacitance) obtained from 0.1% DMSO (n=8) and 1µM physalin F (n=8) treated sensory neurons are summarized in Figure 6B. When compared with the vehicle, physalin F treated sensory neurons had lower total calcium current density with a ~62% decrease in peak current density (Figure 6B, C). Thus, a significant fraction of the total ICa blocked by physalin F is via R-type channels. We investigated the consequence of physalin F on the biophysical properties of voltage-contingent activation and inactivation of R-type calcium channels. Data is shown with representative Boltzmann fits for ACS Paragon Plus Environment


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either DMSO or physalin F treatment (Figure 6D, E). The V0.5 and k, for the single fits are shown in Figure 6D, E and are summarized in Table 1; these results showed that physalin F induced a left shift in the V0.5 activation but had no effect on inactivation properties of CaV2.3 sensory neurons (Table 1).

Physalin F does not affect L-type calcium currents in DRG sensory neurons Finally, we isolated L-type (CaV1.x) channels using blockers/toxins to the other subtypes (Figure 7A). A representative recording from a DMSO- or physalin F-treated neuron is illustrated in Figure 7A (voltage protocol shown to the left of the traces). The current-voltage relations for the L-type ICa (normalized to cell capacitance) obtained from 0.1% DMSO (n=9) and 1µM physalin F (n=8) treated sensory neurons are summarized in Figure 7B. When compared with the vehicle, the calcium current density in physalin F treated sensory neurons was indifferent from control-neurons (Figure 7B, C). We investigated the consequence of physalin F on the biophysical properties of voltage-contingent activation and inactivation of L-type calcium channels. Data is shown with representative Boltzmann fits for either DMSO or physalin F treatment (Figure 7D, E). The V0.5 and k, for the single fits are shown in Figure 7D, E and are summarized in Table 1; these results showed that physalin F did not affect gating properties of CaV1.x currents in sensory neurons (Table 1).

Physalin F does not bind to µ, κ, or δ opioid receptors Since inhibition of Ca2+ influx in DRGs may occur downstream of engagement of G protein coupled receptors such as the opioid receptors, we next examined if physalin F had any off-target activity on these receptors. Thus, we tested if opioid receptors bound to physalin F using in vitro competition radioligand binding assays on human mu opioid receptor (MOR), kappa opioid receptor (KOR), or delta opioid receptor (DOR). We competed physalin F and appropriate positive controls (naloxone for MOR; Naloxone for DOR; and U50,488 for KOR) against a constant amount of tritiated diprenorphine in heterologous Chinese Hamster Ovary cells stably expressing human µ, κ, or δ opioid receptors. Physalin F, even at a maximum concentration of 10 M, did not bind to the orthosteric site of any of the opioid receptors (Figure 8A-C). As expected, Naloxone and ACS Paragon Plus Environment

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U50,488 bound to the three opioid receptor subtypes. Thus, these results suggest a non-opioidergic mechanism for physalin F’s inhibition of CaV2.2 channels.

Physalin F has no effects on sodium or potassium currents As sodium and potassium ions are critical components in generating action potentials and modulating neuronal excitability and by extension, propagating nociceptive signaling, we used whole cell voltage-clamp electrophysiology to assess the effects of physalin F on Na+ and K+ currents in DRG neurons. Typical families of sodium currents from DRG neurons treated with DMSO or physalin F are shown in Figure 9A. Treatment with physalin F (10µM, overnight) showed no effects on peak sodium current density (Figure 9B, C); data is displayed as normalized by cell capacitance. We next investigated the effect of physalin F on biophysical properties of voltage-contingent activation and inactivation of DRG sodium currents. Steady-state activation showed a right shift of the V0.5 (Figure 9D) while inactivation properties (Figure 9E) of sodium currents remained unchanged after treatment with physalin F (see also Table 1). We also measured tetrodotoxinsensitive (TTX-S) currents which activate at low thresholds and are fast-inactivating. These are largely carried by NaV1.7, NaV1.6, NaV1.3, and NaV1.1 in DRGs and shape the action potential and consequent requirement for initial depolarization. As distinctive inactivation kinetics distinguish TTX-R from TTX-S Na+channels, a fast-inactivation protocol (see Methods) was used to electrically isolate TTX-R (current available following a −40mV prepulse) from total current (current left after a −120mV prepulse), as previously described28. Neurons were treated overnight with 1 μM of physalin F, or control (0.1% DMSO) as indicated and TTX-resistant and TTX-sensitive Na+ currents were subsequently recorded and isolated. Physalin F did not significantly inhibit TTX-sensitive Na+ currents (Figure 9F, 0.1% DMSO: −629.9 ± 71.8 mV (n=10); physalin F: −490.6 ± 68.9 mV (n=10), p=0.58; Mann Whitney test). Based on different properties of the DRG TTX-S and TTX-R Na+ currents1, TTX-R currents was estimated by a 200 ms test pulse to 10mV following 1000ms prepulse at -40mV to inactivate the TTX-S component. Physalin F did not significantly inhibit TTX-resistant currents (Figure 9G, 0.1% DMSO: −598.3 ± 141.5 mV (n=10); physalin F: −677.4 ± 201.9 mV (n=10), p=0.32; Mann Whitney test). This results shows that the physalin F does not inhibit Na+ channels. ACS Paragon Plus Environment


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Next, we tested the possible action of physalin F on potassium currents (IK). Figure 10A shows representative family of current traces of total IK as well as fast inactivating IKA and slowly inactivating IKS from neurons following an overnight treatment with 1 μM of physalin F, or control (0.1% DMSO) as indicated. Treatment with physalin F did not alter IKA or IKS current or peak densities (Figure 10D, E, H, I). We noted IKA activation and inactivation were left shifted after treatment with 1µM physalin F (Figure 10K, L, and Table 1). Thus, we conclude that Physalin F has no effect on potassium currents densities but may affect potassium channel gating.

Physalin F reduces the frequency of spontaneous excitatory post-synaptic transmission in spinal cord slice recordings CaV2.2 coordinates spinal nociceptive neurotransmission29 via release of pre-synaptic nociceptive neurotransmitter from C-fibers.29-32 Since physalin F blocked CaV2.2 channels, we next asked if physalin Fmediated inhibition of CaV2.2 in DRG neurons could, as a result, reduce spontaneous excitatory post-synaptic currents (sEPSCs) examined in laminae I-II neurons in the substantia gelatinosa of the dorsal horn of the spinal cord. Figure 11A shows representative recordings of sEPSCs from both experimental groups (0.1% DMSO and 1µM physalin F). Physalin F had no effect on the amplitude of the sEPSCs (Figure 11B) but resulted in a significant decrease in sEPSCs frequency (Figure 11C). The decrease of sEPSC frequency suggests that physalin F inhibits glutamatergic excitatory inputs via a presynaptic mechanism.

Physalin F shows antinociceptive effects in Paclitaxel and SNL-induced models of pain in rats. The above data reveal CaV2.2 as a potential target of physalin F. CaV2.2 is a key channel in nociceptive signaling,33 we asked whether treatment with physalin F would show efficacy in in-vivo tests of neuropathic pain. Two neuropathic pain models were selected to test physalin F’s potential antinociceptive activity – (1) paclitaxel-induced peripheral neuropathy and (2) spinal nerve ligation (SNL). In chemotherapy-induced neuropathy, rats injected intraperitoneally with paclitaxel (2mg/kg) developed mechanical allodynia as evaluated by a decreased paw withdrawal threshold. These rats were then injected intrathecally with saline ACS Paragon Plus Environment

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(vehicle) or physalin F (2 μg/5 μL) (See Methods). Paw withdrawal thresholds in rats injected with physalin F increased compared to saline control rats, beginning at 1-hour post injection and lasting for four hours (Figure 12A). Coherent with the time-window of reversal of allodynia, the area under the curve value for rodents treated with physalin F was also increased (Figure 12B). We subsequently tested the efficacy of physalin F in the SNL model which produces partial denervation of the sciatic nerve sensory zone through damage of the sciatic nerve.34 Six adult male rats were subject to SNL surgery (see Methods), and paw withdrawal threshold was measured 15 days post-surgery. In this allodynic state, the rats were intrathecally treated with saline (vehicle) or Physalin F (2 μg/5 μL). Paw withdrawal thresholds in SNL rats treated with physalin F were increased at 1-hour post-treatment. Significant reversal of allodynia lasted for about three hours post injection (Figure 12C). These data were supported by an increased area under the curve as a result of treatment with physalin F in comparison to control-treated rats (Figure 12D). Together, these data demonstrate the antinociceptive potential of physalin F in experimentally induced neuropathic states.

CONCLUSIONS The two salient findings of our work are: (i) small-molecule natural product, physalin F, derived from Physalis acutifolia, has antinociceptive potential in neuropathic pain, and (ii) physalin F targets high-voltage activated R-type (CaV2.3) and N-type (CaV2.2) channels to achieve this effect. We arrived at this mechanistic specificity conclusion by noting the lack of effect of physalin F on low-voltage activated T-type (CaV3.x) calcium channels, or sodium (both tetrodotoxin-sensitive and resistant), or rapidly or slowly inactivating potassium channels in sensory neurons. Equally importantly, our data demonstrates that block of high-voltage gated calcium signaling is not through engagement of the opioid G protein coupled receptor family. Also, for the first time, we established the utility of this phytochemical in relief of mechanical allodynia in experimentally induced neuropathic pain models. Since their discovery in the 1980s, voltage-gated Ca2+ channels have been portrayed as significant players in nociceptive transmission. As reviewed by Zamponi33 and colleagues, voltage-gated Ca2+ channels ACS Paragon Plus Environment


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allow for neurotransmitter and hormone release in excitable cells. Specifically, CaV2.x high-voltage-gated channels are thought to drive evoked synaptic transmission, opening in response to incoming action potentials. Subsequent calcium influx then allows for rapid neurotransmitter release. Due to its role in propagating synaptic transmission, it is then no surprise that CaV2.x channels, and specifically CaV2.2 and CaV2.3 channels, are involved in nociceptive transmission. Evidence of this was demonstrated by the decreased pain hypersensitivity in neuropathic and inflammatory pain models in mice lacking CaV2.2.35, 36 Because of its critical role in neurotransmitter release from axonal projections into dorsal horn terminals in the spinal cords, CaV2.2 is proposed to be a potential target for analgesic therapies.33 In fact, three CaV2.2 antagonists – gabapentin, pregabalin, and ziconotide have been sanctioned as therapy for chronic pain by the US FDA.37 However, both pregabalin and gabapentin have serious side effects38 and low efficacy, while ziconotide is encumbered with a bevy of severe side effects and a difficult route of administration (spinally).39, 40 A novel use-dependent and activation state-selective small-molecule – the N-triazole oxindole (TROX-1) – inhibitor of HVA calcium channels (including CaV2.2) was recently reported and may have a therapeutic window safer than Prialt and is currently in clinical trials.27, 41 Other state-dependent blockers of CaV2.2 with antinociceptive promise have been reported, including ZC8842 and A-126408743 and studies to progress them to the clinic are ongoing. Hence, developing CaV2.2-targeted drugs that lack tolerance, have fewer side effects, and have an easy route of administration are still highly desirable. These considerations make way for potential new therapies like physalin F which target CaV2.2, as shown here. Since CaV2.2 is blocked by activation of GPCRs (e.g., Gi/o protein-coupled μ-opioid receptors) and its activity modulated by interactions with other proteins (e.g. collapsin response mediator protein 2 (CRMP2)44-46, presynaptic transmitter release associated proteins47-49), a cautionary note in designing CaV2.2-targeted drugs would be to fully take into account how CaV2.2’s extensive network of protein-protein interactions are affected. CaV2.3 (R-type) channels have also been implicated in pain pathways14 as evidence by their localization to nociceptive cells - in somatosensory neurons of the peripheral ganglia - and genetic work establishing that mice lacking the R-type channel have reduced pain perception.15, 16 Elegant studies from the Adams laboratory have also highlighted the regulation of CaV2.3 by mu, kappa, and delta opioid receptors in a voltageACS Paragon Plus Environment

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independent manner via the G subunits of these receptors.50 Remarkably, a polymorphism reported in the Rtype channel in a small group of Japanese patients who underwent painful cosmetic surgery is associated with a lesser need for the opioid fentanyl for postoperative pain management. This link to opioid analgesia and a clear demonstration of analgesia caused by SNX-482 firmly establish the importance of targeting R-type channels for pain management. In this regard, development of a mixed N-/R-type blocker (like Physalin F) may be advantageous. Phytochemicals have been used worldwide for their medicinal properties. For example, phytochemicals isolated from Withania somnifera have been proposed as workable therapeutic agents for immune modulation, infection (microbial), anxiety, cancer, and neurodegenerative disorders.51 Combination of phytochemicals piperine, sulforaphane, and thymoquine isolated from black pepper, cruciferous vegetables, and black seed, respectively, exhibit enhanced efficacy in combating breast cancer.52 More specifically and relevant to our studies, physalins isolated from Physalis plants, including physalin F exhibit antibacterial,53 anticancer,7, 10, 54, 55 antitumor,56 antimalarial,57 anti-inflammatory,58 and antimycobacterial effects.59 The structural features of physalin F may afford a scaffold for designing or refining its selectivity and affinity towards CaV2.2 for chronic pain. To date, 58 steroids have been reported from Physalis speciesL..60 Of these, 50 are seco-steroids termed “physalins” which contain 13,14-seco-16,24-cycloergostane scaffold. The structural diversity beyond this seco-steroid framework include cyclization, modifications in relative amount of unsaturation, and changes in ring substituents. Relevant to our interests in finding novel opioid-independent therapeutics, a limited SAR of Physalins B, D, F, and G has been reported, allowing us to glean some key information as to parts of this natural product that may possess antinociceptive activity.5 Though structurally different in only as many as 2 carbon-site moieties, these physalins have differing profiles in their inflammatory pain-relieving capability, and even in their efficacy in potentially treating non-pain related diseases. Previous work showed the superior antinoceptive profile of physalin F over physalin B, D, and G. After concentration-dependent, intraperitoneal administration of each physalin compound in acetic-acid induced mice, only physalin F reduced the number of writhes in animals at the initial concentration of 25 mg/kg and maintained through doses of 50 and 100 mg/kg, while all ACS Paragon Plus Environment


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other physalins began to decrease number of writhes only at 50mg/kg or higher doses. Additionally, in the formalin test, only physalin F was able to offer animals pain relief in the early phase of this test; the early phase is thought to be due C-fiber recruitment by the peripheral stimulus, while the late phase is driven by peripheral tissue inflammation and functional remodeling of the dorsal horn of the spinal cord (i.e., central sensitization). In further testing, it was noted that though physalins B and G showed efficacy in improving tail flick response only, and physalins D and G were effective in increasing hot plate latency, only physalin F had efficacy in both tests. In fact, of all the physalins tested, physalin F had the greatest efficacy in these assays, second only to treatment with morphine. Along these lines, only physalin F reversed the allodynia induced by complete Freund’s adjuvant injection in an experimental model of inflammation-related pain. A single epoxide structure distinguishes physalin F from its other Physalin counterparts and may offer clues as to the antinociceptive moiety. Where physalin F’s structure possesses an epoxide, Physalin B holds no substituents, physalin D possesses 2 polar hydroxyl groups, and Physalin G only a single hydroxyl group. Perhaps this epoxide group of physalin F is what specifically endows physalin F with a superior antinociceptive efficacy over the other physalins. These structure-based assessments and data characterizing the antinociceptive profiles of physalins B, D, F, and G offer evidence-based potential for similar and seemingly simple improvements in physalin F’s structure to tailor it for use in chronic pain via targeting CaV2.2. A limitation of our study is that because of the complex, polyoxygenated skeleton of Physalins (13 chiral centers; Figure 13A), synthesis of SAR analogs seems extremely challenging. Structure optimization of physalin F based on synthetic chemistry is likely to be be quite limited as the synthesis steps required would be ~20 steps and total yields would be typically low (~ 1-5%). The A and B rings of physalins have been suggested to be involved in a wide range of biological activities. Visual inspection of structure of Physalin F (Figure 13A) reveals at least two reactive motifs within its complex structure: -unsaturated ketone (Michael acceptor) in the ring A, and -epoxide in the ring B (positions 5 and 6). The former electrophilic group was suggested to react with cysteine residues of IKK (ref 2, [ref12]). A more attainable approach might be isolation of analog compounds from the Physalis plant.3 Since inhibition characteristics of Physalin F indicate that it behaves as an irreversible N- and R-type channel inhibitor, the hypothesis of covalent modification of rings A and/or B could ACS Paragon Plus Environment

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be explored by analogs that are devoid of at least one such group, i.e., the -epoxide. For example, Physalin J contains -epoxide, which is sterically hindered for an attack of nucleophilic protein residues (Cys, Lys) (Figure 13B). Physalins B, O, and N have double bond instead of epoxide and would be expected to show weaker inhibition (Figure 13C). Alternatively, 20 minutes treatment of Physalin F with 5 mM mercaptoethanol (a simple surrogate for GSH-glutathione) should abolish the inhibition or show a reversible mode of inhibition (Figure 13D, E). Should the hypothesis of covalent modification as the cause for irreversible binding of Physalin F be confirmed, less complex molecules (e.g. terpenoids, chromanes) that still feature those reactive motifs could be selected and tested. In our study, we add to the evidence supporting physalin F’s pain-relieving potential with data from both SNL and chemotherapy-induced models of neuropathic pain, thus supporting both its antinociceptive properties and relevance to chronic pain treatment from nerve injury as well as in cancer treatment. The identification of an ion channel target as a mechanism of action for the activity of physalin F positions this natural compound for further optimization for the eventual production of a selective, highly-efficacious, and non-addictive therapy for chronic pain.

METHODS Animals As done previously,61 adult male Sprague Dawley rats (Pathogen-free; 225-250g; Envigo) were kept in temperature (23±3˚C)-controlled and light-controlled conditions (12-h dark and 12-h light; with lights coming on 07:00-19:00), fed with rodent chow and water freely as needed. All procedures/experiments were conducted per the regulations of the University of Arizona’s College of Medicine Institutional Animal Care and Use Committee and the NIH-published “Guide for Care and Use of Laboratory Animals”, as well as the ethical regulations sanctioned by the International Association for the Study of Pain. With regard to experimental design, behavioral experiments were done with random assignment of animals to both treatment and control conditions; experimenters were blinded to both treatments and experimental groups. Changes in housing were made such that initial housing included three rats per cage and subsequently housed individually after ACS Paragon Plus Environment


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intrathecal catheterization.

Materials All utilized chemicals/reagents were purchased from Sigma (St. Louis, MO). Procedures for extraction, isolation and characterization of physalin F from Physalis acutifolia (family: Solanaceae) are described in the Supplementary Methods section.

Isolation and culture of rat dorsal root ganglia sensory neuron cultures Dorsal root ganglia were cultured using methods as described previously28, 31, 61-73. Dissociated DRG neurons were subsequently plated onto 12- or 15-mm laminin and poly-D-lysine-coated coverslips. Cultures were utilized before 2-days’ time.

Calcium imaging of rat dorsal root ganglionic cultures (acutely dissociated) Calcium imaging was done as described previously61, 63. In the veratridine evoked calcium influx assay, elevation of the calcium concentration secondary to the sodium influx is driven by a combination of the following events: opening of voltage gated calcium channels, sodium/calcium exchangers (NCX) functioning in reverse mode and mitochondrial calcium release through the mitochondrial NCX74. The contribution of voltagegated calcium channels in the veratridine evoked calcium influx is in the initial phase of the calcium influx and contributes to a submicromolar increase of intracellular calcium. Plasma membrane NCX working in reverse mode and calcium release by mitochondrial NCX are responsible for most of the increase in calcium intracellular concentration in our assay and thus we do not expect Physalin F do have a dramatic effect on this.

Whole-cell electrophysiological recordings of sodium currents in acutely dissociated DRG neurons Recordings were obtained from acutely dissociated DRG neurons as described by us before.66, 75, 76 The neurons were subjected to current-density (I-V) and activation/inactivation voltage protocols as previously described and shown in the figures.28, 62 Because of differential inactivation kinetics of TTX-R and TTX-S ACS Paragon Plus Environment

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channels, the fast inactivation protocol permitted separation of electrically isolated TTX-R (current available following a –40 mV prepulse) from the total current (current left after a –120 mV prepulse), to get TTX-S currents, as previously reported.28 TTX-R current was estimated by a 200 ms test pulse to 10mV following 1000ms prepulse at –40mV to inactivate the TTX-S component.77

Whole-cell electrophysiological recordings of K+ currents in acutely dissociated DRG neurons Recordings of total IK, fast inactivating IKA, slowly inactivating IKS and inactivation IKA were obtained using recording solutions and protocols (also illustrated in the figures and Table 2) as previously described.78

Whole-cell electrophysiological recordings of calcium currents in acutely dissociated DRG neurons Recordings of total calcium currents were obtained using recording solutions and protocols (also illustrated in the figures and Table 2) described earlier.28 To isolate N-type specific calcium currents, toxins/compounds were used: SNX482 (200 nM, R-type Ca2+ channel blocker), TTA-P2(1 µM, T-type Ca2+ channel blocker), ω-agatoxin (200 nM, P/Q-type Ca2+ channel blocker), and nifedipine (10 µM, L-type Ca2+ channel blocker). Protocol for N-type Ca2+ currents under hyperpolarized and depolarized conditions: the neurons were first held at -90 mV for hyperpolarized (to ensure channels were in the closed state) or at -50mV for depolarized condition, then the voltage jumped to –50 mV for 100 milliseconds (this allowed for inactivation of T-type channels) followed by a 50-ms holding at 10 mV. Steps were repeated once every 10s and were run constantly to activate CaV2.2 channels.26 The protocol for isolating T-type calcium currents was used exactly as previously described by Choe et al.25

Competition radioligand binding cell culture and cell lines Competition radioligand binding was performed as described previously, using the same Chinese hamster ovary cells staby expressing the human MOR, DOR, and KOR.61 ACS Paragon Plus Environment


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Preparation of spinal cord slices and recording of spontaneous excitatory post-synaptic transmission in substantia gelatinosa neurons of the spinal cord Culture of spinal cord slices and recording from spinal cord substantia gelatinosa neurons was done as described previously.61

Intrathecal catheterization of rodents Adult male rats were intrathecally catheterized as described by Yaksh and Rudy.79 Technical procedure of catheterization was as done previously.61

Assessment of allodynia Allodynia was tested as described previously.61 Data was analyzed as reported by Chaplan et al80 using Dixon’s nonparametric method.

Chemotherapy-induced neuropathy model of pain Adult male rats were administered paclitaxel (Cat# P-925-1, Goldbio, Olivette, MO) as described by Polomano et al.81 Rats were intraperitoneally injected with Taxol – resuspended at 2 mg/ml in 30% 1:1 Cremophor EL: ethanol – at 2 mg/kg four separate time, every other day for a total dose of 8 mg/kg. Mechanical allodynia developed after 10 days of post-initial paclitaxel injection.

Spinal nerve ligation-induced rodent model of pain Injury (nerve ligation) was performed as described previously.63, 66 Animals assigned to the sham group underwent the same operation but without nerve ligation. Nerve operations were done 5 days post-intrathecal catheterization. Animals exhibiting signs of motor deficiency were not used in the study.

Statistical analyses ACS Paragon Plus Environment

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All statistical analyses were done as described previously.61

AUTHOR INFORMATION Corresponding author Email address: [email protected]

Author contributions Z.S., S.C., and J.Y. conducted electrophysiology experiments. G-B.X. and Y-M. X. isolated and identified the compound. T.G.M.V., M.J.S., A.M.T., N.Y.N-P., Y.Z., A.M. and S.S.B. performed calcium imaging experiments. S.L. performed the behavioral experiments. J.M.S. supervised the opioid receptor binding experiments. A.A.L.G. and R.K. conceived the study. A.A.L.G., Z.Z., and R.K. designed/supervised the overall project and R.K. wrote the manuscript.

Funding Sources This work is supported by a National Institutes of Health awards (R01NS098772, R01DA042852, and R01AT009716) to R.K. and grants from the Medical Scientific Research Foundation of Guangdong Province, China. (No. A2017047) to Z.S. and grants from the National Natural Science Foundation of China (81603088) and National Key Project of Research and Development of China (2018YFC1705501) to J.Y. Visit of G-B.X. to A.A.L.G.s lab is supported by the China Scholarship Council.

Conflict of interest R.K. is a stakeholder in Regulonix Holding Inc.

ABBREVIATIONS AUC, area under the curve; CaV1.x, L-type voltage-gated calcium channel; CaV2.1, P/Q-type voltage-gated calcium channel; CaV2.2, N-type voltage-gated calcium channel; CaV2.3, R-type voltage-gated calcium ACS Paragon Plus Environment


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channel; CHO, Chinese Hamster Ovary; DOR, delta opioid receptor; DRG, dorsal root ganglion; GPCR, G protein coupled receptor; KCl, potassium chloride; KOR, kappa opioid receptor; MOR, mu opioid receptor; NaV1.x, voltage-gated Na+ channel isoform 1.x; sEPSCs, spontaneous excitatory postsynaptic currents; TTX, tetrodotoxin; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-sensitive

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FIGURE LEGENDS Figure 1. Physalin F inhibits calcium influx via voltage-gated calcium, but not sodium, channels in sensory neurons. (A) Chemical structure of physalin F. Normalized peak responses of sensory neurons incubated overnight with 0.1% DMSO (control) or 1 µM physalin F in response to 90 mM KCl (to trigger opening of low- and high-voltage activated calcium channels) (n= 276 to 381 neurons) (B) or 30 M veratridine (to trigger opening of sodium channels) (n= 93 to 613 neurons) (C). Asterisks indicate statistical significance compared with cells treated with the vehicle (p

Reversal of peripheral neuropathic pain by the small-molecule natural

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