(E)-3-Furan-2-yl-N-p-tolyl-acrylamide and its Derivative DM489 Decrease Neuropathic Pain in Mice Predominantly by α7 Nicotinic Acetylcholine Receptor Potentiation

pubs.acs.org/chemneuro Research Article (E)‑3-Furan-2-yl‑N‑p‑tolyl-acrylamide and its Derivative DM489 Decrease Neuropathic Pain in Mice Predominantly by α7 Nicotinic Acetylcholine Receptor Potentiation Downloaded via UNIV DEGLI STUDI DI FIRENZE on December 10, 2020 at 11:39:17 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Hugo R. Arias,* Carla Ghelardini, Elena Lucarini, Han-Shen Tae,* Arsalan Yousuf, Irina Marcovich, Dina Manetti, Maria Novella Romanelli, Ana Belén Elgoyhen, David J. Adams, and Lorenzo Di Cesare Mannelli Cite This: ACS Chem. Neurosci. 2020, 11, 3603−3614 ACCESS Read Online Metrics & More Article Recommendations ABSTRACT: The main objective of this study was to determine whether (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) and its structural derivative DM489 produce anti-neuropathic pain activity using the streptozotocin (STZ)- and oxaliplatin-induced neuropathic pain animal models. To assess possible mechanisms of action, the pharmacological activity of these compounds was determined at α7 and α9α10 nicotinic acetylcholine receptors (nAChRs) and CaV2.2 channels expressed alone or coexpressed with G protein-coupled GABAB receptors. The animal results indicated that a single dose of 3 mg/kg PAM-2 or DM489 decreases STZ-induced neuropathic pain in mice, and chemotherapy-induced neuropathic pain is decreased by PAM-2 (3 mg/ kg) and DM489 (10 mg/kg). The observed anti-neuropathic pain activity was inhibited by the α7-selective antagonist methyllycaconitine. The coadministration of oxaliplatin with an inactive dose (1 mg/kg) of PAM-2 decreased the development of neuropathic pain after 14, but not 7, days of cotreatment. The electrophysiological results indicated that PAM-2 potentiates human (h) and rat (r) α7 nAChRs with 2−7 times higher potency than that for hCaV2.2 channel inhibition and an even greater difference compared to that for rα9α10 nAChR inhibition. These results support the notion that α7 nAChR potentiation is likely the predominant molecular mechanism underlying the observed anti-nociceptive pain activity of these compounds. KEYWORDS: Neuropathic pain, positive allosteric modulators, nicotinic acetylcholine receptors, CaV2.2 channels For instance, intraplantar injections of Complete Freund’s Adjuvant, but not chronic constriction nerve injury, increased hyperalgesia and allodynia in α7 KO mice compared to wildtype mice, and the anti-nociceptive effects of systemic nicotine were observed only in wild-type animals.10 Although α7 KO mice did not show differences in thermal and mechanical hypersensitivity, they were less susceptible to the antinociceptive activity of choline (an agonist with relatively higher selectivity for α7 nAChRs) infusions compared to wildtype mice.11 INTRODUCTION Nicotinic acetylcholine receptors (nAChRs) such as the α7 and α9α10 subtypes are members of the Cys-loop ligand-gated ion channel superfamily, which also comprises the γ-aminobutyric acid type A (GABAA), glycine, and serotonin type 3 (5HT3) receptors.1 Both α7 and α9α10 subtypes have been reported to be involved in pain-related processes, including neuropathic pain and chronic inflammation. α9α10 nAChRs are expressed mainly in outer hair cells of the cochlea,2 endocrine cells,3 and various immune cells,4 and its inhibition decreases neuropathic pain in animal models.5,6 On the contrary, α7 nAChRs are abundantly expressed in the brain and peripheral tissues, including immunocompetent cells and nociceptive dorsal root ganglion (DRG) neurons, and different studies confirmed the role of this receptor subtype in inflammatory and neuropathic pain.6−9 Although the use of α7 knockout (KO) mice in principle supported the role of α7 nAChRs in pain processes, they are somehow controversial. ■ © 2020 American Chemical Society Received: July 26, 2020 Accepted: October 5, 2020 Published: October 19, 2020 3603 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Previous attempts to alleviate pathological conditions involving chronic pain were focused on selective α7 nAChR agonists.8,12 However, their therapeutic efficacies have been limited due to potential side effects and increased receptor desensitization. Several laboratories have focused on the use of positive allosteric modulators (PAMs) with high selectivity for α7 nAChRs (e.g., PNU-120596 and PAM-2), which induce anti-nociceptive and anti-inflammatory effects in various animal models.6,13,14 In this regard, we compared the antineuropathic pain activity of PAM-2 [(E)-3-furan-2-yl-N-ptolyl-acrylamide]15 with that of a novel structural derivative with relatively higher brain penetration, DM489 [(E)-3-(furan2-yl)-1-(indolin-1-yl)prop-2-en-1-one)] (see 3D structures in Figure 1), by using the oxaliplatin- and streptozotocin (STZ)induced neuropathic pain models.5,16−18 Research Article Scheme 1. Chemical Synthesis of PAM-2 [(E)-3-Furan-2-ylN-p-tolyl-acrylamide] and DM489 [(E)-3-(Furan-2-yl)-1(indolin-1-yl)prop-2-en-1-one]a a Et3N, triethylamine; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole hydrate. Brain Permeability Prediction for PAM-2 and DM489. To predict whether PAM-2 and DM489 can penetrate the brain, their lipophilicity (LogP) and blood−brain barrier penetration (LogBB) values were calculated (Table 1). The Table 1. Lipophilicity (LogP) and Blood−Brain Barrier Permeability (LogBB) of PAM-2 and DM489 Figure 1. 3D molecular structure of PAM-2 [(E)-3-furan-2-yl-N-ptolyl-acrylamide] and DM489 [(E)-3-(furan-2-yl)-1-(indolin-1-yl)prop-2-en-1-one] with the van der Waals surface surrounding the molecule (stick model) and colored according to atom type: O (red), N (blue), H (white), and C (gray). Representations were generated using DSViewer Pro 6.0 (Accelrys, San Diego, CA). compound LogP LogBB PAM-2 DM489 2.96 2.76 0.09 0.17 results indicated that, although both compounds have similar lipophilicity (LogP > 2), DM489 has a relatively higher brain permeability (0.17) than that of PAM-2 (0.09). This is a consequence of the higher polarity of the secondary amine of PAM-2, which has a H-bond donor, whereas DM489 is a tertiary amine with no H-bond donor (see Figure 1). Previous in vivo animal studies showed that intraperitoneal (ip) injections of PAM-2 induced several behavioral effects in mice,21−23 thus suggesting an active brain permeability of PAM-2. PAM-2 or DM489 Decreased Drug-Induced Neuropathic Pain in Mice. The pain-relieving properties of PAM-2 and DM489 were evaluated using different animal models of neuropathic pain. In Figure 2, the effects of these PAMs against streptozotocin (STZ)-induced diabetic neuropathic pain are shown. STZ-induced hypersensitivity to cold noxious stimuli (i.e., allodynia-like measurements using the cold plate test) was developed, where STZ decreased the pain threshold (i.e., increased pain sensitivity) to 9.8 ± 0.4 s in comparison to those of vehicle-treated animals (18.5 ± 0.2 s, n = 10) (oneway ANOVA; P < 0.01). Interestingly, the treatment with 3 mg/kg, but not 1 mg/kg PAM-2, was able to significantly (P < 0.05) decrease neuropathic pain between 15 and 45 min, with complete reversal at 30 min. On the contrary, DM489 was active at 3 (P < 0.05) and 10 (P < 0.01) mg/kg compared to vehicle-treated animals. No statistical differences were observed between 3 mg/kg PAM-2 and DM489 (P > 0.05). To study the drug’s profile against a different model of chronic pain, neuropathic conditions were evoked using the neurotoxic anti-cancer agent oxaliplatin.5,16 Treatment (ip) with 2.4 mg/kg oxaliplatin progressively developed neuropathic pain in mice as evaluated by the cold plate test. The licking latency decreased to 9.5 ± 0.2 s in comparison to in vehicle-treated animals (18.7 ± 0.2 s, n = 10) (P < 0.01) To assess the molecular mechanisms underlying the observed anti-neuropathic pain activity of PAM-2 and DM489, their pharmacological activities were determined at α7 and α9α10 nAChRs heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage clamp recording technique. Given that voltage-gated N-type calcium (CaV2.2) channels modulated by G protein-coupled GABA type B receptors (GABABRs) have been implicated in pain-related processes,5,19,20 the activity of PAM-2 and DM489 was also investigated on CaV2.2 channels expressed alone or coexpressed with GABABRs using the whole-cell patch clamp recording technique. This study clearly demonstrated that both PAM-2 and DM489 induce anti-neuropathic pain activity in mice and that α7 nAChR potentiation, rather than α9α10 nAChR and/or CaV2.2 channel inhibition, is likely the predominant molecular mechanism underlying this activity. The results also suggested that PAM-2 and DM489 might be used for the prevention and treatment of (chronic) neuropathic pain, especially in chemotherapy and diabetes conditions. RESULTS AND DISCUSSION Chemical Synthesis. DM489 was synthesized by reacting (E)-3-(furan-2-yl)acrylic acid with indoline, using N-(3dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and 1hydroxybenzotriazole hydrate (HOBt) as coupling reagents (Scheme 1). For convenience, the same method was also applied to the preparation of PAM-2, using p-toluidine as a reactant. This procedure gave high yields for both compounds and better purity (>99.5% for PAM-2 and >95% for DM489) than those obtained by previous methods.15 ■ 3604 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Figure 2. Effect of (A) PAM-2 and (B) DM489 on streptozotocin (STZ)-induced neuropathic pain in mice. Mice (n = 10/condition) were treated with a single injection (ip) of 100 mg/kgSTZ (□) to develop neuropathic pain. On day 15, (A) PAM-2 [1 (◊) and 3 mg/ kg (Δ)] or (B) DM489 [3 (Δ) and 10 (○) mg/kg] was administered per os (p.o.), and the response to a thermal stimulus was subsequently evaluated by the cold plate test. The licking latency (mean ± SEM in seconds) for the first signs of pain-related behavior was recorded before and 15, 30, 45, and 60 min after treatment. Bonferroni post hoc analyses indicated that STZ + vehicle (□) induced neuropathic pain during the whole testing time (i.e., 0−60 min) [P < 0.01 vs vehicle + vehicle-treated mice (●)] and that 3 mg/kg PAM-2 (Δ) decreased drug-induced neuropathic pain at 15 (P < 0.05) and 30 (P < 0.01) min period (A), whereas 3 (Δ) and 10 (○) mg/kg DM489 decreased neuropathic pain during the 15−30 and 15−45 min periods (P < 0.01), respectively, compared to that for STZ + vehicle (□). Research Article Figure 3. Effect of (A) PAM-2 and (B) DM489 on oxaliplatininduced neuropathic pain in mice. Mice (n = 10/condition) were treated (ip) with 2.4 mg/kg oxaliplatin to induce neuropathic pain. On day 15, (A) PAM-2 [1 (Δ) and 3 mg/kg (○)] or (B) DM489 [10 (Δ) and 30 (○) mg/kg] was administered (p.o.), and the response to a thermal stimulus was subsequently evaluated by the cold plate test. The licking latency (mean ± SEM) for the first signs of pain-related behavior was recorded before and 15, 30, 45, and 60 min after treatment. Bonferroni post hoc analyses indicated that oxaliplatin + vehicle induced neuropathic pain (□) during the whole testing time [P < 0.01 vs vehicle + vehicle-treated mice (●)] and that 3 (○) (P < 0.01), but not 1 mg/kg (Δ) (P > 0.05), PAM-2 decreased druginduced neuropathic pain during the 15−45 min period (A), whereas 10 mg/kg DM489 decreased neuropathic pain during the 15−30 min period (Δ) and 30 mg/kg DM489 decreased neuropathic pain during the 15−45 min period (○) (P < 0.01 for both) (B), compared to oxaliplatin + vehicle-treated mice (□). (Figure 3). The effect elicited by a single p.o. administration of PAM-2 (Figure 3A) or DM489 (Figure 3B) was subsequently evaluated on day 15. Bonferroni post hoc analyses indicated that a dose of 3 mg/kg (P < 0.01), but not 1 mg/kg, PAM-2 induced pain relief, starting 15 min after treatment and lasting for 45 min. In addition, DM489 produced pain relief at doses of 10 and 30 mg/kg (P < 0.01), and the effect was quicker (peak at 15 min) than that for PAM-2 (peak at 30 min). These results indicated that both compounds at different doses are capable of inducing anti-nociceptive effects but that the onset of PAM-2’s action is double that of DM489. Anti-Neuropathic Pain Activity of PAM-2 and DM489 is Mediated by α7-Containing nAChRs. To determine whether the anti-neuropathic pain activity of PAM-2 and DM489 was mediated by α7-containing nAChRs, the effect of 6 mg/kg methyllycaconitine citrate (MLA), an α7-selective antagonist,24 was determined on mice treated with an active dose of PAM-2 (3 mg/kg) (Figure 4A) or DM489 (30 mg/kg) (Figure 4B). Statistical analyses of the results indicated that the anti-neuropathic pain effect elicited by PAM-2 and DM489 was completely blocked by MLA (P < 0.01), whereas MLA did not change per se the neuropathic pain effect elicited by oxaliplatin. PAM-2 Decreased the Development of OxaliplatinInduced Neuropathic Pain. To determine whether PAM-2 decreases the development of oxaliplatin-induced neuropathic pain, an inactive dose of PAM-2 (1 mg/kg) was coadministered with 2.4 mg/kg oxaliplatin using the same protocol as for oxaliplatin alone, and cold plate tests were performed on day 15. Bonferroni post hoc analyses indicated that PAM-2 + oxaliplatin-treated animals have a significantly higher pain threshold compared to oxaliplatin-treated animals after 14, but not 7, days of treatment (P < 0.01) (Figure 5A). Interestingly, an additional administration of PAM-2 30 min after the described cotreatment, produced a higher pain reversion in the 0−30 min time regime compared to the same period in the previous cotreatment (P < 0.01; Figure 5B). The animal results indicated that that a single dose of either 3 mg/kg PAM-2 or DM489 was able to decrease STZ-induced diabetic neuropathic pain. In addition, PAM-2 decreased oxaliplatin-induced neuropathic pain at a lower dose (3 mg/ kg) than that used with DM489 (10 mg/kg); however, the latter produced a faster onset of action than that for PAM-2. Since electrophysiology results showed that DM489 and PAM2 enhance ACh-evoked rα7 activity with similar potency and efficacy, a possible explanation for the anti-nociceptive differences observed between these compounds might be based on the inhibitory component observed for DM489, but not for PAM-2, and/or distinct outcomes between druginduced neuropathic models (i.e., chemotherapy-induced neuropathy is in general more painful than that elicited by diabetic drugs). In addition, the faster onset observed for DM489 compared to PAM-2 could be due to the relatively greater ability of the former drug to cross the blood−brain barrier. Additional animal experiments also showed that PAM-2 can decrease the development of oxaliplatin-induced neuropathic pain in mice, which could be of clinical relevance in the prevention of chemotherapy-induced chronic pain in cancer 3605 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Figure 4. Effect of methyllycaconitine (MLA) on the anti-neuropathic pain activity elicited by (A) PAM-2 and (B) DM489. Mice (n = 10/ condition) were treated (ip) with 2.4 mg/kg oxaliplatin to develop neuropathic pain. On day 15, 6 mg/kg MLA was administered (ip) 15 min before (A) 3 mg/kg PAM-2 (p.o.) (□) or (B) 30 mg/kg DM489 (□). The response of MLA to the respective effect elicited by (A) PAM-2 (□)and (B) DM489 (□) was subsequently compared to that for PAM-2 (◊) and DM489 (◊) by the cold plate test. The licking latency (mean ± SEM) for the first signs of pain-related behavior was recorded before and 15, 30, 45, and 60 min after treatment. Bonferroni post hoc analysis indicated that MLA inhibited the antineuropathic pain effect elicited by PAM-2 during the 15−45 min period (□) and by DM489 during the 15−30 min period (□) (P < 0.01 for both), compared to the respective drug + vehicle-treated animals (◊), whereas MLA + oxaliplatin-treated animals (Δ) were not different from oxaliplatin + vehicle treated animals (P > 0.05) (○). Research Article Figure 5. PAM-2 decreased the development of oxaliplatin-induced neuropathic pain. Mice (n = 10/condition) were treated (ip) with 2.4 mg/kgoxaliplatin alone (gray) or coadministered with an inactive dose (1 mg/kg; p.o.) of PAM-2 (black). (A) Effect of PAM-2 after 7 and 14 days of cotreatment with oxaliplatin. Cold plate tests were determined 24 h after the last administration and the pain threshold values (mean ± SEM) compared between drug- and vehicle-treated animals (white). Bonferroni post hoc analyses indicated that oxaliplatin + vehicle induced neuropathic pain after 7 and 14 days of treatment [**P < 0.01 vs vehicle + vehicle-treated mice (white)], and that PAM2 + oxaliplatin decreased neuropathic pain after 14, but not 7, days of treatment [P̂̂ < 0.01 vs oxaliplatin + vehicle-treated mice (gray)]. (B) Thirty minutes after an additional administration of 1 mg/kg PAM-2, the pain threshold (mean ± SEM) was recorded at 0, 15, 30, 45, and 60 min. Bonferroni post hoc analyses indicated that oxaliplatin + vehicle-treated mice developed neuropathic pain during the whole testing time (□) [P < 0.01 vs vehicle + vehicle-treated mice (○)], and that PAM-2 decreased neuropathic pain (●) during the 0−30 min period (P < 0.01 vs oxaliplatin + vehicle-treated mice). patients, especially considering that PAM-2 has a higher efficacy than that of DM489. Our study based on drug-induced neuropathic pain models and especially the chemotherapybased model, which mainly induces neuropathy toxicity without an inflammatory component, expanded previous results showing that PAM-2 reduces neuropathic pain induced by sciatic nerve ligation,25 which has a strong inflammatory component.16 PAM-2 and DM489 Potentiated ACh-Activated α7 nAChRs in a Concentration-Dependent Manner. The activity of PAM-2 and DM489 was determined at human and rat α7 nAChRs (>90% amino acid sequence homology) heterologously expressed in X. laevis oocytes using the twoelectrode voltage clamp recording technique. At 10 μM, PAM2 and DM489 potentiated ACh-evoked hα7 currents by 30 ± 4% and 27 ± 6% (n = 5−6), respectively, in a reversible manner, indicating that receptors are not continuously stimulated upon washout (Figure 6A). Similarly, 10 μM PAM-2 and DM489 potentiated rα7 nAChR-mediated currents by 15 ± 4% and 30 ± 8% (n = 5−8), respectively (Figure 7A). The concentration−response relationships for PAM-2 and DM489, relative to that induced by ACh at 20% maximal effective concentrations (EC20) [i.e., 30 μM for hα7, and 160 μM for rα7 (see Figure 7B)], indicated that both compounds enhanced ACh-evoked currents at hα7 (Figure 6B) and rα7 (Figure 7C) nAChRs with comparable potencies (Table 2). Although both PAM-2 and DM489 enhanced rα7 nAChRs with 3- to 4-fold higher potency than that at hα7 nAChRs, their efficacies (i.e., ΔEmax) were higher at hα7 nAChRs (120%−150%) compared to that at rα7 nAChRs (∼30%) (Table 2). The calculated nH values (i.e., nH > 1.5) (Table 2) support a cooperative mechanism at both hα7 and rα7 nAChRs. By using the two-component Hill equation [see eq.] 1], we were able to discriminate the potentiating and inhibitory effects of both PAM-2 and DM489 at α7 nAChRs. The results indicated that concentrations above 100 μM PAM-2 and 30 μM DM489 started to inhibit, probably by a negative allosteric modulatory mechanism at rα7 (Figure 7C), but not hα7 (Figure 6B), nAChRs. Comparison of the half-maximal effective and inhibitory concentrations (EC50 and IC50, respectively) (Table 2) showed that PAM-2 potentiated rα7 nAChRs with >8-fold higher potency than that elicited by inhibition (12 vs >100 μM), whereas DM489 potentiated the rα7 nAChR with ∼6-fold higher potency than that elicited by inhibition (9 vs 56 μM). PAM-2 and DM489 Inhibited rα9α10 nAChRs with Different Potencies and in a Voltage-Independent Manner. To determine the inhibitory activity of PAM-2 and DM489 at rα9α10 nAChRs, two-electrode voltage clamp experiments were performed on nAChRs expressed in Xenopus oocytes. Both compounds inhibit ACh (10 μM)-evoked currents in a concentration-dependent manner (Figure 8A), and the concentration−response analyses (Figure 8B) showed that the inhibitory potency for DM489 was 4-fold higher than 3606 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 6. Effect of PAM-2 and DM489 at ACh-activated hα7 nAChRs expressed in Xenopus oocytes. (A) Representative ACh (30 μM)-evoked hα7 currents obtained at −80 mV in the absence and presence of 10 μM PAM-2 or DM489 and after washout. ▽, ACh alone; ▼, coapplication of ACh + compound after 5 min incubation () with compound alone; ▽, ACh alone after washout. (B) Concentration−response relationships obtained for PAM-2 (○) and DM489 (▲)-induced potentiation of ACh-evoked current amplitude at hα7 nAChRs. Current amplitudes (mean ± SEM; n = 5−6) were normalized to the response elicited by 30 μM ACh alone (corresponding to its EC20 at hα7 nAChRs). The calculated potentiating EC50, ΔEmax, and nH values are summarized in Table 2. Depolarization-activated Ba2+ currents (IBa) recorded from HEK293T cells transiently cotransfected with GABABR and hCaV2.2 channels were reversibly inhibited by PAM-2 and DM489 but were not antagonized by 1 μM CGP55845, a selective GABABR antagonist. In contrast, the inhibition of IBa by 50 μM baclofen, a GABABR agonist, was antagonized by CGP55845 (Figure 9A). However, in HEK293T cells expressing hCaV2.2 alone, both PAM-2 (Figure 9B) and DM489 (Figure 9C) inhibited whole-cell IBa values in a concentration-dependent manner, and the calculated IC50 values were 89 ± 15 and 122 ± 25 μM (n = 4), respectively (Table 2). These results demonstrated that both PAM-2 and DM489 inhibit hCaV2.2 channels directly without involving the G protein-coupled GABABR. Thus, these compounds must have a mechanism distinct to that of analgesic α-conotoxins (e.g., Vc1.1 and Rg1A), which inhibit GABABR coupled CaV2.2 channels.19 PAM-2 and DM489 appear to act similar to coronaridine congeners, which also exhibit anti-neuropathic pain activity and inhibit CaV2.2 channels directly.5 Previous studies clearly identified CaV2.2 channels in painrelated mechanisms and as targets for the anti-nociceptive activity of a variety of selective inhibitors.5,19,20,27 Moreover, both CaV2.2 channels and α7 nAChRs are expressed in DRG neurons, where they modulate the release of glutamate onto dorsal horn neurons, enhancing excitatory signaling in these neurons.6,9 Although this overlapping expression opens the possibility for complementary mechanisms, our study demonstrated that PAM-2 and DM489 inhibit CaV2.2 channels with that for PAM-2 (Table 2). Given that the observed nH values were close to 2 (Table 2) indicated that the inhibitory process is mediated by a cooperative mechanism. Moreover, the voltage-dependence of rα9α10 inhibition, as shown in the representative current−voltage (I−V) curves (Figure 8C), indicated that DM489 inhibition was voltageindependent. Specifically, responses were equally inhibited (Paired Student’s t test; P = 0.38) by both negative (−90 mV) hyperpolarized potentials (% I/Imax = 63 ± 8%, n = 6) and positive (+40 mV) depolarized potentials (57 ± 6%, n = 6) (Figure 8C), suggesting that the inhibition is voltageindependent. Although this result supports a competitive inhibitory mechanism, we cannot rule out the possibility of channel pore blockade at higher concentrations. Since PAM-2 was >7-fold more potent enhancing rα7 than inhibiting rα9α10 nAChRs, it is unlikely that the latter subtype is involved in the anti-neuropathic pain activity elicited by these compounds. PAM-2 and DM489 Inhibited CaV2.2 Channels Alone but Not Those Coupled to GABABRs. A class of αconotoxins that exhibit potent anti-nociceptive activity have been shown to not only inhibit α9α10 nAChRs but also CaV2.2 channels via G protein-coupled GABAB receptor activation.19,26 Given that neither PAM-2 nor DM489 have been tested on either GABABRs or CaV2.2 channels, we investigated the activity of these compounds on HEK293T cells expressing hCaV2.2 channels alone or coexpressing GABABRs and CaV2.2, respectively. 3607 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 7. Effect of PAM-2 and DM489 on ACh-activated rα7 nAChRs expressed in Xenopus oocytes. (A) Representative ACh (160 μM)-evoked rα7 currents obtained at −80 mV in the absence and presence of 10 μM PAM-2 or DM489 and after compound washout. ▽, ACh alone; ▼, coapplication of ACh + compound after 5 min incubation () with compound alone; ▽, ACh alone after washout. (B) Concentration−response relationships for ACh-evoked current amplitudes at rα7 nAChRs. Current amplitudes (mean ± SEM; n = 5) were normalized to the response elicited by 30 mM ACh, and an ACh EC50 value of 575 ± 32 μM was calculated. (C) Concentration−response relationships obtained for PAM-2 (○) and DM489 (▲)-induced potentiation of ACh-evoked current amplitude at rα7 nAChRs. Current amplitudes (mean ± SEM; n = 5−8) were normalized to the response elicited by 160 μM ACh alone [corresponding to its EC20 at rα7 nAChRs; see (B)]. The calculated EC50, ΔEmax, IC50, and nH values are summarized in Table 2. Table 2. Pharmacological Activity of PAM-2 and DM489 at α7 and α9α10 nAChR Subtypes and CaV2.2 Channels potentiation molecular target hα7a rα7b rα9α10c hCaV2.2d compound EC50 (μM) PAM-2 DM489 PAM-2 DM489 PAM-2 DM489 PAM-2 DM489 39 ± 6 34 ± 7 12 ± 3 9±3 no activity no activity no activity no activity nH 1.50 1.62 2.28 2.81 ± ± ± ± 0.26 0.45 1.41 3.47 inhibition ΔEmax (% control) IC50 (μM) nH 155 ± 9 126 ± 14 35 ± 4 32 ± 6 no activity no activity >100e 56 ± 14e 174 ± 18 39 ± 2 89 ± 15 122 ± 25 2.25 ± 0.60e 2.17 ± 0.30 2.41 ± 0.27 1.2 ± 0.2 1.0 ± 0.1 a Values were calculated from Figure 6B. bValues were calculated from Figure 7C. cValues were obtained from Figure 8B. dValues were obtained from Figure 9C. eValues obtained by using two-component Hill eq 1. activity when α7 KO mice are used10,11 support the concept that alterations in peripheral and central mechanisms involving α7 nAChRs could be also involved in neuropathic pain. In particular, α7-selective agonists produced neuroprotective effects by preventing oxaliplatin-induced damages in peripheral nerves and DRG neurons,8,12 suggesting that α7 nAChRs expressed in DRG neurons are involved in nociception mechanisms.9 Moreover, oxaliplatin treatment induced α7 nAChR downregulation in the peripheral and central nervous systems, whereas the repeated treatment with selective agonists potencies relatively lower than that determined for α7 nAChR potentiation (see Table 2), suggesting that the latter is the predominant mechanism underlying the observed anti-neuropathic activity in mice. The widespread expression of α7 nAChRs in neurons, microglia, astrocytes, and lymphocytes suggests their preeminent role in neuro-immune crosstalk.7 Accordingly, a protective α7-mediated microglial function has been detected in hippocampal cultures under brain ischemic conditions.28 Studies showing that nAChR ligands lose anti-nociceptive 3608 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 8. Effect of DM489 and PAM-2 on ACh-activated rα9α10 nAChRs expressed in Xenopus oocytes. (A) Peak current amplitude of α9α10 nAChRs elicited by 10 μM ACh is diminished by either DM489 or PAM-2 in a concentration-dependent manner. (B) Concentration−response curves (mean ± SEM; n = 3−6) for the inhibitory activity of DM489 (□) and PAM-2 (■). Response was normalized to that elicited by 10 μM ACh (control set as 100%). The IC50 and nH values were obtained by nonlinear least-squares fit and summarized in Table 2. (C) Current−voltage relationship for DM489 was obtained by applying 2 s voltage ramps from −120 to +50 mV, 10 s after the peak response to 10 μM ACh from a holding potential (Vhold) of −70 mV, in the absence (black line) and presence (gray line) of 30 μM DM489. Paired Student’s t test analysis showed that DM489’s inhibition is not significantly different (P = 0.38) between −90 mV (63 ± 8%, n = 6) and +40 mV (57 ± 6%, n = 6). 3609 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Research Article Figure 9. Inhibitory activity of PAM-2 and DM489 at hCaV2.2 channels and GABABRs heterologously expressed in HEK293T cells. (A) Timedependent plot of peak Ba2+ current (IBa) amplitudes before, during, and upon washout of 50 μM (±)-baclofen (Bac), 100 μM PAM-2, and 100 μM DM489, in the continuous presence of the selective GABABR antagonist, CGP55845 (1 μM). (B) Representative depolarization-activated IBa elicited from a holding potential of −80 mV to a test potential of −10 mV (50 ms duration; 0.1 Hz) from HEK293T cells expressing CaV2.2 channels in the (a) absence and presence of (b) 30 and (c) 300 μM PAM-2 or DM489. (C) Concentration−response relationship for PAM-2 (○) and DM489 (Δ) inhibition of hCaV2.2 channels expressed alone in HEK293T cells (mean ± SEM; n = 4). The calculated IC50 and nH values are summarized in Table 2. prevented receptor dysregulation and upregulated α7 nAChRs in peripheral nerves and spinal cord as well as increased the ̈ density of glial cells in spinal cord and brain areas of naive 8 rats. In conclusion, the PAM-induced α7 nAChR potentiation observed in our experiments could beneficially affect glial signaling, leading to pain control along with a significant neuroprotective effect. Since the repeated administration of α7-PAMs does not change the levels of α7 nAChRs in the brain,29 a mechanism involving PAM-induced up-regulation as part of the anti-neuropathic pain effects elicited by PAM-2 and DM489 can be ruled out. Considering that PAM-2 delays receptor desensitization,30 corresponding to the definition of type II PAMs, the possibility that α7 nAChR desensitization accounts for the anti-neuropathic pain effect of PAM-2 can also be dismissed. ■ CONCLUSIONS The main objective of this study was to determine whether PAM-2 and its structural derivative DM489 have antineuropathic pain activity in mice by using the STZ- and oxaliplatin-induced neuropathic pain models. To define the mechanisms of action of these compounds, their pharmacological activities were determined at α7 and α9α10 nAChRs, as well as CaV2.2 channels alone or coupled with GABABRs by electrophysiological recordings. This study clearly demonstrates that PAM-2 and DM489 reduce drug-induced neuropathic pain in mice and that α7 nAChR potentiation is likely the predominant molecular mechanism underlying this activity. Our results also suggest that PAM-2 and DM489 might be used for the prevention and treatment of chronic neuropathic pain, especially in chemotherapy and diabetes conditions. 3610 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience ■ pubs.acs.org/chemneuro Research Article Co., Waltham, MA). The scale for LogBB ranges from values ≥0.7 (very high brain penetration), 0.0−0.7 (high penetration), and < 0.0 to −0.52 (medium penetration) to ≤ −0.52 (low penetration). The use of this software to estimate drug LogBB values has been validated by the observed correlation between calculated and experimental values.31 Animals. Male CD-1 albino mice (Envigo, Varese, Italy), 2−3 months old (22−25 g), were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their arrival. Ten mice were housed per cage (size 26 × 41 cm2); animals were fed a standard laboratory diet and tap water ad libitum and kept at 23 ± 1 °C with a 12 h light/dark cycle, with light at 7 am. All animal manipulations were carried out according to the Directive 2010/63/EU of the European parliament and of the European Union council (September 22, 2010) on the protection of animals used for scientific purposes. Formal approval to conduct animal experiments was obtained from the Animal Subjects Review Board of the University of Florence, in compliance with the Guide for the Care and Use of Laboratory Animals of the NIH (USA). All efforts were made to minimize animal suffering and to reduce the number of animals used. A randomization of animals between groups and treatments was carried out. The investigators responsible for data analysis were blind to which animals represented treatments and controls. Female X. laevis were sourced from Nasco (Fort Atkinson, WI), and a maximum of three frogs were kept in purpose-built 15 L aquarium at 20−26 °C with a 12 h light/dark cycle. Oocytes were obtained from three frogs (five years old) anaesthetized with 1.7 mg/ mL ethyl 3-aminobenzoate methanesulfonate (pH 7.4 with NaHCO3), and for recovery, post-surgery animals were placed in fresh water at a level below the nostrils. Frogs were allowed to recover for a minimum of four months between surgeries. Terminal anesthesia with 5 mg/mL ethyl 3-aminobenzoate methanesulfonate (pH 7.4 with NaHCO3) was performed on frogs at the sixth surgery. All procedures were approved by the Animal Ethics Committees from University of Wollongong, University of Sydney, and Universidad de Buenos Aires. Treatment of PAM-2 and DM489 on StreptozotocinInduced Neuropathic Pain. To develop neuropathic pain, mice (n = 10/condition) were treated with a single intraperitoneal (ip) injection of 100 mg/kg streptozotocin (STZ) [dissolved in 0.1 M citrate buffer, pH 4.5 (vehicle)] on day 1 after overnight fasting as described previously.17,18 The solution of STZ was prepared immediately before the injection. Control animals received an equivalent volume of vehicle. Three days later, glycemia was measured by blood sampling from the caudal vein and analyzed with the AccuCheck Aviva planar sensor (glucose oxidase method; Roche, Italy). STZ-treated animals with blood glucose levels <270 mg/dL were rejected from the study. Animals were then group-housed with full access to food and water for 2 weeks. To maintain cleanliness and avoid the development of any infection due to excessive urination, animal bedding was frequently changed. On day 15, the pain threshold was evaluated by the cold plate test at 0, 15, 30, 45, and 60 min, revealing significant hypersensitivity of STZ-treated animals. Mice were then per os (p.o.) administrated with PAM-2 (1 and 3 mg/ kg) or DM489 (3 and 10 mg/kg) [each suspended in 1% carboxymethylcellulose (i.e., vehicle)], and the anti-nociceptive activity of these compounds was subsequently evaluated by the cold plate test. Treatment of PAM-2 and DM489 on Oxaliplatin-Induced Neuropathic Pain. To develop neuropathic pain, mice (n = 10/ condition) were administered (ip) with 2.4 mg/kgoxaliplatin [dissolved in 5% glucose solution (vehicle)] on days 1−3, 6−10, and 13−14.3,5 Control animals received an equivalent volume of vehicle. On day 15, the pain threshold was evaluated by the cold plate test at 0, 15, 30, 45, and 60 min, revealing significant hypersensitivity of oxaliplatin-treated animals. Mice were then administrated (p.o.) with PAM-2 (1 and 3 mg/kg) or DM489 (10 and 30 mg/kg) [each suspended in 1% carboxymethyl cellulose (i.e., vehicle)], and the pain threshold was subsequently determined by the cold plate test. METHODS Chemicals. Acetylcholine chloride (ACh), (±)-baclofen, ethyl 3aminobenzoate methanesulfonate and BAPTA-AM were obtained from Sigma-Aldrich (St. Louis, MO), Streptozotocin (STZ), glucose, carboxymethyl cellulose, (E)-3-(furan-2-yl)acrylic acid, 1-hydroxybenzotriazole hydrate (HOBT), p-toluidine, indoline, and methyllycaconitine citrate (MLA) were obtained from Sigma-Aldrich SRL (Milan, Italy). Oxaliplatin was obtained from Carbosynth (Berkshire, UK). CGP55845 hydrochloride was purchased from Tocris Bioscience (Bristol, UK). HAM’s F10 medium, horse serum, and hygromycin B were purchased from Thermo Fisher Scientific (Waltham, MA). Fetal bovine serum (FBS) was obtained from Bovogen (East Keilor, VIC, Australia) and Thermo Fisher Scientific. DMEM, GlutaMAX, penicillin, and streptomycin were purchased from Invitrogen Life Technologies (Carlsbad, CA). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was obtained from Fluorochem (Glossop, UK). Salts and other compounds were of analytical grade. Synthesis of PAM-2 and Its Novel Derivative DM489. For the synthesis of PAM-2 and DM489, a modification of the method described previously was used.15 (E)-3-(Furan-2-yl)acrylic acid (1.0 mmol) was dissolved in anhydrous dichloromethane (5 mL), and the solution was cooled to 0 °C. Then, triethylamine (2.0 mmol) and the proper amine (p-toluidine for PAM-2 or indoline for DM489, 1.0 mmol) was added (see Scheme 1). The mixture was maintained at 0 °C and stirred for 1 h. Then, 1-hydroxybenzotriazole hydrate (HOBt) (2.0 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC HCl) (2.0 mmol) were sequentially added, and the mixture was warmed to room temperature (RT) and stirred for 18 h. The mixture was next treated with dichloromethane (15 mL), and the organic layer was washed twice with 6 N HCl (5 mL). After drying with sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by flash silica gel chromatography [dichloromethane (99.9)/methanol (0.1), v:v], giving each purified compound. Chemical Characterization. The uncorrected melting point of each compound was measured using a SMP3 apparatus (StuartBioCote, Staffordshire, UK). 1H NMR and 13C NMR spectra were recorded using a Bruker NMR 400 AVANCE spectrophotometer (Faellanden, Switzerland). Samples were dissolved in CDCl3 and placed in 5 mm NMR tubes. Measurements were taken using a CDCl3 lock and tetramethylsilane as the internal reference. ESI-MS spectra were obtained using a Varian 1200L triple quadrupole system (Palo Alto, CA), equipped with an electrospray ionization source (ESI). Chromatographic separations were performed on a silica gel column (40, 0.040−0.063 mm; Merck, Darmstadt, Germany) by flash chromatography. Compounds were named following IUPAC rules as applied by ChemBioDraw (version 14.0.0.117) software by PerkinElmer (Waltham, MA). PAM-2 [(E)-3-(Furan-2-yl)-N-(p-tolyl)acrylamide] (Figure 1). 1H NMR (CDCl3, 400 MHz): δ 2.30 (s, 3H, CH3), 6.43 (dd, J = 3.3, 1.8 Hz, 1H), 6.50 (d, J = 19.1 Hz, 1H), 6.51 (s, 1H), 7.10 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 1.3 Hz, 1H), 7.47−7.51 (m, 3H), 7.73 (bs, 1H, NH) ppm. 13C NMR (CDCl3, 400 MHz): δ 20.9, 112.2, 114.2, 118.7, 120.0, 128.7, 129.5, 144.2, 151.3, 164.0 ppm. ESI-LC/MS (C14H13NO2): m/z 228.0 [M + H]+; mp 161−162 °C; yield 76.6%. DM489 [(E)-3-(Furan-2-yl)-1-(indolin-1-yl)prop-2-en-1-one] (Figure 1). 1H NMR (CDCl3, 400 MHz): δ 3.15−3.21 (m, 2H), 4.21 (t, J = 8.3 Hz, 2H), 6.46 (dd, J = 3.3, 1.8 Hz, 1H), 6.58 (d, J = 3.3 Hz, 1H), 6.71−6.77 (m, 1H), 7.00 (t, J = 7.4 Hz, 1H), 7.16−7.25 (m, 2H), 7.46 (d, J = 1.0 Hz, 1H), 7.56 (d, J = 15.1 Hz, 1H), 8.32 (bs, 1H) ppm. 13C NMR (CDCl3, 400 MHz): δ 27.9, 48.1, 112.3, 114.5, 116.6, 117.4, 123.8, 124.5, 127.5, 129.6, 144.2, 151.6, 164.1 ppm. ESILC/MS (C15H13NO2): m/z 240.1 [M + H]+; mp 113−114 °C; yield 87.3%. Lipophilicity and Brain Permeability Properties of PAM-2 and DM489. To predict whether PAM-2 and DM489 cross the blood−brain barrier, the lipophilicity (i.e., LogP) and brain permeability (LogBB = Log[brain]/[plasma]) parameters were calculated using Biovia Discovery Studio software (Dassault Systèmes 3611 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro Coadministration of PAM-2 and Oxaliplatin. To determine whether PAM-2 decreases the development of neuropathic pain induced by oxaliplatin, mice (n = 10/condition) were coadministered with 2.4 mg/kg oxaliplatin and an inactive dose of PAM-2 (1 mg/kg; p.o.) following the same protocol as that for oxaliplatin alone. Pain threshold values corresponding to 7 and 14 days of cotreatment were determined 24 h after the last administration by using the cold plate test. To determine whether an additional administration of PAM-2 increases its efficacy, animals were administered with 1 mg/kg PAM-2 after 30 min of the last cotreatment/cold plate test, and the pain threshold was subsequently assessed. Effect of Methyllycaconitine on the Anti-Neuropathic Pain Activity Mediated by PAM-2 and DM489. To determine whether the anti-neuropathic pain activity elicited by 3 mg/kg PAM-2 or 30 mg/kg DM489 is mediated by α7 nAChRs, mice (n = 10/condition) were administered (ip) with 6 mg/kg methyllycaconitine (MLA), an α7-selective antagonist,24 15 min before the tested drug or vehicle. Pain threshold measurements were performed at 15, 30, 45, and 60 min after treatment by using the cold plate test. Cold Plate Test. The animals were placed in a stainless steel box (12 cm × 20 cm × 10 cm) with a cold plate as the floor. The temperature of the cold plate was kept constant at 4 ± 1 °C. Painrelated behavior (licking of the hind paw) was observed, and the time (s) of the first sign was subsequently recorded (i.e., licking latency). The cutoff time of the latency of paw lifting or licking was set at 60 s.5 Comparative Activity of PAM-2 and DM489 at Human and Rat α7 nAChRs Heterologously Expressed in Xenopus laevis Oocytes. Stage V−VI oocytes (Dumont’s classification; 1200−1300 μm in diameter) were obtained from X. laevis, defolliculated with 1.5 mg/mL collagenase Type II at RT (21−24 °C) for 1−2 h in OR-2 solution containing (in mM) 82.5 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, at pH 7.4. Oocytes were injected with 10 ng of either human (h) α7 or rat (r) α7 cRNAs (concentration confirmed spectrophotometrically and by gel electrophoresis) using glass pipettes (3-000-203 GX, Drummond Scientific Co., Broomall, PA). For the synthesis of α7 nAChR subunit cRNAs, plasmid pMXT construct of hα7 and plasmid pNKS2 construct of rα7 were linearized with BamHI (NEB, Ipswich, MA) and XbaI (NEB), respectively, for in vitro SP6 mMessage mMachine-cRNA transcription (AMBION, Foster City, CA). Both α7 nAChR constructs were sourced from Prof. J. Lindstrom (University of Pennsylvania, Philadelphia, PA). Oocytes were incubated at 18 °C in sterile ND96 solution composed of (in mM) 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4, supplemented with 5% FBS, 0.1 mg/mL gentamicin, and 100 U/mL penicillin−streptomycin. Electrophysiological recordings were carried out 2−7 days post-cRNA microinjection. Twoelectrode voltage clamp recordings of X. laevis oocytes expressing α7 nAChRs were performed at room temperature (RT) using a GeneClamp 500B amplifier and pClamp9 software interface (Molecular Devices, San Jose, CA) at a holding potential of −80 mV. Voltage-recording and current-injecting electrodes were pulled from GC150T-7.5 borosilicate glass (Harvard Apparatus, Holliston, MA) and filled with 3 M KCl, giving resistances of 0.3−1 MΩ. Oocytes expressing α7 nAChRs were perfused with ND96 solution at a rate of 2 mL/min. Initially, oocytes were briefly washed with ND96 solution followed by three applications of ACh at concentrations corresponding to the 20% maximal effective concentration (i.e., EC20) for hα7 (30 μM) and rα7 (160 μM) nAChRs (Figure 7B), respectively, and 3 min washout between ACh applications. The ACh EC20 value used for hα7 was calculated from reported ACh EC50,32 whereas the rα7 ACh EC20 (160 μM) was calculated from the concentration−response curve obtained for this subtype (see Figure 7B). Peak current amplitudes before and after incubation were measured using Clampfit 10.7 software (Molecular Devices), and the relative current amplitude, IACh+compound/IACh (Λ), was used to assess the compound’s activity at nAChRs. Solutions containing the compound were prepared in ND96 + 0.1% FBS. Oocytes were incubated with PAM-2 or DM489 for 5 min with the perfusion system stopped, followed by coapplication of ACh plus the compound with flowing Research Article bath solution. Incubation with 0.1% FBS was performed to ensure that the FBS and the pressure of the perfusion system had no effect on nAChRs. Voltage Clamp Recordings of Oocytes Expressing rα9α10 nAChRs. For the expression studies, rα9 and rα10 subunits were subcloned into a modified pGEMHE vector. Capped cRNAs were in vitro transcribed from linearized plasmid DNA templates using RiboMAX Large Scale RNA Production System (Promega, Madison, WI). The maintenance of X. laevis and the preparation and cRNA injection of stages V and VI oocytes have been described in detail elsewhere.33 Typically, oocytes were injected with 50 nL of RNasefree water containing 0.01−1.0 ng of cRNA (at a 1:1 molar ratio when pairwise combined) and maintained in Barth’s solution at 18 °C. Electrophysiological recordings were performed 2−6 days after cRNA injection under two-electrode voltage clamp with an Oocyte Clamp OC-725B or C amplifier (Warner Instruments Corp., Hamden, CT). Recordings were filtered at a cutoff frequency of 10 Hz using a 900BT Tunable Active Filter (Frequency Devices Inc., Ottawa, IL). Data acquisition was performed using a Patch Panel PP-50 LAB/1 interphase (Warner Instruments Corp., Hamden, CT) at a rate of 10 points/s. Both voltage and current electrodes were filled with 3 M KCl and had resistances of ∼1 MΩ. Data were analyzed using Clampfit from the pClamp 6.1 software. During electrophysiological recordings, oocytes were continuously superfused (∼15 mL/min) with normal frog saline containing (in mM) 115 NaCl, 2.5 KCl, 1.8 CaCl2, and 10 HEPES, pH 7.2. ACh and PAM-2 or DM489 were added to the perfusion solution. Unless otherwise indicated, the membrane potential was held at −70 mV. To minimize endogenous Ca2+-dependent chloride currents, oocytes were incubated with the Ca2+ chelator BAPTA-AM (100 μM) for 3 h before electrophysiological recordings. PAM-2 and DM489 concentration-inhibition curves were performed on ACh-activated α9α10 nAChRs, using a concentration of ACh (i.e., 10 μM) corresponding to its EC50 value. Oocytes were preincubated for 2 min with the compound under study before adding ACh + compound. Current−voltage (I−V) relationships in the absence and presence of 30 μM DM489 were obtained by applying 2 s voltage ramps from −120 to +50 mV, 10 s after the peak response to 10 μM ACh from a holding potential (Vhold) of −70 mV. Leakage correction was performed by subtraction of the I−V curve obtained by the same voltage ramp protocol before the application of ACh. Data were analyzed using Clampfit from the pClamp 6.1 software. Whole-Cell Patch Clamp Recording of HEK293T Cells Expressing CaV2.2 Channels Alone or Coexpressing GABABRs. HEK293T cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, and 1× GlutaMAX at 37 °C in 5% CO2. Cells were plated on 12 mm glass coverslips and transiently transfected using the calcium phosphate method. The tricistronic plasmid cDNA3.1 construct containing human GABABR1 and GABABR2 subunits, and the green fluorescent protein (GFP), was cotransfected with plasmid cDNA3.1 constructs encoding hCaV2.2 α1B, α2δ1, and β3 subunits (all constructs were inhouse generated) at 2 μg each. The plasmid construct of GFP (0.5 μg) was only cotransfected with pCDNA3.1 constructs of CaV2.2 α1B, α2δ1, and β3 subunits for the identification of transfected cells. Whole-cell patch clamp recordings were performed within 24−48 h post-transfection at RT (21−23 °C). Cells were constantly superfused using a gravity flow perfusion system (AutoMate Scientific, Berkeley, CA) with extracellular solution containing (in mM) 110 NaCl, 10 BaCl2, 1 MgCl2, 5 CsCl, 30 TEA-Cl, 10 glucose, and 10 HEPES (pH 7.35 with TEA−OH; ∼310 mOsmol/kg). Fire-polished borosilicate patch pipettes (2−3 MΩ) were filled with intracellular solution containing (in mM) 125 K-gluconate, 5 NaCl, 2 MgCl2, 5 EGTA, and 10 HEPES (pH 7.2 with KOH; ∼290 mOsmol/kg). Inward Ba2+ currents were elicited by a test depolarization to −10 mV (50 ms duration) from a holding potential of −80 mV applied at 0.1 Hz. Solutions containing PAM-2 or DM489 were prepared in the external solution. Statistical Analysis of Data. The concentration−response relationship for PAM-2 and DM489 was analyzed using the Prism 7 3612 https://dx.doi.org/10.1021/acschemneuro.0c00476 ACS Chem. Neurosci. 2020, 11, 3603−3614 ACS Chemical Neuroscience pubs.acs.org/chemneuro software (GraphPad, La Jolla, CA) according to one- and twocomponent (see eq 1) Hill equation: David J. Adams − Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522, Australia; orcid.org/0000-0002-7030-2288 Lorenzo Di Cesare Mannelli − Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA) and, Section of Pharmacology and Toxicology, University of Florence, Florence 50019, Italy Icomp = 100 + ΔEmax × [comp]nH /([comp]nH + EC50 nH) + (Isat − ΔEmax ) × [comp]nH /([comp]nH + IC50 nH) (1) where Icomp is the current in the presence of the compound (expressed as% of control current), ΔEmax is the maximal potentiating effect (compound efficacy; considering control 100%), Isat is the current at saturating compound concentration (expressed as% control), [comp] is the compound concentration, nH is the Hill coefficient, EC50 is the compound concentration to produce 50% potentiation, and IC50 is the compound concentration to produce 50% inhibition. The inhibition of rα9α10 nAChRs determined at different voltage values was compared by paired Student’s t test. Values of P < 0.05 were considered statistically significant. One-way ANOVA analyses and the subsequent Bonferroni post hoc comparison of the behavioral data were assessed by using the Origin 9 software (OriginLab Corp., Northampton, MA). Values of P < 0.05 were considered statistically significant. ■ Research Article Complete contact information is available at: https://pubs.acs.org/10.1021/acschemneuro.0c00476 Author Contributions H.R.A. developed the concept and wrote the overall manuscript. D.M. and M.N.R. synthesized the compounds and wrote the synthesis method. E.L. performed the animal studies. H.S.T., I.M., and A.Y. performed the electrophysiological experiments. A.B.E. and D.J.A. supervised the electrophysiological experiments. H.R.A., E.L., H.-S.T., I.M., A.Y., C.G., and L.D.C.M. performed data analyses. H.-S.T., A.Y., and L.D.C.M. wrote the methods and results of the electrophysiological and animal studies, respectively. H.R.A., C.G., D.J.A., and L.D.C.M. contributed to critical comments on the manuscript and discussion. AUTHOR INFORMATION Corresponding Authors Funding Hugo R. Arias − Department of Pharmacology and Physiology, College of Osteopathic Medicine, Oklahoma State University Center for Health Sciences, Tahlequah, Oklahoma 74464, United States; Phone: (918) 525-6324; Email: hugo.arias@ okstate.edu; Fax: (918) 280-1847 Han-Shen Tae − Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522, Australia; orcid.org/0000-0001-8961-7194; Phone: (61) 2 4221 5426; Email: hstae@uow.edu.au; Fax: (61) 2 4221 8130 This work was supported by grants from OVPR Pilot/Seed Grant (Oklahoma State University Center for Health Sciences) (to H.R.A.), Italian Ministry of Instruction, University and Research (MIUR), and University of Florence, Italy (to C.G.), National Agency for Scientific and Technologic Promotion, Argentina (to A.B.E.), and Australian Research Council (Discovery Project Grant DP150103990 to D.J.A.). Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors would like to thank Prof. J. Lindstrom for the α7 nAChR constructs and Dr. M. Ortells for the calculation of the LogBB values. ■ Authors Carla Ghelardini − Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA) and, Section of Pharmacology and Toxicology, University of Florence, Florence 50019, Italy Elena Lucarini − Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA) and, Section of Pharmacology and Toxicology, University of Florence, Florence 50019, Italy Arsalan Yousuf − Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522, Australia Irina Marcovich − Instituto de Investigaciones en Ingenieriá Genética y Biologiá Molecular “Dr. Héctor N. Torres” ́ (INGEBI), Consejo Nacional de Investigaciones Cientificas y Téchnicas (CONICET), Universidad de Buenos Aires, Bueno Aires C1121, Argentina Dina Manetti − Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Florence 50019, Italy; orcid.org/0000-0002-5881-6550 Maria Novella Romanelli − Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Florence 50019, Italy; orcid.org/0000-0002-5685-3403 Ana Belén Elgoyhen − Instituto de Investigaciones en Ingenieriá Genética y Biologiá Molecular “Dr. Héctor N. Torres” ́ (INGEBI), Consejo Nacional de Investigaciones Cientificas y Téchnicas (CONICET) and Instituto de Farmacologia,́ Facultad de Medicina, Universidad de Buenos Aires, Bueno Aires C1121, Argentina ABBREVIATIONS ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; GABABR, γ-aminobutyric acid type B receptor; CaV2.2, voltage-gated N-type calcium channel; STZ, streptozotocin; RT, room temperature; PAM, positive allosteric modulator; NAM, negative allosteric modulator; PAM-2, (E)-3-furan-2-ylN-p-tolyl-acrylamide; DM489, (E)-3-(furan-2-yl)-1-(indolin-1yl)prop-2-en-1-one; MLA, methyllycaconitine; EC50, ligand concentration that produces half-maximal potentiation; EC20, ligand concentration that produces 20% maximal potentiation; ΔEmax, maximal potentiation compared to control (100%); IC 50, ligand concentration that produces half-maximal inhibition; nH, Hill coefficient; FBS, fetal bovine serum ■ ■ REFERENCES (1) Arias, H. R. 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