marine drugs

Article A Pharmacological Comparison of Two Isomeric Nicotinic Receptor Agonists: The Marine Toxin Isoanatabine and the Alkaloid Anatabine

Hong Xing, Sunil Keshwah, Anne Rouchaud and William R. Kem *

Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL 32610, USA; hong.xing@ufl.edu (H.X.); [email protected] (S.K.); [email protected] (A.R.) * Correspondence: wrkem@ufl.edu; Tel.: +1-352-392-0669

 Received: 10 December 2019; Accepted: 7 February 2020; Published: 11 February 2020 

Abstract: Many organisms possess “secondary” compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other “minor” compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4β2 nicotinic receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand binding experiments revealed similar α4β2 nAChR binding affinities for the isoanatabines, but R-anatabine affinity was twice that of S-anatabine. While the two anatabines and S-isoanatabine were highly efficacious agonists at α7 nAChRs, R-isoanatabine was only a weak partial agonist. The four compounds share an ability to stimulate both α4β2 and α7 nAChRs, a property that may be useful in developing more efficacious drugs to treat neurodegenerative and other medical disorders.

Keywords: acetylcholine; alkaloid; Alzheimer’s disease; ; anabasine; anatabine; inflammation; isoanatabine; nemertine; ; nicotinic acetylcholine receptor; tobacco; toxin

1. Introduction Due to its many effects on the human organism, (S)-nicotine, the most abundant pharmacologically active constituent of the commercial tobacco plant , is one of the most thoroughly investigated natural products. It is also one of the most addictive of drugs and, when administered by smoking, is the major cause of preventable human death. In recent decades, much has been learned about the myriad central nervous system nicotinic acetylcholine receptors (nAChRs) that are activated by nicotine and similar “nicotinoid” compounds [1–5]. It has been demonstrated that nicotine and some other nAChR agonists have pro-cognitive, anxiolytic, analgesic, and anti-inflammatory properties that might be useful in the treatment of certain neurodegenerative, neurodevelopmental, inflammatory, and drug addiction disorders [4–6]. Several drug candidates targeting nAChRs have undergone clinical tests for some of these potential therapeutic indications, but only one, , has reached the clinic [7–10]. The mammalian organism contains more than fifteen different nAChRs, which are expressed in neurons, skeletal muscles, glia, macrophages, keratinocytes, lung epithelia, and immune cells [1].

Mar. Drugs 2020, 18, 106; doi:10.3390/md18020106 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 106 2 of 12

Each nAChR is composed of five homologous, protein subunits, which form a barrel-like membrane protein complex. When this membrane protein is naturally activated by the neurotransmitter ACh, endogenous , or nicotine, its ion channel opens, causing a membrane potential change that may be sufficient to transiently generate a nerve or muscle impulse in an electrically excitable cell or elevate its intracellular calcium concentration. The nAChRs are conveniently classified according to their subunit compositions. Some are homo-oligomeric, containing five copies of a single subunit. Most receptors containing the α7 subunit are homo-oligomeric and their ion channel displays selectivity for calcium ions, which activate intracellular signaling cascades [11]. Hetero-oligomeric nAChRs are more common and include several brain, autonomic nervous system, and neuromuscular nAChRs. The α4β2 receptors are the major heteromeric receptor subtype in the brain and spinal cord. Nicotine has an exceptionally high affinity for β2-containing nAChRs, which mediate many of its physiological and behavioral effects, including tobacco addiction. Recently, other nAChRs have been found to participate in certain brain circuits involved in nicotine and other drug addictions [12]. Although nAChRs have been extensively investigated during the past three decades, once they were cloned and expressed in cell lines, much remains to be learned about their in vivo functions and their potential as therapeutic targets. In the 1980s, it was reported by many laboratories that the membrane concentrations of some nAChRs, especially the α4β2 subtypes, are greatly reduced in cognitive circuits of the brain during the progression of neurodegenerative diseases (Alzheimer’s and Parkinson’s) and are not expressed normally in some other central nervous system disorders [13]. Thus, initial pharmaceutical interest targeting α4β2 or α7 nAChRs focused on behavioral effects of nicotinoids, including enhancement of cognition and the treatment of tobacco addiction. Efforts to design and develop drugs that can selectively stimulate or antagonize these central nervous system receptors are still in progress. In addition, recent investigations demonstrating the involvement of nAChRs in acute (sepsis, pancreatitis, etc.) and chronic (rheumatoid arthritis, multiple sclerosis, etc.) inflammatory diseases have opened a new therapeutic frontier that ultimately may lead to better treatments of these medical conditions [14,15]. Nicotine, anabasine, and anabaseine (Figure1) are potent but unselective agonists at most nAChRs, which limits their therapeutic use. However, many drug candidates, including analogs of nicotine and anabaseine, have been reported to selectively affect a single nAChR subtype, which reduces the possibility of adverse effects. In spite of numerous compounds being developed and the recent appearance of selective allosteric modulators, nAChR-targeted drug design is still an immature field of investigation. It has benefited greatly from the availability of a large number of potent natural products (“molecular probes”) that act upon nAChRs. These include natural agonists such as anatoxin [16], anabaseine [17,18], and [19], as well as competitive antagonists such as Erythrina [20] and Delphinium alkaloids [21]. This paper will describe in vitro studies of two “minor” nicotinoid natural products: (1) isoanatabine, which we isolated from a nemertine (nemertean) worm, Amphiporus angulatus, and (2) anatabine, the most abundant minor alkaloid of the commercial tobacco plant (Figure1). Both compounds are isomers differing only in the position of a single double bond in an otherwise saturated piperidine ring in the “parent” compound anabasine, which is present in wild tobacco [22]. The pharmacological properties of anabasine [23,24] and the related anabaseine [25,26] have been investigated, but not as extensively as those of nicotine. The hoplonemertine A. angulatus contains many pyridine alkaloids. We initially isolated two major compounds, 2,30-bipyridyl and a tetrapyridyl we named nemertelline [27,28]. The worm inhabits the northwestern Atlantic, North American Arctic, and both northern Pacific coasts. It lives under rocks in the lower intertidal and sublittoral zones and preys upon amphipod crustaceans. Its potential predators include crabs and blenny fishes that live under the same rocks. While 2,30-bipyridyl rapidly produces convulsions and paralysis of crustaceans, nemertelline causes a less rapid, sleep-like paralysis in crustaceans. Many (>10) “minor” compounds, including isoanatabine, have been isolated from A. angulatus by high-pressure liquid chromatography and identified by nuclear magnetic resonance and Mar. Drugs 2020, 18, 106 3 of 12

Mar. Drugs 2019, 17, x 3 of 12 mass spectrometric methods (Kem et al., in preparation). The presence of 3-methyl-2,30-bipyridyl and isoanatabine has already been reported [29–31].

Figure 1. Structures of the two alkaloids, tobacco anatabine and nemertine isoanatabine, compared with anabasine and another tetrahydropyridyl ring isomer, anabaseine. Anatabine and anabasine are known to largely occur as (S)-enantiomers; the possible chirality of natural isoanatabine is not yet Figure 1. Structures of the two alkaloids, tobacco anatabine and nemertine isoanatabine, compared known. Both anabaseine and anabasine have been found in marine (nemertine) worms and in ants. with anabasine and another tetrahydropyridyl ring isomer, anabaseine. Anatabine and anabasine are knownOver ato century largely ago,occur anatabineas (S)-enantiomers; was isolated the possible from tobacco chirality leaves, of natural and isoanatabine its structure is reportednot yet by Spath’sknown. laboratory Both anabaseine [32]. It isand the anabasine most abundant have been minor found alkaloid in marine in (nemertine) tobacco, representing worms and in about ants. 4% of the alkaloids present in tobacco leaves; nicotine represents >90% of the alkaloids present [33]. Due to it is relative resistance to metabolism, it has become a useful biomarker of tobacco use [34]. Several This paper will describe in vitro studies of two “minor” nicotinoid natural products: (1) anatabine syntheses and in vivo pharmacological studies have been published, but its in vitro effects isoanatabine, which we isolated from a nemertine (nemertean) worm, Amphiporus angulatus, and (2) on nAChRs have not been published. For that reason, we investigated the effects of anatabine and anatabine, the most abundant minor alkaloid of the commercial tobacco plant (Figure 1). Both isoanatabine on the two major central nervous system (CNS) nAChRs. compounds are isomers differing only in the position of a single double bond in an otherwise saturated2. Results piperidine ring in the “parent” compound anabasine, which is present in wild tobacco [22]. The pharmacological properties of anabasine [23,24] and the related anabaseine [25,26] have been investigated,2.1. Experiments but withnot as Human extensively nAChRs as Expressed those of nicotine. in Xenopus Oocytes TheComplete hoplonemertine concentration–response A. angulatus contains curves for many the stimulation pyridine alkaloids. of human Weα4 βinitially2 nAChRs isolated are shown two major compounds, 2,3′-bipyridyl and a tetrapyridyl we named nemertelline [27,28]. The worm in Figure2. Table1 provides e fficacy (current response, Imax) and potency (EC50, the concentration inhabitsproducing the halfnorth thewestern maximal Atlantic, response North for American the compound) Arctic, and estimates both northern from the Pacific curves coast ins Figure. It lives2; untheseder estimatesrocks in the are lower arranged intertidal to facilitate and sublittoral comparisons zones betweenand preys di uponfferent amphipod enantiomers crustaceans of the same. Its potentialcompound predators (top half) includ ande between crabs and similar blenny enantiomers fishes that of live the diunderfferent the compounds same rocks (bottom. While half). 2,3′- bipyridylThe two isoanatabinesrapidly produces displayed convulsions significantly and paraly greatersis of cr effiustaceanscacies than, nemertelline the two anatabines causes a less (Table rapid,1). sleepR-isoanatabine’s-like paralysis potency in crustaceans was greater. Many than (>1 that0) “minor” of S-isoanatabine compounds, (Student’s including t-test isoanatabine, P = 0.020). have The beenefficacy isolated of S-anatabine from A. angulatus was larger by than high that-pressure of R-anatabine liquid chromatography (P = 0.029). Comparing and identified the S-enantiomers by nuclear magneticof the two resonance compounds, and mass S-isoanatabine’s spectrometric effi methodscacy was (Kem significantly et al., in greaterpreparation than). thatThe ofpresence S-anatabine of 3- methyl-2,3′-bipyridyl and isoanatabine has already been reported [29–31]. (P = 0.0061). Comparing the R-enantiomers of the two compounds, R-isoanatabine’s efficacy was also Over a century ago, anatabine was isolated from tobacco leaves, and its structure reported by greater than that of S-anatabine (P < 0.001). Spath’s laboratory [32]. It is the most abundant minor alkaloid in tobacco, representing about 4% of the alkaloids present in tobacco leaves; nicotine represents >90% of the alkaloids present [33]. Due to it is relative resistance to metabolism, it has become a useful biomarker of tobacco use [34]. Several anatabine syntheses and in vivo pharmacological studies have been published, but its in vitro effects on nAChRs have not been published. For that reason, we investigated the effects of anatabine and isoanatabine on the two major central nervous system (CNS) nAChRs.

2. Results

2.1. Experiments with Human nAChRs Expressed in Xenopus Oocytes Complete concentration–response curves for the stimulation of human α4β2 nAChRs are shown in Figure 2. Table 1 provides efficacy (current response, Imax) and potency (EC50, the concentration

Mar. Drugs 2019, 17, x 4 of 12

producing half the maximal response for the compound) estimates from the curves in Figure 2; these estimates are arranged to facilitate comparisons between different enantiomers of the same compound (top half) and between similar enantiomers of the different compounds (bottom half). The two isoanatabines displayed significantly greater efficacies than the two anatabines (Table 1). R- isoanatabine’s potency was greater than that of S-isoanatabine (Student’s t-test P = 0.020). The efficacy of S-anatabine was larger than that of R-anatabine (P = 0.029). Comparing the S-enantiomers of the Mar. Drugs 2019, 17, x 4 of 12 Mar.two Drugs compounds 2019, 17, x , S-isoanatabine’s efficacy was significantly greater than that of S-anatabine4 of 12(P = Mar. Drugs 2019, 17, x 4 of 12 0.0061). Comparing the R-enantiomers of the two compounds, R-isoanatabine’s efficacy was also Mar.greater Drugs than2020, 18that, 106 of S-anatabine (P < 0.001). 4 of 12 Mar. Drugs 2019, 17, x 120 5 of 12

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Log [Compound],Log [Compound], M M Figure 2. Activation of human α4β2 neuronal nicotinic acetylcholine (nACh) receptors expressed in Figure 2. Activation of human α4β2 neuronal nicotinic acetylcholine (nACh) receptors expressed in Figure Xenopus2.Figure Activation 2.oocytes.Activation of human All peak of α human4 responsesβ2 neuronalα4β2 were neuronal nicotinic normalized nicotinic acetylcholine with acetylcholine respect (nACh) to the (nACh)receptors response receptors expressed of the expressed cells in to 100 in Figure 3. ActivationXenopus of human oocytes. α 7All (right) peak neuronal responses nACh were receptorsnormalized expressed with respect in Xenopus to the responseoocytes. R- of the cells to 100 XenopusμXenopus Moocytes. ACh.oocytes. AllR-Isoanatabine peak All responses peak ( responsesBlue were, ▲ ),normalized wereS-Isoanatabine normalized with respect( withRed, respect to), R-Anatabinetheto response the response (ofGreen the of cells,the ), cells toS-Anatabine 100 to 100 µM μM ACh.▲ R-Isoanatabine (Blue, ▲), S-Isoanatabine (Red, ), R-Anatabine (Green, ), S-Anatabine Isoanatabine (BlueFigure, ), S-Isoanatabine 2. Activation of(Red human, ), αR-Anatabine4β2 neuronal (Green nicotinic, ), -Anatabine (PurplenACh), receptors). expressed in μM ACh.(ACh.Purple R-Isoanatabine R-Isoanatabine, ). SEM bars (Blue (areBlue, ▲included.,),N ),S-Isoanatabine S-Isoanatabine (Red (Red, ,),), R-Anatabine R-Anatabine ( (GreenGreen,, ), S-AnatabineS-Anatabine (Purple, All peak responses(PurpleXenopus were, normalized ).oocytes. SEM bars All with are peak respectincluded. responses to the were response normalized of the cells with to respect 1 mM (toα7) the ACh. response SEM of the cells to 100 (Purple,H ).). SEM SEM bars bars are are included. included. values are included. ▲   TableμM 1.ACh. Comparison R-Isoanatabine of Xenopus (Blue oocyte-expressed, ), S-Isoanatabine human (Red α, 4β),2 RnAChR-Anatabine efficacy (Green (Imax, ) and), S -potencyAnatabine Table 1. Comparison of Xenopus oocyte-expressed human α4β2 nAChR efficacy (Imax) and potency Table(Purple 1. Comparison, ). SEM bars of Xenopusare included.oocyte-expressed human α4β2 nAChR efficacy (Imax) and potency Table 1.(EC Comparison50) estimates of for Xenopus the four oocyte-expressed compounds. These human efficacy α4β2 nAChRand potency efficacy estimates (Imax) and were potency obtained by (EC50) estimates for the four compounds. These efficacy and potency estimates were obtained by Table 2. Comparison(EC50 of) estimatesXenopus oocyte for the human four compounds. α7 nAChR These efficacy effi (Icacymax) and potencypotency estimates(EC50) estimates were obtained by Prism (EC50) estimatesPrism analysis for the of thefour data compounds. used to construct These efficacythe curv esand in potencyFigure 2 nestimates = number were of oocytes obtained tested. by Tables for the four compounds.Prism analysis These of efficacy the data and used potency to construct estimates the werecurv esobtained in Figure by 2 Prism n = number analysis of oocytesof the tested. Tables 1–3analysisTable are organized 1. of Comparison the data to facilitate used of to Xenopus construct comparison oocyte the of curves- expressedthe tw ino Figureenantiomers human2n α4β2= number(above) nAChR of of ea oocytesefficacych isomer tested. (Imax and) andTables the potencysame1–3 Prism analysis1–3 are organized of the data to used facilitate to construct comparison the curv of thees in tw Figureo enantiomers 2 n = number (above) of oocytes of each tested.isomer Tablesand the same data used to constructare(EC organized 50the) e curvesstimates to in facilitate forFigure the 2. comparisonfour n = numbers compounds of of the oocytes. These two enantiomers tested. efficacy SEM and (above)values potency are of estimates eachincluded. isomer were and obtained the same by 1–3 are enantiomersorganized to offacilitate the two comparison isomers (below). of the twSEMo enantiomers values are included. (above) of each isomer and the same enantiomersenantiomersPrism analysis of of the the of two the two dataisomers isomers used (b (below). toelow). construct SEM SEM thevalues values curves are are inincluded. included. Figure 2 n = number of oocytes tested. Tables Compoundenantiomers of the twoI maxisomers (% ACh) (below). SEM valuesP are included.EC50 (µM) P n Compound IMax (%ACh) P EC50 (µM) P N 1Compound–3 are organized to facilitateIMax (%ACh) compar ison of the P two enantiomers EC50 (µM) (above) of each P isomer and Nthe same S-Isoanatabine Compound 81.9 ± 9.1 IMax (%ACh) 0.0007 *** P 52.3 ± EC1350 (µM) 0.61 P 9 N CompoundS-isoanatabineenantiomers ofI Maxthe (%ACh)two 78.7 isomers ± 1.9 (below). P SEM 0.02 values * EC 50are (µM) 1.01included. ± 0.33 P 0.12 N 4 R-Isoanatabine S-isoanatabine 35.4 ± 5.3 78.7 1.9 0.02 * 61.3 ± 1.0112 0.33 0.12 9 4 S-isoanatabineR-isoanatabine 78.7 ± 1.9 102 ±± 5.7 0.02 * 1.01 ± 0.33 0.31 ± 0.09 0.12 4 4 S-AnatabineR-isoanatabine R-isoanatabineCompound 113 ± 20 102I 102Max ± (%ACh) 5.75.7 0.788 P 69.7 ± 0.31 0.3130EC ±50 0.09 (µM) 0.569 P 9 4 4 N R-isoanatabineS-anatabine 102 ± 5.7 43.2 ± 4.3 0.029 * 0.31 ± 0.09 2.65 ± ± 1.4 0.3 4 3 S-anatabineS-isoanatabineS-anatabine 43.2 43.278.7 ± 4.3 4.3± 1.9 0.029 0.0290.02 * * 2.65 2.651.01 ± 1.4± 0.3 3 0.3 0.30.12 3 3 4 R-Anatabine 105 ± 16 ± 51.8 ± 6.5 ± 8 S-anatabineR-anatabineR-anatabine 43.2 ± 25.04.3 25.0 ± 5.25.2 0.029 * 2.65 ± 0.74 0.741.4 ± 0.21 0.3 3 8 8 S-Isoanatabine R-isoanatabine 81.9 ± 9.1 102± ± 5.7 0.193 ŧŧ 52.3 ± 130.31 ± ± 0.09 0.599 9 4 R-anatabineS-isoanatabine 25.0 ± 5.2 78.7 ± 1.9 0.0061 ŧŧ 0.74 ± 0.21 1.01 ± 0.33 0.36 8 4 S-AnatabineS-isoanatabine S-isoanatabineS-anatabine 113 ± 20 78.7 78.743.2 ± 1.9 1.9± 4.3 0.0061 0.00610.029 * 69.7 ± 1.01 1.01302.65 ± 0.33 ± 1. 4 0.36 0.360.3 9 4 4 3 S-anatabine 43.2 ± 4.3 ŧŧ 2.65± ± 1.4 3 S-isoanatabineS-anatabineS-anatabine 78.7 ± 43.21.9 43.2 ± 4.34.3 0.0061 ŧŧ 1.01 ± 0.33 2.65 2.65 ± 1.4 0.36 4 3 3 R-Isoanatabine R-anatabine 35.4 ± 5.3 25.0± ± 5.20.003 ŧŧŧ 61.3 ± 120.74 ± ± 0.21 0.50 9 8 S-anatabineR-isoanatabineR-isoanatabine 43.2 ± 4.3 102 102 ± 5.75.7 <0.001<0.001 ŧŧŧ 2.65 ± 0.310.311.4 ± 0.09 0.099 0.099 3 4 4 R-AnatabineR-isoanatabine S-isoanatabine 105 ± 16 102 78.7 ±± 5.7 ± 1.9 <0.0010.0061 51.8 ŧŧ ± 0.316.51.01 ± 0.09 ± 0.33 0.0990.36 8 4 4 R-isoanatabineR-anatabineR-anatabine 102 ± 5.7 25.0 25.0 ± 5.25.2 <0.001 ŧŧŧ 0.31 ± 0.09 0.74 0.74 ± 0.21 0.099 4 8 8 R-anatabineS-anatabine 25.043.2 ±± 5.2 ± 4.3 0.742.65 ± 0.21 ± 1. 4 8 3 * P value fromR-anatabine t-test** P P value value for fromS-from andt -testt -testR-enantiomers between25.0 between ± 5.2 S- andS- ofand R-enantiomers the R-enantiomers same compound of the sameof 0.74 the (S-/R-Isoanatabine compound± same 0.21 compound (S-/R-Isoanatabine and(S-/R-Isoanatabine S-/R- and S- 8/ R-Anatabine): and S- * P value fromŧ t-test between S- and R-enantiomers of ŧŧŧ the same compound (S-/R-Isoanatabine and S- Anatabine): *** p* << 0.001.0.05.R-isoanatabine ValueValue fromfrom t -testt-testŧ comparing 102comparing ± 5.7 the the same same<0.001 (S- (S- or R-)or enantiomersR-) enantiomers0.31 ± of0.09 di ff oferent different compounds:0.099 p < 40.01, * P value/R-Anatabine): from t-test between * < 0.05. S- ŧ andValue R-enantiomers from t-test comparing of the same the compound same (S- (S-/R-Isoanatabine or R-) enantiomers and of S-different ŧŧ /R-Anatabine):p

Mar. Drugs 2019, 17, x 4 of 12 Mar. Drugs 2019, 17, x 5 of 12

120 120 M μ 100 100 80 80 60 60 40 40 Response) ACh Response) ACh 20 20 0 0 100 of (% Response Peak

Peak Response (% of 1mM ACh 1mM (% of Response Peak -20 -20 -9 -8 -7 -6 -5 -4 -7 -6 -5 -4 -3 Log [Compound], M Log [Compound], M

Figure 3. Activation of human α7 (right) neuronal nACh receptors expressed in Xenopus oocytes. R- Figure 2. Activation of human α4β2 neuronal nicotinic acetylcholine (nACh) receptors expressed in Isoanatabine (Blue, ▲), S-Isoanatabine (Red, ), R-Anatabine (Green, ), S-Anatabine (Purple, ). Xenopus oocytes. All peak responses were normalized with respect to the response of the cells to 100 All peak responses were normalized with respect to the response of the cells to 1 mM (α7) ACh. SEM μM ACh. R-Isoanatabine (Blue, ▲), S-Isoanatabine (Red, ), R-Anatabine (Green, ), S-Anatabine values are included. Mar.(Purple Drugs 2020, ,).18 SEM, 106 bars are included. 5 of 12

Table 2. Comparison of Xenopus oocyte human α7 nAChR efficacy (Imax) and potency (EC50) estimates Table 1. Comparison of Xenopus oocyte-expressed human α4β2 nAChR efficacy (Imax) and potency for the four compounds.Table 2. Comparison These efficacy of Xenopus and potencyoocyte estimates human α 7were nAChR obtained efficacy by (IPrismmax) andanalysis potency of the (EC 50) estimates (EC50) estimates for the four compounds. These efficacy and potency estimates were obtained by data used to constructfor the four the compounds.curves in Figure These 2. n e ffi= numberscacy and potencyof oocytes estimates tested. SEM were values obtained are byincluded. Prism analysis of the Prism analysis of the data used to construct the curves in Figure 2 n = number of oocytes tested. Tables data used to construct the curves in Figure2. n = numbers of oocytes tested. SEM values are included. Compound1–3 are organized toI maxfacilitate (% ACh) comparison ofP the two enantiomersEC50 (µM) (above) of eachP isomer andn the same S-Isoanatabineenantiomers Compound of the two 81.9 isomers I±max 9.1(% (b ACh)elow). 0.0007 SEM values*** P are included. 52.3 EC ± 5013( µM) 0.61 P 9 n R-IsoanatabineS-Isoanatabine 35.4 ± 81.95.3 9.1 0.0007 *** 61.3 52.3± 12 13 0.61 9 9 Compound IMax (%ACh)± P EC50 (µM)± P N S-Anatabine R-Isoanatabine 113 ± 35.420 5.3 0.788 69.7 61.3± 30 12 0.569 9 9 S-isoanatabine 78.7 ±± 1.9 0.02 * 1.01 ± ±0.33 0.12 4 S-Anatabine 113 20 0.788 69.7 30 0.569 9 R-Anatabine 105 ± 16 ± 51.8 ± 6.5± 8 R-isoanatabineR-Anatabine 102 105 ± 5.716 0.31 51.8 ± 0.096.5 8 4 S-IsoanatabineS-anatabine 81.9 43.2± 9.1 ± ± 4.3 0.193 0.029 * 52.3 2.65 ± 13 ±± 1.4 0.599 0.3 9 3 S-Isoanatabine 81.9 9.1 0.193 52.3 13 0.599 9 S-AnatabineR-anatabine 113 25.0± 20 ±± 5.2 69.7 0.74 ± 30± ± 0.21 9 8 S-Anatabine 113 20 ŧŧ 69.7 30 9 R-Isoanatabine 35.4 ± 5.3 ± 0.003 61.3 ± 12± 0.50 9 S-isoanatabineR-Isoanatabine 78.7 35.4 ± 1.95.3 0.00610.003 ŧŧ 1.0161.3 ± 0.3312 0.50 0.36 9 4 ± ± R-AnatabineS-anatabine R-Anatabine 105 43.2± 16 105 ± 4.316 51.8 2.65 51.8± 6.5 ± 1.46.5 8 8 3 ± ± * P value from*R-isoanatabine Pt-test value for from S- and t-test R-enantiomers for S- 102and R-enantiomers± 5.7 of the same <0.001 of the compound same ŧŧŧ compound 0.31(S-/R-Isoanatabine ± (S-0.09/R-Isoanatabine and 0.099 S-/R- and S-/R-Anatabine): 4 Anatabine): ****** pR-anatabinep << 0.001.0.001. ŧ Value from from t-test25.0 t-test comparing ± comparing5.2 the samethe same (S- or R-)(S- enantiomersor 0.74R-) enantiomers± 0.21 of different of compounds:different p < 80.01. ŧŧ compounds:* Pp value< 0.01. from t-test between S- and R-enantiomers of the same compound (S-/R-Isoanatabine and S- Table 3. Isoanatabine and anatabine binding to rat brain α4β2 nAChRs measured by displacement of /R-Anatabine): * < 0.05. ŧ Value from t-test comparing the same (S- or R-) enantiomers of different The two isoanatabines[3H]--specific were more binding. efficacious There wereand the no statisticallytwo anatabines significant were di fflesserences efficacious (P > 0.05) at between compounds: ŧŧ p < 0.01, ŧŧŧ p < 0.001. human α4β2 nAChRsisoanatabine compared and with anatabine S-anabasine or for S-anatabine (Imax = 78%, versus Kem et R-anatabine. al., in preparation). SEM values However, are shown. By all compounds exceptcomparison, R-isoanatabine S- and R-anabasine still had efficacies Ki values at for human rat brain α4βα24 nAChRsβ2 nAChRs that measured were inferior under similar to that of nicotineconditions (Xing et al., were submitted). 1100 and 910 All nM, four respectively compounds [24]. displayed n = total number potencies of replicates. at human α4β2 nAChRs that were greater than that for S-anabasine (EC50 = 4 μM, Kem et al., in preparation). Compound Ki (nM) P Hill Slope P n The two anatabines displayed the highest efficacies at α7 receptors (Figure 3 and Table 2). S-Isoanatabine 108 14 0.13 1.08 0.15 0.40 12 However, the α7 receptorMar. Drugs potencies 2019, 17, x of all four± compounds were rather ±similar, the EC50s ranging 5 of 12 R-Isoanatabine 136 11 0.94 0.05 12 ± ± from 52 to 70 μM. All fourS-Anatabine compounds possessed 249 32 potencies 0.001 that ** were 0.98similar0.09 to that of 0.70 nicotine (EC 12 50 The two isoanatabines w±ere more efficacious and the two± anatabines were less efficacious at = 65 μM, Xing et al., submitted).humanR-Anatabine α4β2 nAChRs compared 119 16 with S-anabasine (Imax = 0.94 78%, Kem0.06 et al., in preparation 12). However, Mar. Drugs 2019, 17, x ± ± 4 of 12 all S-Isoanatabinecompounds except R-isoanatabine 108 14 still 0.005had efficac **ies1.08 at human0.15 α4β2 nAChR 0.59s that we 12re inferior ± ± to thatS-Anatabine of nicotine (Xing et 249 al., submitted32). All four compounds 0.98 0.09 displayed potencies at human 12 α4β2 ± ± nAChRsR-Isoanatabine that were greater 136 than that11 for S-anabasine 0.39 (EC50 0.94 = 4 µM,0.05 Kem et al., 0.99 in preparation) 12 . 120 ± ± R-AnatabineThe two anatabines 119displayed16 the highest efficacies 0.94 at α70.06 receptors (Figure 3 and 12 Table 2). M ± ± However, the α7 receptor potencies of all four compounds were rather similar, the EC50s ranging μ t * P value100 from -test for S- and R-enantiomers of the same compound (S-/R-Isoanatabine and S-/R-Anatabine): ** < 0.01.from 52Value to 70 from µMt.-test All comparingfour compounds the same possessed (S- or R-) potencies enantiomers that of were different similar compounds: to that of nicotinep < 0.01. (EC50 = 65 µM, Xing et al., submitted). 80 120 60 100 40 80

ACh Response) ACh 20 60

0 40

Peak Response (% of 100 of (% Response Peak Response)

-20 20 -9 -8 -7 -6 -5 -4 0

Peak Response (% of ACh 1mM Response Peak Log [Compound], M -20 -7 -6 -5 -4 -3

Log [Compound], M Figure 2. Activation of human α4β2 neuronal nicotinic acetylcholine (nACh) receptors expressed in

Xenopus oocytes.Figure All 3. peakActivation responses of humanwere normalizedα7 (right) with neuronal respect nACh to the receptors response expressed of the cells in toXenopus 100 oocytes. Figure 3. Activation of human α7 (right) neuronal nACh receptors expressed in Xenopus oocytes. R- μM ACh. R-Isoanatabine (Blue, ▲), S-Isoanatabine (Red, ), R-Anatabine (Green, ), S-Anatabine R-IsoanatabineIsoanatabine (Blue (,BlueN),, S-Isoanatabine▲), S-Isoanatabine (Red (Red, , ),),R-Anatabine R-Anatabine (Green (Green, ,), S),-Anatabine S-Anatabine (Purple (Purple, ). , H). (Purple, ).All SEM peak bars responsesAll are peak included. responses were normalized were normalized with with respect respect to to the the response response of the the cells cells to to1 mM 1 mM (α7) ( αACh.7) ACh. SEM SEM values arevalues included. are included. Table 1. Comparison of Xenopus oocyte-expressed human α4β2 nAChR efficacy (Imax) and potency Table 2. Comparison of Xenopus oocyte human α7 nAChR efficacy (Imax) and potency (EC50) estimates (EC50) estimates for the four compounds. These efficacy and potency estimates were obtained by for the four compounds. These efficacy and potency estimates were obtained by Prism analysis of the Prism analysis of the data used to construct the curves in Figure 2 n = number of oocytes tested. Tables data used to construct the curves in Figure 2. n = numbers of oocytes tested. SEM values are included. 1–3 are organized to facilitate comparison of the two enantiomers (above) of each isomer and the same enantiomers of the two Compoundisomers (below). SEMI valuesmax (% ACh) are included. P EC50 (µM) P n S-Isoanatabine 81.9 ± 9.1 0.0007 *** 52.3 ± 13 0.61 9 Compound R-IIsoanatabineMax (%ACh) 35.4 P ± 5.3 EC50 (µM) P61.3 ± 12 N 9 S-isoanatabine S-Anatabine 78.7 ± 1.9 0.02113 * ± 20 1.01 ± 0.330.788 0.1269.7 ± 30 4 0.569 9

R-isoanatabine R-Anatabine 102 ± 5.7 105 ± 16 0.31 ± 0.09 51.8 ± 6.5 4 8 S-Isoanatabine 81.9 ± 9.1 0.193 52.3 ± 13 0.599 9 S-anatabine 43.2 ± 4.3 0.029 * 2.65 ± 1.4 0.3 3 S-Anatabine 113 ± 20 69.7 ± 30 9 R-anatabine R-Isoanatabine 25.0 ± 5.2 35.4 ± 5.3 0.74 ± 0.210.003 ŧŧ 61.3 ± 12 8 0.50 9 S-isoanatabine R-Anatabine 78.7 ± 1.9 0.0061105 ŧŧ± 16 1.01 ± 0.33 0.3651.8 ± 6.5 4 8 S-anatabine 43.2 ± 4.3 2.65 ± 1.4 3 R-isoanatabine 102 ± 5.7 <0.001 ŧŧŧ 0.31 ± 0.09 0.099 4 * P value from t-test for S- and R-enantiomers of the same compound (S-/R-Isoanatabine and S-/R- R-anatabine 25.0 ± 5.2 0.74 ± 0.21 8 Anatabine): *** p < 0.001. ŧ Value from t-test comparing the same (S- or R-) enantiomers of different * P value from t-test betweencompounds: S- andŧŧ p < 0.01.R-enantiomers of the same compound (S-/R-Isoanatabine and S- /R-Anatabine): * < 0.05. ŧ Value from t-test comparing the same (S- or R-) enantiomers of different compounds: ŧŧ p < 0.01, ŧŧŧ p < 0.001.

Mar. Drugs 2020, 18, 106 6 of 12 Mar. Drugs 2019, 17, x 6 of 12

2.2.2.2. Radioligand Radioligand Binding Binding Experiments Experiments with with Rat Rat Brain Brain nAChRs nAChRs WeWe utilized utilized [3H]-cytisine [3H]-cytisine displacement displacement to measureto measure the the relative relative affi affinitiesnities of theof the isoanatabines isoanatabines and and anatabinesanatabines for for rat rat brain brain high-nicotine-a high-nicotineffi-affinitynity receptors, receptors, which which have have been been shown shown to beto >be90% >90% of theof the 3 α4βα4β22 subtype. subtype At. theAt the low low [3H]-cytisine [ H]-cytisine concentration concentration (0.3 nM)(0.3 employednM) employed in these in experiments,these experiments one can, one can be confident that binding to the high-sensitivity α42β23 stoichiometry receptors is being be confident that binding to the high-sensitivity α42β23 stoichiometry receptors is being measured, sincemeasured, the low-sensitivity since the lowα4-βsensitivity2 receptors α4β2 have receptors a much have lower a amuchffinity lower (~50X) affinity for cytisine (~50X) [ 35for]. cytisine [35]. CompoundCompound affi affinitiesnities for forα4 βα4β22 nAChRs, nAChRs, measured measured by by radioligand radioligand binding binding methods, methods, are are shown shown in Figurein Figure4, and4, and the the binding binding constants constants are are presented presented and and compared compared in in Table Table3. 3. The The a affinitiesffinities of of the thefour four compounds compounds were were rather rather similar, similar, being being in in the the 100100–250–250 nM range. The The binding affinitaffinityy of S- S-isoanatabineisoanatabine was was slightly slightlyhigher higher thanthan thatthat ofof thethe S-anatabineS-anatabine (P(P = 0.005).0.005). Although Although t thehe affinities affinities of of the Mar. Drugs 2019, 17, x 4 of 12 two isoanatabines were not statistically different, the R-anatabine Ki was half of the S-anatabine Ki (P the two isoanatabines were not statistically different, the R-anatabine Ki was half of the S-anatabine Ki = 0.001). By comparison, the nicotine Ki for rat brain receptors is approximately 2 nM (Xing et al., (P = 0.001). By comparison, the nicotine Ki for rat brain receptors is approximately 2 nM (Xing et al., 120 submitted).submitted). The The affi affnitiesinities measured measured by by displacement displacement of cytisineof cytisine binding binding were were approximately approximately 100 100× 50 ×

M higher than would be predicted from the EC values of the compounds. This is not surprising since higher than would be predicted from the EC50 values of the compounds. This is not surprising μ 100 sinceother other binding binding studies studies using using cytisine cytisine and and related related radioligands radioligands (nicotine (nicotine and and epibatidine) epibatidine) have have found foundmuch much higher higher affinities affinities than than would would be be inferred from from functional functional estimates. estimates. Radioligand Radioligand binding binding 80 experimentsexperiments measure measure ligand ligand interaction interaction under under steady-state steady-state conditions, conditions, unlike unlike functional functional experiments experiments and,and thus,, thus are, are greatly greatly aff ectedaffected by by high high affi affinitynity for for the the desensitized desensitized state state of theof theα4 βα4β22 receptors. receptors. Thus, Thus, one cannot expect a strict proportionality between EC50 and Ki estimates. 60 one cannot expect a strict proportionality between EC50 and Ki estimates.

40 120 120 S-Isoanatabine S-Anatabine 100 100 ACh Response) ACh R-Isoanatabine R-Anatabine 20 80 80

0 60 60

Peak Response (% of 100 of (% Response Peak %of Control %of Control 40 40 -20 -9 -8 -7 -6 20 -5 -4 20

Log [Compound],0 M 0 -8 -7 -6 -5 -8 -7 -6 -5

Log[ Isoanatabine], M Log [Anatabine], M

Figure 2. Activation of human α4β2 neuronal nicotinic acetylcholine (nACh) receptors expressed in FigureFigure 4. Competition 4. Competition binding binding assay assay of (Sof and(S and R)-Isoanatabines R)-Isoanatabines (left) (left) and and (S and(S and R)-Anatabines R)-Anatabines (right). (right). Xenopus oocytes. All peak responses were normalized with respect to the response3 of the cells to 100 DisplacementDisplacement of [ ofH]-cytisine [3H]-cytisine from from rat brain rat brain membranes. membranes. R-Isoanatabine R-Isoanatabine (Blue, N(Blue), S-Isoanatabine, ▲), S-Isoanatabine (Red, μM ACh. R-Isoanatabine (Blue, ▲), S-Isoanatabine (Red, ),), R-Anatabine R-Anatabine ( (GreenGreen,, ), S-AnatabineS-Anatabine (Purple, ). Each point represents the mean of 12 replicates  (Red, ), R-Anatabine (Green, ), S-AnatabineH (Purple, ). Each point represents the mean of 12 (Purple, ). SEM bars are included. (three experiments, identical concentrations tested in quadruplicate); SEM bars are shown. replicates (three experiments, identical concentrations tested in quadruplicate); SEM bars are shown. 125 Table 1. Comparison of Xenopus oocyte-expressed human Ourα4β2 bindingnAChR efficacy data for(Imax) rat and brain potencyα7 receptors were limited to single [ I]-α-bungarotoxin Our binding data for rat brain α7 receptors were limited to single [125I]-α-bungarotoxin (EC50) estimates for the four compounds. These efficacydisplacement and potency experiments estimates were (ten obtained compound by concentrations, each with four identical samples) for displacement experiments (ten compound concentrations, each with four identical samples) for S- Prism analysis of the data used to construct the curves inS-anatabine Figure 2 n = (Knumberi = 236 of oocytes20 nM) tested. and Tables R-anatabine (Ki = 680 120 nM). anatabine (Ki = 236 ± 20 nM) and R-anatabine (Ki = 680 ±± 120 nM). 1–3 are organized to facilitate comparison of the two enantiomers (above) of each isomer and the same enantiomers of the two isomers (below). SEM values are3. included. Discussion Table 3. Isoanatabine and anatabine binding to rat brain α4β2 nAChRs measured by displacement of 3 Compound IMax (%ACh) P ECInsertion50[ H](µM)-cytisine of the-specific double P binding. bond There N in an were otherwise no statistically piperidine significant ring flattensdifferences this (P part> 0.05) of between the ring, S-isoanatabine 78.7 ± 1.9 0.02 * preventing 1.01isoanatabine ± 0.33 the 3,4 (isoanatabine) and 0.12 anatabine or or 4 for 4,5 S position-anatabine (anatabine) versus R-anatabine. carbons SEM from values taking are a half-chair shown. By or R-isoanatabine 102 ± 5.7 half-boat 0.31comparison, ± conformation.0.09 S- and Our R-anabasine main 4observations Ki values for are rat that brain the α4β2 presence nAChRs of ameasured double bondunder in similar either S-anatabine 43.2 ± 4.3 0.029 * position 2.65conditions increases ± 1.4 were agonist 1 0.3100 potency and 910 nM at 3α , 4respectivelyβ2 nAChRs [24 but]. n decreases= total number potency of replicates. at α7 nAChRs, relative to S-anabasine. Anabasine displays about a 10-fold lower potency than nicotine for α4β2 nAChRs, but an R-anatabine 25.0 ± 5.2 0.74 ± 0.21 Compound 8K i (nM) P Hill Slope P n approximately threefold higher potency at α7 nAChRs (Xing et al., in preparation). Anabasine binds S-isoanatabine 78.7 ± 1.9 0.0061 ŧŧ 1.01 ± 0.33S -Isoanatabine 0.36 4108 ± 14 0.13 1.08 ± 0.15 0.40 12 about fivefold less tightly to rat α4β2 nAChRs than to α7 nAChRs [25]. The larger ring size of anabasine S-anatabine 43.2 ± 4.3 2.65 ± 1.4 R-Isoanatabine 3136 ± 11 0.94 ± 0.05 12 seems less favorable for binding and activation of α4β2 nAChRs [35]. Addition of the double bond R-isoanatabine 102 ± 5.7 <0.001 ŧŧŧ 0.31 ± 0.09 S-Anatabine 0.099 4249 ± 32 0.001 ** 0.98 ± 0.09 0.70 12 appears to partially compensate for the enlargement of the ring. Crystal structures of nicotine bound to R-anatabine 25.0 ± 5.2 0.74 ± 0.21 R-Anatabine 8119 ± 16 0.94 ± 0.06 12 * P value from t-test between S- and R-enantiomers of the same compoundS-Isoanatabine (S-/R-Isoanatabine and108 S- ± 14 0.005 ** 1.08 ± 0.15 0.59 12 /R-Anatabine): * < 0.05. ŧ Value from t-test comparing the same (S- or R-) enantiomers of different compounds: ŧŧ p < 0.01, ŧŧŧ p < 0.001.

Mar. Drugs 2020, 18, 106 7 of 12

Lymnaea acetylcholine binding protein (AChBP, Protein Data Bank IUW6 [36]) and anabaseine bound to Aplysia AChBP (Protein Data Bank 2WNL [37]) indicate that these nicotinoid agonists occupy most of the volume of the binding site. Introduction of the 3,4-double bond in isoanatabine enhances efficacy at α4β2 receptors relative to anabasine, but 4,5-double bond introduction in anatabine reduces its efficacy at these receptors relative to anabasine. Perhaps this is due to the proximity of the 3,4-double bond to the 2-position carbon, to which the pyridyl ring is connected; the lone sp2 hydrogen at the 3-position may improve the relative orientations of the two rings for isoanatabine binding at the α4β2 binding site. Crystal structures of anabasine, isoanatabine, and anatabine bound to nAChRs or to AChBPs could provide a firmer basis for interpreting the pharmacological effects of an added double bond in the two isomeric compounds. The S- and R-enantiomers for each compound did not display large differences in their interactions with the two nAChRs, as was also observed for the anabasine enantiomers [23, and (Kem et al., in preparation)]. An exception was the much lower efficacy of R-isoanatabine at α7 nAChRs (Figure3 and Table2) relative to S-isoanatabine. S-anatabine accounts for approximately 85% of tobacco anatabine [33]. Anabasine also occurs predominantly as the S-form, as does nicotine [33]. Biologically, the presence of a distal double bond in isoanatabines and anatabines provides the organism (nemertine and tobacco plant) with a toxin capable of interacting more potently and efficaciously with α4β2 nAChRs than either anabasine or anabaseine [25]. Thus, the organism probably acquires an enhanced ability to neutralize predators and prey (nemertine) or herbivores (tobacco) by targeting an additional subtype of nAChR more effectively than anabasine or anabaseine. Until now, the in vitro agonist properties of anatabine have not been investigated. However, anatabine has been shown to have anti-inflammatory actions, both in vivo and in vitro [38,39]. Racemic anatabine was marketed as an herbal supplement called Anatabloc by Rock Creek Pharmaceuticals. Several studies supported by this company have shown that anatabine, in addition to its anti-inflammatory actions, has procognitive [40–43], neuro-protective [44], immune disease modulating [45,46], and smoking cessation actions. Anatabine partially substitutes for nicotine in drug discrimination behavioral assays but does not have the addictive potency of nicotine [47,48]. Racemic anatabine released dopamine from striatal slices obtained from adult mice [49] but failed to reduce the threshold for intracranial electrical stimulation, a useful measure of its reinforcing ability [50]. A recent investigation also found that anatabine has anti-depressant effects in mice [51]. Most (if not all) of these studies apparently used synthesized, racemic anatabine. Ours is the first study where both enantiomers were tested separately. While similar in vivo studies have yet to be carried out with an isoanatabine, the similarity of its in vitro properties with those of anatabine suggest that isoanatabine will also have similar in vivo actions. Both isomeric analogs of anabasine—isoanatabine and anatabine—have therapeutic potential and merit further investigation. It was recently reported that agonists that co-stimulate both of the major brain nAChRs have greater efficacy in protecting brain cells from β-amyloid [52]. Similarly, the precognitive effects of varenicline seem to depend on stimulation of both α4β2 and α7 nAChRs [53]. These interesting findings will stimulate further investigations of agonists that have this co-stimulatory ability at a common concentration. Isoanatabine and anatabine are compounds which should be capable of co-stimulating these two receptors, particularly since it has been shown that α7 nAChRs are maximally stimulated at concentrations that are as much as 10 lower than are found to be × effective when measured by peak current measurements such as the ones we used in this study. It will also be important to evaluate the effects of isoanatabine and anatabine on autonomic ganglion and neuromuscular nAChRs, which are potential sites for their adverse effects. Mar. Drugs 2020, 18, 106 8 of 12

4. Materials and Methods

4.1. Isoanatabine and Anatabine Syntheses Isoanatabine synthesis was achieved via a procedure using simple and inexpensive starting materials [31]. The key step of our synthesis was introduction of the pyridyl group via a Bruylants reaction, reacting 1-methyl-2-cyano-3-piperideine [54] with 3-pyridyl magnesium chloride. N-Demethylation of the N-oxide of the N-Me isoanatabine by FeSO4 in MeOH at 10 ◦C (non-classical Polonovski reaction) led to isoanatabine. The 1H and 13C NMR spectra of racemic isoanatabine were identical to those of natural isoanatabine (Kem et al., in preparation]. Racemic anatabine analytical data were also in agreement with published data. Enantio-separation was achieved by chiral-phase HPLC (Chiralcel OJ-H column, 1 cm 25 cm, Daicel Chemical Industries Ltd.; Affiliate: × Chiral Technologies, Inc., West Chester, PA, USA). For anatabine, an initial 10 min development with hexane-2.5% ethanol-0.1% trifluoroacetic acid-0.1% diethylamine was followed by a 40 min linear gradient attaining hexane-10% ethanol-0.1% TFA-0.1% DEA after 40 min. The S-anatabine peak occurred at 43 min and the R-anatabine peak at 47 min. For isoanatabine, an initial 10 min development with 98% (v/v) hexane-2.0% ethanol-0.15% trifluoroacetic acid-0.1% diethylamine was followed by a linear gradient attaining 80% hexane-10% ethanol-0.1% TFA-0.1% DEA after 40 min. The S-isoanatabine peak occurred at 33 min and the R-anatabine peak at 40 min. See Tang et al. [55] for more details regarding chiral separations of nicotine alkaloids with this column.

4.2. Two-Electrode Voltage-Clamp Analysis of Human Brain nAChRs Because the two nAChRs of interest often occur on the cell membranes of the same neurons, we chose to separately investigate the functional effects of the two alkaloids using the frog (Xenopus) oocyte expression system, which has been widely used to investigate the agonistic or antagonistic effects of many compounds acting on nAChRs. It has been especially useful for investigating α7 nAChRs, due to the difficulties that were initially encountered in functional expression of this particular homomeric nAChR in mammalian cells. We measured the peak current response of an oocyte to a given concentration of the compound and expressed its amplitude with respect to the mean response to ACh before and after the compound test concentration. These EC50 values are minimal estimates of α7 receptor potency, as it has been shown that the peak current is not a direct measure of receptor activation by the concentration being tested, since the rate of desensitization of this receptor exceeds the rate of equilibration of the compound with the receptors on the oocyte surface [56]. The actual EC50 values of the compounds may be as much as 10-fold lower than our peak current estimates due to this difference in the method of estimating the α7 receptor responses [56]. However, since we were interested in comparing the relative efficacies and potencies of the four isomeric compounds and their rates of diffusion to the receptors are expected to be almost identical, our peak current measurements should allow quantitative comparisons. Oocytes were harvested from Xenopus laevis frogs obtained from NASCO Scientific Co. (Ft. Atkinson, WI, USA) according to an accepted Kem laboratory Institutional Animal Care Committee (IACUC) approved protocol. We have already described the methods of oocyte injection, perfusion, rapid solution changes, and recording in considerable detail [57]. Oocytes were injected with identical amounts (usually 12 ng) of human α4 and β2 mRNAs for α4β2 nAChR expression, or 20 ng of human α7 mRNAs for α7 nAChR expression. The two-electrode voltage-clamp method was used for recording peak responses (holding Em-50 mV for α4β2 and-60 mV for α7 receptors). For convenience, we only measured peak current responses to various concentrations of the two compounds and normalized the data relative to ACh responses before and after the test pulse; the standard ACh concentrations were 100 µM for α4β2 nAChRs and 1000 µM for α7 nAChRs, since the latter receptor is less sensitive to ACh (Xing et al., submitted). The oocyte was no longer used if the current response to ACh decreased more than 20% during the experiment. Compounds dissolved in Barth’s saline (pH 7.3) were rapidly administered by an AutoMate perfusion system. Compound applications were separated by a 5 min Mar. Drugs 2020, 18, 106 9 of 12 wash period. We utilized GraphPad Prism software to analyze the data, always assuming a single homogeneous receptor population.

4.3. Radioligand Binding [3H]-Cytisine was purchased from New England Nuclear and iodinated α-bungarotoxin [125I]-BTX from Amersham. Unstripped whole male Sprague-Dawley rat brains obtained from Pel-Freez Biologicals (Rogers, AZ, USA) were prepared in Tris binding saline (pH = 7.4) containing 2 mg/mL BSA. [3H]-Cytisine binding experiments were performed according to [57] with several modifications [58]. 3 Rat brain membranes were incubated with [ H]-cytisine at 4 ◦C for 4 h. Then, 200 µg of membrane protein was incubated in a final volume of 500 µL binding saline at 4 ◦C for 4 h, while binding assays with 125 I-bungarotoxin were incubated at 37 ◦C for 3 h to assure that equilibrium was reached. Nonspecific binding was measured in the presence of 1 mM (S)-nicotine hydrogen tartrate salt. Reactions were terminated by vacuum filtrations through Whatman GF/C filters (presoaked in PEI) using a Brandel cell harvester. Bound [3H]-cytisine and 125I-bungarotoxin were measured with liquid scintillation and gamma counters, respectively. Data were analyzed using GraphPad Prism software (San Diego, CA, USA). The data were fit by nonlinear regression analyses to a sigmoidal dose-response with variable slope. Ki values were calculated using the Cheng-Prusoff equation (K i = IC50/(1 + [RL]/Kd RL)) with a Kd RL value for each radioligand that had been previously determined. IC50 is the median inhibition concentration of the experimental compound and [RL] is the concentration of the radioligand.

5. Conclusions Both isoanatabine enantiomers were more potent α4β2 agonists than the two anatabines; R-isoanatabine was the most efficacious and potent agonist. Both anatabine enantiomers and S-isoanatabine were strong partial agonists at the human α7 receptor relative to R-isoanatabine. Further pharmacological analysis of these two isomeric piperideine analogs of anabasine may provide structural explanations for the effects of the added double bond and differences between isoanatabines and anatabines. The ability of these isomeric natural products to co-stimulate the two major brain nAChRs may provide a means of increasing therapeutic efficacy beyond what would be achieved by solely stimulating one of these nAChRs.

Author Contributions: Conceptualization, W.R.K.; methodology,W.R.K., H.X., and A.R.; software, H.X.; validation, H.X. and W.R.K.; formal analysis, H.X. and W.R.K.; investigation, W.R.K., H.X., S.K., and A.R.; resources, W.R.K.; data curation, H.X.; writing—original draft preparation, W.R.K.; writing—review and editing, W.R.K. and H.X.; visualization, H.X.; supervision, W.R.K.; project administration, W.R.K.; funding acquisition, W.R.K. All authors have read and agree to the published version of the manuscript. Funding: This research was funded by a Florida Sea Grant (R/LR-MB-20; PI: WR Kem). Acknowledgments: We thank Jon Lindstrom, Dept. of Neuroscience, University of Pennsylvania, for the human α4 and β2 subunit plasmids, and Ron Lukas, Barrow Research Institute, Phoenix, Arizona, for the human α7 plasmid. Conflicts of Interest: The authors declare no conflict of interest.

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