1 Ivermectin binding sites in human and invertebrate Cys-loop receptors

2 Timothy Lynagh1, Joseph W. Lynch2 3 4 1 Neurosensory Systems Group, Technical University of Darmstadt, Schnittspahnstrasse 3, 64287 5 Darmstadt, Germany 6 2 Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, 7 Brisbane, QLD 4072, Australia 8 9 Corresponding author: Lynch, J.W. ([email protected]). Phone: +617 33466375 10 11

1 12 Abstract

13 Ivermectin is a gold-standard antiparasitic drug that has been used successfully to treat billions of 14 humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had 15 been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular 16 modelling now explain how ivermectin binds to these receptors and reveal why it is selective for 17 invertebrate members of the Cys-loop receptor family. Combining this with emerging genomic 18 information, we are now in a position to predict species sensitivity to ivermectin and better 19 understand the molecular basis of ivermectin resistance. An understanding of the molecular 20 structure of the ivermectin binding site, which is formed at the interface of two adjacent subunits 21 in the transmembrane domain of the receptor, should also aid the development of new lead 22 compounds as both anthelmintics and as therapies for a wide variety of human neurological 23 disorders. 24

25 New insight into a wonder drug

26 Orally- or topically-administered ivermectin is used to eliminate parasitic nematodes and 27 arthropods from animals and humans[1]. It is effective yet well-tolerated due to the potency with 28 which it activates a uniquely invertebrate member of the Cys-loop receptor family of ligand-gated 29 ion channels[2]. Although the activation of Cys-loop receptors by neurotransmitter agonists has 30 been studied in molecular detail for 20 years, the ivermectin interaction with these receptors has 31 received relatively little attention. This interaction requires attention, given the evidence that 32 ivermectin resistance can occur via mutations that affect the ivermectin sensitivity of Cys-loop 33 receptors [3,4]. In the last year, the binding sites of ivermectin in nematode and human Cys-loop 34 receptors have been identified, revealing key molecular determinants of ivermectin sensitivity. 35 Coinciding with these discoveries, a growing number of complete genomes have revealed the 36 Cys-loop receptor complements of species of known ivermectin susceptibility. Combining this 37 knowledge should allow the prediction of ivermectin sensitivity, help understand resistance 38 mechanisms and inform the development of broader spectrum antiparasitic drugs. Recent insights 39 in this area are reviewed here, following a brief introduction concerning the use of ivermectin and 40 the structural basis of Cys-loop receptor function.

2 41

42 Origins of ivermectin

43 Intensive screening of soil-dwelling microbes in the 1970s led to the isolation from the 44 bacterium, Streptomyces avermectinius, of a group of natural macrocyclic lactones with potent 45 nematicidal activity, named avermectins [5]. Saturation of the avermectin 22-23 double bond 46 produced 22,23-dihydroavermectin B1, or ivermectin. Ivermectin consists of a mixture of B1a 47 and B1b components in a 80:20 ratio. These compounds vary in structure only at C25, a moiety 48 that is not involved in binding. As the B1a component is more lethal to nematode, insect and 49 acarid parasites [1], it is conventional to refer to either the mixture or the B1a component as 50 ‘ivermectin’. Much attention has been paid to the use of ivermectin against Onchocerca volvulus, 51 a mosquito-transmitted parasitic nematode that causes river blindness, but its use now extends to 52 other human diseases including lymphatic filariasis, strongyloidiasis, scabies and head lice [1]. 53 Despite its broad antiparasitic spectrum, ivermectin is ineffective against platyhelminths [6,7], 54 including the blood fluke Schistosoma mansoni which causes bilharzia [8]. 55

56 Ivermectin targets Cys-loop receptors

57 Early investigations into the lethal effect of ivermectin on arthropods and nematodes showed that 58 ivermectin increased chloride conductance in neurons and thus decreased responses of neurons to 59 excitatory input [9,10]. The search for the identity of this conductance led to the cloning of two 60 novel subunits from the free-living nematode Caenorhabditis elegans that formed a recombinant 61 glutamate-gated chloride channel (GluClR) that was activated irreversibly by nanomolar 62 ivermectin concentrations [2]. Numerous GluClR subunits have since been cloned in various 63 ivermectin-susceptible invertebrates [11,12]. The identification of GluClRs as the target of 64 ivermectin was confirmed by their high ivermectin sensitivity when expressed recombinantly, 65 their expression in tissues sensitive to low doses of ivermectin, and the development of 66 ivermectin resistance upon their mutation [3,4,12-15]. GluClRs belong to the Cys-loop receptor 67 family of ligand-gated channels, which includes human excitatory nicotinic acetylcholine and

68 type-3 5-hydroxytryptamine receptors (nAChRs and 5-HT3Rs) and inhibitory GABA type-A and

69 glycine receptors (GABAARs and GlyRs).

3 70 The Cys-loop receptor family is large, with 102 subunits in C. elegans and 45 in humans [16,17]. 71 Numerous inhibitory receptors in this family are activated by ivermectin, including arthropod 72 histamine-gated chloride channels (HisCls) and pH-gated chloride channels (pHCls) and, perhaps

73 unexpectedly, human GABAARs and GlyRs. A deciding factor in the combined lethality in 74 nematode/arthropod parasites and safety in mammalian hosts is the differential potency with

75 which ivermectin activates these receptors. α GluClRs are typically activated by low (< 20)

76 nanomolar concentrations [2,14,18,19], whereas GABAARs and GlyRs require concentrations in 77 the low (1-10) micromolar range [20-23]. HisCls and pHCls respond to micromolar ivermectin, 78 but the dose dependency has not been established [24-26]. At several other receptors, ivermectin 79 activates no current but modulates (inhibits or potentiates) the responses to the neurotransmitter

80 agonist. These include some invertebrate GABAARs and the human α7 nAChR (below)..

81 The sensitivity of human Cys-loop receptors to micromolar ivermectin, along with the 82 preliminary findings of anti-spasticity efficacy in humans [28], begs the question as to what 83 concentration reaches in the brain. Therapeutic doses in mice, dogs and humans produce plasma 84 levels of 5-10 nM that persist for days, with ~2 nM in the brain [29-31], which is insufficient to

85 activate GABAARs and GlyRs. When α GluClRs are exogenously expressed in the mouse brain,

86 animals respond to systemic ivermectin [32], indicating that ivermectin safety rests on the 87 reduced sensitivity of mammalian Cys-loop receptors. P-glycoprotein, which transports 88 avermectins out of the nervous system, is also crucial to ivermectin potency in mammals and 89 nematodes, evidenced by the increased ivermectin toxicity that results upon its mutation [33,34]. 90

91 Structure-activity relationships in Cys-loop receptors and ivermectin

92 Before describing in detail the structural features that determine ivermectin sensitivity, we briefly 93 outline the structural basis of function in Cys-loop receptors and the determinants of efficacy 94 within the ivermectin molecule. Functional Cys-loop receptor pentamers are formed by five of 95 the same or, as is usually the case for native receptors, two to three different subunits (Figure 96 1a,b). A single subunit consists of: a large extracellular N-terminal domain (ECD) and four 97 membrane-bound helices (M1-M4) that constitute the transmembrane domain (TMD). Two 98 highly conserved ECD Cys residues are crosslinked within a functional receptor, forming the

4 99 eponymous Cys-loop, and the apposition of five M2 helices forms the central channel pore 100 (Figure 1b,c). Neurotransmitter ligands bind at the extracellular interface of two adjacent subunits 101 (red segments in Figure 1b), resulting in the closure of binding domain loop C around the agonist. 102 This triggers structural rearrangements at the ECD-TMD interface (purple segments in Figure 103 1b), and ultimately the opening of the channel gate (Figure 1c,d), allowing ions to flow through 104 the channel [35]. This chemoelectric signalling mechanism underlies most rapid synaptic 105 transmission in the brain. 106 The structural basis of activity of avermectins and milbemycins (Streptomyces spp-derived 107 compounds with similar structures and antiparasitic spectra to avermectins) has been extensively 108 reviewed[36]. Briefly, all compounds share a macrocyclic backbone and differ at the 109 disaccharide, spiroketal and benzofuran moieties, respectively located at the three “corners” of 110 the molecule (Figure 2). Evidence indicates that the benzofuran is the most important potency 111 determinant at GluClRs. For example, substitution of the hydroxyl at the C5 position on the 112 benzofuran ring ("C5-OH") for oxime or ketone in several avermectins reduces nematicidal 113 activity 100- and 10000-fold, respectively [37], implying that either an H-bond donating or 114 generally hydrophilic C5-substituent is required for high potency. In contrast, changes to the 115 spiroketal group have little effect on nematicidal potency [36,37]. Similarly, hydrogenation of 116 double bonds C10-C11 (in the macrocyclic backbone) and 22-23 (in the spiroketal) does not 117 reduce acaricidal potency, whereas hydrogenation of double bonds C3-C4 and C8-C9 (in or

118 adjacent to the benzofuran) reduces both acaricidal potency and activity at mouse GABAARs 119 [38,39]. The sugar moiety is not essential for nematicidal potency [36,37]. However, given that 120 epimerisation of the dissacharide decreases activity at GluClRs [40,41], polar C13 substitutions 121 yield less biologically active avermectins than lipophillic C13 substitutions [42], and size of the 122 sugar moiety correlates with affinity for P-glycoprotein [43], it is evident that the sugar may 123 modulate avermectin potency in vivo. 124

125 The binding site in anionic Cys-loop receptors

126 Hibbs and Gouaux recently solved the crystal structure of the C. elegans α GluClR complexed

127 with ivermectin [44]. This gives a clear picture of the ivermectin binding site and sheds light on 128 previous functional studies. Furthermore, with over 30 % amino acid sequence identity with the

5 129 α1 GlyR, it provides a high resolution template for the modelling of vertebrate GABAARs and

130 GlyRs, which had previously relied on nAChR and prokaryotic receptor structures with much 131 lower homologies [45-47]. Their structure shows ivermectin binding between the M3 of one 132 subunit (the principal or (+) face) and the M1 of an adjacent subunit (the complementary or (-) 133 face), with the benzofuran moiety contacting the M2 helix of the (+) subunit (Figure 3a). The 134 structure implies the existence of three H-bonds: (i) between the benzofuran C5-OH and the

135 hydroxyl sidechain of (+)M2-Ser15' (Ser260 in the α GluClR), (ii) between a spiroketal oxygen

136 and the hydroxyl sidechain of (+)M3-Thr285, and (iii) between the benzofuran C7-OH and the 137 backbone carbonyl oxygen of (-)M1-Leu218 (Figure 3b). Leu218, Ser260 and Thr285 are 138 highlighted in yellow in Figure 3c, as are residues at the equivalent positions in various other 139 Cys-loop receptors. Twelve other sidechains, highlighted in orange or red in Figure 3c, contribute 140 potential Van der Waals interactions. One of these is (+)M3-Gly281, a position in anionic Cys- 141 loop receptors we will henceforth refer to as "M3-Gly". M3-Gly is entirely conserved in GluClRs

142 that show nanomolar ivermectin sensitivity (Figure 3c). In the crystal structure, the α carbon of

143 M3-Gly is approximately 4.5 Å from each corner of the ivermectin macrocycle, such that any 144 larger sidechain at this position - that is, any amino acid other than Gly - would hinder the access 145 of ivermectin to its binding site or alter the binding conformation of ivermectin (Figure 3b).

146 Hence, in the Haemonchus contortus α3B GluClR, the mutation of M3-Gly to Ala (the human α1

147 GlyR equivalent) shifts the ivermectin EC50 for activation from 39 nM up to 1.2 µM - exactly that

148 of the human α1 GlyR [48]. Indeed, the α3B GluClR and the α1 GlyR can be tuned to

149 remarkably similar ivermectin sensitivities by introducing a common residue at the M3-Gly 150 position [48]. Consistent with this, the Gly323Asp mutation (at the M3-Gly position) in the 151 Tetranychus urticae (two-spotted spider mite) GluClR was shown to increase by 18-fold the 152 avermectin dose required for half-maximal lethality [4]. Indeed, the equivalent Gly-Asp

153 substitution abolished binding of a tritiated milbemycin to the α3B GluClR [49]. We have not

154 found in the literature any example of a highly ivermectin-sensitive receptor without a Gly at the 155 M3-Gly position.

6 156 The binding site at the α1 GlyR has also been investigated using mutagenesis and molecular

157 modelling. Ivermectin was modeled as wedging into the same TMD interface, with the 158 benzofuran oriented towards the (+)M2 [50]. Surprisingly, the docking of ivermectin to the

159 Ala288Gly α1 GlyR (this is the M3-Gly position) did not differ greatly from the unmutated

160 receptor, suggesting that the on-rate of ivermectin, rather than the ultimate binding conformation 161 of ivermectin, is changed by this substitution [50]. Alternately, the glycine substitution may 162 increase the gating equilibrium constant, thus facilitating receptor activation by ivermectin. Bulky 163 substitutions at only two positions decreased both agonist and modulatory effects of ivermectin, 164 indicative of a decisive role in binding. These positions are (+)Ala288 and (-)Pro230. Both of 165 these positions are highlighted in red in Figure 3c, revealing the apparent requirement of a small 166 residue at the M3-Gly position and the conservation of the "M1-Pro". M1-Pro is in close 167 proximity to the crucial benzofuran moiety of ivermectin (Figure 3b), providing a possible steric 168 explanation for decreased binding at this mutant. In addition, this highly conserved residue 169 disrupts M1 helical structure thereby exposing a backbone carbonyl that is thought to form a H- 170 bond with the ivermectin C7-OH [44]. However, the existence of this bond requires confirmation, 171 given that C7-OH is equidistant from both the M1 backbone carbonyl and the endogenous 172 ivermectin C1 carbonyl. 173 A significant role for the third putative H-bond (between O14 and T285 – see Figure 3b) can be

174 eliminated on the basis of site-directed mutagenesis experiments in the Ala288Gly α1 GlyR, and

175 the fact that the three GluClRs with nanomolar ivermectin affinity do not contain H-bonding

176 sidechains at the corresponding M3 position [50]. Moving deeper into the pore, substitution of α1

177 GlyR (+)M3-Leu291, (+)M2-Thr264 or (-)M1-Leu233 for bulky Trp sidechains does not reduce 178 ivermectin sensitivity but converts ivermectin from an agonist to an antagonist of glycine- 179 activated currents [50]. Still further removed from the binding site, mutations in the conserved 180 Cys-loop, the pre-M1 domain and the M2-M3 linker (Figure 1b) decrease ivermectin potency 181 most likely by impairing its gating efficacy [14,18,49-51]. 182 Native Cys-loop receptors are often heteromeric and thus do not necessarily possess the five 183 binding sites available to ivermectin in homomers. The following evidence at the GlyR, however, 184 suggests that the binding of two molecules of ivermectin is sufficient for either direct activation

7 185 or potentiation. First, ivermectin activates α1β heteromeric and α1 homomeric GlyRs with equal

186 potency [21], even though heteromers possess a β-α1-β-α1-β arrangement [52] and ivermectin

187 only binds to the (+)α1/(-)β interface [48]. Second, the β-Ile312Gly substitution (at the M3-Gly

188 position) confers sensitivity to potentiation on the otherwise ivermectin-insensitive α1-

189 Ala288Phe/β heteromer, presumably by introducing a (+)β/(-)α1 ivermectin binding site [48].

190

191 M2 H-bonds

192 The putative H-bond between the ivermectin C5-OH and the M2-15' Ser of the GluClR and GlyR 193 seems a logical ivermectin binding interaction, as (i) it links the ivermectin molecule to the pore- 194 lining M2 helix[44] and (ii) a H-bond donor at the C5-OH is crucial to avermectin nematicidal 195 potency (above). However, because various GluClRs with nanomolar ivermectin sensitivities 196 contain either an Ala or Ile at this position (Figure 3c), it is evident this H-bond is not required for 197 high ivermectin potency [4,18,53]. In support of this, the mutant M2-Ser15'Ile substitution in the

198 α1 GlyR causes no change to the agonist or modulatory effects of ivermectin [50]. However,

199 most anionic Cys-loop receptor subunits possess several nearby residues that could act as 200 potential H-bond acceptors in the absence of one at the M2-15' position. These include (+)M2- 201 12', (-)M2-14', (+)M2-19' and also an (-)M1 position (Figure 4). The proximity of these

202 sidechains to the ivermectin molecule is suggested by site-directed mutagenesis data from the α1

203 GlyR: the Thr12'Trp receptor is not activated by ivermectin, the Gln226Cys receptor has 204 decreased ivermectin sensitivity and the Gln266Trp receptor has increased sensitivity [50]. The 205 conservation of polarity in this domain of ivermectin-sensitive receptors is illustrated by the 206 boxed residues in Figure 3c. 207

208 A clear pattern?

209 We now group the above ivermectin sensitivity determinants together and consider their chemical 210 properties in several receptors with known high ivermectin sensitivity. Dividing M2-12', M2-14', 211 M2-15', M2-19' and the M1-Gln residue into their (+) and (-) components gives a "(+)Thr-Ser-

212 Arg/(-)Gln-Gln" arrangement, in the case of the α1 GlyR (Figure 4). The following five 8 213 homomeric Cys-loop receptors are particularly sensitive to ivermectin activation (i.e., activated

214 by 1-20 nM ivermectin): Ala288Gly α1 GlyRs, C. elegans α, H. contortus α and α3B and

215 Drosophila α GluClRs. Respectively, these subunits have (+)Thr-Ser-Arg/(-)Gln-Gln, (+)Thr-

216 Ser-Asp/(-)Gln-Gln, (+)Thr-Ala-Asn/(-)Gln-Gln, (+)Thr-Ala-Asn/(-)Gln-Gln and (+)Ala-Thr- 217 Asn/(-)Gln-Gln arrangements, and thus present an overwhelmingly polar environment to the

218 ivermectin C5-OH moiety. Given that the α1 GlyR possesses a similar polarity in this region to

219 GluClRs, we infer that its large M3-Gly sidechain is solely responsible for its reduced ivermectin

220 sensitivity. UNC-49B/UNC-49C heteromeric GABAARs possess an M3-Gly at the (+) faces of 221 UNC-49C, yet are not activated by ivermectin [54,55]. However, with a (+)Thr-Met-Asn/(-)Gln- 222 Leu arrangement, they are distinct from the GluClRs and GlyRs in that both the M2-14' and M2- 223 15' positions are occupied by larger hydrophobic residues (Figure 3c), perhaps limiting H-bonds 224 with and channel activation by ivermectin [44]. Similarly, the Dermacentor variabilis (American 225 dog tick) homomeric RDL receptor possesses an M3-Gly but is not sensitive to ivermectin 226 (Figure 3), and its M2-14' and M2-15' positions are occupied by hydrophobic residues [56]. By 227 contrast, MOD-1 is insensitive to ivermectin [57], despite its ostensibly suitable (+)Thr-Ser- 228 Arg/(-)Gln-Ile arrangement; MOD-1 insensitivity is likely due to a Val residue at the M3-Gly

229 position preventing access to the site [50]. Curiously, although mammalian GABAAR π subunits

230 possess an M3-Gly, incorporation of the π subunit actually decreased the ivermectin sensitivity of

231 α1β2 GABAARs [48]. Again we suggest that the ivermectin-insensitivity results from

232 hydrophobic residues at both M2-14' and M2-15' positions in the π subunit [58]. Finally,

233 functional homomeric β GluClRs from C. elegans and C. onchophora are also completely

234 insensitive to ivermectin [59], and studies with radiolabelled ivermectin show that it does not

235 even bind to the H. contortus β GluClR [60]. These receptors contain a (+)Thr-Gln-Asn/(-)Gln-

236 Met arrangement and an M3-Gly. We thus conclude that the (-)M1-Met is responsible for 237 disrupting ivermectin sensitivity. In the above analysis, we have used the GluClR crystal 238 structure to infer that the five polar residues are essential for high ivermectin affinity. However, it 239 is also possible that the overwhelmingly polar environment facilitates receptor gating by 240 ivermectin.

9 241 An alternate possibility in GABAARs is that ivermectin binds to a different site. For example, 242 GAB-1/HG1A heteromers and UNC-49B homomers possess Val, Leu or Asn residues at the M3- 243 Gly position, and these receptors are potentiated but not activated by ivermectin [54,55,61], 244 perhaps reflecting the exclusion of ivermectin from the interfacial site and the existence of an 245 alternate site. Moreover, HG1A and HG1E differ by only four amino acids - in the ECD and in 246 M4 - and, whereas GAB1/HG1A heteromers are potentiated by ivermectin, GAB1/HG1E 247 heteromers are inhibited [61]. An alternative site, consistent with HG1A/E structural differences,

248 might be similar to that proposed for the mammalian α7 nAChR (see below). The possibility of

249 distinct ivermectin binding sites in various GABAARs, together with the large diversity in

250 GABAAR subunits, might underlie the differential effects of ivermectin on various native

251 GABAAR preparations [62]. 252

253 An alternative site for potentiation of nAChRs

254 Due to the differing M2-M3 domain lengths in anionic and cationic Cys-loop receptors, it is

255 difficult to determine which amino acid occupies the M3-Gly position in the mammalian α7

256 nAChR [63]. Most alignments suggest that Ala275 in the α7 nAChR and Leu279 in the Torpedo

257 marmorata (marbled electric ray) nAChR α subunit occupy the M3-Gly position. Although the 4

258 Å resolution structure of the T. marmorata nAChR places this Leu279 in the (+)M3 helix, it faces 259 the outside of the membrane, 1-2 helical turns above the level of (-)M1Pro (Figure 5a). In the T. 260 marmorata nAChR structure, the (+)M3 residue physically opposite (-)M1-Pro is either Met282,

261 Ile283 or Ile286, which equate with little ambiguity to Met278, Ile279 and Gly282 in the α7

262 nAChR (Figure 5a,b). Thus, the α7 nAchR structural equivalent to the M3-Gly might Gly282,

263 and the selectivity of ivermectin for α7 nAChRs over insensitive cationic Cys-loop receptors may

264 be due to Val, Ile and Thr sidechains at this position (e.g., in nematode ACR-16 receptors and

265 mammalian 5-HT3Rs [64,65]). Accordingly, the lack of H-bond propensity at M2-14', M2-15' and

266 M2-19' positions in the α7 nAChR might limit the direct agonist efficacy of ivermectin.

267 However, if the structural equivalent in α7 nAChR is Met278 or Ile279, access of ivermectin to

268 the interfacial site is likely precluded, and ivermectin might bind to a distinct site.

10 269 Computer docking simulations from two separate laboratories have shown that the lowest energy

270 docking of ivermectin to the α7 nAChR was in an intrasubunit cavity, as opposed to the

271 interfacial site in anionic receptors [64,66]. In both studies, the ivermectin molecule wedges 272 between M1 and M4 from the same subunit, partly in contact with the surrounding lipids, with

273 the benzofuran moiety oriented towards either M2 or M3 of the same subunit. Substitution of α7

274 nAChR TMD residues for the non-conserved equivalents from the 5-HT3AR resulted in decreased 275 potentation by ivermectin or conversion of potentiation to inhibition [64]. Three of the four 276 mutations that simply decrease potentiation line a space between M1 and M4 of the one subunit, 277 albeit two helical turns below the docked ivermectin molecule. Consistent with putative existence

278 of this site in the α7 nAChR and not in GluClRs and GlyRs, the latter possess large sidechains

279 that might obscure this cavity (Figure 5). Finally, the effects of ivermectin at nAChRs are 280 reversible and are decreased by ECD-acting competitive antagonists [67], which differs from 281 results at most anionic receptors and supports a different site or mechanism of ivermectin at 282 nAChRs. 283

284 Structural bases of ivermectin resistance

285 There are several reports that ivermectin resistance occurs nematodes in natural settings [1], in 286 some cases due to mutations in the coding sequences or changes in expression levels of GluClRs

287 or GABAARs [3,68,69]. In laboratory-induced mutant strains, single mutations in GluClRs have 288 been clearly shown to cause avermectin resistance. For example, mutation of the M3-Gly position

289 in the T. urticae GluClR causes an 18-fold increase in abamectin LD50 [4]. (A C22-C23 double 290 bond in the spiroketal of abamectin is the only difference from ivermectin.) It is also possible that 291 ivermectin insensitivity may be caused by mutations that allosterically disrupt the efficacy with 292 which ivermectin activates the receptors, without necessarily affecting ivermectin affinity. This 293 was first suggested by an M2-M3 linker mutation in a Drosophila GluClR that caused a four-fold

294 increase in ivermectin LD100 [14]. Disruptions to the efficacy of GluClR gating by ivermectin 295 most likely account for the effects of mutations identified in several resistant H. contortus strains 296 [18,49]. Of course, if a GluClR mutation converts ivermectin to an inhibitor, as demonstrated at 297 recombinant GlyRs and nAChRs (above), the usual CNS depression elicited by ivermectin could

11 298 be converted to excitation. This may underlie the resistance seen in nematodes with an up-

299 regulation of HG1E GABAAR subunits that are inhibited by ivermectin [61,68]. 300

301 Predicting species susceptibility to ivermectin

302 The discovery of molecular determinants of ivermectin sensitivity in Cys-loop receptors provides 303 an opportunity to investigate parasites of known ivermectin susceptibility to establish the 304 molecular basis for this susceptibility. The complete genome of the trematode S. mansoni reveals 305 13 Cys-loop receptor subunits [70], much fewer than the 102 found in C. elegans and 45 in 306 humans [16,17]. It is therefore likely that ivermectin has fewer potential Cys-loop receptor targets 307 in S. mansoni than in C .elegans which might help explain why S. mansoni is insensitive to 308 ivermectin. On the other hand, arthropods also possess few GluClRs but are ivermectin- 309 susceptible, suggesting that there must be some feature of S. mansoni GluClRs (other than a small 310 number in the genome) that renders them insensitive to ivermectin. An amino acid sequence 311 alignment of recently available transcripts in several trematodes suggests that they indeed possess 312 GluClRs (Figure 6). Although their functional properties remain to be investigated, these

313 transcripts possess ~30 % homology with α GluClRs and α1 GlyRs, suggesting a functional

314 relationship. Furthermore, they possess a Gly residue on the (+) face of the ECD neurotransmitter 315 binding domain (Figure 6), at a position where small residues are correlated with Glu binding and 316 larger residues are correlated with Gly, GABA or His binding[71]. These putative GluClRs 317 include largely polar (+)Ile-Ser-Ala/(-)Ser-Gln arrangements (as per the scheme above), but large 318 residues at the M3-Gly position undoubtably preclude ivermectin binding (Figure 6). Thus, large 319 sidechains at the M3-Gly position, as opposed to a lack of GluClRs, may well underlie the 320 ivermectin-insensitivity of trematodes. The free-living trematode Schmidtea mediterranea also 321 possesses GluClR-like transcripts with large sidechains at the M3-Gly position (Figure 6). The 322 recent sequencing of Ascaris suum (pig roundworm), Anopheles gambiae (mosquito) and 323 Daphnia pulex (water flea), by contrast, reveal one or more M3-Gly GluClR transcripts, as 324 expected for ivermectin-susceptible phyla [72-74]. This reiterates the importance of the M3-Gly 325 position. Of course, species susceptibility also depends on if and where the relevant subunits are 326 expressed, and the functionality with which an M3-Gly subunit heteromerises with other 327 subunits.

12 328

329 Concluding remarks

330 Ivermectin binds to nematode GluClRs and mammalian GlyRs at the interface of adjacent (+) and 331 (-) subunits in the TMD of the receptor. High affinity for this site is controlled by an M3 Gly 332 residue on the (+)M3 helix that allows rapid access to the site. The C5 hydroxyl of the ivermectin 333 benzofuran likely forms an H-bond within the subunit interface cleft, leading to potent channel 334 activation. Potent activity requires polar sidechains at either M2-14', M2-15' or M2-19' (and 335 perhaps other) positions. Scanning Cys-loop receptor amino acid sequences for these 336 determinants predicts ivermectin sensitivity reasonably well, and scanning genomes for these 337 transcripts may enable the prediction of species susceptibility to ivermectin. However, several 338 questions are yet to be answered, including the structural basis for modulation (not activation) by

339 ivermectin of certain GABAARs and nAChRs, and the inability of ivermectin to activate β

340 GluClRs. Attempts to answer these questions will be aided by the structural template provided by 341 the GluClR crystal structure. Given the emergence of ivermectin resistance in parasitic nematode 342 pests [75,76], the information presented here may help in the design of novel anthelmintics 343 targeting GluClRs or other nematode Cys-loop receptors. Similarly, because ivermectin binding 344 sites also exist in human Cys-loop receptors, understanding the ivermectin binding site structure 345 may help identify new therapeutic pharmacophores for a wide variety of human neurological 346 disorders. 347

13 348 Figure legends

349 Figure 1. Molecular architecture of Cys-loop receptors. (a) The C.elegans α GluClR crystal

350 structure, viewed from the extracellular space (top panel) and from within the lipid membrane 351 (lower panel) [44]. (b) Single subunit from (a). Red indicates segments that contribute to the 352 neurotransmitter binding domain, which is formed between the (+) face of one subunit and the (-) 353 face of an adjacent subunit; magenta indicates Loop 2, the conserved Cys-loop, the pre-M1 354 domain, and the M2-M3 linker, which couple neurotransmitter binding to channel movement; 355 helices M1-M4 are indicated; M2-Leu9' sidechains that contribute to the channel gate are shown 356 as blue spheres [35]. (c) Crystal structure of the closed bacterial channel ELIC [46] and (d) 357 crystal structure of the open bacterial channel GLIC [45], both viewed from the extracellular 358 space with ECDs removed for clarity; the hydrophobic M2-9' sidechain is moved from the 359 channel axis in models of channel activation. 360 361 Figure 2. The ivermectin molecule. (a) Natta projection of ivermectin, including the C atom

362 numbering referred to in the text. (b) 3D structure of ivermectin, as used for docking to the α1

363 GlyR model[50]. Certain atoms/substituents are labelled, for comparison with (a) or for their 364 significance in the text. Image in (b) prepared with PyMOL Molecular Graphics System 365 (Schrödinger, LLC). 366 367 Figure 3. Ivermectin binding sites and sensitivity determinants in anionic Cys-loop receptors. (a)

368 The ivermectin binding site in the C. elegans α GluClR [44]. Adjacent (+) and (-) subunits are

369 shown in grey and black, respectively, with M2 domains from the remaining three subunits 370 shown in blue and all ECDs removed for clarity. Yellow lines indicate H-bonds with indicated 371 residues. Orange positions indicate residues that form Van der Waals interactions with the 372 ivermectin molecule. (b) The same site as in (a), but viewed from (+)M2-Ser260 (15'). Red lines 373 indicate proximity of Pro230 (3.4 Å) and Gly281 (4.0 and 4.5 Å) to ivermectin; yellow lines 374 indicate H-bonds with (-)Leu218 and (+)Thr285. (c) Amino acid sequence alignment of TMD 375 portions of Cys-loop receptor subunits of known ivermectin sensitivity. Orange and red (Van der 376 Waals interactions) and yellow (H-bonds) highlighting indicates residues that line the site, as in

14 377 (a) and (b); boxed residues constitute the polar binding site for the ivermectin benzofuran in 378 ivermectin-sensitive receptors (see text and Figure 4). Cel, Caenorhabditis elegans; Hco, 379 Haemonchus contortus; Dme, Drosophila melanogaster; Tur, Tetranychus urticae; Hsa, Homo 380 sapiens; Ssc, Sacrcoptes scabiei; Dme, Dermacentor variabilis; Mmu, Mus musculus. "nM" and

381 "µM" (nanomolar and micromolar) indicate the concentration range in which receptors of that

382 subunit are robustly activated by ivermectin. "mod" (modulatory) means that the receptor is not 383 activated but is potentiated or inhibited by micromolar concentrations of ivermectin. *The T.

384 urticae α GluClR confers ivermectin susceptibility on the organism [4]; it is therefore considered

385 highly ivermectin-sensitive although the recombinant receptor has not been tested. **The 386 recombinant D. variabilis RDL has only been exposed to 100 nM ivermectin, which had no 387 agonist or modulatory effect [56]. Lines joining subunits indicate that these form heteromeric 388 receptors. References for all subunits listed are provided in the main text, except for Cel GluClR

389 α2B [77] and Cel GLC-3 [78].

390 391 Figure 4. Possible H-bond partners of the ivermectin C5-hydroxyl. The ivermectin binding site in

392 an α1 GlyR homology model [50], viewed from the (+)M3 helix, with the (+)M3 helix removed.

393 Distances (yellow lines) are 8.1, 2.9, 4.0, 3.3 and 10.1 Å, clockwise from M2-12'. 394 395 Figure 5. Comparison of an intrasubunit site in nAChRs and anionic Cys-loop receptors. (a)

396 TMD portions of adjacent (+)α and (-)γ subunits of the Torpedo marmorata ("Tma") nAChR

397 (left; electron microscopy structure [47]) and of adjacent subunits of the C. elegans α GluClR

398 (right, [44]), seen from within the membrane plane. For each receptor, the (+) subunit is shown in

399 light grey, the (-) subunit in dark. nAChR α Leu279 (green) aligns with the GluClR M3-Gly

400 position in most amino acid sequence alignments, but Met282, Ile283 (yellow) and Ile286(red) 401 are structurally more equivalent to GluClR M3-Gly, indicated by the proximity to M1-Pro (red in 402 both receptors). Shown in magenta are several residues that line a space between M1 and M4 of 403 each subunit (these are equivalent in amino acid alignments but were selected simply due to their 404 apparent structural equivalence). Shown in cyan are residues equivalent to those whose

405 substitution in the rat α7 nAChR decreases potentiation by ivermectin [64]; only one of these is 15 406 indicated in the GluClR structure, as it is one of the aligned residues in (b). (b) Amino acid 407 sequence alignment. Colours indicate the residues shown in (a). Circled residues are smaller in

408 the α7 nAChR than in other cationic receptors, providing a possible explanation for selectivity of

409 ivermectin for the α7 nAChR over other cationic receptors. Boxed residues are smaller in the α7

410 nAChR than in anionic Cy-loop receptors, providing a possible explanation for the selectivity of 411 ivermectin for the M1-M4 site in the former. 412 413 Figure 6. Putative ivermectin-insensitive receptor subunits from trematodes. An amino acid

414 sequence alignment of characterised GluClR, GlyR, HisCl and GABAAR (UNC-49B) subunits 415 from vertebrates, insects and nematodes (black) and uncharacterised subunits from trematodes 416 (purple). Sma, Schistosoma mansoni; Sja, Schistosoma japonicum; Sha, Schistosoma 417 haematobium; Csi, Clonorchis sinensis; Sme, Schmidtea mediterranea. Loop B is part of the 418 neurotransmitter binding domain (see Figure 1). A Glu residue at the cyan position is important to 419 the binding of the agonists glycine, histamine and GABA [71]. Although the putative trematode 420 subunits possess several features of the ivermectin binding site, such as the M1-Pro (first red 421 residue) and several polar sidechains (boxed residues), they possess large hydrophobic sidechains 422 at the M3-Gly position (second red residue), indicative of ivermectin insensitivity. Smp_104890 423 and Smp_096480 [70] and Sjp_0079260 [79] were retrieved at GeneDB. Sha_300716 was 424 retrieved from the original publicaton [80]. GAA36399.1 [81] was retrieved from GenBank. 425 mk4.001914 [82] was retrieved from SmedGD. 426

16 427 References

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23

M1-Pro 12' 15' 19' M3-Gly M1 M2 M3

Loop B M1 M2 M3 GluClR a Cel ASYAY LLQLYIPSCM LTMTAQSAGIN DVWIGACMTFIF GlyR a Hsa ESFGY LIQMYIPSLL LTMTTQSSGSR DIWMAVCLLFVF HisCl1 Dme ESLSH LFHTYIPSAL LTLATQNTQSQ DVWMSSCSVFVF UNC-49B Hco ESYCY SMNIVIPSML LTMTTLITTTN DVFLNFCFVMVF Smp_104890 Sma GSYGY LIYAYLPSLL LALITQSAAIL DIWLFVCLAFVV Smp_096480 Sma GSYGY LASTYIPNIL LGVITQSTSYA DIWTIACITFNS Sjp_0079260 Sja GSYGY LASTYIPNIL LGVITQSTSYA DIWTIACITFNS Sha_300716 Sha GSYGY LASTYIPNIL LGVITQSTSYA DIWTIACITFNS GAA36399.1 Clo GSYGY LASTYIPNTL LGVITQTTSYA DVWTIACIAFNS mk4.001914 Sme GSYGY LASTYIPNVL LGILTQATGMS DIWIIACIIFVI