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

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Page 1: Ivermectin binding sites in human and invertebrate Cys ...284027/UQ284027_postprint.pdf · 1 1 Ivermectin binding sites in human and invertebrate Cys-loop receptors 2 Timothy Lynagh1,

1

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

Timothy Lynagh1, Joseph W. Lynch2 2

3 1 Neurosensory Systems Group, Technical University of Darmstadt, Schnittspahnstrasse 3, 64287 4

Darmstadt, Germany 5 2 Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, 6

Brisbane, QLD 4072, Australia 7

8

Corresponding author: Lynch, J.W. ([email protected]). Phone: +617 33466375 9

10

11

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Abstract 12

Ivermectin is a gold-standard antiparasitic drug that has been used successfully to treat billions of 13

humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had 14

been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular 15

modelling now explain how ivermectin binds to these receptors and reveal why it is selective for 16

invertebrate members of the Cys-loop receptor family. Combining this with emerging genomic 17

information, we are now in a position to predict species sensitivity to ivermectin and better 18

understand the molecular basis of ivermectin resistance. An understanding of the molecular 19

structure of the ivermectin binding site, which is formed at the interface of two adjacent subunits 20

in the transmembrane domain of the receptor, should also aid the development of new lead 21

compounds as both anthelmintics and as therapies for a wide variety of human neurological 22

disorders. 23

24

New insight into a wonder drug 25

Orally- or topically-administered ivermectin is used to eliminate parasitic nematodes and 26

arthropods from animals and humans[1]. It is effective yet well-tolerated due to the potency with 27

which it activates a uniquely invertebrate member of the Cys-loop receptor family of ligand-gated 28

ion channels[2]. Although the activation of Cys-loop receptors by neurotransmitter agonists has 29

been studied in molecular detail for 20 years, the ivermectin interaction with these receptors has 30

received relatively little attention. This interaction requires attention, given the evidence that 31

ivermectin resistance can occur via mutations that affect the ivermectin sensitivity of Cys-loop 32

receptors [3,4]. In the last year, the binding sites of ivermectin in nematode and human Cys-loop 33

receptors have been identified, revealing key molecular determinants of ivermectin sensitivity. 34

Coinciding with these discoveries, a growing number of complete genomes have revealed the 35

Cys-loop receptor complements of species of known ivermectin susceptibility. Combining this 36

knowledge should allow the prediction of ivermectin sensitivity, help understand resistance 37

mechanisms and inform the development of broader spectrum antiparasitic drugs. Recent insights 38

in this area are reviewed here, following a brief introduction concerning the use of ivermectin and 39

the structural basis of Cys-loop receptor function. 40

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41

Origins of ivermectin 42

Intensive screening of soil-dwelling microbes in the 1970s led to the isolation from the 43

bacterium, Streptomyces avermectinius, of a group of natural macrocyclic lactones with potent 44

nematicidal activity, named avermectins [5]. Saturation of the avermectin 22-23 double bond 45

produced 22,23-dihydroavermectin B1, or ivermectin. Ivermectin consists of a mixture of B1a 46

and B1b components in a 80:20 ratio. These compounds vary in structure only at C25, a moiety 47

that is not involved in binding. As the B1a component is more lethal to nematode, insect and 48

acarid parasites [1], it is conventional to refer to either the mixture or the B1a component as 49

‘ivermectin’. Much attention has been paid to the use of ivermectin against Onchocerca volvulus, 50

a mosquito-transmitted parasitic nematode that causes river blindness, but its use now extends to 51

other human diseases including lymphatic filariasis, strongyloidiasis, scabies and head lice [1]. 52

Despite its broad antiparasitic spectrum, ivermectin is ineffective against platyhelminths [6,7], 53

including the blood fluke Schistosoma mansoni which causes bilharzia [8]. 54

55

Ivermectin targets Cys-loop receptors 56

Early investigations into the lethal effect of ivermectin on arthropods and nematodes showed that 57

ivermectin increased chloride conductance in neurons and thus decreased responses of neurons to 58

excitatory input [9,10]. The search for the identity of this conductance led to the cloning of two 59

novel subunits from the free-living nematode Caenorhabditis elegans that formed a recombinant 60

glutamate-gated chloride channel (GluClR) that was activated irreversibly by nanomolar 61

ivermectin concentrations [2]. Numerous GluClR subunits have since been cloned in various 62

ivermectin-susceptible invertebrates [11,12]. The identification of GluClRs as the target of 63

ivermectin was confirmed by their high ivermectin sensitivity when expressed recombinantly, 64

their expression in tissues sensitive to low doses of ivermectin, and the development of 65

ivermectin resistance upon their mutation [3,4,12-15]. GluClRs belong to the Cys-loop receptor 66

family of ligand-gated channels, which includes human excitatory nicotinic acetylcholine and 67

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

glycine receptors (GABAARs and GlyRs). 69

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The Cys-loop receptor family is large, with 102 subunits in C. elegans and 45 in humans [16,17]. 70

Numerous inhibitory receptors in this family are activated by ivermectin, including arthropod 71

histamine-gated chloride channels (HisCls) and pH-gated chloride channels (pHCls) and, perhaps 72

unexpectedly, human GABAARs and GlyRs. A deciding factor in the combined lethality in 73

nematode/arthropod parasites and safety in mammalian hosts is the differential potency with 74

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

nanomolar concentrations [2,14,18,19], whereas GABAARs and GlyRs require concentrations in 76

the low (1-10) micromolar range [20-23]. HisCls and pHCls respond to micromolar ivermectin, 77

but the dose dependency has not been established [24-26]. At several other receptors, ivermectin 78

activates no current but modulates (inhibits or potentiates) the responses to the neurotransmitter 79

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

The sensitivity of human Cys-loop receptors to micromolar ivermectin, along with the 81

preliminary findings of anti-spasticity efficacy in humans [28], begs the question as to what 82

concentration reaches in the brain. Therapeutic doses in mice, dogs and humans produce plasma 83

levels of 5-10 nM that persist for days, with ~2 nM in the brain [29-31], which is insufficient to 84

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

animals respond to systemic ivermectin [32], indicating that ivermectin safety rests on the 86

reduced sensitivity of mammalian Cys-loop receptors. P-glycoprotein, which transports 87

avermectins out of the nervous system, is also crucial to ivermectin potency in mammals and 88

nematodes, evidenced by the increased ivermectin toxicity that results upon its mutation [33,34]. 89

90

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

Before describing in detail the structural features that determine ivermectin sensitivity, we briefly 92

outline the structural basis of function in Cys-loop receptors and the determinants of efficacy 93

within the ivermectin molecule. Functional Cys-loop receptor pentamers are formed by five of 94

the same or, as is usually the case for native receptors, two to three different subunits (Figure 95

1a,b). A single subunit consists of: a large extracellular N-terminal domain (ECD) and four 96

membrane-bound helices (M1-M4) that constitute the transmembrane domain (TMD). Two 97

highly conserved ECD Cys residues are crosslinked within a functional receptor, forming the 98

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eponymous Cys-loop, and the apposition of five M2 helices forms the central channel pore 99

(Figure 1b,c). Neurotransmitter ligands bind at the extracellular interface of two adjacent subunits 100

(red segments in Figure 1b), resulting in the closure of binding domain loop C around the agonist. 101

This triggers structural rearrangements at the ECD-TMD interface (purple segments in Figure 102

1b), and ultimately the opening of the channel gate (Figure 1c,d), allowing ions to flow through 103

the channel [35]. This chemoelectric signalling mechanism underlies most rapid synaptic 104

transmission in the brain. 105

The structural basis of activity of avermectins and milbemycins (Streptomyces spp-derived 106

compounds with similar structures and antiparasitic spectra to avermectins) has been extensively 107

reviewed[36]. Briefly, all compounds share a macrocyclic backbone and differ at the 108

disaccharide, spiroketal and benzofuran moieties, respectively located at the three “corners” of 109

the molecule (Figure 2). Evidence indicates that the benzofuran is the most important potency 110

determinant at GluClRs. For example, substitution of the hydroxyl at the C5 position on the 111

benzofuran ring ("C5-OH") for oxime or ketone in several avermectins reduces nematicidal 112

activity 100- and 10000-fold, respectively [37], implying that either an H-bond donating or 113

generally hydrophilic C5-substituent is required for high potency. In contrast, changes to the 114

spiroketal group have little effect on nematicidal potency [36,37]. Similarly, hydrogenation of 115

double bonds C10-C11 (in the macrocyclic backbone) and 22-23 (in the spiroketal) does not 116

reduce acaricidal potency, whereas hydrogenation of double bonds C3-C4 and C8-C9 (in or 117

adjacent to the benzofuran) reduces both acaricidal potency and activity at mouse GABAARs 118

[38,39]. The sugar moiety is not essential for nematicidal potency [36,37]. However, given that 119

epimerisation of the dissacharide decreases activity at GluClRs [40,41], polar C13 substitutions 120

yield less biologically active avermectins than lipophillic C13 substitutions [42], and size of the 121

sugar moiety correlates with affinity for P-glycoprotein [43], it is evident that the sugar may 122

modulate avermectin potency in vivo. 123

124

The binding site in anionic Cys-loop receptors 125

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

with ivermectin [44]. This gives a clear picture of the ivermectin binding site and sheds light on 127

previous functional studies. Furthermore, with over 30 % amino acid sequence identity with the 128

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

GlyRs, which had previously relied on nAChR and prokaryotic receptor structures with much 130

lower homologies [45-47]. Their structure shows ivermectin binding between the M3 of one 131

subunit (the principal or (+) face) and the M1 of an adjacent subunit (the complementary or (-) 132

face), with the benzofuran moiety contacting the M2 helix of the (+) subunit (Figure 3a). The 133

structure implies the existence of three H-bonds: (i) between the benzofuran C5-OH and the 134

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

and the hydroxyl sidechain of (+)M3-Thr285, and (iii) between the benzofuran C7-OH and the 136

backbone carbonyl oxygen of (-)M1-Leu218 (Figure 3b). Leu218, Ser260 and Thr285 are 137

highlighted in yellow in Figure 3c, as are residues at the equivalent positions in various other 138

Cys-loop receptors. Twelve other sidechains, highlighted in orange or red in Figure 3c, contribute 139

potential Van der Waals interactions. One of these is (+)M3-Gly281, a position in anionic Cys-140

loop receptors we will henceforth refer to as "M3-Gly". M3-Gly is entirely conserved in GluClRs 141

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

M3-Gly is approximately 4.5 Å from each corner of the ivermectin macrocycle, such that any 143

larger sidechain at this position - that is, any amino acid other than Gly - would hinder the access 144

of ivermectin to its binding site or alter the binding conformation of ivermectin (Figure 3b). 145

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

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

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

remarkably similar ivermectin sensitivities by introducing a common residue at the M3-Gly 149

position [48]. Consistent with this, the Gly323Asp mutation (at the M3-Gly position) in the 150

Tetranychus urticae (two-spotted spider mite) GluClR was shown to increase by 18-fold the 151

avermectin dose required for half-maximal lethality [4]. Indeed, the equivalent Gly-Asp 152

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

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

M3-Gly position. 155

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

modelling. Ivermectin was modeled as wedging into the same TMD interface, with the 157

benzofuran oriented towards the (+)M2 [50]. Surprisingly, the docking of ivermectin to the 158

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

receptor, suggesting that the on-rate of ivermectin, rather than the ultimate binding conformation 160

of ivermectin, is changed by this substitution [50]. Alternately, the glycine substitution may 161

increase the gating equilibrium constant, thus facilitating receptor activation by ivermectin. Bulky 162

substitutions at only two positions decreased both agonist and modulatory effects of ivermectin, 163

indicative of a decisive role in binding. These positions are (+)Ala288 and (-)Pro230. Both of 164

these positions are highlighted in red in Figure 3c, revealing the apparent requirement of a small 165

residue at the M3-Gly position and the conservation of the "M1-Pro". M1-Pro is in close 166

proximity to the crucial benzofuran moiety of ivermectin (Figure 3b), providing a possible steric 167

explanation for decreased binding at this mutant. In addition, this highly conserved residue 168

disrupts M1 helical structure thereby exposing a backbone carbonyl that is thought to form a H-169

bond with the ivermectin C7-OH [44]. However, the existence of this bond requires confirmation, 170

given that C7-OH is equidistant from both the M1 backbone carbonyl and the endogenous 171

ivermectin C1 carbonyl. 172

A significant role for the third putative H-bond (between O14 and T285 – see Figure 3b) can be 173

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

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

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

GlyR (+)M3-Leu291, (+)M2-Thr264 or (-)M1-Leu233 for bulky Trp sidechains does not reduce 177

ivermectin sensitivity but converts ivermectin from an agonist to an antagonist of glycine-178

activated currents [50]. Still further removed from the binding site, mutations in the conserved 179

Cys-loop, the pre-M1 domain and the M2-M3 linker (Figure 1b) decrease ivermectin potency 180

most likely by impairing its gating efficacy [14,18,49-51]. 181

Native Cys-loop receptors are often heteromeric and thus do not necessarily possess the five 182

binding sites available to ivermectin in homomers. The following evidence at the GlyR, however, 183

suggests that the binding of two molecules of ivermectin is sufficient for either direct activation 184

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

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

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

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

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

190

M2 H-bonds 191

The putative H-bond between the ivermectin C5-OH and the M2-15' Ser of the GluClR and GlyR 192

seems a logical ivermectin binding interaction, as (i) it links the ivermectin molecule to the pore-193

lining M2 helix[44] and (ii) a H-bond donor at the C5-OH is crucial to avermectin nematicidal 194

potency (above). However, because various GluClRs with nanomolar ivermectin sensitivities 195

contain either an Ala or Ile at this position (Figure 3c), it is evident this H-bond is not required for 196

high ivermectin potency [4,18,53]. In support of this, the mutant M2-Ser15'Ile substitution in the 197

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

most anionic Cys-loop receptor subunits possess several nearby residues that could act as 199

potential H-bond acceptors in the absence of one at the M2-15' position. These include (+)M2-200

12', (-)M2-14', (+)M2-19' and also an (-)M1 position (Figure 4). The proximity of these 201

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

GlyR: the Thr12'Trp receptor is not activated by ivermectin, the Gln226Cys receptor has 203

decreased ivermectin sensitivity and the Gln266Trp receptor has increased sensitivity [50]. The 204

conservation of polarity in this domain of ivermectin-sensitive receptors is illustrated by the 205

boxed residues in Figure 3c. 206

207

A clear pattern? 208

We now group the above ivermectin sensitivity determinants together and consider their chemical 209

properties in several receptors with known high ivermectin sensitivity. Dividing M2-12', M2-14', 210

M2-15', M2-19' and the M1-Gln residue into their (+) and (-) components gives a "(+)Thr-Ser-211

Arg/(-)Gln-Gln" arrangement, in the case of the α1 GlyR (Figure 4). The following five 212

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homomeric Cys-loop receptors are particularly sensitive to ivermectin activation (i.e., activated 213

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

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

Ser-Asp/(-)Gln-Gln, (+)Thr-Ala-Asn/(-)Gln-Gln, (+)Thr-Ala-Asn/(-)Gln-Gln and (+)Ala-Thr-216

Asn/(-)Gln-Gln arrangements, and thus present an overwhelmingly polar environment to the 217

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

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

sensitivity. UNC-49B/UNC-49C heteromeric GABAARs possess an M3-Gly at the (+) faces of 220

UNC-49C, yet are not activated by ivermectin [54,55]. However, with a (+)Thr-Met-Asn/(-)Gln-221

Leu arrangement, they are distinct from the GluClRs and GlyRs in that both the M2-14' and M2-222

15' positions are occupied by larger hydrophobic residues (Figure 3c), perhaps limiting H-bonds 223

with and channel activation by ivermectin [44]. Similarly, the Dermacentor variabilis (American 224

dog tick) homomeric RDL receptor possesses an M3-Gly but is not sensitive to ivermectin 225

(Figure 3), and its M2-14' and M2-15' positions are occupied by hydrophobic residues [56]. By 226

contrast, MOD-1 is insensitive to ivermectin [57], despite its ostensibly suitable (+)Thr-Ser-227

Arg/(-)Gln-Ile arrangement; MOD-1 insensitivity is likely due to a Val residue at the M3-Gly 228

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

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

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

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

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

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

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

Met arrangement and an M3-Gly. We thus conclude that the (-)M1-Met is responsible for 236

disrupting ivermectin sensitivity. In the above analysis, we have used the GluClR crystal 237

structure to infer that the five polar residues are essential for high ivermectin affinity. However, it 238

is also possible that the overwhelmingly polar environment facilitates receptor gating by 239

ivermectin. 240

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An alternate possibility in GABAARs is that ivermectin binds to a different site. For example, 241

GAB-1/HG1A heteromers and UNC-49B homomers possess Val, Leu or Asn residues at the M3-242

Gly position, and these receptors are potentiated but not activated by ivermectin [54,55,61], 243

perhaps reflecting the exclusion of ivermectin from the interfacial site and the existence of an 244

alternate site. Moreover, HG1A and HG1E differ by only four amino acids - in the ECD and in 245

M4 - and, whereas GAB1/HG1A heteromers are potentiated by ivermectin, GAB1/HG1E 246

heteromers are inhibited [61]. An alternative site, consistent with HG1A/E structural differences, 247

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

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

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

GABAAR preparations [62]. 251

252

An alternative site for potentiation of nAChRs 253

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

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

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

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

Å resolution structure of the T. marmorata nAChR places this Leu279 in the (+)M3 helix, it faces 258

the outside of the membrane, 1-2 helical turns above the level of (-)M1Pro (Figure 5a). In the T. 259

marmorata nAChR structure, the (+)M3 residue physically opposite (-)M1-Pro is either Met282, 260

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

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

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

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

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

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

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

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

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

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

interfacial site in anionic receptors [64,66]. In both studies, the ivermectin molecule wedges 271

between M1 and M4 from the same subunit, partly in contact with the surrounding lipids, with 272

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

nAChR TMD residues for the non-conserved equivalents from the 5-HT3AR resulted in decreased 274

potentation by ivermectin or conversion of potentiation to inhibition [64]. Three of the four 275

mutations that simply decrease potentiation line a space between M1 and M4 of the one subunit, 276

albeit two helical turns below the docked ivermectin molecule. Consistent with putative existence 277

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

that might obscure this cavity (Figure 5). Finally, the effects of ivermectin at nAChRs are 279

reversible and are decreased by ECD-acting competitive antagonists [67], which differs from 280

results at most anionic receptors and supports a different site or mechanism of ivermectin at 281

nAChRs. 282

283

Structural bases of ivermectin resistance 284

There are several reports that ivermectin resistance occurs nematodes in natural settings [1], in 285

some cases due to mutations in the coding sequences or changes in expression levels of GluClRs 286

or GABAARs [3,68,69]. In laboratory-induced mutant strains, single mutations in GluClRs have 287

been clearly shown to cause avermectin resistance. For example, mutation of the M3-Gly position 288

in the T. urticae GluClR causes an 18-fold increase in abamectin LD50 [4]. (A C22-C23 double 289

bond in the spiroketal of abamectin is the only difference from ivermectin.) It is also possible that 290

ivermectin insensitivity may be caused by mutations that allosterically disrupt the efficacy with 291

which ivermectin activates the receptors, without necessarily affecting ivermectin affinity. This 292

was first suggested by an M2-M3 linker mutation in a Drosophila GluClR that caused a four-fold 293

increase in ivermectin LD100 [14]. Disruptions to the efficacy of GluClR gating by ivermectin 294

most likely account for the effects of mutations identified in several resistant H. contortus strains 295

[18,49]. Of course, if a GluClR mutation converts ivermectin to an inhibitor, as demonstrated at 296

recombinant GlyRs and nAChRs (above), the usual CNS depression elicited by ivermectin could 297

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

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

300

Predicting species susceptibility to ivermectin 301

The discovery of molecular determinants of ivermectin sensitivity in Cys-loop receptors provides 302

an opportunity to investigate parasites of known ivermectin susceptibility to establish the 303

molecular basis for this susceptibility. The complete genome of the trematode S. mansoni reveals 304

13 Cys-loop receptor subunits [70], much fewer than the 102 found in C. elegans and 45 in 305

humans [16,17]. It is therefore likely that ivermectin has fewer potential Cys-loop receptor targets 306

in S. mansoni than in C .elegans which might help explain why S. mansoni is insensitive to 307

ivermectin. On the other hand, arthropods also possess few GluClRs but are ivermectin-308

susceptible, suggesting that there must be some feature of S. mansoni GluClRs (other than a small 309

number in the genome) that renders them insensitive to ivermectin. An amino acid sequence 310

alignment of recently available transcripts in several trematodes suggests that they indeed possess 311

GluClRs (Figure 6). Although their functional properties remain to be investigated, these 312

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

relationship. Furthermore, they possess a Gly residue on the (+) face of the ECD neurotransmitter 314

binding domain (Figure 6), at a position where small residues are correlated with Glu binding and 315

larger residues are correlated with Gly, GABA or His binding[71]. These putative GluClRs 316

include largely polar (+)Ile-Ser-Ala/(-)Ser-Gln arrangements (as per the scheme above), but large 317

residues at the M3-Gly position undoubtably preclude ivermectin binding (Figure 6). Thus, large 318

sidechains at the M3-Gly position, as opposed to a lack of GluClRs, may well underlie the 319

ivermectin-insensitivity of trematodes. The free-living trematode Schmidtea mediterranea also 320

possesses GluClR-like transcripts with large sidechains at the M3-Gly position (Figure 6). The 321

recent sequencing of Ascaris suum (pig roundworm), Anopheles gambiae (mosquito) and 322

Daphnia pulex (water flea), by contrast, reveal one or more M3-Gly GluClR transcripts, as 323

expected for ivermectin-susceptible phyla [72-74]. This reiterates the importance of the M3-Gly 324

position. Of course, species susceptibility also depends on if and where the relevant subunits are 325

expressed, and the functionality with which an M3-Gly subunit heteromerises with other 326

subunits. 327

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328

Concluding remarks 329

Ivermectin binds to nematode GluClRs and mammalian GlyRs at the interface of adjacent (+) and 330

(-) subunits in the TMD of the receptor. High affinity for this site is controlled by an M3 Gly 331

residue on the (+)M3 helix that allows rapid access to the site. The C5 hydroxyl of the ivermectin 332

benzofuran likely forms an H-bond within the subunit interface cleft, leading to potent channel 333

activation. Potent activity requires polar sidechains at either M2-14', M2-15' or M2-19' (and 334

perhaps other) positions. Scanning Cys-loop receptor amino acid sequences for these 335

determinants predicts ivermectin sensitivity reasonably well, and scanning genomes for these 336

transcripts may enable the prediction of species susceptibility to ivermectin. However, several 337

questions are yet to be answered, including the structural basis for modulation (not activation) by 338

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

GluClRs. Attempts to answer these questions will be aided by the structural template provided by 340

the GluClR crystal structure. Given the emergence of ivermectin resistance in parasitic nematode 341

pests [75,76], the information presented here may help in the design of novel anthelmintics 342

targeting GluClRs or other nematode Cys-loop receptors. Similarly, because ivermectin binding 343

sites also exist in human Cys-loop receptors, understanding the ivermectin binding site structure 344

may help identify new therapeutic pharmacophores for a wide variety of human neurological 345

disorders. 346

347

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Figure legends 348

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

structure, viewed from the extracellular space (top panel) and from within the lipid membrane 350

(lower panel) [44]. (b) Single subunit from (a). Red indicates segments that contribute to the 351

neurotransmitter binding domain, which is formed between the (+) face of one subunit and the (-) 352

face of an adjacent subunit; magenta indicates Loop 2, the conserved Cys-loop, the pre-M1 353

domain, and the M2-M3 linker, which couple neurotransmitter binding to channel movement; 354

helices M1-M4 are indicated; M2-Leu9' sidechains that contribute to the channel gate are shown 355

as blue spheres [35]. (c) Crystal structure of the closed bacterial channel ELIC [46] and (d) 356

crystal structure of the open bacterial channel GLIC [45], both viewed from the extracellular 357

space with ECDs removed for clarity; the hydrophobic M2-9' sidechain is moved from the 358

channel axis in models of channel activation. 359

360

Figure 2. The ivermectin molecule. (a) Natta projection of ivermectin, including the C atom 361

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

GlyR model[50]. Certain atoms/substituents are labelled, for comparison with (a) or for their 363

significance in the text. Image in (b) prepared with PyMOL Molecular Graphics System 364

(Schrödinger, LLC). 365

366

Figure 3. Ivermectin binding sites and sensitivity determinants in anionic Cys-loop receptors. (a) 367

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

shown in grey and black, respectively, with M2 domains from the remaining three subunits 369

shown in blue and all ECDs removed for clarity. Yellow lines indicate H-bonds with indicated 370

residues. Orange positions indicate residues that form Van der Waals interactions with the 371

ivermectin molecule. (b) The same site as in (a), but viewed from (+)M2-Ser260 (15'). Red lines 372

indicate proximity of Pro230 (3.4 Å) and Gly281 (4.0 and 4.5 Å) to ivermectin; yellow lines 373

indicate H-bonds with (-)Leu218 and (+)Thr285. (c) Amino acid sequence alignment of TMD 374

portions of Cys-loop receptor subunits of known ivermectin sensitivity. Orange and red (Van der 375

Waals interactions) and yellow (H-bonds) highlighting indicates residues that line the site, as in 376

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(a) and (b); boxed residues constitute the polar binding site for the ivermectin benzofuran in 377

ivermectin-sensitive receptors (see text and Figure 4). Cel, Caenorhabditis elegans; Hco, 378

Haemonchus contortus; Dme, Drosophila melanogaster; Tur, Tetranychus urticae; Hsa, Homo 379

sapiens; Ssc, Sacrcoptes scabiei; Dme, Dermacentor variabilis; Mmu, Mus musculus. "nM" and 380

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

subunit are robustly activated by ivermectin. "mod" (modulatory) means that the receptor is not 382

activated but is potentiated or inhibited by micromolar concentrations of ivermectin. *The T. 383

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

highly ivermectin-sensitive although the recombinant receptor has not been tested. **The 385

recombinant D. variabilis RDL has only been exposed to 100 nM ivermectin, which had no 386

agonist or modulatory effect [56]. Lines joining subunits indicate that these form heteromeric 387

receptors. References for all subunits listed are provided in the main text, except for Cel GluClR 388

α2B [77] and Cel GLC-3 [78]. 389

390

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

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

Distances (yellow lines) are 8.1, 2.9, 4.0, 3.3 and 10.1 Å, clockwise from M2-12'. 393

394

Figure 5. Comparison of an intrasubunit site in nAChRs and anionic Cys-loop receptors. (a) 395

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

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

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

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

position in most amino acid sequence alignments, but Met282, Ile283 (yellow) and Ile286(red) 400

are structurally more equivalent to GluClR M3-Gly, indicated by the proximity to M1-Pro (red in 401

both receptors). Shown in magenta are several residues that line a space between M1 and M4 of 402

each subunit (these are equivalent in amino acid alignments but were selected simply due to their 403

apparent structural equivalence). Shown in cyan are residues equivalent to those whose 404

substitution in the rat α7 nAChR decreases potentiation by ivermectin [64]; only one of these is 405

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indicated in the GluClR structure, as it is one of the aligned residues in (b). (b) Amino acid 406

sequence alignment. Colours indicate the residues shown in (a). Circled residues are smaller in 407

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

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

nAChR than in anionic Cy-loop receptors, providing a possible explanation for the selectivity of 410

ivermectin for the M1-M4 site in the former. 411

412

Figure 6. Putative ivermectin-insensitive receptor subunits from trematodes. An amino acid 413

sequence alignment of characterised GluClR, GlyR, HisCl and GABAAR (UNC-49B) subunits 414

from vertebrates, insects and nematodes (black) and uncharacterised subunits from trematodes 415

(purple). Sma, Schistosoma mansoni; Sja, Schistosoma japonicum; Sha, Schistosoma 416

haematobium; Csi, Clonorchis sinensis; Sme, Schmidtea mediterranea. Loop B is part of the 417

neurotransmitter binding domain (see Figure 1). A Glu residue at the cyan position is important to 418

the binding of the agonists glycine, histamine and GABA [71]. Although the putative trematode 419

subunits possess several features of the ivermectin binding site, such as the M1-Pro (first red 420

residue) and several polar sidechains (boxed residues), they possess large hydrophobic sidechains 421

at the M3-Gly position (second red residue), indicative of ivermectin insensitivity. Smp_104890 422

and Smp_096480 [70] and Sjp_0079260 [79] were retrieved at GeneDB. Sha_300716 was 423

retrieved from the original publicaton [80]. GAA36399.1 [81] was retrieved from GenBank. 424

mk4.001914 [82] was retrieved from SmedGD. 425

426

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M3M1 M215'

M1-Pro

M3-Gly

12'19'

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ASYAYESFGYESLSHESYCYGSYGYGSYGYGSYGYGSYGYGSYGYGSYGY

DVWIGACMTFIFDIWMAVCLLFVFDVWMSSCSVFVFDVFLNFCFVMVFDIWLFVCLAFVVDIWTIACITFNSDIWTIACITFNSDIWTIACITFNSDVWTIACIAFNSDIWIIACIIFVI

CelHsaDmeHcoSmaSmaSjaShaCloSme

GluClR aGlyR aHisCl1

UNC-49BSmp_104890Smp_096480Sjp_0079260Sha_300716

GAA36399.1mk4.001914

LLQLYIPSCMLIQMYIPSLLLFHTYIPSALSMNIVIPSMLLIYAYLPSLLLASTYIPNILLASTYIPNILLASTYIPNILLASTYIPNTLLASTYIPNVL

LTMTAQSAGINLTMTTQSSGSRLTLATQNTQSQLTMTTLITTTNLALITQSAAILLGVITQSTSYALGVITQSTSYALGVITQSTSYALGVITQTTSYALGILTQATGMS

Loop B M1 M2 M3