The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinarian importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like receptor subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel and is more responsive to oxantel than the ACR-16 receptor from A. suum. These finding most likely explain the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as valuable screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds. Author Summary The human whipworm, Trichuris trichiura, is an intestinal parasitic nematode infecting approximately 289.6 million people globally, primarily children living in developing countries. Chronic T. trichiura infection may cause dysentery, growth stunting and decreased cognitive performance. Whipworm infections are notoriously difficult to control with most available anthelmintics, including those commonly used in mass drug administration programs. Recently performed randomised controlled trials with whipworm-infected humans, have reported superior efficacies of oxantel, a classic, narrow-spectrum anthelmintic, developed for the treatment of Trichuris infections. Despite this knowledge, the molecular target(s) of oxantel within the whipworm has not been identified. In this study, we used the whipworm from pigs as a model and identified a receptor, which was explored using the Xenopus oocyte expression system. We demonstrated that this receptor is highly responsive to oxantel, and therefore a major target of oxantel within Trichuris. In addition, we discovered that this receptor-type is distinctive and only present in the ancient group of parasitic nematodes, Clade I, which also includes the important zoonotic parasite Trichinella. Our findings, explain the specific mode of action of oxantel and open the way for additional characterization of similar receptor subtypes in other medically or veterinary important parasitic nematodes of Clade I.

we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a 26 functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and 27 cloned a novel ACR-16-like receptor subunit and successfully expressed the corresponding 28 homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this 29 receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence 30 we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype 31 moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We 32 demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel and is more 33 responsive to oxantel than the ACR-16 receptor from A. suum. These finding most likely explain 34 the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of 35 additional distinct receptor subunit types within Trichuris that can be used as valuable screening 36 tools to evaluate the effect of new synthetic or natural anthelmintic compounds. 37 38 Author Summary 39 The human whipworm, Trichuris trichiura, is an intestinal parasitic nematode infecting 40 approximately 289.6 million people globally, primarily children living in developing countries. 41 Chronic T. trichiura infection may cause dysentery, growth stunting and decreased cognitive 42 performance. Whipworm infections are notoriously difficult to control with most available 43 anthelmintics, including those commonly used in mass drug administration programs. Recently 44 performed randomised controlled trials with whipworm-infected humans, have reported superior 45 efficacies of oxantel, a classic, narrow-spectrum anthelmintic, developed for the treatment of 46 Trichuris infections. Despite this knowledge, the molecular target(s) of oxantel within the 47 whipworm has not been identified. In this study, we used the whipworm from pigs as a model and 48 identified a receptor, which was explored using the Xenopus oocyte expression system. We

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The human whipworm, Trichuris trichiura, is a Clade I parasitic nematode [1] and one of the Soil 58 Transmitted Helminths (STHs) that is estimated to infect 289.6 million people globally, primarily 59 those living in the tropics and subtropics [2]. Trichuriasis is rarely fatal, but chronically affects the 60 health and nutritional status of the host [3,4], and is known to be notoriously difficult to treat using 61 current anthelmintic drugs (e.g. albendazole and mebendazole) [5][6][7][8][9][10][11][12]. The extensive use of 62 anthelmintics in livestock has led to widespread anthelmintic resistance (AR) to all the major drug 63 classes [13]. Therefore AR in human parasitic nematodes is a concern where decreased susceptibility 64 to albendazole has already been reported for both T. trichiura [14] and the human roundworm, Ascaris 65 lumbricoides [15]. 66 Oxantel, is a cholinergic agonist [16], and a m-oxyphenol analogue of pyrantel which was developed 67 in 1972 [17] and marketed as a veterinary anthelmintic in 1974 for the treatment of Trichuris [18]. 68 Early clinical trials reported oxantel to be effective against T. trichiura infections [19,20] and recent 69 studies show that oxantel is superior to single-dose albendazole and mebendazole [21,22], which are 70 currently recommended by the WHO for the control of STHs [23]. Cholinergic agonists [16] exert 71 their effect by paralyzing the worms, which are subsequently killed or expelled from the host [24]. 72 This effect is mediated by nicotinic acetylcholine receptors (nAChRs) [24] that are either heteromeric 4 73 or homomeric five-subunit ligand-gated ion channels expressed in neuronal, muscle and non-neuronal 74 cell membranes [25,26]. nAChRs of parasitic nematodes have been separated into different 75 pharmacological subtypes based on their sensitivities to a range of cholinergic anthelmintics. Patch-76 clamp recordings of muscle cells isolated from the pig roundworm A. suum, have revealed that their 77 muscle nAChRs are preferentially activated either by levamisole (L), nicotine (N) or bephenium (B), 78 and correspondingly are described as L-, N-, and B-AChRs subtypes [27]. Oxantel is classified as 79 an agonist which is selective for the N-AChR subtypes [16]. The N-AChR subtypes from the model 80 nematode Caenorhabditis elegans and the distantly related pig parasite A. suum are homomeric 81 receptors made of the ACR-16 subunits [28,29]. Both of these ACR-16 receptors have a low, but 82 significant sensitivity to oxantel [29,30].

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The high sensitivity of Trichuris spp. to oxantel has previously been speculated to be due to an 84 nAChR subtype present in Trichuris spp. that differs from nAChRs present in other intestinal parasitic 85 nematodes [16]; we hypothesized that a potential homolog of ACR-16 in Trichuris could be a target 86 of oxantel within this species.

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Here we describe the functional characterization of a novel AChR subtype from the pig whipworm 88 T. suis with a high sensitivity to oxantel and distinctive pharmacological properties. In the present study, based on the current knowledge of the original mode of action of oxantel [16,29], 115 we hypothesized that the divergent ACR-16-like from Trichuris spp. could represent a preferential 116 target for this narrow-spectrum anthelmintic. Using T. suis as a model, we cloned its acr-16-like full-   anthelmintics, morantel and levamisole, were the least potent. The rank order potency series for the 157 agonist drugs on the Tsu-ACR-16-like receptor when normalized to control oocytes exposed to 100 158 µM ACh was: oxantel > ACh >>> pyrantel >>> epibatidine > nicotine ~ 3-bromocytosine ~ DMPP 159~ morantel ~ cytosine ~ levamisole. Taken together, these observations provide strong evidence that 160 the Tsu-ACR-16-like receptor represents the preferential molecular target for oxantel. Interestingly, 161 when exposed to 100 μM ACh for 1-3 min the Tsu-ACR-16-like receptor did not show a fast-162 desensitization kinetics (Suppl. Fig. 2) which is a distinctive characteristic of the N-AChR of 163 nematode such as Asu-ACR-16 [29] and the ACR-16 from Parascaris equorum (Peq-ACR-16) [33]. Oxantel and pyrantel have similar chemical structures (Fig. 5A), but their potencies on the Tsu-ACR- 167 16-like receptor were found to be significantly different (Fig. 4). We performed a dose-response study 168 on oxantel and pyrantel to determine their EC 50 values, their relative maximum current responses, 169 I max, and their Hill slopes, n H. The mean current response of positive control oocytes exposed to 300 170 µM ACh was used for normalization. Fig. 5B shows representative traces and dose-response 171 relationships of the normalized inward currents (mean ± SEM) induced by different concentrations 172 of oxantel or pyrantel. The EC 50 ± SE for oxantel (9.49 ± 1.13 µM) was significantly lower than that 173 of pyrantel (148.5 ± 1.19 µM, P < 0.001). The relative maximum current response, I max (mean ± SE) 174 was significantly larger for oxantel (86.85 ± 4.63%) than pyrantel (29.41 ± 1.95%, P = 0.003), 175 whereas no significant difference was found between the Hill slopes, n H (mean ± SE), of oxantel (2.51 176 ± 1.31) and pyrantel (3.13 ± 1.07). receptor along with representative current responses. The initial ACh (100 µM) current response of 183 each oocyte was used for normalization, to measure the reduced current responses in the presence of 184 the antagonists. For DHβE, the mean ± SEM inhibition was very small (i.e. 7.60 ± 1.6%) and no 185 inhibition was observed for derquantel (0.16 ± 1.6%). The effect of α-BTX is given in Figure 7A 186 which shows the response-inhibition of the Tsu-ACR-16-like receptor to 100 μM ACh when 10 μM 187 α-BTX is applied 10 s before the second application of 100 µM ACh (test oocytes). Fig. 7B shows 188 the effect of the Tsu-ACR-16-like receptor when exposed to 100 μM ACh, only (control oocytes).

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The first current response of 100 µM ACh (ACh1) was used for normalization. Representative current 9 190 traces along with a bar chart presenting the normalized mean ± SEM of the second and third drug 191 application of test-and control oocytes are given in Fig. 7A and 7B. The 10 μM α-BTX significantly 192 (P < 0.0001) reduced the current response of ACh to 8.3 ± 2.4% of the test oocytes (Fig. 7A). This 193 reduction was not observed in the control oocytes (Fig. 7B), thus, when α-BTX was applied 10 s 194 before the second application of ACh, the current response was significantly reduced in test oocytes 195 as compared to control oocytes ( Fig. 7A and 7B , P < 0.0001).

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In the present study, we report the identification and the functional expression of the Tsu-ACR-  like receptor, a novel AChR subtype corresponding to the first specific drug target for oxantel to be Use of screens for C. elegans mutants that survive exposure to the broad-spectrum anthelmintics 207 provided a means to decipher their molecular targets in a wide range of nematode species. However, 208 this approach was not helpful for oxantel because the C. elegans is insensitive to this drug [34]. The 209 weak, but measurable activity of oxantel on recombinant N-AChR from C. elegans and A. suum, 210 supported the hypothesis that a putative ACR-16 homologs in Trichuris species could be involved in 211 an oxantel-sensitive receptor. Williamson et al. [35] reported that only two members from the ACR- Trichuris species investigated in the present work. The first one corresponded to the highly conserved 215 AChR subunit encoded by the acr-19 gene; the second one corresponded to a highly divergent subunit 216 specific to Clade I nematode species designated as ACR-16-like.

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When co-expressed in the X. laevis oocytes with the ancillary protein RIC-3, the ACR-16-like 218 subunits from T. suis formed a functional homomeric channel (O-AChR) with unexpected 219 pharmacological properties. Indeed, this receptor was highly sensitive to oxantel which is in contrast 220 to the Asu-ACR-16 for which a low agonist effect of oxantel has been reported (i.e. <10% of the 221 control ACh current) [29] and to the Cel-ACR-16 on which oxantel has an antagonistic effect [30].

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Likewise, pyrantel had a relatively high effect on the Tsu-O-AChR, whereas pyrantel had no agonist 223 effect on Asu-ACR-16 [29] or Cel-ACR-16, but in contrast, showed an antagonistic effect on the 224 latter, which was ascribed to pyrantel acting as an open channel blocker [28]. In accordance with this 225 assumption, patch-clamp recording studies from isolated A. suum muscle cells, show that both oxantel 226 and pyrantel to act as agonists and open channel blockers [36,37]. Another surprising difference 227 between the Tsu-O-AChR and the ACR-16 receptors from A. suum, C. elegans and P. equorum is the 228 lack of sensitivity to nicotinic agonists [28,29,33]. Since oxantel has been characterised as an agonist 229 selective for the N-subtypes of the ionotropic AChRs [16] which include the ACR-16 receptors, we 230 expected the Tsu-O-AChR receptor to be highly sensitive to nicotine, cytosine, 3-bromocytosine, 231 epibatidine and DMPP, but only small current responses were observed using these agonists. Another 232 feature of the Tsu-O-AChR receptor is its slow desensitization kinetics, which contrasts with the faster 233 desensitization of the N-AChR from C. elegans [38], A. suum [29] and P. equorum [33]. 234 Interestingly, we also showed that the antagonist, α-BTX had a potent inhibitory effect on the ACh 235 induced current responses of the Tsu-O-AChR whereas A. suum [29] and C. elegans N-AChR [28] 236 are nearly insensitive to α-BTX. We point out however, that α-BTX only induced a strong inhibitory 237 effect when α-BTX was applied 10 s before the application of ACh suggesting a slow association This also contrasts with the Asu-N-AChR which is moderately sensitive to DHβE (~ 65% inhibition) 240 and derquantel (~ 60% inhibition) [29] and the Cel-N-AChR which is highly sensitive to DHβE [28]. affinity of nicotinic agonists [39]. Perhaps it is not surprising that pyrantel which was modified by 256 replacing the 2-thiophene moiety with the an m-oxyphenol group [17] to produce oxantel has a 257 different pharmacology. Thus, the anthelmintic spectrum of oxantel and pyrantel is very different.

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Pyrantel is a broad-spectrum anthelmintic with no effect on adult Trichuris [17] whereas oxantel is 259 a narrow-spectrum anthelmintic with a potent and selective effect on adult Trichuris [19][20][21][22]. This  The worm material used in this study was obtained during a previous described study [40]    Signal peptide was predicted using the SignalP 4.1 server [42] and the transmembrane regions were 304 predicted using the TMHMM version 2 server [43]. Deduced amino-acid sequences were aligned 305 using MUSCLE Phylogenetic analysis was performed on deduced amino-acid sequence. Maximal   The effects of the antagonists DHβE, derquantel and α-BTX (10 μM) were examined in the presence 405 100 µM ACh as previously described for Asu-ACR-16 [29]. In short, X. laevis oocytes co-injected 406 with Tsu-acr-16-like-and Xle-ric-3 cRNA were sequentially superfused with ACh for 10 s, then ACh 407 + antagonist for 10 s, and finally with ACh for 10 s. For α-BTX a five-step protocol including a pre-408 incubation (10 s) with the antagonist (10 μM) was used with ACh (100 μM). Xenopus laevis oocytes, 409 co-injected with Tsu-acr-16-like-and Xle-ric-3 cRNAs, were exposed to: i) a control application of 410 100 µM ACh for 10 s (first application); ii) followed by a wash-off period of 5 min; iii) then by an 411 application of 10 μM α-BTX for 10 s, immediately followed by 100 µM ACh and the continued 412 presence of α-BTX for 10 s (second application); iv) then a wash-off period of 5 min; v) and finally 413 an application of ACh for 10 s (third application). Control oocytes were exposed to ACh for 10 s in ACh was set to 100%, and all other responses were normalized to this. The relative means were 422 statistical analysed using One-Way ANOVA with a Dunnett's Test and P < 0.05 was considered 423 significant. For the drug-potency-test, peak currents of drugs were normalized to the peak current 19 424 measured in the presence of 100 µM ACh and was expressed as mean ± SEM. Data was tested for 425 normality using the D'Agostino-Pearson normality test. Drug-group means were statistical analysed 426 using One-Way ANOVA with a Turkey's Multiple Comparison Test where P < 0.05 was considered 427 significant.

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For the dose-response relationships, and for each experiment, 6 oocytes were exposed to ACh (300       Dose-response curves for oxantel (oxa) and pyrantel (pyr). The current response on Tsu-ACR-  like receptor is normalised to current responses induced by 300 µM ACh and given as mean ±  block of the Tsu-ACR-16-like receptor mediated ACh response (i.e. 7.60 ± 1.61%) and no effect 516 was observed for der (0.16 ± 1.61%).