Abstract
The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A.
SIGNIFICANCE STATEMENT Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.
Introduction
Paraherquamide A (Fig. 1A) is a polycyclic oxindole alkaloid, first isolated as a toxic metabolite from Penicillium paraherquei (Yamazaki et al., 1981). Its anthelmintic activity was demonstrated using gerbils (Meriones unguiculatus) infected with a parasitic nematode Trichostrongylus colubriformi (Ostlind et al., 1990) and in studies on the nematode genetic model organism Caenorhabditis elegans (Ondeyka et al., 1990). Subsequently, paraherquamide A and related compounds were tested on a variety of parasitic nematodes, thereby establishing their broad-spectrum anthelmintic activity (Lee et al., 2002).
Paraherquamide A and its effects on C. elegans N-type and L-type nicotinic acetylcholine receptors (nAChRs) expressed in X. laevis oocytes. Paraherquamide A (A) was bath-applied at 10 μM and 100 nM to oocytes expressing the N-type (B) and L-type (C) nAChR, respectively, for 1 min and then co-applied with 100 μM ACh (B, C). Paraherquamide A reduced the ACh-response of N-type and L-type nAChRs (n = 5). (D, E) Concentration-inhibitory action relationships for paraherquamide A on the N-type (D) and L-type (E) nAChRs. (F, G) Effects of paraherquamide A on the concentration-agonist action relationships for ACh on the N-type (F) and L-type (G) nAChRs. (H−K) Concentration-agonist action relationships of ACh (H, I) and concentration-inhibitory antagonist actions of paraherquamide A (J, K) on C. elegans 2.1 and 2.2 L-type nAChRs. Efach data plot represents the mean ± S.E.M. (n = 5).
Paraherquamide A induced rapid flaccid paralysis in the parasitic nematode Haemonchus contortus without affecting ATP concentration, indicating a possible action on nervous system or neuromuscular receptors (Thompson et al., 1996). In the case of Ascaris suum, 2-deoxy-paraherquamide A (derquantel) showed a higher paralytic activity than paraherquamide A and thus the mechanism of action of the deoxy-derivative was further examined using an in vitro assay. It reduced ACh-induced contraction of an A. suum muscle strip in a manner similar to that seen with mecamylamine and methyllycaconitine, pointing to an action on cholinergic neuromuscular transmission (Zinser et al., 2002). Robertson et al. (2002) tested both paraherquamide A and derquantel for their capacity to block agonist-induced contraction of A. suum muscle. Both compounds induced parallel shifts to the right of the ACh concentration-response curve, i.e., increased the EC50 values, indicating a competitive type of antagonism at nematode nAChRs (Robertson et al., 2002). The nicotine-sensitive (N-type), levamisole-sensitive (L-type), and bephenium-sensitive (B-type) nAChRs expressed on A. suum body wall muscles were characterized and the blocking action of paraherquamide A and derquantel were investigated (Robertson et al., 1999). These studies confirmed that paraherquamide A blocked L-type receptors more effectively than N-type receptors in A. suum, whereas derquantel was most effective in blocking the B-type receptors, albeit with lower potency than paraherquamide A.
C. elegans is a genetic model organism with a fully sequenced genome and a comprehensive genetic toolkit (C. elegans Sequencing Consortium, 1998). The complete C. elegans nAChR subunit gene family has been identified (Jones et al., 2007), and the N-type (Ballivet et al., 1996; Raymond et al., 2000; Boulin et al., 2008) and L-type nAChRs (Boulin et al., 2008) have been expressed successfully in Xenopus laevis oocytes. A C. elegans receptor pharmacologically similar to the native L-type nAChR of A. suum results from the co-expression of 8 genes (5x nAChR subunits UNC-38, UNC-29, UNC-63, LEV-1, and LEV-8 co-expressed with 3x auxiliary proteins, RIC-3, UNC-50, and UNC-74), while a C. elegans equivalent of the A. suum N-type nAChR is obtained by co-expressing the nAChR ACR-16 with the expression-enhancing cofactor RIC-3 (Boulin et al., 2008; Bennett et al., 2012). Using recombinant nAChRs expressed functionally with the aid of auxiliary proteins, the actions of agonists levamisole, pyrantel, and tribendimidine as well as the antagonist derquantel have been described for nAChRs from the important porcine nematode parasite Oesophagostomum dentatum (Buxton et al., 2014). Changes in both EC50 and maximum nAChR response were observed by adding UNC-38 and LEV-8 to the UNC-63/UNC-29 nAChR. The C. elegans L-type 2.1 (UNC-38/UNC-29/UNC-63/LEV-1/ACR-8) and 2.2 (UNC-38/UNC-29/UNC-63/ACR-8) nAChRs were employed to show that the ACR-8 subunit has the capacity to substitute for LEV-8 in the L-type nAChR when expressed in X. laevis oocytes (Blanchard et al., 2018). Binding of [3H]paraherquamide A to C. elegans membranes was displaced by phenothiazines, members of a previous generation of anthelmintics, suggesting the possibility of a similar site of action (Schaeffer et al., 1992). Omitting ACR-8 from UNC-38/UNC-29/UNC-63/ACR-8 nAChRs was shown to change the action of derquantel from competitive to noncompetitive (Buxton et al., 2014). However, the molecular mechanism underpinning the higher efficacy of these ligands for the L-type than the N-type nAChR remains unknown.
Here we have investigated the mechanism of actions of paraherquamide A on the C. elegans N-type (ACR-16) and L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChRs expressed in X. laevis oocytes to address its subtype selectivity and to examine whether it shows competitive or non-competitive actions on C. elegans nAChRs. Also, we co-crystallized paraherquamide A with the acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) to elucidate how the ligand-binding, orthosteric site interacts with the compound. We found that paraherquamide A is a much more potent antagonist of the L-type compared with the N-type C. elegans nAChR and explored structural features of the nematode nAChR subunits contributing to the L-type nAChR selectivity of the compound.
Materials and Methods
Chemicals
Paraherquamide A was purified de novo from the fermentation products of Penicillium sp. OK188 strain as follows. The Penicillium culture was inoculated with 8 kg of the okara medium and incubated at 25°C for 1 week. The fermentation products were soaked in methanol for 1 week. The methanol extract was partly concentrated and partitioned with an equal volume of ethyl acetate. The ethyl acetate layer was evaporated to yield 34.2 g of residue. The residue was dissolved in a minimal amount of methanol, and water (0.1 volume of methanol) was added to the methanol solution. The aqueous methanol solution was partitioned with hexane (equal volume of the aqueous methanol solution). Methanol was removed from the aqueous methanol layer by evaporation. The remaining aqueous solution was partitioned with ethyl acetate (equal volume of the aqueous solution), and the ethyl acetate layer was evaporated to dryness. The residue (5.06 g) was purified using silica gel column chromatography (Wakogel C-200, Wako Pure Chemical Industries, Japan) with ethyl acetate-methanol mixture [stepwise increase of methanol concentration (5%, 10%, 20%, and 50%)]. Fractions eluted by ethyl acetate containing methanol 0%, 5%, and 10% were collected and evaporated to yield 2.49 g residue, which was purified by silica gel column chromatography using a hexane–acetone mixture with stepwise increase of the acetone concentration (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%). Fractions eluted with hexane containing 50% and 60% acetone were evaporated to obtain an 800 mg residue, which was further purified by silica gel column chromatography using a chloroform–methanol mixture with stepwise increase of methanol concentration (0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 5%, 10%). Fractions eluted with chloroform containing 0.2%, 0.3%, 0.4%, and 0.5% methanol were collected, and the solvents were evaporated. The resultant solid was recrystallized in ethanol (yield, 157.6 mg), and this recrystallized material was dissolved in methanol to further purify by a preparative HPLC (VP-10 series, Shimadzu, Japan) using a Unison US-C18 column (20 × l50 mm, Imtakt, Japan) with methanol–water mixture (4:1) at a flow rate of 7.5 mL min−1 (yield, 25. 3 mg; purity was confirmed to be > 99% by HPLC; Supplemental Fig. 1).
Spectral Data of Paraherquamide A
Specific rotation ([α]D25) of paraherquamide A was determined using a SEPA-300 polarimeter (Horiba, Japan) to be = −17.3 (c = 0.2 dL−1, methanol). Electrospray ionization–mass of the compound was measured using a Q-Tof Premier (Waters, USA) with a 2-propanol/50 mM aqueous NaOH solution containing 0.5% formic acid = 9/1). The observed mass of the protonated molecular ion was 494.2660 (calculated to be 494.2665 for C28H36O5N3). The 1H- and 13C-NMR spectra were measured in CDCl3 using an Avance System, UltraShield 400 Plus (Bruker BioSpin, USA) (Supplemental Figs. 2 and 3). Chemical shifts δ (ppm) [splitting patterns and coupling constants (Hz)] of the 1H-NMR spectrum (Supplemental Fig. 2) were 0.83 (3H, s, H-23), 1.07 (3H, s, H-22), 1.41 (3H, s, H-28), 1.42 (3H, s, H-27), 1.62 (3H, s, H-17), 1.73∼1.91 (4H, m, H-10b, H-15a, H-19a, H-19b), 2.23 (1H, ddd, J = 4.7, 9.1, 10.8 Hz, H-16b), 2.35 (1H, ddd, J= 4.5, 10.8, 13.3 Hz, H-15b), 2.55 (1H, dd, J= 1.4, 11.2 Hz, H-12b), 2.63 (1H, br s, 14-OH), 2.68 (1H, d, J= 15.3 Hz, H-10a), 3.00 (1H, dd, J=1.5, 11.1 Hz, H-20), 3.03 (3H, s, H-29), 3.21 (1H, ddd, J= 4.5, 9.0, 9.1 Hz, H-16a), 3.60 (1H, d, J=11.2, H-12a), 4.87 (1H, d, J=7.7 Hz, H-25), 6.30 (1H, d, J=7.6 Hz, H-24), 6.67 (1H, d, J=8.0 Hz, H-5), 6.79 (1H, d, J=8.1 Hz, H-4), 7.62 (1H, s, H-N1). Chemical shifts δ (ppm) of the 13C-NMR spectrum (Supplemental Fig. 3) were 19.1 (C-17), 20.4 (C-22), 22.0 (C-19), 23.6 (C-23), 25.9 (C-29), 29.7 (C-28), 29.9 (C-27), 37.0 (C-10), 38.0 (C-15), 46.3 (C-21), 51.4 (C-20), 51.8 (C-16), 59.0 (C-12), 63.0 (C-3), 65.2 (C-11), 71.3 (C-13), 78.0 (C-14), 79.7 (C-26), 115.0 (C-25), 117.2 (C-5), 120.3 (C-4), 124.9 (C-9), 132.4 (C-8), 135.2 (C-7), 138.9 (C-24), 146.0 (C-6), 171.3 (C-18), 182.6 (C-2). These NMR spectral data were in agreement with those reported previously (Blanchflower et al., 1991).
ACh chloride, levamisole, and (-)-nicotine were purchased from MilliporeSigma (USA). Derquantel was purchased from Santa Cruz Biotechnology (USA). These compounds were >95% pure.
cRNA Preparation
The cRNAs encoding the C. elegans nAChR subunits and auxiliary proteins RIC-3, UNC-50 and UNC-74 were prepared using the mMESSAGE mMACHINE T7 ULTRA kit (Thermo Fisher Scientific, USA) from their cDNAs (Accession number: ACR-16, AY523511.1; UNC-38, X98600.1; UNC-29, NM_059998.4; UNC-63, AF288374.1; LEV-1, X98601.1; LEV-8, NM_077531.4; ACR-8, NM_001375112; RIC-3, NM_068898.4; UNC-50, NM_066878.3; UNC-74, accession number not determined). The cDNAs encoding the L-type nAChR subunits UNC-38, UNC-29, UNC-63, LEV-1, LEV-8, as well as those encoding the auxiliary proteins RIC-3, UNC-50, and UNC-74 were gifts from Prof. Thomas Boulin. The cRNAs were dissolved in RNase-free water and mixed at a final concentration of 50 ng µL−1 of each subunit and auxiliary protein. Then 50 nL of this RNA mixture solution was injected into X. laevis oocytes. When measuring the effects of (-)-nicotine and levamisole on the YPSCC mutant of the N-type nAChR, the RNA concentration was 500 ng µL−1 to confirm that they had no agonist action on this nAChR (see later discussion of the results).
Expression of C. elegans nAChRs in Xenopus laevis Oocytes
An ethical statement for experiments using the frogs is not required in Japan but, as a UK scientist was involved, all our experiments followed the standards of the UK legislation. Oocytes (stage V or VI) were excised from female X. laevis anesthetized by benzocaine according to the UK Animals (Scientific Procedures) Act, 1986. We also minimized the use of frogs as much as possible. After treating the excised oocytes for 15 min with 2.0 mg mL−1 collagenase (Type IA, MilliporeSigma) in Ca2+-free standard oocyte saline (SOS) containing100 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES 5.0 (pH 7.6), they were moved into SOS consisting of 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES 5.0 (pH 7.6). The follicle cell layer was removed and the cytoplasm of defolliculated oocytes was injected with 50 nL of the cRNA solution mix encoding either the N-type nAChR (ACR-16 with accessory protein RIC-3) or the L-type nAChR (UNC-38, UNC-29, UNC-63, LEV-1, LEV-8 with the accessory proteins RIC-3, UNC-74, and UNC-50) and incubated at 18°C in SOS supplemented with penicillin (100 units mL−1), streptomycin (100 μg mL−1), gentamycin (50 μg mL−1), 2.5 mM sodium pyruvate, and 4% horse serum (Thermo Fisher Scientific, #26050–070) for 2 to 5 days prior to electrophysiological experiments.
Voltage-Clamp Electrophysiology
The ACh-induced nAChR responses were recorded in SOS containing 0.5 μM atropine (SOS-A), which was added to suppress any possible endogenous muscarinic responses, at 18–23°C (Matsuda et al., 1998). Oocytes were secured in a recording chamber and perfused with SOS-A at a flow rate of 7 to 10 mL min−1. Membrane currents were recorded at a holding potential of −100 mV. The electrodes were filled with 2 M KCl and had a resistance of 0.3 to 5 MΩ in SOS-A. Signals were digitized at a frequency of 1 kHz, recorded, and analyzed using with pCLAMP software (Molecular Devices, USA). ACh was dissolved in SOS-A, while paraherquamide A was dissolved in dimethyl sulfoxide at 10 mM and then diluted with SOS-A immediately prior to experiments. DMSO concentrations in test solutions were 0.1% or lower, at which the solvent had no effect on the nAChR response to ACh and the actions of paraherquamide A. ACh was applied to oocytes for 3 to 5 s, with an interval of 3 min between applications. When testing the blocking action of paraherquamide, ACh was first applied several times until the oocyte response amplitude became stable. Then paraherquamide A was applied for 1 min prior to co-applications with ACh.
Analysis of Current Data
The amplitude of the ACh-induced current was normalized to that of the response at which it reached a plateau (Imax). The ACh concentration–response curves in the presence and absence of paraherquamide A and the paraherquamide A concentration-inhibition curves were fitted with Eq. (1) and (2), respectively, using Prism 9 (GraphPad Software, USA):
where Y is the normalized response, Imax is the normalized maximum ACh response, EC50 (M) is the half maximal effective concentration, IC50 (M) is the half inhibition concentration (M), [L] is the logarithm of the concentration of ligand (M), and nH is the Hill coefficient.
Toxicity of Paraherquamide A on C. elegans
C. elegans [Strains N2 (wild-type), RB918 acr-16 (ok789) V (N-type nAChR mutant), ZZ20 unc-38 (x20) I (L-type nAChR mutant), ZZ37 unc-63 (x37) I (L-type nAChR mutant)] were obtained from the Caenorhabditis Genetics Center. Effects of the compounds on swimming (thrashing movements) of the worms were investigated as previously reported (Ondeyka et al., 1990). pLC50 values (= −logLC50), where LC50 is the half lethal concentration (M) for paraherquamide A on the wild-type and mutant worms, were determined by nonlinear regression with Prism 9 according to Eq. (2) in which IC50 is replaced by LC50.
Crystallization of Ls-AChBP Complexed with Paraherquamide A
The wild-type Ls-AChBP was expressed in the yeast Pichia pastoris X-33 strain as described previously (Ihara et al., 2014). Secreted proteins were concentrated using a Vivaflow 200 Cross Flow Cassette (Sartorius, Germany) and purified with a Source 30Q column (Cytiva, USA). The Ls-AChBP was treated overnight at 37°C with His-tagged endo-β-N-acetylglycosidase H (Endo H, gene accession number K02182), where Endo H was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA column chromatography. The protein was further purified with a Mono Q column followed by a Superdex 200 (Cytiva). Purified Ls-AChBP was dialyzed over 20 mM Tris-HCl buffer (pH 7.5) and its concentration adjusted to 5.0 mg/mL. The Ls-AChBP complexed with paraherquamide A was crystallized by sitting drop vapor diffusion method. Crystals of the complex were flash cooled in liquid nitrogen and stored in liquid nitrogen prior to X-ray diffraction data collection.
X-Ray Crystallography
X-ray diffraction data were collected at 100 K with a RAYONIX MX225HE detector at BL26B1 located at SPring-8, the third-generation synchrotron facility in Harima Science Park City, Hyogo, Japan. Diffraction data were processed using the Aimless (CCP4: supported program) (Winn et al., 2011; Evans and Murshudov, 2013) along with XDS (Kabsch, 2010). The initial phase was obtained by molecular replacement with PHASER (McCoy, 2007) using a protein coordinate: 2ZJU. Refinement of the structure model was performed using Refmac5 (Murshudov et al., 2011), and manual model building was performed with Coot (Emsley and Cowtan, 2004). Two-dimensional figures and cartoon/stick models of the crystal structure of the Ls-AChBP-paraherquamide A complex were illustrated by LIGPLOT (Wallace et al., 1995) and PyMoL (Schrödinger, USA), respectively.
Modeling Proteins in Complex with Ligands
Prior to modeling, structure coordinates of paraherquamide A and derquantel were prepared using Chem3D software and AutoDock Tools 1.5.7. Derquantel was docked into Ls-AChBP after removing water and paraherquamide A from the X-ray crystal structure of the protein complexed with paraherquamide A. For modeling the ACR-16 protein complexed with paraherquamide A, ACR-16 was aligned with Ls-AChBP using MAFFT 7.308 (Katoh et al., 2002; Katoh and Standley, 2013). Then the aligned region corresponding to amino acid number 23 − 228 of C. elegans ACR-16 was modeled with MODELER 10.1 using the AutoModel algorithm (Webb and Sali, 2016). Paraherquamide A was docked into the homology model of wild-type ACR-16 using AutoDock Vina 1.1.2 (Trott and Olson, 2010).
Statistical Analysis
Differences of the means were analyzed by parametric methods [t test (two-tailed) or one-way ANOVA (Dunnett test)] in which post hoc tests were conducted only when F values were <0.05. The difference compared was judged significant at P < 0.05 level.
Results
Actions of Paraherquamide A on N-type and L-type C. elegans nAChRs
First we tested paraherquamide A alone on oocytes expressing either the N-type (ACR-16) or the L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) C. elegans nAChRs. Paraherquamide A had no detectable effect on the membrane currents when applied at 10 μM (Fig. 1, B and C), indicating that it had no detectable agonist action on either receptor at this concentration. Next we perfused nAChR-expressing oocytes with paraherquamide A (N-type, 10 μM; L-type, 100 nM) for 1 min prior to applying ACh at 100 μM. These paraherquamide A exposures reduced the ACh response of N-type and L-type nAChRs by 31.6 [95% confidence interval (CI)]: 14.5–48.8) and 76.4 (95% CI, 68.0–84.8), respectively (Fig. 1, B and C). We evaluated the antagonist potency of paraherquamide A by treating oocytes expressing the N-type nAChR with the alkaloid for 1 minute and then co-applying ACh at 10 μM, which is close to the EC50 for ACh (Table 1). Paraherquamide A reduced the peak ACh response of the N-type nACh with a pIC50 [= −logIC50 (M)] of 4.84 (95% CI, 4.76 − 4.91) (Fig. 1D; Table 1).
Agonist actions of acetylcholine and antagonist actions of paraherquamide A on acetylcholine for the Caenorhabditis elegans N-type and L-type nAChRs expressed in Xenopus laevis oocytesa
In a similar way, we also treated oocytes expressing the L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChR with paraherquamide A for 1 minute prior to co-application with 30 μM ACh. Paraherquamide A also reduced the peak current amplitude of the ACh response of the L-type nAChR with a pIC50 of 7.58 (95% CI, 7.51 − 7.64) (Fig. 1E; Table 1). The nAChR blocking potency measured as pIC50 was higher for the L-type nAChR compared with the N-type nAChR. Derquantel also blocked the ACh-responses of the N-type (ACR-16) and L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChRs [N-type, pIC50 values of 4.76 (95% CI, 4.63 − 4.89)]; L-type 6.03 (95% CI, 5.94 − 6.11) with preference for L-type over N-type nAChRs (Supplemental Fig. 4). Since paraherquamide A was 35.5-fold more potent on the L-type nAChR than derquantel, we focused on the mechanism of selectivity of paraherquamide A to the C. elegans L-type over N-type nAChRs.
Prior to investigating the effects of paraherquamide A, we first measured the concentration-response curves for ACh for the N-type (ACR-16) and L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChRs. ACh activated the N-type nAChR with pEC50 (= −logEC50) of 4.67 (95% CI, 4.63 − 4.70) (Fig. 1F; Table 1), whereas it activated the L-type nAChR with pEC50 of 4.39 (95% CI, 4.34 − 4.44) (Fig. 1G; Table 1). These data are comparable to those reported previously (Boulin et al., 2008). We then investigated the effects of 10 μM paraherquamide A on the ACh concentration-response curve for the N-type nAChR. The compound shifted the ACh concentration-response curve to the right [pEC50 = 4.35 (95% CI, 4.18 − 4.52)], P < 0.05, two-tailed t test) and reduced the amplitude of the normalized maximum response [Imax = 0.453 (95% CI 0.395 − 0.510), P < 0.05, two-tailed t test] (Fig. 1F), suggesting mixed competitive and noncompetitive interactions with the N-type nAChR. On the other hand, 100 nM paraherquamide A reduced the maximum response of the L-type nAChR [Imax = 0.300 (95% CI, 0.190 − 0.409), P < 0.05, two-tailed t test], while hardly shifting pEC50 [4.27 (95% CI, 3.75 − 4.79)] (Fig. 1G), indicating noncompetitive interactions with the L-type nAChR.
To examine the role of the L-type nAChR subunits UNC-38, UNC-29, UNC-63, LEV-1, and LEV-8 in determining paraherquamide A sensitivity, we tested the compound on the L-type 2.1 (UNC-38/UNC-29/UNC-63/LEV-1/ACR-8) and 2.2 (UNC-38/UNC-29/UNC-63/ACR-8) nAChRs. Replacing the LEV-8 subunit by the ACR-8 subunit had a minimal impact on the concentration-response curve for ACh (Fig. 1H). However, omitting the LEV-1 subunit shifted it (Fig. 1I). The 2.1 nAChR showed lower paraherquamide A sensitivity than the L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChR but > 100-fold higher sensitivity than the N-type (ACR-16) nAChR (Fig. 1J; Table 1). Omitting the LEV-1 subunit from the 2.2 nAChR had a minimal impact on the paraherquamide A sensitivity (Fig. 1K; Table 1).
Actions of Paraherquamide A on Wild-type and Mutant C. elegans
We investigated the effects of paraherquamide A on thrashing of wild-type and mutant C. elegans to confirm its selectivity for the L-type nACR in vivo. The compound suppressed thrashing of the wild-type worms with pLD50 of 5.02 (95% CI, 4.91 − 5.12) (Fig. 2A). Paraherquamide A inhibited thrashing of C. elegans with a mutation in the ACR-16 subunit [RB918 acr-16 (ok789) V] with pLC50 of 5.61 (95% CI, 5.52 − 5.69) (Fig. 2B), showing higher paraherquamide A sensitivity than the wild-type worms. We failed to determine the activity on the UNC-38 mutant [ZZ20 unc-38 (x20) I] because of its considerably reduced motility even in the absence of the compound, although we could measure activity on worms with a mutation in the unc-63 gene [ZZ37 unc-63 (x37) I]. The UNC-63 mutant worms were highly resistant to paraherquamide A (Fig. 2C), confirming a preference of the compound for L-type over N-type AChRs.
Actions of paraherquamide A on motility of wild-type and mutant C. elegans. (A) Wild-type (N2). (B) Mutant (RB918 acr-16 (ok789) V). (C) Mutant (ZZ37 unc-63 (x37) I). Each data plot represents the mean ± S.E.M. (n = 5).
X-Ray Crystal Structures of Ls-AChBP in Complex With Paraherquamide A
The AChBPs have been used to investigate the mechanism of action of various ligands interacting with the orthosteric sites of nAChRs. Having observed the competitive paraherquamide A interactions with ACh at the N-type nAChR (Fig. 1F), we co-crystallized paraherquamide A with the Ls-AChBP. When crystallized in 14.1% to 15.6% PEG 4000 in sodium citrate buffer (pH 5.0) at 20°C, the Ls-AChBP-paraherquamide A complex resulted in a crystal with a space group of P65 diffracting at a resolution of 2.2 Å (Table 2). We therefore refined its crystal structure with the amino acid sequence shown in Fig. 3A. The electron densities for the ligand were observed at all five interfaces between protomers (Fig. 3B; see Supplemental Fig. 5 for detailed omit maps). Paraherquamide A interacted with the Ls-AChBP at ligand binding loops A, B, C, D, E and F (Fig. 3C). The Ser186 and main chain of Cys187 in loop C, as well as the hydroxy group of Tyr164 in loop F formed hydrogen bonds via a water with the carbonyl group in the amide bond of paraherquamide A (Fig. 3, C and D). The mainchain of Trp143 in loop B formed a hydrogen bond with the bridgehead nitrogen of paraherquamide A (Fig. 3C, arrowed). Tyr192 in loop C and the main chain carbonyl of Trp143 in loop B formed hydrogen bonds with the hydroxy group of paraherquamide A (Supplemental Fig. 6). Trp53 (loop D), Tyr89 (loop A), Met114 (loop E), Tyr185 (loop C), and Tyr192 (loop C) made hydrophobic contacts with the compound (Fig. 3, C and E). In loop C, UNC-38, UNC-63, and LEV-8 of the L-type nAChR have a proline, while ACR-16 N-type nAChR subunit has an aspartate (Fig. 3A). In loop E, UNC-38 of the L-type nAChR has Glu142, which corresponds to Met114 in the Ls-AChBP (Fig. 3A), and has a negative charge favorable for interactions with a nitrogen of paraherquamide A (Fig. 3, C and E), whereas ACR-16 has Val138 with no negative charge at the corresponding position.
Crystal structure of Ls-AChBP complexed with paraherquamide A. (A) Amino sequences of the N-terminal domain of C. elegans nAChR subunits and the Ls-AChBP. Positions of amino acids unique to the L-type nAChR subunits are indicated by ▼ (B) Side view of the complex in which paraherquamide A molecules bound at five subunit interfaces. Paraherquamide A is shown with electron density maps (omit map at contour level of root mean square deviation 3.0). (C) LigPlot of paraherquamide A interactions with amino acid residues or main chains in the Ls-AChBP. Carbons, nitrogens, oxygens, and sulfur are colored black, blue, red, and yellow, respectively, while water is colored cyan. Tyr89 (loop A), Trp53 (loop D), and Tyr192 (loop C) made hydrophobic contacts. Tyr164 (loop F), Ser186 (loop C), and the main chain nitrogen of Cys187 (loop C) formed hydrogen bonds with a carbonyl oxygen in paraherquamide A. Dashed lines indicate hydrogen bonds. (D, E) Close views of the interactions of the Ls-AChBP with paraherquamide A. (D) Hydrogen bonds shown by dashed lines were observed between Tyr164, Ser186, the main chain of Cys187, and one of the carbonyl groups in paraherquamide A via water. (E) Met114 (loop E) located in the vicinity of the bridgehead nitrogen arrowed in paraherquamide A. The principal and complementary chains are colored green and cyan, respectively, while carbons, nitrogens, oxygens, sulfurs, and water are colored gray, blue, red, yellow, and sky blue, respectively. The bridgehead nitrogen is arrowed.
X-ray diffraction data for the Ls-AChBP-paraherquamide A complex
The Effects of Mutations in Loop C and E of N- and L-type nAChRs on the Actions of Paraherquamide A, (-)-Nicotine, and Levamisole
To examine whether loop C interacts with paraherquamide A as in the crystal structure of the Ls-AChBP complex, we investigated the effects of replacing its YDCC sequence in loop C of ACR-16 (N-type nAChR) by the YPSCC sequence of UNC-38 (L-type nAChR) on paraherquamide A actions (see Fig. 3A for amino acid sequence comparisons). Switching this loop C segment lowered the pEC50 value of ACh to 4.01 (95% CI, 3.95 − 4.06) (Fig. 4A; Table 1). The pIC50 of paraherquamide A determined in terms of reduction of the response to 100 µM ACh was increased significantly by the mutation to 5.28 (95% CI, 5.20 − 5.36) (Fig. 4B; Table 1). A reciprocal loop C switch in the UNC-38 of the L-type nAChR, replacing the YPSCC sequence by the YDCC sequence of the N-type (AChR-16) nAChR subunit, had a minimal effect on the agonist potency of ACh (Fig. 4C; Table 1). However, the mutation reduced the antagonist potency of paraherquamide A to 7.37 (95% CI, 7.28 − 7.46) (Fig. 4D; Table 1). Further, loop C of ACR-16 was mutated from YDCC to YPCC, as seen in UNC-63, resulting in enhanced paraherquamide A sensitivity to pIC50 of 5.54 (95% CI, 5.43 − 5.65) (Fig. 4B; Table 1). In contrast, replacing the loop C of UNC-63 L-type nAChR subunit from YPCC by YDCC seen in AChR-16 subunit reduced sensitivity [pIC50 = 7.31 (95% CI, 7.23 − 7.38), Fig. 4D; Table 1].
Effects of mutations in loop C and loop E on agonist actions of ACh and antagonist actions of paraherquamide A on the ACh-induced response of the C. elegans N-type and L-type nAChRs expressed X. laevis oocytes. (A and B) Effects of the mutations in loop C on the actions of ACh (A) and paraherquamide A (B) on the N-type nAChR. (C and D) Effects of the mutations in loop C on the actions of ACh (C) and paraherquamide A (D) on the L-type nAChR. (E and F) Effects of the mutations in loop E on the actions of ACh (E) and paraherquamide A (F) on the N-type nAChR. (G and H) Effects of the mutations in loop E on the actions of ACh (G) and paraherquamide A (H) on the L-type nAChR. Each plotted point is mean ± S.E.M. (n = 5). Each data plot indicates the mean ± S.E.M. (n = 5). ACh concentrations: For wild-type N-type nAChRs, 10 µM; for YPSCC mutant N-type nAChR,100 μM; for V138E mutant N-type nAChR and YPCC mutant N-type nAChR, 500 μM; for wild-type and mutant L-type nAChRs, 30 μM.
As described previously, Met114 in loop E was in the vicinity of the bridgehead nitrogen of paraherquamide A in the crystal structure of the Ls-AChBP (Fig. 3E). Since Met114 corresponds to Val138 and Glu142 in the ACR-16 (N-type nAChR) and UNC-38 (L-type nAChR) subunits, respectively (Fig. 3A), Val138 was mutated to glutamate in the ACR-16 subunit. As a result, the antagonist potency in terms of pIC50 of paraherquamide A on the N-type nAChR was increased to 5.21 (95% CI, 5.11 − 5.31), while the agonist potency, monitored as pEC50 of ACh, was reduced to 3.24 (95% CI, 3.16 − 3.33) (Fig. 4, E and F; Table 1). By contrast, an inverse mutation E142V in the UNC-38 subunit reduced the antagonist potency of paraherquamide A on the L-type nAChR [pIC50 = 7.15 (95% CI, 7.03 − 7.27)], while scarcely influencing the agonist potency of ACh (Fig. 4, G and H; Table 1).
We also tested the effects of these mutations on the agonist actions of (-)-nicotine and levamisole and on the antagonist actions of paraherquamide A in response to these ligands for both the N-type and L-type nAChRs (Fig. 5). (-)-Nicotine activated the wild-type N-type nAChR with pEC50 of 4.73 (95% CI, 4.64 − 4.83) (Fig. 5A; Supplemental Fig. 7A), and paraherquamide A inhibited the nicotine-induced response with pIC50 of 5.48 (95% CI, 5.35 − 5.61) (Supplemental Fig. 7B). However, all the mutations tested abolished the agonist action of (-)-nicotine on the N-type (ACR-16) nAChR (Fig. 5A) and did not make it an agonist of the L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChR (Fig. 5B).
Effects of mutations in loop C and loop E on the actions of (-)-nicotine and levamisole on the C. elegans N- and L-type nAChRs expressed X. laevis oocytes and antagonist actions of paraherquamide A on the levamisole induced response of the L-type nAChR. (A) Actions of (-)-nicotine on the wild type and mutant N-type nAChRs. (B) Actions of (-)-nicotine on the wild type and mutant L-type nAChRs. (C) Actions of levamisole on the on the wild-type and mutant N-type nAChRs. (D) Actions of levamisole on the wild-type and mutant L-type AChRs. (E, F) Effects of mutations in loop C (E) and loop E (F) on the agonist action of levamisole on the L-type nAChR. (G, H) Effects of mutations in loop C (G) and loop E (H) on the antagonist action of paraherquamide A for the levamisole-induced response of the L-type nAChRs. The antagonist potency of paraherquamide A was determined for the nAChR response to 10 μM levamisole. In (E), (F), (G), and (H), each plot shows the mean ± S.E.M. (n = 5).
Levamisole did not activate the wild-type or the mutant N-type nAChRs (Fig. 5C) while activating not only the wild-type but also the mutant L-type nAChRs with no significant difference in pEC50 (Fig. 5, D−H). However, the YDCC mutation in loop C of UNC-63 and the E142V mutation in loop E of UNC-38 reduced Imax (Fig. 5, E and F; Supplemental Table 1). The mutations tested hardly affected pIC50 of paraherquamide A on the response to levamisole (Fig. 5, G and H).
The effects of the mutations in loop C and loop E on IC50 values for paraherquamide A acting on the L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChR, albeit significant, were not as large as those observed for the corresponding mutations in the N-type (ACR-16) nAChR (Fig. 4; Table 1). Therefore, it is conceivable that other structural features may also underlie the L-type nAChR selectivity of the compound. Referring to the homology model of the N-type nAChRs in complex with paraherquamide A (Fig. 6), we postulated that Asp210 in loop C and Phe184 in loop F, both of which are only seen in the ACR-16 subunit (Fig. 3A), may contact each other, indirectly interrupting loop C−paraherquamide A interactions. No such steric interaction occurs between the proline residue in the α subunits and valine residues in the non-α subunits (UNC-29, LEV-1) in the L-type nAChR (Fig. 3A), strengthening interactions with the alkaloid. To test this hypothesis, we mutated Phe184 to valine in loop F of ACR-16 and measured the blocking potency of paraherquamide A on the mutant N-type nAChR (Fig. 7). We found that the mutation hardly affected the potency of ACh, while increasing pIC50 of paraherquamide A for the N-type nAChR (Fig. 7; Table 1). By contrast, both the V193F mutation in UNC-29 and the V201F mutation in LEV-1 reduced the blocking potency of paraherquamide A (Fig. 7; Table 1). On the other hand, the F184V mutation in the N-type nAChR and the V193F mutation (UNC-29) and V201F mutation (LEV-1) in the L-type nAChR had a limited impact on the agonist activity of (-)-nicotine and levamisole, respectively (Supplemental Fig. 8).
Homology model of the C. elegans wild-type N-type nAChR in complex with paraherquamide A. The main chain in the principal and complementary side of the N-type nAChRs are colored green and cyan, respectively, while carbons, nitrogens, oxygens, and sulfurs are colored gray, blue, red, and yellow, respectively. In paraherquamide A, carbons, nitrogens, and oxygens are colored gray, blue, and red, respectively.
Effects of mutations in loop F on agonist actions of ACh and antagonist actions of paraherquamide A on the Ach-induced responses of C. elegans N-type and L-type nAChRs expressed X. laevis oocytes. Each plotted point is mean ± S.E.M. (n = 5). (A, B) Effects of F184V mutation in loop F of the ACR-16 subunit on the actions of ACh (A) and paraherquamide A (B) on the N-type nAChR. (C, D) Effects of V193F and F201F mutations in loop F of the UNC-29 and LEV-1 subunits, respectively, on the actions of ACh (C) and paraherquamide A (D) on the L-type nAChR. Each plot represents the mean ± S.E.M. (n = 5). In B and D, antagonist actions of paraherquamide A were determined for the responses to 10 μM ACh of the N-type nAChRs and 30 μM ACh of the L-type nAChRs.
Discussion
We have investigated the antagonist actions of pararherquamide A on recombinant C. elegans N-type (ACR-16) and L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChRs expressed in Xenopus laevis oocytes. The L-type nAChR showed a higher paraherquamide A sensitivity compared with the N-type nAChR with a difference in pIC50 of 2.74 (an approximately 550-fold change in IC50; Fig. 1, D and E; Table 1), consistent with findings for the A. suum N- and L- type nAChRs (Robertson et al., 2002). Also, the tests of the compound on the L-type 2.1 and 2.2 nAChRs (Fig. 1, J and K) and the wild-type and mutant C. elegans worms (Fig. 2) suggest that the higher paraherquamide sensitivity of the L-type nAChR compared with the N-type nAChR appears to hold for nAChRs of a parasitic (A. suum) and a free-living (C. elegans) nematode.
Paraherquamide A interacted competitively with ACh on the N-type nAChRs of C. elegans (Fig. 1F), as is the case of native A. suum nAChRs (Robertson et al., 2002), suggesting that the compound binds to the orthosteric site. Its noncompetitive antagonist action on the C. elegans L-type nAChR does not exclude interactions with the orthosteric site, because such an action can result from binding to a distinct orthosteric site from that to which ACh is bound among the orthosteric α subunit/α subunit and α subunit/non-α subunit interfaces in the L-type nAChR. Alternatively, paraherquamide A may lock the nAChR to an inactive state and prevent its activation by ACh, resulting in an apparent noncompetitive interaction as in the case of the α7 nAChR interactions with α-bungarotoxin, where the toxin allosterically inhibits the ACh-induced activation of the nAChR even though both ligands share the orthosteric site (daCosta et al., 2015).
Given the similarity to the orthosteric site of the N-type (ACR-16) nAChR in forming a homo-pentameric structure and the competitive interaction of the ACR-16 homomer with paraherquamide A, Ls-AChBP was used as a nAChR ligand binding domain surrogate for co-crystallization studies with the fungal alkaloid paraherquamide A. The crystal structure showed that hydrophobic interactions as well as cation-π interactions with aromatic amino acid residues appeared to play a major role in the binding of paraherquamide A to Ls-AChBP (Fig. 3C). However, amino acids involved in such interactions (Trp53 in loop D, Tyr89 at loop A, Trp143 in Loop B, and Tyr185 and Tyr192 in loop C) are conserved through the ACR-16, UNC-38, UNC-29, UNC-63, LEV-8, and LEV-1 subunits (Fig. 3A). Therefore, such interactions might underpin the L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChR selectivity only when the overall conformation of the orthosteric site is the dominant determinant of the affinity of the compound.
Paraherquamide A interacts via water with the hydroxy group of Ser186 and the main chain of Cys187 in loop C as well as the hydroxy group of Tyr164 in loop F in the crystal structure (Fig. 3, C and D). In accord with this finding, exchanging loop C between the ACR-16 (N-type nAChR) and UNC-38 (L-type nAChR) or UNC-63 subunits (L-type nAChR) led to a change of paraherquamide A potency on the N- and L-type nAChRs (Fig. 4, B and D), suggesting a contribution of the proline in loop C to determining the paraherquamide A actions. The V138E mutation in ACR-16 and E142V mutation in UNC-38 respectively enhanced and reduced the blocking potency of paraherquamide A on the N-type and L-type nAChRs (Fig. 4, F and H), demonstrating that the bridgehead nitrogen of paraherquamide A, when protonated, interacts electrostatically with the negatively charged glutamate in loop E of the UNC-38 subunit in the L-type nAChR, thereby strengthening the binding of the ligand. Derquantel was much less potent than paraherquamide A on the C. elegans L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8) nAChR (Supplemental Fig. 4) in accordance with the homology model of the Ls-AChBP in complex with the compound, where the lack of the carbonyl group resulted in a loss of hydrogen bonds with loop C and loop F (Supplemental Fig. 9).
The model of the N-type nAChR in complex with paraherquamide A (Fig. 6) indicated that Phe184 in loop F of ACR-16 may prevent the compound interactions with the orthosteric site by steric contacts with Asp210. Hence, we examined the effects of the F184V mutation in the ACR-16 subunit of the N-type nAChR and inverse mutations of the corresponding valines in the UNC-29 and LEV-1 subunits (V193F mutation in UNC-29 and V201F mutation in LEV-1) in the L-type nAChR on the blocking potency of paraherquamide A. We found that the F184V mutation in ACR-16 increased pIC50, while the V193F mutation in UNC-29 and V201F mutation in LEV-1 decreased it (Fig. 7; Table 1), supporting our hypothesis. It is therefore conceivable that loop C, loop E, and loop F cooperatively determine the antagonist actions of the compound.
We investigated the actions of (-)-nicotine and levamisole on the wild-type and mutant N-type and L-type nAChRs (Fig. 5; Supplemental Fig. 8). All the mutations in loop C and E tested abolished the agonist activity of (-)-nicotine on the N-type nAChR (Fig. 5, A and B), while the mutation in loop F had no clear impact on the agonist activity of the compound (Supplemental Fig. 8A). On the other hand, none of the mutations in loop C and loop E of the UNC-38 and UNC-63 subunits as well as of the mutations in loop F of the UNC-29 and LEV-1 enabled activation by (-)-nicotine of the L-type nAChR (Fig. 5B; Supplemental Fig. 8B). Also, all the mutations of the N-type nAChRs failed to make levamisole an agonist (Fig. 5C), and the mutations of the L-type nAChR had a limited impact on the agonist activity of levamisole and the antagonist potency paraherquamide A for the agonist action of levamisole (Fig. 5, D–H; Supplemental Fig. 8B; Table S1), suggesting differences in the modes of actions at the orthosteric site between (-)-nicotine, levamisole, and paraherquamide A.
In conclusion, we have co-crystallized the Ls-AChBP with paraherquamide A to elucidate determinants underpinning the L-type nAChR selectivity of the anthelmintic compound. We have shown for the first time that structural features of loop C, loop E, and loop F account for the L-type nAChR selectivity of paraherquamide A. Although other features, notably interactions either with noncompetitive site or differential interactions with α/α versus α/non-α subunit interfaces, cannot be ruled out from the mechanism of selectivity, the results offer new insights into the mode of action of paraherquamide A and a platform to assist in the design of new drugs targeting cholinergic neurotransmission of parasitic nematodes.
Acknowledgments
The authors are indebted to Steven Buckingham, School of Biological & Chemical Sciences, Queen Mary University of London, for advice on oocyte expression. The authors acknowledge Thomas Boulin for the gift of the cDNAs of C. elegans L-type nAChR subunits and auxiliary proteins RIC-3, UNC-50, and UNC-74. The synchrotron radiation experiments were performed at the BL26B1 of SPring-8, with approvals of the Japan Synchrotron Radiation Research Institute (proposal numbers 2017A2514, 2018A2566).
Note Added in Proof: The author corrected the Imax data for (-)-Nicotine to 3 decimal places in Supplemental Table 1 in the Fast Forward version published March 22, 2023. Supplemental Table 1 has now been corrected.
Authorship Contributions
Participated in research design: Koizumi, Otsubo, Furutani, Niki, Ihara, Sattelle, Matsuda.
Conducted experiments: Koizumi, Otsubo, Furutani, Niki, Takayama, Fujimura, Maekawa, Koyari, Ihara, Kai, Hayashi, Ali, Kage-Nakadai, Matsuda.
Performed data analysis: Koizumi, Otsubo, Furutani, Niki, Koyari, Ihara, Matsuda.
Wrote or contributed to the writing of the manuscript: Koizumi, Otsubo, Furutani, Niki, Ihara, Kai, Hayashi, Kage-Nakadai, Sattelle, Matsuda.
Footnotes
- Received July 29, 2022.
- Accepted January 27, 2023.
K.M. and M.I. were supported by Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science [Grant 21H04718 (K.M.); 22H02350 (M.I.)]. D.B.S. was supported by an MRC Programme grant [Grant MR/N024842/1] on which he is co-principal investigator.
No author has an actual or perceived conflict of interest with the contents of this article.
↵*These authors contributed equally to this study.
↵
This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- ACh
- acetylcholine
- AChBP
- acetylcholine binding protein
- CI
- confidence interval
- nAChR
- nicotinic acetylcholine receptor
- SOS
- standard oocyte saline
- SOS-A
- SOS containing 0.5 μM atropine
- Copyright © 2023 by The Author(s)
This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.