Molecular Basis for Allosteric Inhibition of Acid-Sensing Ion Channel

Article pubs.acs.org/jmc

Molecular Basis for Allosteric Inhibition of Acid-Sensing Ion Channel 1a by Ibuprofen Timothy Lynagh,† José Luis Romero-Rojo,† Camilla Lund, and Stephan A. Pless* Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 160, 2100 Copenhagen, Denmark S Supporting Information *

ABSTRACT: A growing body of evidence links certain aspects of nonsteroidal antiinflammatory drug (NSAID) pharmacology with acid-sensing ion channels (ASICs), a small family of excitatory neurotransmitter receptors implicated in pain and neuroinflammation. The molecular basis of NSAID inhibition of ASICs has remained unknown, hindering the exploration of this line of therapy. Here, we characterized the mechanism of inhibition, explored the molecular determinants of sensitivity, and sought to establish informative structure−activity relationships, using electrophysiology, site-directed mutagenesis, and voltageclamp fluorometry. Our results show that ibuprofen is an allosteric inhibitor of ASIC1a, which binds to a crucial site in the agonist transduction pathway and causes conformational changes that oppose channel activation. Ibuprofen inhibits several ASIC subtypes, but certain ibuprofen derivatives show some selectivity for ASIC1a over ASIC2a and vice versa. These results thus define the NSAID/ASIC interaction and pave the way for small-molecule drug design targeting pain and inflammation.

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INTRODUCTION A hallmark of neuronal function is rapid electrical signaling in response to synaptic activity or other changes in extracellular environment. This is achieved by ligand-gated ion channels, membrane proteins with an extracellular ligand-sensing domain and an intrinsic membrane-spanning ion channel. In the central nervous system, even subtle increases in the extracellular concentration of protons constitute sensory information that is converted into excitatory neuronal activity by acid-sensing ion channels (ASICs).1−3 ASICs show a predominantly neuronal distribution and mediate a variety of central and peripheral physiological responses, some of which are attributed to different combinations of subunits (ASIC1a, -1b, -2a, -2b, and -3) into trimeric complexes.4 In the hippocampus and the amygdala, protons are synaptically released, triggering ASIC1a activation, which enhances depolarization and contributes to synaptic plasticity and learning.1,5,6 In numerous other scenarios, however, ASIC1a activation is intricately linked to negative experiences, both physiological and pathophysiological. For example, evidence suggests that ischemia involves enhanced ASIC1a activation,7,8 and pharmacological inhibition and genetic disruption of ASIC1a are neuroprotective against ischemic injury.8,9 ASIC1a is also implicated in pain. Painful responses to acid on human skin involve ASICs,10 and activation of ASIC1a by peripherally injected snake toxin MitTx rapidly induces pain in mice,11 implicating ASIC1a in early, sensory aspects of pain. ASIC1a is also up-regulated in inflammatory and chronic pain, 12,13 and these central aspects of pain can be pharmacologically reduced by venom toxins that selectively inhibit ASIC1a.14,15 Given this role of ASIC1a in inflammation © 2017 American Chemical Society

and pain, it is remarkable that perhaps the most common antiinflammatories, nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, were shown to inhibit ASIC1a, reduce painful responses to acid, diminish inflammation, and exert neuroprotection.13,16,17 Although small-molecule inhibitors of ASICs are generally less potent and subtype-selective than venom toxin inhibitors,18 the suitability of small-molecules for targeting central mechanisms of pain and/or neuroinflammation is undeniable.19 Together with the promising pharmacological outcomes of ASIC1a inhibition, this calls for a thorough understanding of ASIC inhibition by NSAIDs. At present, our knowledge of both the mechanism of inhibition and the potency at different ASIC subtypes is scant, hindering both the potential development of therapeutics and the understanding of intrinsic ASIC function. In the present study, we therefore sought to establish the mechanism of ibuprofen inhibition of ASIC1a, the structure− activity relationships underlying inhibitory potency, and the ibuprofen binding site in ASIC1a.

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RESULTS Allosteric Inhibition of ASIC1a by Ibuprofen. We expressed rat ASIC1a in Xenopus laevis oocytes and measured the effects of ibuprofen (1, (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid) on proton-gated currents using twoelectrode voltage clamp. Co-application of 1 (1 mM) with pH 6.7 (a 50% effective concentration, or “pH50”, at ASIC1a) caused 92 ± 2% inhibition in peak current amplitude that was Received: July 21, 2017 Published: September 26, 2017 8192

DOI: 10.1021/acs.jmedchem.7b01072 J. Med. Chem. 2017, 60, 8192−8200

Journal of Medicinal Chemistry

Article

current amplitude was through enhanced desensitization of channels during activation or through trapping of channels in a desensitized state and slowing recovery to the resting state. However, 1 caused no change in the rate of current decay during prolonged proton application (Figure 1C) and caused no delay in recovery from desensitization (Figure 1D), arguing against those possibilities. Very small increases in proton concentration, without activating ostensible current, decrease subsequent responses to activating proton concentrations at ASIC1a, a phenomenon called steady-state desensitization (SSD). ASIC1a inhibitors that enhance SSD, e.g., PcTx1, cause a shift in the proton concentration−response relationship for SSD to more basic pH.22 We observed that 1 caused no such change in ASIC1a SSD (Figure 1E, Supporting Information, Figure S1B), indicating that 1 does not enhance SSD. However, we observed that 1 caused a significant acidic shift in the proton concentration−response relationship for activation (Figure 1F, Supporting Information, Figure S1C), with a significant change in pH50, from 6.81 ± 0.01 to 6.60 ± 0.06 (t(15) = 3.19; p = 0.006) and cooperativity coefficient, from 7.5 ± 1.1 to 2.7 ± 0.7 (t(15) = 3.89; p = 0.002). These observations are consistent with an allosteric effect of 1 on ASIC1a transitions between closed and open states,23 where in the presence of 1, additional agonist (decrease in pH) is required to achieve maximal activation. Reciprocally, the presence of agonist should hinder the activity of the allosteric inhibitor, and we tested this by measuring inhibition by 1 in different activating pH (Figure 1G, Supporting Information, Figure S1D). Indeed, 1 inhibited pH 6.9-gated currents with a pIC50 value of 3.87 ± 0.11 (EC50, 135 μM), and this was significantly decreased to 3.53 ± 0.05 (295 μM) at pH 6.7 and 3.29 ± 0.08 (513 μM) at pH 6.4 (Supporting Information, Figure S1E). This trend continued down to pH 4.5, at which inhibition was practically abolished (Figure 1G). Although this trend was clear even above pH 6.0 and estimates of 1 pKa are generally below 5.0,24−26 we considered the possibility that the decreased inhibitory potency of 1 at increased agonist concentrations (lower pH) could be due to protonation of its carboxylate moiety rather than through inability to inhibit activation by increased agonist concentrations. To verify that the decreases in ibuprofen potency with increased proton concentration were indeed due to increased agonist concentration, we tested the inhibitory potency of an analogue with a lower pK a , where increased proton concentrations should be unlikely to protonate the more acidic carboxylate. 2-Fluoro-[4-(2-methylpropyl)phenyl]propanoic acid (2) has an estimated pKa of well below 4.0, considering propanoic acid (present in 1) pKa = 4.87 and 2-fluoroacetic acid (present in 2) pKa = 2.59.27 We observed that the inhibitory potency of this drug decreased progressively in the pH range from 6.9 to 5.5 much like 1 (Figure 1H, Supporting Information, Figure S1D,E), suggesting that the decreased potency of 1 in low pH is indeed due to the presence of additional agonist preventing the inhibitory effects of 1. Together, these experiments show that 1 allosterically inhibits ASIC1a activation by binding to a site outside of the membrane-spanning domain of ASIC1a. Identification of an Ibuprofen Binding Site. To dissect the interactions between 1 and ASIC1a, we next turned to structure−activity relationships. We first considered the carboxylic acidic moiety of the drug, as it is a common feature of the NSAIDs shown to inhibit ASIC1a,13 and when inhibiting

rapidly reversible (n = 6; Figure 1A). In contrast, 30 s application of 1 immediately before pH activation caused no

Figure 1. Characteristics of ASIC1a inhibition by ibuprofen (1). (A) Example (left) and averaged (right, n = 4) pH50-gated currents during co-application (Co.) or after preapplication (Pre.) of 1 (1 mM). As in all subsequent panels, data are mean ± SEM. (B) Inhibition of protongated currents at different membrane potentials by 1 (1 mM; n = 6) or 300 μM amiloride (Ami, n = 5) at indicated holding potentials (mV). * P < 0.05 in one-way ANOVA with Tukey’s test. (C) Time constant (τ) for desensitization of proton-gated current in the absence (black) or presence (blue) of 1 (400 μM). Dashed line indicates segment for exponential fitting. (D) Recovery of proton-gated current amplitude from desensitization in the absence of 1 (400 μM), in the presence of 1 during recovery, or in the presence of 1 throughout both proton application and recovery (n = 4). (E) Steady-state desensitization measured with pH 5.5-gated currents in the absence (n = 4) or presence (n = 6) of 1 (400 μM) during 30 s conditioning at various pH (indicated by bars, inset). (F) Proton concentration−response relationships in the absence or presence of 1 (400 μM; n = 8−9). Gray scale bars: x, 30 s (except for (C) as indicated); y, 2 μA. (G,H) Inhibition by 1 (G) or 2 (H) of ASIC1a currents gated by different pH. This figure relates to Supporting Information, Figure S1.

significant inhibition (n = 7; Figure 1A). Similarly, combined pre- and coapplication of 1 (300 μM) caused no greater inhibition than co-application only (Figure S1A). These results are consistent with previous 1/ASIC1a experiments13,20 and provide tentative evidence that 1 has a fast off-rate and does not modulate after partitioning into the membrane, unlike other similarly hydrophobic ion channel modulators.21 The level of inhibition did not differ at membrane potentials of −60, −20, or 20 mV, in stark contrast to the ASIC pore blocker amiloride (Figure 1B), further suggesting that 1, negatively charged at physiological pH, does not inhibit via a site within the membrane plane. We next considered if inhibition of peak 8193



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methionine, or asparagine, respectively. The resulting 27 charge-neutralizing mutants were then tested for inhibition of pH50-gated currents by 1 (1 mM; Figure 3A,B). Only three

ASIC1a above pH 5.0, this moiety is most probably charged. As evident in experiments with compounds analogous to 1, substitution of the carboxylic acid moiety with uncharged nitro (in compound 3), methylketone (in compound 4), or alcohol (in compound 5) groups practically abolished inhibition (Figure 2A,C). In contrast, drugs retaining carboxylic acid

Figure 2. Structure−activity relationships at ASIC1a. (A,B) Example recordings of ASIC1a inhibition by increasing concentrations of analogues (pictured). Gray scale bars: x, 10 s; y, 1 μA. (C) Analogue structures (left) and concentration−inhibition relationships (right) for inhibition of pH50-gated currents at ASIC1a (mean ± SEM, n = 5−8).

groups, (R)-ibuprofen (6), (S)-ibuprofen (7), and the simpler compound, 4-(2-methylpropyl)benzoic acid (8), retained similar inhibitory potency to 1 (Figure 2B,C), suggesting that the presence of a carboxylate “head” atop the hydrophobic “body” of the molecule is crucial for the interaction with ASIC1a. This also demonstrates for the first time that ASIC1a inhibition by ibuprofen enantiomers is similar to that of the racemate. We also sought to test for decreased inhibitory potency of 1 upon protonation at low pH, using mutant R190Q/D237N channels, which showed a pH50 for activation of 4.6 (Supporting Information, Figure S1F). 1 inhibited pH 4.8-gated currents with a pIC50 of 3.69 ± 0.03 (207 μM) and pH 4.4-gated currents with a pIC50 of ∼2.5 (∼3 mM, Supporting Information, Figure S1G). This is a decrease in potency of over 1 order of magnitude upon an increase in agonist concentration of only 0.4 pH units (4.8−4.4), compared to a smaller decrease in potency at WT channels upon an increase in agonist concentration of 0.5 pH units (Supporting Information, Figure S1E), suggesting that protonation (or decreased solubility) of 1 at pH < 5.0 decreases its inhibitory potency. On the basis of this structure−activity relationship, the finding that 1 binds to ASIC1a outside of the membrane plane (above) and the fact that 1 binds well-characterized target cyclooxygenase (COX) via a carboxylate/arginine interaction,28 we hypothesized that ASIC1a inhibition involves an interaction between the drug carboxylate and a positively charged side chain in the ASIC1a extracellular domain (ECD). We used sitedirected mutagenesis to individually mutate each apparently surface-accessible arginine, lysine, or histidine in the ASIC1a ECD (based on the chick ASIC1 structure29) to glutamine,

Figure 3. Mutagenesis screen of basic residues in the ASIC1a extracellular domain. (A) Example recordings at charge-neutralizing mutants for inhibition of pH50-gated currents by 1 (1 mM, filled bars), contributing to data in (B). (B) Percentage inhibition of pH50-gated currents by 1 (1 mM, black symbols) and pH50 for proton-gated currents (gray symbols) at charge-neutralizing ASIC1a mutants (mean ± SEM, n = 4−7). Blue text indicates mutants at which inhibition was significantly lower than WT (F = 76, p < 0.001, one-way ANOVA with Dunnett’s test). (C) Position of R64, K76, and K422 in ASIC1a model. (D) Example (left) and mean (±SEM, n = 4−6; right) inhibition of indicated ASIC1a mutants by increasing concentrations of 1. Gray scale bars: x, 10 s, y, 2 μA. This figure relates to Supporting Information, Figure S2.

mutants were inhibited significantly less than WT: R64Q, K76M, and K422M (Figure 3B). Strikingly, R64, K76, and K422 side chains share a common location, just external to the membrane (Figure 3C), identifying a pocket of basic residues contributing to sensitivity. To establish which of these side chains contributes most importantly to interactions with 1, we generated R64A, R64K, K76A, K76R, K422A, and K422R mutants, reasoning that alanine substitution of a carboxylate-binding residue would abolish sensitivity, whereas arginine or lysine substitution might retain the interaction and thus a WT-like sensitivity to the drug. R64A and K76A substitutions caused no significant reduction in sensitivity (Figure 3D; Supporting Information, Figure S2C), arguing against a direct role for these residues in binding. However, alanine substitution of K422 caused a significant reduction in sensitivity (Figure 3D; Supporting Information, Figure S2C), implicating K422 in drug recognition. This was 8194



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Figure 4. Site-directed mutagenesis implicates the ECD−TMD interface in ASIC1a inhibition. (A) Rat ASIC1a model showing side chains in the vicinity of K422. (magenta) Side chains oriented to an intersubunit pocket. (green) Side chains oriented to an intrasubunit site. (B) Concentration− inhibition data (mean ± SEM, n = 4−7) for 1 at rat ASIC1a mutations involving the pictured positions. (C) Mean ± SEM pIC50 values for inhibition of WT and mutants (n = 4−7 except A427W: see Supporting Information, Figure S2 caption). Dashed line for comparison to WT value. One-way ANOVA with Dunnett’s test: F = 33.0, ** p = 0.002, *** p = 0.001. This figure relates to Supporting Information, Figure S2.

cysteine, and labeled this with the fluorescent dye AF546 and measured changes in fluorescence (ΔF) induced by protons or 1. ΔF of fluorescent E425C labels is already established to track conformational changes contributing to channel activation in ASIC1a, rather than desensitization or SSD.31,33 We observed that activation with pH 6.0 induced a reversible decrease in fluorescence (ΔF = −1.8 ± 0.4%, n = 6, Figure 5A). Remarkably, the application of 1 alone at a resting pH of 7.4 induced a reversible increase in fluorescence (ΔF = 0.9 ± 0.2%, n = 6, Figure 5A), suggesting that, indeed, 1 induces a

confirmed by the observation that combining R64A and K76A substitutions with K422A in a triple mutant caused no additional decrease in sensitivity (Figure 3D; Supporting Information, Figure S2C). The K422R substitution also caused a significant decrease in sensitivity, demonstrating that positive charge alone at this position is not sufficient for inhibition. We note that the modest reduction in sensitivity with K422 mutants contrasts the more severe loss of inhibitory potency in compounds lacking the carboxylate (Figure 2). Thus, the precise nature of the 1/ASIC1a interaction requires further elucidation and likely involves other ASIC1a side chains. To identify other side chains that could interact with the drug, we mutated residues vicinal to K422 to tryptophan, hypothesizing that such bulky substitution would sterically hinder drug binding and decrease inhibitory potency. The single tryptophan residue in this region, W287, was mutated to alanine. After examining this region of the chick ASIC1 structure, side chains can be loosely divided into those facing an intersubunit cavity (magenta in Figure 4A) and those facing an external, intrasubunit site (green in Figure 4A). Of 16 substitutions, only two caused significant changes in sensitivity, Y68W and W287A (Figure 4B,C; Supporting Information, Figure S2B,C). Remarkably, Y68, at the extracellular end of the first membrane-spanning helix (M1) and W287, in the ECD, are both part of the intrasubunit site, within 5−6 Å of each other and 3−7 Å of K422 (Figure 4A). This provides strong evidence that this site is crucial for sensitivity, possibly through hydrophobic interactions between Y68/W287 and the drug benzene/aliphatic moieties and between K422 and the drug carboxylate. Ibuprofen Induces Conformational Changes Opposing Proton-Induced Activation. The intrasubunit site in ASIC1a is understood to contribute to proton-induced activation by physically coupling proton-induced ECD conformational changes to the membrane-spanning channel.30−32 We reasoned that 1 binding to this site could hinder such transduction and/or induce opposing conformational changes. To test this notion, we replaced E425, ∼5 Å from K422 but oriented into the intersubunit site (Figure 4A), with

Figure 5. Voltage-clamp fluorometry reveals conformational changes induced by 1. (A) (left) Current (black) and fluorescence (red) recordings from an oocyte expressing mouse ASIC1a E425C channels labeled with AF546, exposed to pH 6.0 or 1 (3 mM). (right) Mean ± SEM (n = 6) pH 6.0- and 1-induced current (I) and fluorescence changes (ΔF). (B) Mean ± SEM (n = 4) inhibition of unlabeled and AF546-labeled E113C and E425C channels by increasing concentrations of 1. (C) (left) I and ΔF recordings from an oocyte expressing mouse ASIC1a E113C channels labeled with AF546, exposed to pH 6.0 or 1 (3 mM). (right) Mean ± SEM (n = 5) pH 6.0- and 1-induced I and ΔF. 8195



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Table 1. Parameters for Inhibition of pH50-Gated Currents at ASIC1a, ASIC2a, and ASIC3 ASIC2aa

ASIC1a pIC50 1 (ibuprofen) 2 6 7 8 9 10 11 12

a

3.53 ± 0.05 3.02 ± 0.04*** 3.54 ± 0.11 3.46 ± 0.05 3.38 ± 0.08 10 mM (pIC50 < 2). For ASIC2a, saturating concentrations of drug were generally not reached due to poor solubility at the pH50 of 4.0, and pIC50 could therefore not be calculated. b Maximum percent inhibition of pH50-gated currents at ASIC1a (inhibition at 3 mM), ASIC2a (inhibition at 1 mM) and ASIC3 (inhibition at 3 mM). *Significantly different compared to 1 for that ASIC subtype and parameter (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Dunnett’s test).

conformational change in this region that is different from that of proton-induced activation. Notably, 1 inhibited proton-gated currents at labeled E425C channels much like unlabeled E425C and WT channels (Figure 5B), indicating that labeling E425C with the fluorophore has no confounding effect on the ability of 1 to alter ASIC1a function. Conformational change induced by 1 was not limited to this region of ASIC1a, as cysteine substitution and AF546 labeling of E113, near the extracellular exterior of the ECD,33 also resulted in robust ΔF signals in response to 1 alone (Figure 5C). ASICs Are Broadly Sensitive to Ibuprofen and Certain Analogues Show Moderate Subtype Selectivity. Despite results showing inhibition by 1 of native ASICs13,16 and recombinant ASIC1a,13,20 the activity of 1 at other specific ASIC subtypes has, to our knowledge, not yet been tested. We therefore returned to structure−activity experiments, testing 1 and analogues at rat ASIC1a, -1b, -2a, and -3 homomers. We observed that 1 inhibited peak pH50-gated currents at each subtype, although this was somewhat weaker at ASIC2a (Figure S3B, Table 1). However, our ASIC2a experiments used the pH50 of ∼4.0, where drug solubility decreased, and it was difficult to exclude drug protonation and/or solubility as a reason for decreased potency. Regarding enantiomers, at ASIC1a, -2a, and -3, the effects of 6 and 7 were practically indistinguishable from each other and from the racemate (Table 1, Supporting Information, Figure S3B). Together with our observation that 8 also inhibited each subtype with similar potency to 1 (Table 1), this reiterates that although the presence of the drug carboxylate is important for inhibition, its precise orientation relative to the hydrophobic tail of the molecule is not. We next measured potency of ibuprofen analogues differing in size at the benzene para substituent or the C2 substituent (Figure 6A-C). A decrease in size at the benzene para substituent selectively decreased potency at ASIC1a, as 2-(ptolyl)propanoic acid (9) inhibited ASIC2a 70 ± 4.3% (n = 3) and ASIC3 68 ± 3.6% (n = 7) while inhibiting ASIC1a only 18 ± 2.4% (n = 6; Figure 6A). Conversely, increased molecular weight at this position had the opposite effect, as flurbiprofen (10), with an additional benzene group (and a meta fluorine), showed significantly greater potency than 1 at ASIC1a, significantly less inhibition than 1 of ASIC2a, and similar potency as 1 at ASIC3 (Figure 6B, Table 1). When testing the relationship between C2 substituent size and inhibitory potency

Figure 6. Subtype specificity of compounds. (A,B) Example recordings and concentration−response data for inhibition of homomeric ASIC1a, -2a, and -3 subtypes by 9 (n = 5−6) and 10 (n = 6−8). Data in each panel are mean ± SEM. (C) Inhibition of ASIC1a by ibuprofen analogues containing different C2 (R in inset structure) substituents (n = 6−8). (D) Inhibition of homomeric ASIC1a, -2a, and -3 subtypes by C2 substituted analogue 12 (n = 4−7; dashed line is ASIC1a/12 fit from (C). (E) Amino acid sequence alignment of major ASIC subtypes showing upper-M1, extracellular domain β9-α4 loop, and upper-M2 segments (ASIC1bM3 is described in Supporting Information, Figure S3). Determinants of drug sensitivity in ASIC1a are indicated in cyan (basic residues, Figure 3) or green (aromatic residues, Figure 4), along with equivalent ASIC1b, -2a and -3 residues.

at ASIC1a, a modest trend emerged, with potency increasing subtly with substituent size from ibufenac (11; H), through 1 (CH3) to butibufen (12; C2H5; Figure 6C, Table 1). However, the additional size (C3H7) in the C2-isopropyl analogue (13) did not increase potency at ASIC1a further, with similar 8196



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Identification of an Allosteric Modulatory Site in ASIC1a. We tested the effects of 1 on numerous aspects of ASIC function, including activation, SSD, desensitization, and recovery from desensitization. These experiments revealed that the presence of 1 inhibits the agonist-induced activation of the channel in an allosteric mechanism of inhibition. This was complemented by our voltage-clamp fluorometry experiments, which showed that agonist (protons) and 1 exert opposing conformational changes at the top of the channel-forming M2 helix. Our extensive mutagenesis data point toward a binding site that seems ideally placed to exert these effects. In an initial scan of 27 positively charged residues throughout the ASIC1a ECD, only three emerged as important for sensitivity to 1, and these are each situated in close proximity, just above the membrane. Moreover, extensive mutagenesis in and around this pocket showed that three residues, Y68, W287, and K422, contribute most significantly to sensitivity. These side chains are within 7 Å of each other in the low-pH, desensitized ASIC1 structure (Figure 4A) and also in ASIC1 structures capturing other states.35 Thus, they form an ostensibly suitable site for 1 to form hydrophobic interactions with aromatic side chains and a polar interaction with the K422 amine and the Y68 hydroxyl, which is reminiscent of interactions in the 1/COX-2 crystal structure.36 As the role of ASICs in the nervous system has emerged relatively recently compared to other neurotransmitter receptors,1,4,37 our knowledge on molecular mechanisms of ASIC activation and modulation has also lagged behind, although a pharmacological repertoire is developing.18 This includes drugs acting in the pore, where current is blocked directly;35,38 at the ECD interface of adjacent subunits, where various toxins and endogenous modulators bind and alter activation or desensitization,39−41 and in the central vestibule of the extracellular domain, through which small molecules have been shown to inhibit42 or activate ASICs.43 Here, we show that the intrasubunit interface of extracellular and channel domains, a site that links proton-sensing to channel activation, also constitutes a site by which small-molecule drugs can inhibit ASIC activation. Structure−Activity Relationships. We tested a selection of closely related analogues differing from 1 in either carboxylic acid, C2, or the benzene para moieties for inhibition of ASIC1a. An essential role for the carboxylic acid moiety emerged, as uncharged substituents showed little or no inhibition, and when tested on an ASIC1a mutant that only responds to very high proton concentrations, the likely protonation of 1 seemed to decrease its inhibitory potency. A modest correlation between C2 substituent length and inhibitory potency at ASIC1a emerged, although we observed that a cyclopropyl C2 substituent did not show significantly increased potency compared to ibuprofen. This suggests that the nanomolar potency reported for 1534 is likely to originate from the additional differences from ibuprofen: a benzene meta fluorine atom and a dichlorophenyl tail (as opposed to the 2methylpropyl tail in 1). Together with the increased potency of 10 (benzene tail) and decreased potency of 9 (methyl tail), this suggests that size and lipophilicity of the tail can enhance potency at ASIC1a. We also tested the effects of 1 and several analogues at other ASIC subtypes, showing that ASIC1b, ASIC2a, and ASIC3 are all inhibited by 1. This also showed that some subtype selectivity is possible with these compounds. 10 and 12 inhibited ASIC1a and ASIC3 more potently than ASIC2a

potency to 1 (Figure 6C). Notably, the C2-cyclopropane analogue (14) did not show greater potency than 1 either (Figure 6C), suggesting that the nanomolar potency observed with the 10 analogue [1-(3′,4′-dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic acid] (CHF5074, 15),34 derives more from differences in the benzene para substituents and added halogen atoms. Given the increased potency of 12 at ASIC1a, we tested its effects on ASIC2a and ASIC3 and observed a selective decrease in potency at ASIC2a (Figure 6D; Table 1). Thus, although the analogues tested showed neither remarkable ASIC1a selectivity nor a dramatic increase in general potency, these results show that differences at C2 and para moieties strongly affect ASIC1a/ASIC2a selectivity. Finally, we attempted to reconcile these results with the molecular determinants of sensitivity to 1 that we identified in ASIC1a at the interface of extracellular and channel domains. An amino acid sequence alignment shows that Y68 and W287 (rat ASIC1a numbering) are present in ASIC1a, -1b, -2a, and 3 (Figure 6E), consistent with some inhibition of each subtype by most compounds. However, K422, apparently the most important basic residue for sensitivity in ASIC1a, is essentially replaced by an alanine residue in ASIC3, raising the question of why these compounds inhibit ASIC3 with similar potency to ASIC1a. Perhaps the absence of K422 in ASIC3 could contribute to the slightly weaker maximum inhibition by most compounds of ASIC3 compared to ASIC1a (Table 1). Additionally, we hypothesize that each of these hydrophobic drugs binds within the aromatic side chains surrounding K422 where several polar moieties could potentially engage the drug carboxylate (e.g., positions R64, Y68, K/T76, Q420, K421, Figure 6E). Given these potentially unspecific interactions, we considered that 1 could modulate other ligand-gated ion channels possessing peripheral aromatic clusters in the proximity of basic side chains such as trimeric P2X receptors (Supporting Information, Figure S3C). We therefore expressed trimeric P2X1, P2X2, and P2X4 receptors in oocytes and measured the effects of 1 (3 mM) on currents activated by EC50 agonist concentrations. 1 had no effect on current amplitude of P2X2 and P2X4 receptors but caused reversible, weak inhibition of P2X1 (Supporting Information, Figure S3D). The notion that P2X1 inhibition by 1 is via a similar site to that in ASIC1a is merely speculative, but it is curious that the P2X1 subunit (unlike P2X2 and P2X4, which were not inhibited) has an additional aromatic side chain in this site, vaguely resembling that of ASIC1a (W272 in zP2X4, pictured in Supporting Information, Figure S3C). In conclusion, however, the mere presence of several aromatic and basic side chains in other channels is not sufficient for the level of inhibition that 1 exerts on ASICs.

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DISCUSSION ASIC1a is strongly implicated in pain and neuroinflammation8,9,14,15 and some of the most widely used antiinfammatories, including 1, have been shown to reduce pain and inflammation through ASIC1a.13,16 Despite this apparent potential for targeting these conditions with established, welltolerated drugs, the molecular basis for ASIC inhibition by NSAIDs has remained unknown. In the present study, we have characterized the mechanism of ASIC1a inhibition by 1, identified a likely binding site in ASIC1a, and performed a structure−activity analysis using both analogues of 1 and various ASIC subtypes. 8197



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10 (flurbiprofen; (RS)-2-(3-fluoro-4-phenylphenyl)propanoic acid) were purchased from Sigma-Aldrich (≥98%, GC). 2 (2-fluoro-2-[4(2-methylpropyl)phenyl]acetic acid), 3 (1-(2-methylpropyl)-4-(1nitroethyl)benzene), and 4 (3-(4-(2-methylpropyl))butan-2-one) were purchased from Chiroblock (custom synthesis, ≥95%, HPLC and 1H NMR). 5 (ibuprofen alcohol; 2-[4-(2-methylpropyl)phenyl]propan-1-ol), 6 ((R)-ibuprofen), 7 ((S)-ibuprofen), 9 (2-(p-tolyl)propionic acid), and 12 (butibufen; 2-[4-(2-methylpropyl)phenyl]butanoic acid) were purchased from Toronto Research Chemicals (≥98%, 1H NMR). 8 (4-(2-methylpropyl)benzoic acid) (≥95%, GC) and 11 (ibufenac; 2-[4-(2-methylpropyl)phenyl]acetic acid) were purchased from Fluorochem (≥98%, GC). 13 (2-[4-(2-methylpropyl)phenyl]-3-methylbutanoic acid) and 14 (1-[4-(2-methylpropyl)phenyl]cyclopropanecarboxylic acid) were purchased from SynChem (custom synthesis, ≥95%, HPLC and 1H NMR). Stocks were generally prepared as 100−300 mM in DMSO and dissolved in bath solution (below) immediately before experiments. AF546 (Alexa Fluor 546 C5 maleimide) was purchased from Thermo Fisher Scientific. All other chemicals were purchased from VWR or Sigma-Aldrich. Molecular Biology. Plasmid DNAs used were rat ASIC1a, ASIC1bM3 (a clone in which the first two methionines are deleted, improving cell surface expression49), ASIC2a, and ASIC3 (provided by Stefan Gründer, RWTH Aachen University) in the pRSSP6009 vector, mouse ASIC1a in the pSP64 vector (Marcelo Carattino, University of Pittsburgh), and rat P2X2 (Ralf Hausmann, RWTH Aachen University) and P2X1 and P2X4 (Annette Nicke LMU, Munich) in the pNKS2 vector. Site-directed mutagenesis was performed with customized oligomers (Eurofins Genomics) and Pfu Ultra II Fusion HS DNA Polymerase (Agilent Technologies). Plasmids were linearized and transcribed with the Ambion mMESSAGE mMACHINE RNA transcription kit (Thermo Fisher Scientific). Two-Electrode Voltage Clamp and Fluorometry. Xenopus laevis oocytes were prepared and recorded from as described previously50 and under license 2014-15-0201-00031 from the Danish Veterinary and Food Administration. Briefly, oocytes were injected with 0.8−40 ng of rat ASIC mRNA (detailed in Supporting Information, Figure S2C) or 4 ng of rat P2X receptor mRNA for electrophysiological experiments, and ∼80 ng of mouse ASIC1a mRNA for voltage-clamp fluorometry. After 24−48 h (or 48−72 h for rat P2X1 and for fluorometry experiments), these were transferred to a recording chamber continuously perfused with bath solution containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, and either 5 HEPES (pH > 5.5) or 5 MES (pH ≤ 5.5). The control solution was pH 7.4, and solutions of varying pH and/or drug concentration were rapidly exchanged with ValveBank 8 perfusion system (AutoMate Scientific). Oocytes were clamped at −40 mV with a OC-725C amplifier (Warner Instruments) and Digidata 1550 digitizer (Molecular Devices). For voltage-clamp fluorometry, oocytes were incubated for 30 min in 10 μM AF546 (in OR2: in mM, 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4), washed twice with OR2, and transferred to the recording chamber with a replaceable coverslip base in an inverted IX73 microscope with XLUMPLFLN 20×, NA 1.0 objective (Olympus). With the oocyte animal pole toward the excitation source, the fluorophore was excited at 550 nm with a pE-100 LED source (CooLED) and Rhodamine Red filter set (Chroma) and detected with a P25C-19 photomultiplier tube (Sens-Tech) and photon-to-voltage converter (IonOptix), via the microscope side port. Light exposure of the oocytes was kept to a minimum to minimize bleaching. Current and fluorescence were recorded with pClamp 10.6 software (Molecular Devices), and peak amplitudes were used for statistical analysis (below). Time constants (Figure 1C) were calculated with single exponential fits to current decay in pClamp. Experimental Design and Statistical Analysis. As is usual in the field, we sought to repeat experiments both at multiple oocytes on a given day and at multiple batches of oocytes. Thus, n values are generally ≥4. All fits and statistical analyses were performed with Prism 6 or 7 (Graphpad Software). Peak current amplitude in response to increasing proton concentrations was plotted against pH and fit with four-parameter Hill equation to establish pH50 values (half-maximal effective proton concentration). Peak current amplitude

(although hydrophobicity of these compounds led to issues with solubility at ASIC2a, where low pH was required for activation). Compound 9, in contrast, with a shorter tail (and comparatively quite soluble), showed markedly greater potency at ASIC2a than at ASIC1a. A striking result from our structure−activity analysis emerged when we considered that other channels with similar sites to that identified in ASIC1a might also show sensitivity to 1. Our experiments with P2X receptors showed that the P2X1 subtype, possessing an intrasubunit site reminiscent of that in ASIC1a, was also inhibited by 1 (Supporting Information, Figure S3C,D). At this stage, the notion that this site in P2X1 actually determines NSAID sensitivity is, of course, speculative. Therapeutic Implications for NSAIDs in Pain and Inflammation. The widely accepted targets of 1 and most other NSAIDs are COX-1 and COX-2, which are expressed in numerous tissues and inhibited by low micromolar concentrations of 1.44,45 ASICs on the other hand, show a primarily neuronal distribution4 and require high micromolar concentrations for inhibition. Are ASICs relevant to the therapeutic effects of 1 and other analogues? Jones et al.16 showed that normal, topical doses of 1 eased pain induced by transdermal iontophoresis of acid on human skin, as did microinjection of the ASIC blocker amiloride. This provides compelling evidence that 1 indeed acts through ASICs in the periphery, where 1 reaches high micromolar concentrations with common doses.46 Our finding that 1 inhibits ASIC1b similarly to ASIC1a is also consistent with actions of 1 in the periphery, where ASIC1b plays a significant role in nociception.14 However, significant inhibition of ASICs in the central nervous system is less likely, where NSAID concentrations are 10−50 times lower.46,47 Nonetheless, some evidence links brain ASICs with the neuroprotective effects of other propanoic acid NSAIDs.17,34 Additionally, the neuroprotection afforded by 1 occurs with either (R) or (S) enantiomer, which is not consistent with COX inhibition that occurs via the (R) entantiomer only,48 whereas we have shown that ASIC inhibition is enantiomer nonspecific. We also showed that at lower agonist concentrations, the potency of 1 increases, raising the possibility that in physiological cases of a small drop in pH, ASICs may become more sensitive to the drug.

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CONCLUSION Despite tantalizing evidence that NSAID inhibition of ASICs is a significant aspect in a common line of therapy, the molecular basis for an NSAID/ASIC interaction was lacking. We explored this interaction by dissecting the mechanism by which 1 inhibits ASIC1a, identifying a potential binding site in ASIC1a, and performing structure−activity relationships. Together, the results suggest that the drug binds to a site in the agonist transduction pathway, where it allosterically inhibits protoninduced activation. While the carboxylic acid moiety is crucial for inhibition, changes to the hydrophobic tail of the molecule can increase or decrease potency and alter subtype selectivity. This paves the way both for functional/computational work aimed at precisely elucidating the binding site in ASIC1a and for medicinal chemistry aimed at anti-inflammatory and neuroprotective drugs targeting ASICs.

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EXPERIMENTAL SECTION

Chemicals. All drugs were purchased as follows, with suppliers’ purity and method to determine purity indicated in brackets. 1 (ibuprofen; (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid) and 8198



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in response to pH50 with increasing concentrations of inhibitor was plotted against log inhibitor concentration and fit with four-parameter Hill equation to establish IC50 value for inhibition. Mean ± SEM pH50 and pIC50 (−log(IC50)) values were compared with either unpaired t test or one-way ANOVA with Dunnett’s test (for comparisons to a particular mean) or Tukey’s test (for comparing multiple differences). p values, together with F values or t values (with degrees of freedom in brackets), are reported where appropriate. Molecular Models and Amino Acid Sequence Alignments. In illustrating the approximate positions of selected amino acids in rat ASIC1a, no genuine homology model was generated, but a simple “model” was made by taking the closed-channel chick ASIC1 structure in PDB 2QTS29 and substituting any divergent side chains for the rat ASIC1a equivalent in PyMOL (Schrödinger). Supporting Information, Figure S3C, uses the zebrafish P2X4 structure from PDB 3H9V.51 All visualizations performed with PyMOL. Amino acid sequence alignments were performed with MUSCLE.52

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(4) Wemmie, J. A.; Taugher, R. J.; Kreple, C. J. Acid-sensing ion channels in pain and disease. Nat. Rev. Neurosci. 2013, 14 (7), 461− 471. (5) Kreple, C. J.; Lu, Y.; Taugher, R. J.; Schwager-Gutman, A. L.; Du, J.; Stump, M.; Wang, Y.; Ghobbeh, A.; Fan, R.; Cosme, C. V.; Sowers, L. P.; Welsh, M. J.; Radley, J. J.; LaLumiere, R. T.; Wemmie, J. A. Acidsensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity. Nat. Neurosci. 2014, 17 (8), 1083−1091. (6) Wemmie, J. A.; Chen, J.; Askwith, C. C.; Hruska-Hageman, A. M.; Price, M. P.; Nolan, B. C.; Yoder, P. G.; Lamani, E.; Hoshi, T.; Freeman, J. H., Jr.; Welsh, M. J. The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory. Neuron 2002, 34 (3), 463−477. (7) Wang, Y. Z.; Wang, J. J.; Huang, Y.; Liu, F.; Zeng, W. Z.; Li, Y.; Xiong, Z. G.; Zhu, M. X.; Xu, T. L. Tissue acidosis induces neuronal necroptosis via ASIC1a channel independent of its ionic conduction. eLife 2015, 4, e05682. (8) Xiong, Z. G.; Zhu, X. M.; Chu, X. P.; Minami, M.; Hey, J.; Wei, W. L.; MacDonald, J. F.; Wemmie, J. A.; Price, M. P.; Welsh, M. J.; Simon, R. P. Neuroprotection in ischemia: blocking calciumpermeable acid-sensing ion channels. Cell 2004, 118 (6), 687−698. (9) Chassagnon, I. R.; McCarthy, C. A.; Chin, Y. K.; Pineda, S. S.; Keramidas, A.; Mobli, M.; Pham, V.; De Silva, T. M.; Lynch, J. W.; Widdop, R. E.; Rash, L. D.; King, G. F. Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (14), 3750−3755. (10) Ugawa, S.; Ueda, T.; Ishida, Y.; Nishigaki, M.; Shibata, Y.; Shimada, S. Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J. Clin. Invest. 2002, 110 (8), 1185−90. (11) Bohlen, C. J.; Chesler, A. T.; Sharif-Naeini, R.; Medzihradszky, K. F.; Zhou, S.; King, D.; Sanchez, E. E.; Burlingame, A. L.; Basbaum, A. I.; Julius, D. A heteromeric Texas coral snake toxin targets acidsensing ion channels to produce pain. Nature 2011, 479 (7373), 410− 414. (12) Duan, B.; Liu, D. S.; Huang, Y.; Zeng, W. Z.; Wang, X.; Yu, H.; Zhu, M. X.; Chen, Z. Y.; Xu, T. L. PI3-kinase/Akt pathway-regulated membrane insertion of acid-sensing ion channel 1a underlies BDNFinduced pain hypersensitivity. J. Neurosci. 2012, 32 (18), 6351−6363. (13) Voilley, N.; de Weille, J.; Mamet, J.; Lazdunski, M. Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammationinduced expression of acid-sensing ion channels in nociceptors. J. Neurosci. 2001, 21 (20), 8026−8033. (14) Diochot, S.; Baron, A.; Salinas, M.; Douguet, D.; Scarzello, S.; Dabert-Gay, A. S.; Debayle, D.; Friend, V.; Alloui, A.; Lazdunski, M.; Lingueglia, E. Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature 2012, 490 (7421), 552−555. (15) Mazzuca, M.; Heurteaux, C.; Alloui, A.; Diochot, S.; Baron, A.; Voilley, N.; Blondeau, N.; Escoubas, P.; Gelot, A.; Cupo, A.; Zimmer, A.; Zimmer, A. M.; Eschalier, A.; Lazdunski, M. A tarantula peptide against pain via ASIC1a channels and opioid mechanisms. Nat. Neurosci. 2007, 10 (8), 943−945. (16) Jones, N. G.; Slater, R.; Cadiou, H.; McNaughton, P.; McMahon, S. B. Acid-induced pain and its modulation in humans. J. Neurosci. 2004, 24 (48), 10974−10979. (17) Mishra, V.; Verma, R.; Singh, N.; Raghubir, R. The neuroprotective effects of NMDAR antagonist, ifenprodil and ASIC1a inhibitor, flurbiprofen on post-ischemic cerebral injury. Brain Res. 2011, 1389, 152−160. (18) Baron, A.; Lingueglia, E. Pharmacology of acid-sensing ion channels - Physiological and therapeutical perspectives. Neuropharmacology 2015, 94, 19−35. (19) Banks, W. A. Characteristics of compounds that cross the bloodbrain barrier. BMC Neurol. 2009, 9 (1), S3. (20) Dorofeeva, N. A.; Barygin, O. I.; Staruschenko, A.; Bolshakov, K. V.; Magazanik, L. G. Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneurons. J. Neurochem. 2008, 106 (1), 429−441.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01072. Acid-sensing ion channel experimental results (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +45 23649066. E-mail: [email protected]. ORCID

Stephan A. Pless: 0000-0001-6654-114X Author Contributions †

T.L. and J.L.R. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.L. was supported by a Sapere Aude Research Talent award (4092-00348B) from the Danish Council for Independent Research and a Lundbeck Postdoctoral Fellowship (R1712014-558). S.A.P. was supported by Lundbeck Foundation Fellowship (R139-2012-12390). The project was also funded by The Carlsberg Foundation (2013 01 0439) and the Novo Nordisk Foundation (11767).

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ABBREVIATIONS USED ASIC, acid-sensing ion channel; COX, cyclooxegenase (or prostaglandin synthase); ECD, extracellular domain; I, current; NSAID, nonsteroidal anti-inflammatory drug; pH50, halfmaximal effective pH; ΔF, change in fluorescence

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REFERENCES

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Molecular Basis for Allosteric Inhibition of Acid-Sensing Ion Channel

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