Voltage-Sensitive Potassium Kv2 Channels as New Targets for

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Voltage-Sensitive Potassium Kv2 Channels as New Targets for Insecticides Jeffrey R. Bloomquist,*,1 James M. Mutunga,1 Rafique M. Islam,1 Astha Verma,2 Ming Ma,2 Maxim M. Totrov,3 and Paul R. Carlier2 1Department

of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, Gainesville, Florida 32610, U.S.A. 2Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, U.S.A. 3Molsoft LLC, 11199 Sorrento Valley Road, San Diego, California 92121, U.S.A. *E-mail: [email protected]

The diacylhydrazines are a class of insecticides usually thought to act by disrupting the insect endocrine system. However, at field use rates, insects affected by these compounds also show signs of neurotoxicity. Previous research found that a blocking action on Kv2 potassium channels of nerve and muscle was the cause of the observed neurotoxicity. This review will summarize the physiological role of Kv2 channels and their possible exploitation as a new target for insecticide development. Included is a description of a potassium channel homology model to visualize protein structures of mosquito and human, and to facilitate design of molecules that are safe and effective. Also included is a proposed ligand docking example with a substituted catechol as a model potassium channel blocker. Preliminary toxicity studies demonstrate that molecules of this type show greater acute toxicity to mosquitoes than the diacylhydrazines. The overall aim of this project is a new commercial insecticide for use in the fight against global malaria.

© 2014 American Chemical Society In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

Introduction


Our interest in new insecticides is focused on the pressing need for malaria vector control with compounds having contact activity against adult females, because of the growing problem of pyrethroid resistance in Anopheles gambiae (1). Accordingly, this review will discuss Kv2 potassium channels in the context of developing new insecticides for controlling this disease vector. Evidence for potassium channels as a target for insecticide action was originally documented in studies that observed neurotoxic signs following exposure to compounds thought to possess a primary mode of action as ecdysone agonists (2). The experimental compounds were designated RH-5849 and RH-1266, and are shown in Figure 1.

Figure 1. Structures of neurotoxic, potassium channel-directed diacylhydrazines.

However, in addition to endocrine effects, neurotoxic symptoms (tremors and paralysis) were observed in the Colorado potato beetle and Mexican bean beetle at field use rates (3). Neurotoxic signs were also reported in American cockroaches exposed to RH-5849 by 50 µg/g injections, and these effects were traced to hyperactivation of central and motor nerve pathways in electrophysiological studies (4). More detailed investigation in housefly larval muscle (Fig. 2) found that the most sensitive membrane current affected by RH-5849 (IC50 = 59 µM) and RH1266 (IC50 = 40 µM) was the delayed rectifier or IK potassium current (Fig. 2, top row), which is responsible for repolarization of the membrane potential in nerve and muscle cells in order to maintain proper function (5). The compounds also affected the rapidly inactivating IA current, but to a lesser extent, and there was no blockage of these channels at a concentration of ≤ 100 µM (Fig. 2, bottom row). A single study describes diacylhydrazine poisoning in mosquitoes, and it was focused on ecdysone agonist effects (6). In this paper, larval development (premature molt, the main effect of ecdysone agonists) was studied after addition of compounds to the water. Insects were evaluated for toxicity after 10 days of exposure. Anopheles gambiae was the most sensitive species tested, followed by Culex quinquefasciatus, and Aedes aegypti (6). It was also observed that the first effect of these compounds (including RH-5849) was behavioral, the larvae descending to the bottom of the cup within 2 days. Any role for neurotoxicity or potassium channel blockage was not mentioned. 72 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 2. Effects of RH-5849 on delayed rectifier IK (top row) and inactivating IA (bottom row) in housefly larval muscle at 0, 31.6, 100 and 316 µM (left to right). Reproduced with permission from reference (5). Copyright 1992 Wiley.

There are a large number of different subtypes of potassium channels, and it is important to assess which subtype might form the major target site for diacylhydrazines. The available genomic sequences of both An. gambiae and Drosophila melanogaster yields clues to which channel might be involved. The potassium channel α-subunit genes of Drosophila and An. gambiae are well conserved, ranging from 42-98% with a mean of 85% amino acid sequence identity (7). This high degree of sequence conservation suggests that their physiological function is conserved, as well. It has been shown that the Drosophila shab gene (later classified as Kv2) is the primary delayed rectifier (IK channel) of Drosophila nerve and muscle, and is present in all life stages (8). Although more Kv-type potassium channel genes were found in An. gambiae (eight) vs. Drosophila (six), only a single gene for Kv2 apparently exists in each organism, and the sequence homology is 94% (7). In mammals, Kv2.1 is the primary homolog of shab, and is widely distributed within central and peripheral neurons, cardiac tissues, and skeletal muscle (9). The Kv potassium channel genes produce multiple copies of an α-subunit (Fig. 3) that associate post-translationally into a tetrameric complex to form the functional ion channel (10). In addition, there are smaller auxiliary subunits, Kvβ, that modulate the gating and trafficking of the channel, and impart inactivation properties to Kv1 channels (10). Not much is known regarding auxiliary subunits in any insect Kv channel, although one report (11) studied interactions of the Kv1 (IA) channel with the β-subunit gene of the hyperkinetic mutant of D. melanogaster. Given the lack of any definitive information on Kv2 subunits interacting with β-subunits, and the non-inactivating nature of native Kv2-mediated currents in insect nerve and muscle (5, 6), β-subunit influence on Kv2 channel properties is difficult to account for at present. 73 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 3. Membrane topology of Kv superfamily genes. Transmembrane α-helices are shown as cylinders numbered 1-6, and the conserved pore domain is “P.”. Reproduced from reference (7) (2003 Genome Biology).

An important consideration in developing a potassium channel-directed insecticide is its possible interaction with the so-called hERG potassium channel (human ether-a-go-go related gene). This gene encodes for a fast-activating delayed rectifier potassium channel (IKf) (12). The channel resides in human cardiac tissue, and when blocked, mediates serious side effects (QT interval prolongation) of non-antiarrhythmic drugs (Fig. 4), sometimes resulting in sudden cardiac death (12). Interaction of hERG with compounds such as Sertindole requires a positively charged nitrogen in an appropriate molecular locus (Fig. 4), which the diacylhydrazines lack (Fig. 1), and which should be avoided in any insecticidal molecule. Inspection of the diacylhydrazines (Fig. 1) reveals a rather simple chemical scaffold, and with little structural similarity to known hERG channel blockers. Therefore, it would appear these compounds should have little blocking activity at hERG channels. Moreover, the potency of RH-1266 and -5849 might be improved because the essentially un-substituted nature of the phenyl rings suggests numerous possible analogs. The fact that RH-5849 has some contact activity (4) is another potential advantage of the existing diacylhydrazine chemistry. RH-5849 also has an oral LD50 in rats of 435 mg/kg, a relatively high value (4), suggesting that selective toxicity is achievable. Thus, the diacylhydrazines serve as potentially excellent leads for a heretofore unexploited target site of critical importance in excitable membranes; viz., voltage-dependent potassium channels, especially Kv2 (Ik). Action on this site will circumvent the target site (kdr) resistance to pyrethroids present in field populations of Anopheles gambiae (1). 74 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 4. Example of a cationic drug (Sertindole, an antipsychotic) taken off the market due to unwanted side effects arising from hERG channel block (12).

Candidate Ligands for Kv2 Besides the diacylhydrazines, a survey of the literature revealed four additional compound classes with promising potassium channel blocking activity (Fig. 5). The catechol 48F10 is predicted to bind near the external mouth of Kv2, but is ineffective on Kv1 channels (13). Rutaecarpine is also a blocker of delayed rectifier potassium channels in NG-108-15 neuronal cells (14). Substituted biphenylpyridazines (15) block IA and IK with IC50s in the low micromolar range. Flonicamid is a compound active on plant-sucking pests, and there is a single citation ascribing its mode of action to blockage of IA potassium channels (16). Among the lead compounds presented in Figures 1 and 5, three structural types stand out in terms of good drug-like properties and ability to construct diverse libraries: these are the RH compounds, 48F10, and rutaecarpine. All of these have CLogP less than 5, and tPSA less than 60 square angstroms. Those parameters should confer good ability to cross permeability barriers of the mosquito. Passage of the cuticle requires significant hydrophobicity; for reference, the carbamate insecticide propoxur has CLogP = 1.648 and tPSA = 47.56 square angstroms. We view the pyridazine compound as less favorable than the others due to carcinogenicity concerns over the terminal biphenylamine moiety. Flonicamid in turn does not appear hydrophobic enough to cross the cuticle, and the available chemical space around this commercial compound may be limited. 75 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 5. Other known or putative ligands for insect or mammalian Kv2 potassium channels gleaned from the literature. The abbreviations are defined as tPSA (topological polar surface area) and ClogP (calculated log P values; octanol/water partition coefficients).

Molecular Modeling of Kv2 Genomic analysis and molecular modeling evaluated Kv2 gene sequences and x-ray crystallographic information, in addition to the in silico computation of predicted 3-dimensional structures and ligand docking that was also performed. Sequence alignments were made of the relevant Anopheles gambiae and human potassium channel proteins (discussed below). Recently, the X-ray structure of a Kv1.3-Kv2.1 chimeric channel has been reported (PDB 2R9R (17);). The transmembrane pore domain in this structure is immersed in a lipid membrane-like environment and is in the physiologically relevant open channel conformation. This experimental structure provides a closely homologous template for comparative modeling of the target, the mosquito delayed rectifier potassium channel, AgKv2/Shab (Fig. 6). Amino acid identity across the entire transmembrane domain is 50%, and goes up to 59% if only the core channel domain is considered. Furthermore, the alignment for this domain is continuous. Lack of any insertions or deletions greatly facilitates the construction of the model (Fig. 6) and improves our confidence in its accuracy. 76 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 6. Model of the AgKv2 channel core. Three of the four symmetric subunits of the homo-tetrameric channel are shown in white, while regions of the fourth subunit are colored: part of helix S6 (aka, the ‘inner’ helix) lining the central hydrophobic cavity is in green, and the highly conserved selectivity filter in magenta. Four K+ ions inside the selectivity filter are shown as yellow spheres. Extracellular space is at the top and cytoplasm below, while most of the channel is immersed in the membrane. Residue numbering is according to the full mosquito AgKv2 gene sequence.

The initial homology model for mosquito was built based on the X-ray structure of the Kv2.1, PDB ID 2r9r. The model of mosquito Shab was refined by global energy optimization using BPMC sampling in ICM (18). Molsoft’s ICM software was previously applied in modeling of various drug target proteins, such as the nicotinic acetylcholine receptor (19). The established view is that the delayed rectifier potassium channel is a homo-tetramer (10). An internal variable linking method in ICM (19) was used to enforce identical conformations of the four subunits and thus maintain 4-fold symmetry throughout energy refinement. A fifth potassium atom at the exterior entrance of the channel was added, as seen in certain Kv structures, using PDB 2a79 as a template to place the cation. When catechol ligand 48F10 was docked into the model at the extracellular and intracellular sites, the most favorable interaction pose was observed at the extracellular opening of the channel (Fig. 7 a,b). 77 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 7. a) Binding pose of 48F10 (magenta); coordination of potassium ion (yellow) and hydrophobic contacts to Y557 and parts of backbone (green) can be observed. b) Location of the putative binding site of 48F10 on the overall channel structure; blue mesh is the inward facing ‘vestibule’, green is the outward facing surface. c) An example of experimentally observed potassium-catechol coordination in a complex with a catechol crown ether.

78 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Two hydroxyl oxygen atoms coordinated a potassium atom at the channel entrance in a bifurcated manner, consistent with interactions seen in X-ray structures of complexes of certain catechol derivatives with potassium (Fig. 7c). The bulky lipophilic moiety extended towards a hydrophobic groove formed by the Y557 side-chain and parts of the backbone in the adjacent subunit, thus forming additional favorable interactions. This later interaction is consistent with an observation that among human channels, 48F10 is selective towards Kv2.1, which also has a tyrosine in corresponding position (Y384), over Kv1.5, which has R487 (13). We have also developed a preliminary model of human Kv2 for comparison with mosquito. It is quite conserved as compared to mosquito Shab; the closest substitution to the putative binding site for catechols is T559(Shab)/K386(hKv2.1), which is fairly distant. Note that this external pore model for catechols is not proposed as a hypothesis of diacylhydrazine binding.

Figure 8. Gene sequence alignment of helix S6 for human KCNH2 (hERG) and mosquito Shab. Dots indicate indicate amino acid identity and boxes are conservative substitutions, respectively, in the two genes.

The most important undesirable off-target effect of K+ channel blockers is likely to be hERG inhibition. We compared sequences of hERG and AgKv2 in the regions of the selectivity filter and inner cavity portion of S6 (Fig. 8). Homology of the helix S6 portion that lines the hydrophobic cavity is weak between these two channels. Residues adjacent to the extracellular opening are also poorly conserved, e.g., Y557(Shab)/Y384(hKv2) is a serine in hERG. This observation suggests that selective inhibition can be achieved at either site. Successful modeling of hERG and hERG interactions with a number of blocker molecules has been reported (12), and can be used to guide these efforts.

Preliminary Toxicity Data for Kv2 Channel Blockers Preliminary toxicity data for diacylhydrazines and 48F10 analogs to Anopheles gambiae mosquitoes are shown in Table 1. Experiments were preformed on susceptible G3 and the multi-resistant Akron strain, which carries kdr and MACE (altered acetylcholinesterase) (19). The data show that diacylhydrazines are of modest topical toxicity to mosquitoes, with LD50s of about 0.5 µg/female, and treated insects also displayed rapid neuroexcitatory signs of intoxication (twitching and postural collapse). 79 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Table 1. Toxicity of K+ Channel-Directed Compounds against Adult Females of An. gambiae

Two substituted catechols were studied in a more comprehensive series of experiments and we observed the following. The analogs tested were 4-5 fold more toxic than the diacylhydrazines. Moreover, the toxicity was not enhanced by 4 hour pretreatment with 200 ng of the monooxygenase synergist piperonyl butoxide (22), and there was no cross resistance in the Akron strain, as expected (Table 1). Injection increased toxicity 5-6 fold, indicating that the cuticle was not a significant barrier to penetration. For comparison, toxicity of propoxur, a WHOPES approved carbamate for mosquito control, had a topical LD50 of ca. 3 ng/female (23), which makes it about 50-fold more active than the substituted catechols reported here. Nonetheless, these compounds have significant toxicity to mosquitoes, and accordingly voltage sensitive potassium channels, in particular Kv2, appear to be new potential targets for mosquitocide development. 80 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

Acknowledgments The authors thank the Foundation for the National Institutes of Health, Vector Control Translational Research program, for funding this work under project number BLOO11VCTR.

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Voltage-Sensitive Potassium Kv2 Channels as New Targets for

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