Abstract
Dehydroabietic acid derivatives according to Formula Ia or Formula Ib and all stereoisomers thereof, having a linker chain A of 1 to 10 atoms selected from carbon, nitrogen and oxygen to a group X capable of being negatively charged at a physiological pH and covalently attached to the linker chain A, selected from carboxyl, sulfate, sulfonate and phosphate groups. The Dehydroabietic acid derivatives are useful for therapeutic treatment of hyperexcitability diseases
Claims
1. A dehydroabietic acid derivative according to Formula 1a or Formula 1b, or a stereoisomer thereof, wherein R.sub.11, R.sub.12, and R.sub.14 are independently selected from hydrogen, halogen and R.sub.2; R.sub.13 is selected from hydrogen, halogen and R.sub.3; and R.sub.7 is selected from hydrogen, halogen, hydroxyl, carbonyl, and NOR.sub.1; where R.sub.1 is selected from hydrogen, and saturated or unsaturated lower alkyl groups selected from C1-C6 alkyl and C2-C6 alkenyl groups; R.sub.2 and R.sub.3 are independently from each other selected from straight, branched or cyclic saturated or unsaturated hydrocarbons comprising from 1 to 6 carbon atoms; wherein Formula 1a and Formula 1b are: ##STR00019## and wherein A(x) is defined as A-X and comprises a saturated, unsaturated, branched, unbranched, substituted or unsubstituted linker chain A of 1 to 10 atoms selected from carbon, nitrogen and oxygen, between the fused tricyclic moieties of Formula 1a or 1b and at least one group X is capable of being negatively charged at a physiological pH and is selected from carboxyl, sulfate, sulfonate and phosphate groups.
2. The derivative according to claim 1, wherein R.sub.7, R.sub.11, R.sub.12, and R.sub.14 are hydrogen and R.sub.13 is isopropyl.
3. The derivative according to claim 1, wherein the linker chain A is a carbon chain optionally interrupted by one or more atoms selected from nitrogen and oxygen and substituted with one or more of oxo groups, carboxyl groups, lower alkyl groups and halogen groups.
4. The derivative according to claim 1, wherein the linker chain A has 1 or 2 carbon atoms and X is a terminal carboxyl group.
5. The derivative according to claim 4, selected from 2-((1S,4aS)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl)acetic acid (Wu180) and 3-((1S,4aS)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl)propanoic acid (Wu179).
6. The derivative according to claim 1, wherein the linker chain A is a carbon chain, optionally interrupted with a nitrogen or oxygen atom, substituted with at least one of an oxo group and a carboxyl group, and wherein X is a terminal carboxyl group.
7. A derivative according claim 6 being ((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl)-L-aspartic acid (Wu148).
8. The derivative according to claim 1, wherein the linker chain A is a carbon chain comprising 2 to 10 atoms of which at least one atom is nitrogen and X is a terminal carboxyl group.
9. The derivative according to claim 6, selected from group of ((1R4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carbonyl)glycine (Wu117); 3-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)propanoic acid (Wu152); 4-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)butanoic acid (Wu149); 3-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)propanoic acid (Wu152) and 3-(3-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)propanamido)propanoic acid (Wu153); 4-(4-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)butanamido)butanoic acid (Wu151); and 4-((1R,4aR,4bR,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,4b,10,10a-decahydrophenanthrene-1-carboxamido)butanoic acid (Wu157).
10. The derivative according to claim 1, wherein the linker chain A comprises 2 to 5 atoms of which one optionally is nitrogen or oxygen and X is a terminal phosphate, sulfate or sulfonate group.
11. The derivative according to claim 10, selected from the group of ((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl)methyl hydrogen sulfate (Wu161); 2-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)ethane-1-sulfonic acid (Wu154); 3-((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxamido)propane-1-sulfonic acid (Wu150); and ((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl)methyl dihydrogen phosphate (Wu162).
12. The derivative according to claim 1, wherein R.sub.7 is selected from hydrogen, halogen, and NOR.sub.1 and where R.sub.13 is selected from H or halogen.
13. The derivative according to claim 12, wherein the linker chain A is one carbon atom and X is sulfate.
14. (canceled)
15. The derivative according to claim 1, being ((1R,4aS,10aR)-6,7,8-trichloro-1,4a-dimethyl-2,3,4,9,10,10a-hexahydrophenanthrene-1-yl)methyl hydrogen sulfate (Wu181).
16. The derivative according to claim 2, wherein the linker chain A is a carbon chain optionally interrupted by one or more atoms selected from nitrogen and oxygen and substituted with one or more of oxo groups, carboxyl groups, lower alkyl groups and halogen groups.
17. The derivative according to claim 2, wherein the linker chain A has 1 or 2 carbon atoms and X is a terminal carboxyl group.
18. The derivative according to claim 2, wherein the linker chain A is a carbon chain, optionally interrupted with a nitrogen or oxygen atom, substituted with at least one of an oxo group and a carboxyl group, and wherein X is a terminal carboxyl group.
19. The derivative according to claim 2, wherein the linker chain A is a carbon chain comprising 2 to 10 atoms of which at least one atom is nitrogen and X is a terminal carboxyl group.
20. The derivative according to claim 2, wherein the linker chain A comprises 2 to 5 atoms of which one optionally is nitrogen or oxygen and X is a terminal phosphate, sulfate or sulfonate group.
21. A method of treating a hyperexcitability disease selected from epilepsy, pain and cardiac arrhythmia, comprising administering a derivative according to claim 1.
Description
FIGURE LEGENDS
[0162] FIG. 1. Lipoelectric compounds. (A) A compound binds with its hydrophobic anchor in the lipid membrane. The effector (a charged group) electrostatically affects the positively charged voltage sensor (S4). (B) Compounds in A affects the voltage-dependence of the channel opening. The direction of the shift depends on the valence of the charge. (C) Structure and nomenclature of dehydroabietic acid (DHAA). (D) Functional pH dependence for the effect of DHAA on the 3R Shaker Kv channel, from Ottosson, N. E. et al. Sci. Rep. 5, 13278 (2015). The carboxyl group in either protonated or deprotonated form.
[0163] FIG. 2. Effect of DHAA derivatives with different stalk length on the 3R Shaker Kv channel. (A) The Shaker Kv channel, top view of the VSD. Gating charges (arginines) as sticks: the wt Shaker Kv channel has 362R as top charge (=R1); the 3R Shaker Kv channel has two additional charges (M356R and A359R). (B) Nomenclature for the position of the charged group (effector) (P1-11) on the stalk (C) K currents at 10 mV and normalized G(V) curves, before (black) and after (grey) application of 100 M P1-DHAA (shift: 13.0 mV) and P6-Wu149 (shift: 5.3 mV). pH=7.4. (D) G(V) shifting effect of DHAA derivatives with different stalk lengths and carboxyl group as effector. 100 M, pH=7.4. MeanSEM (n=3-11). Data fitted with a one phase exponential decay curve. Length constant=5.1 atoms.
[0164] FIG. 3. Role of stalk length with a fully charged effector. All compounds 100 M. (A) Structures of permanently charged DHAA derivatives with a sulfonic-acid group. (B) G(V) shifting effects of DHAA derivatives with carboxyl groups (from FIG. 2B) at pH=10 (white symbols), and permanently charged groups (from a) at pH=7.4 (black symbols). MeanSEM (n=3-7). Grey dashed line is for DHAA derivatives with carboxyl group at pH=7.4 (adopted from FIG. 2D). (C) Functional pH dependence for G(V) shifts of indicated compounds on the 3R Shaker Kv channel. MeanSEM (n=3-11). pK.sub.a=7.2 (DHAA), 7.3 (Wu180), 7.5 (Wu179). 6.8 (Wu176). (D) Functional pH dependence for the effect of Wu154 on the 3R Shaker Kv channel. MeanSEM (n=4-6). pKa<7.4. (E) G(V) shifting effects on the wt Shaker Kv channel. Symbols as in B. MeanSEM (n=3-7).
[0165] FIG. 4. The cut-off model. (A) Schematic illustration of cut-off model. (B) Electrostatic energy for a charge q.sub.1=1e at (0,4) nergy q2=1e at (x,z) z. (C) Critical stalk length for a stalk fixed at (x,z) z) cal staq.sub.1=1e at d=(0,2), (0,4), (0,6) for aq.sub.2=1e at the end of the stalk with different lengths. (D) Positions for the cut-off point when the stalk length is 4 h/=4 ions for thq.sub.1=1e at d=(0,2), (0,4), (0,6) e cut-q.sub.2=1e at the end of the stalk.
[0166] FIG. 5. The valance of the charge is critical for the effect The 3R Shaker Kv channel. Concentration of compounds=100 the effect is 4). a carboxyl group. Ch(A) A)annel. Concentrate uncharged compounds. (B) Normalized G(V) curves. G(V) shift=0.0 mV. (C) G(V) shifts for DHAA derivatives in A. Mean 0 the efn=3-4). (D) Stalk structures of permanently charged compounds. (E) Normalized G(V) curves. G(V) shift=34.6 mV. (F) G(V) shifts for DHAA derivatives in D. Mean ives (n=5-6).
[0167] FIG. 6. A divalent charge does not increase the G(V) shifting effect (A) Cut-off area for charge-dependent effects for a stalk length of 4 4 al=4 area for chq.sub.1=1e at d=(0,4). q.sub.2=1e (black) and q.sub.2=2e (grey, dashed line). At point X, q.sub.2=1e will be attracted towards q.sub.1=1e (representing the voltage sensor S4) in the membrane, and q.sub.2=2e will be attracted towards the water (with reduced effect on S4). (B) Structure of Wu162 and stalk structure for molecular species with valance 0, 1, 2. (C) Theoretical pH dependence for Wu162 (D) Functional pH dependence for Wu162 with valence 1 or 2. Mean ence (n=3-5). Dashed line: best fit of Eq. 5.
[0168] FIG. 7. Two carboxyl groups on the stalk. 3R shaker Kv channel, 100 M (A) Structure of Wu148. (B) G(V) shifts for Wu148. pH=7.4. Compared to P1-DHAA, P4-Wu117, P5-Wu152. MeanSEM (n=6), dashed lines. (C) Functional pH dependence for Wu148. MeanSEM (n=3-6). G(V) shifts are compared with Wu148 at pH 7.4, one-way ANOVA Dunnett's multiple comparison test, *p<0.05.
[0169] FIG. 8. P3-Wu161 is five times more potent than DHAA. (A) Normalized G(V)-curves. G(V) shift=10.4 mV. The 3R Shaker Kv channel. pH=7.4. (B) Concentration-response curves for P3-Wu161. pH=7.4. MeanSEM (n=3-6). c.sub.1/2=44 M, V.sub.MAX=13.9 mV (wt). c.sub.1/2=36 M, V.sub.MAX=45.5 mV (3R). (C) Wu161 and DHAA on the 3R (10 M) and wt (100 M) Shaker Kv channels respectively. pH=7.4. MeanSEM (n=3-10). Data for DHAA, from Ottosson, N. E. et al. Sci. Rep. 5, 13278 (2015).
[0170] FIG. 9. The role of S4 charges for the effect of Wu161 and other DHAA derivatives. Concentration for all compounds=100 M. pH=7.4. (A) Schematic picture of S4 charges on the Shaker Kv channel. The top gating charge (an arginine, R362 in wt) was moved step-by-step further out on S4 (left), or removed (R362Q; right). (B) Wu161-induced G(V) shifts on the Shaker Kv channel S4 arginine mutants. MeanSEM (n=3-5). Shifts are compared with R362Q (dashed line), one-way ANOVA with Dunnett.s multiple comparison test. Grey: non-significant, p>0.05. White: larger effect than for R362Q, p<0.001. Black: smaller effect than for R362Q, p<0.001. (C) Correlation between G(V) shifts for the PUFA DHA (Data for DHA: from Ottosson, N. E. et al. J. Gen. Physiol. 143, 173-182 (2014)) and for Wu161 on the Shaker Kv channel S4 arginine mutants. Slope (2.10.2) is significantly different from zero (Pearson correlation test and linear regression are both significant). (D) Effects of P1-DHAA, P1-Wu32 (Wu32 is described in WO 2016/114707), P3-Wu161 and PUFA DHA on the R362Q, wt and 3R Shaker Kv channels.
[0171] FIG. 10. Combined stalk and anchor modifications of the DHAA molecule. Effects on the 3R Shaker Kv channel. pH=7.4 (A) DHAA derivatives studied. (B) Concentration-response curves for DHAA derivatives in A. MeanSEM (n=3-9). c.sub.1/2=98 M, V.sub.MAX=26.5 mV (DHAA). c.sub.1/2=36 M, V.sub.MAX=45.5 mV (Wu161). c.sub.1/2=37 M, V.sub.MAX=46.0 mV (Wu50). c.sub.1/2=6.1 M, V.sub.MAX=53.6 mV (Wu181). Data for Wu50 and DHAA, from Ottosson, N. E. et al. Sci. Rep. 5, 13278 (2015) (C) Currents at 10 mV and normalized G(V) curves. G(V) shift=20.4 mV.
[0172] FIG. 11. DHAA derivatives open the human M-channel. (A) Currents before and after application of 100 M Wu161. Steps to voltages between 120 and +20 mV start at t=0.7 s. At t=2.7 the voltage is switched to 10 mV. Traces at voltage=20 mV in red. (B) G(V) curve for the cell in A. G(V) shift=23.8 mV. (C) G(V) shifts for compounds with different stalk lengths (100 M). MeanSEM (n=3-6). P1-DHAA (pH=10). Permanently charged P2-Wu164, P3-Wu161, P5-Wu154 (pH=7.4). (D) Compounds with different valance (100 M). MeanSEM (n=3-5). ***p<0.001, significantly different from 0. (E) Concentration-response curves. MeanSEM (n=3-4). c.sub.1/2=41.8 M, V.sub.MAX=33.7 mV (Wu161). c.sub.1/2=12.4 M, V.sub.MAX=42.1 mV (Wu181)
[0173] FIG. 12. Summary of suggested mechanisms. Proposed binding of four different compounds to the S3/S4 cleft. The three positive charges represent M356R, A359R, and R362R (=R1) in the top of S4. The negative charge represent the charged group of the four different compounds.
[0174] It is established that polyunsaturated fatty acids (PUFAs) bind to at least five different sites in different voltage-gated ion channels, see Elinder F & Liin SI Front. Physiol. 8:43 2017. One site found on the Drosophila Shaker Kv (Kv1-type) channel, and on the human Kv7.1 and Kv7.2/7.3 channels is close to S4. PUFAs anchored to this site, electrostatically open the channel via its negatively charged carboxyl group. The hydrophobic tail seems to act as an anchor for binding, and the charged group as the executor part, altering the S4 movement, the direction depending on the valence of the charge (FIG. 1A). A negatively charged group shifts the conductance-versus-voltage curve, G(V), in negative direction along the voltage axis, and a positively charged group in positive direction (FIG. 1B).
[0175] Hydrophobic resin acids (e.g. dehydroabietic acid (DHAA), FIG. 1C, and abietic acid (AA)), with a three-ring motif and a negatively charged carboxyl group most likely act via the same mechanism. The present inventors assume that the tree-ring motif acts as the anchor, and that the carboxyl group act as the executor. There are two major arguments for an electrostatic effect: (1) Altering the charge of the resin acid, or (2) altering the charge of the voltage sensor both affect the resin acid-induced G(V) shift. These two arguments can be divided in several subarguments: (1a) Altering pH protonated or deprotonated the carboxyl group and thereby altered the G(V) shift; the pKa value was 7.2 for DHAA (FIG. 1D). (1b) Altering the negatively charged carboxyl group to a positively charged amine group altered the sign of the G(V)shift. (2a) Addition of two positively charged arginines in the top of S4 of the Shaker Kv channel (M356R and A359R) increased the resin-acid induced G(V) shift about three-fold (This channel is referred to as the 3R Shaker Kv channel because the added charges together with the top charge in the wild-type (wt) Shaker Kv channel (R362) form the active triad of charges affecting the PUFA-induced shifts. (2b) The resin-acid charge supports or prevents rotation of the voltage sensor S4, depending on which side of S4 the top charge of S4 is located, and on the sign of the charge.
[0176] The inventors have previously performed a systematic exploration of molecular properties of the anchor of resin acids, primarily DHAA derivatives, when side chains on C7 in ring B (FIG. 1C) and halogenation of ring C was altered, see WO 2016/114707. The anchor modifications likely alter properties such as the depth of binding (into the lipid membrane), the affinity, the pKa value, and the solubility, which in turn affect the channel-opening properties. In the present invention, the effect of DHAA derivatives with modifications at the effector-site (carboxyl acid-site) is explored in an effort to maximize the interaction between the resin acid and the voltage sensor. In particular, the distance between the carboxyl group and S4 is considered (by putting a stalk between the carboxyl-group and the anchor with increasing length) and by increasing the valence of charge.
[0177] Increasing the Stalk Length Decreases the Effect on the 3R Channel at Neutral pH
[0178] We hypothesized that a shorter distance between this negatively charged effector (carboxyl group) and the positively charged gating charges (arginines) of the voltage sensor S4 should increase the G(V)-shifting efficacy of the resin acid on the Shaker Kv channel. To explore this, we used the 3R Shaker Kv channel (with two additional arginines in the top of S4, M356R and A359R, FIG. 2A), a modification clearly increasing the resin-acid induced G(V) shift. Synthesized DHAA molecules with the carboxyl group located further and further out from the three-ringed motif (the anchor) were tested at pH 7.4 and 100 M (FIG. 2B-D). When the stalk length increased, the resin-acid induced G(V) shift decreased exponentially (FIG. 2D). The length constant was 5.10.5 atoms. However, the functional pKa value (around 7.2 for DHAA) is sensitive to the local environment, and the local environment might be very different depending on the stalk length, e.g. further into the membrane or closer to extracellular solution. We therefore aimed to minimize the environmental influence (and the theoretical pKa differences) by making the effector fully charged.
EXAMPLE 2
[0179] A Critical Cut-Off Point for Stalks Between Three and Four Atoms
[0180] To explore the effect with a fully charged effector experiments were performed either at pH 10 to make sure that the carboxyl group, regardless of stalk length, was fully charged (see FIG. 1D), or alternatively, changed the carboxyl group to a permanently negatively charged sulfonic-acid group whenever possible (FIG. 3A). For stalk lengths up to three atoms (P1-P3) the effect was clearly potentiated at pH 10 (white symbols, FIG. 3B), or when a permanently charged group was used (black symbols, FIG. 3B), compared with pH 7.4 for the compounds with carboxyl groups (grey dashed curve, FIG. 3B). For the permanently charged compounds the effect was much larger for P3 than for P2. However, for stalks longer than three carbons the effect was radically suppressed (FIG. 3B) and the compounds with a carboxyl group were no longer sensitive to an increased pH. The carboxyl acid-derivatives with one, two- or three-carbon stalks had a functional pK.sub.a value of 7.2 (P1), 7.3 (P2), and 7.5 (P3), respectively, while the permanently charged P3 compound was not pH sensitive, as expected because of its low theoretical pK.sub.a value (FIG. 3C). By altering the stalk geometry it was also possible to shift the pKa value. Wu176 with a double bond in the stalk had a pKa value of 6.8 (FIG. 3C). The P4-P6 compounds had functional pKa values <7.4 as if these carboxyl groups experienced another local environment (FIG. 3D).
[0181] The data on the 3R Shaker Kv channel shows that (i) the largest effect was found for the permanently charged P3 DHAA derivative, and that (ii) there was a drastic decrease in effect beyond three carbons. A critical question is if this behaviour is found also for the wt Shaker Kv channel (with clear sequence similarity in S4 to several human Kv channels) with apparent therapeutic implications, or if it is restricted to the artificial 3R Shaker Kv channel. When the stalk-length series was tested on the wt Shaker Kv channel a noticeable difference, except for the absolute magnitude, was that derivative-induced G(V) shift increased when the stalk length was prolonged from P1 to P3, but the cut-off was not shifted (FIG. 3E). This suggests that the stalk length is a powerful variable to alter the efficacy for a channel-opening pharmaceutical drug. It also opens for a selectivity if, let us say, another channel, which is not intended to be affected by the drug, has a cut-off between P2 and P3.
[0182] If a longer stalk brings the negative charge closer to S4 and thereby increases channel opening and the G(V) shift (FIG. 3B) was corroborated for stalk lengths up to three atoms, most clearly for the wt Shaker Kv channel and in particular for permanently charged compounds at pH 7.4. However, the radically sharp cut-off at longer stalk lengths was a surprise.
EXAMPLE 3
[0183] The Cut-Off Model
[0184] The experimental data presented above showed that the G(V)-shifting effect increased if the stalk was prolonged, as if the negative resin-acid charge was allowed to come closer to the positive S4 charge to more strongly pull the channel open. At a certain length the G(V)-shifting effect disappeared and a sudden break occurred in the curve (FIG. 3B,E). A simple explanation for this behaviour is that when the stalk exceeds a certain length the negative resin-acid charge suddenly find an energetically more suitable position, which is far away from the S4 charge, or even outside the membrane, and thus do not help to open the channel.
[0185] To explore this theoretically and quantitatively we analysed a simple system (FIG. 4A) where a low dielectric medium (the lipid bilayer, relative dielectric constant .sub.r=2) meets a high dielectric medium (water, .sub.r=80). In reality, the dielectric medium varies gradually in the channel's rough structure, but the model can give us some simple guidelines. For simplicity also there are no ions in the solution. A fixed positive charge in the lipid, at a distance d from the water represents the voltage sensor S4, most likely the top charge of S4. If a counter charge (of valence 1) is introduced at specific positions of the system, the total electrostatic energy can be calculated (d=4 in FIG. 4B). Next, if the counter charge instead is attached to a stalk and the other end of the stalk is fixed at a certain anchor point (x in FIG. 4A), the electrostatic interaction will stretch the stalk and the charge will end up in the energetically most favourable position. We call this charge on a stalk the semi-mobile charge. For most of the positions of the anchor points, the semi-mobile charge will be attracted either to the fixed S4 charge or to the high dielectric water, independent of the stalk length l. However, for some anchor positions, the semi-mobile charge will be attracted to the fixed S4 charge (i in FIG. 4A) for short stalk lengths and to the high dielectric water (ii in FIG. 4A) for long stalk lengths. The switch from one direction to the other occurs at a specific stalk length (the cut-off length). For each position of the fixed S4 charge, the area for these anchor points can be calculated and the cut-off lengths colour coded (FIG. 4C for the positions d=2 , d=4 , d=6 ).
[0186] Even though the distance d is not known some general conclusions can be drawn: (i) The shorter the cut-off length of the stalk is, the closer the anchor point is to the surface. (ii) For a specific cut-off length of the stalk, the possible anchor points are relatively independent of the distance d of the gating charge from the surface (FIG. 4D). Experimentally, we found that the cut-off length was roughly 4 . This places the anchor point about 4-6 from the water if the horizontal distance from the S4 charge is less than 10 (FIG. 4D). Having said this, we should be aware of that the quantitative estimations depend on the model and their constants.
EXAMPLE 4
[0187] The Valence of the Charge is Critical
[0188] Above, we have analysed the effect of a single fully charged effector on stalks of different lengths. A critical finding was that, most strikingly for the wt Shaker Kv channel, there was a maximal G(V) shift when the effector group was 3 atoms away from the anchor point. However, for longer stalks, for both the wt and the 3R Shaker Kv channels, the effect was eliminated, probably because of snorkelling of the charge towards the extracellular solution.
[0189] Our next hypothesis was that the G(V)-shifting effect could be increased by increasing the valance of charge on the stalk. To further corroborate that the charge was critical for the effect we introduced a neutral group instead of the carboxyl group. Two different uncharged P3 compounds (FIG. 5A, Wu110 and Wu111) tested on the 3R Shaker Kv channel at 100 M and pH 7.4 did not shift the G(V) curve (FIG. 5B,C).
[0190] Contrary, two different permanently single-charged P3 compounds (FIG. 5D, Wu109 and Wu161) at 100 M and pH 7.4 shifted the G(V) curve of the 3R Shaker Kv channel by approximately 30 mV (FIG. 3; FIG. 5E,F). What happens with a divalent charge? If the divalent charge is in the same position as the monovalent charge and if there is an electrostatic interaction between S4 and the compound, the effect should increase. However, our simple cut-off model also suggests another finding. At some anchor points, the double-charged group on a stalk is expected to find its way towards the water rather than to the S4 charge in the membrane, while the single-charged group on a stalk finds its way towards the S4 charge (FIG. 6A, when the semi-mobile charge is anchored (x) between the two different cut-off lines for the valences 1 and 2). Thus, it is possible that the G(V)-shifting effect also decrease with a divalent charge on the stalk.
[0191] Wu162 (FIG. 6B) has a phosphorous-acid group (P(O)(O.sup.).sub.2) three atoms away from the anchor. It is a strong acid, expected to have one negative charge in the pH range from 3 to 6 (theoretically 90% of the molecules in this range) and with increasing pH the group is expected to have two negative charges (FIG. 6C). At pH 5.5, when nearly all Wu162 molecules are expected have a single negative charge, 100 M Wu162 shifted the G(V) curve by almost 20 mV (FIG. 6D). The oocytes did not tolerate pH 5. When pH instead was increased above 6 the effect significantly decreased (FIG. 6D), suggesting that a stalk with a divalent charge instead finds a position further away from the voltage sensor. The dashed line is the best fit of Eq. 5. In other aspects, the divalent Wu162 molecule at pH 7.4 behaved qualitatively as the monovalent compounds: The effect was smaller on the wt Shaker Kv channel than on the 3R (Table 1). Increasing the stalk beyond four atoms decreased the voltage shift (Wu158 (P4) compared with Wu162 (P3), Table 1). Another possibility to increase the charge on the stalk was to add two monovalent charges on the same stalk, but at different positions. One P4 charge and one P5 charge (carboxyl group), sharing the first three atoms of the stalk, made it possible to add the effects from the single charged P4 and P5 molecules, but because adding two charges only is possible for longer stalks, with small effect on its own, so the sum was not very impressive (FIG. 7A-C).
[0192] In conclusion, a divalent charge does not increase the G(V)-shifting effect compared with a monovalent charge, but decreased it. A possible explanation is that a divalent charge instead finds a more energetic favourable position towards the water rather than the voltage sensor. Therefore, the stalk length is better modifier of the effect than to increase the valance above 1.
EXAMPLE 6
[0193] Wu161a Functional Chimera Between a Fatty Acid and a Resin Acid
[0194] The aim of this investigation was to modify the carboxyl-group of DHAA to enhance the G(V) shift. So far we have described that a three-atom long stalk combined with a decreased pK.sub.a value for the effector group is the most efficient modification. Increasing the valance above one did not increase the effect, but rather decreased it. P3-Wu161 (FIG. 3A) is thus one of the most intriguing compounds in this study and it holds promises for future drug-development efforts. In this section we explored the compound further, both with respect to concentration dependence and to specific channel mutations around S4 to get information about its interaction surface with the channel.
[0195] Wu161 at 10 M, clearly shifted the G(V) of the 3R Shaker Kv channel along the voltage axis by 12.31.0 mV (n=4; FIG. 8A). A concentration-response curve shows a half-maximal concentration, c.sub.1/2, of 369 M and a maximum shift of 45.53.5 mV even though a saturation was not reached experimentally in the concentration range investigated (FIG. 8B, grey symbols). For the wt Shaker Kv channel the concentration dependence was not different (cy.sub.1/2=4410 M) but the amplitude was a factor 3 smaller (13.91.1 mV; FIG. 8B, black symbols). Thus, the two added charges in top of S4 (the 3R channel) does not affect the affinity of Wu161 but increases the efficacy. This 3-fold difference between the wt and the 3R Shaker Kv channel is the same as for DHAA, see Ottosson, N. E. et al. Sci. Rep. 5, 13278 (2015). However, compared to DHAA, the modification at the carboxyl-site for Wu161 (prolongation with two carbons), increased the G(V) shifting effect by a factor of about five, both on the wt and the 3R Shaker Kv channel (FIG. 8C). Data for DHAA, from Ottosson, N. E. et al. Sci. Rep. 5, 13278 (2015).
[0196] For compounds acting close to the voltage sensor, the positions of the gating charges play a large role. We explored a series of single mutations where the top charge of S4 (R362) was moved residue by residue towards the extracellular end of S4 (from R362R to M356R), or explored the effect on a channel where this area was uncharged (R362Q) (FIG. 9A). The amplitude of the effect followed an oscillatory pattern when the positive gating charge, at the top of S4, was moved along S4 (FIG. 9B). This is consistent with previous studies with the PUFA docosahexaenoic acid (DHA), see Ottosson, N. E. et al. J. Gen. Physiol. 143, 173-182 (2014) and for two P1 resin acids (DHAA, Wu32). Our data for P3-Wu161 is very similar to that of the PUFA DHA, except that Wu161 is about twice as effective, as expected from a fully charged compound (Wu161) compared with a partially charged compound (DHA). There is an almost linear relation between the effects of DHA vs. Wu161 (FIG. 9C), suggesting that DHA and Wu161 act in a similar way, probably from about the same position close to S4. In contrast with this, two P1 resin acids (DHAA and Wu32) differed from the PUFA DHA with respect to one of the channel mutations in S4: DHAA and Wu32 have an about twice as large effect on R362Q than on wt R362, while DHA and Wu161 have about the same effects on these two channels (FIG. 9D). This has been interpreted as if the P1 resin acids binds deeper into the VSD (in the S3/S4 cleft). Now, the P3 compounds seems to be more PUFA-like and we suggest that the longer stalk makes the resin acid act more a snake-like fatty acid.
EXAMPLE 7
[0197] Combined Stalk and Anchor Modifications Enhanced the G(V) Shift
[0198] The G(V)-shifting effect of the DHAA derivatives can also be increased by modifications on the three ringed motif (the anchor), not only by modifications of the effector as shown in above. Wu161 and Wu50 (FIG. 10A) have similar effects on the 3R Shaker Kv channel (FIG. 10B), both are clearly more potent than DHAA. They both increased the affinity by a factor of 2.7 (from 9819 M (DHAA) to 369 M (Wu50) and 376 M (Wu161) respectively) and the amplitude by a factor of 1.7 (from 26.52.2 mV to 45.53.5 mV (Wu50) and 46.02.4 mV (for Wu161) respectively) compared to the unmodified DHAA. Data for DHAA and Wu50, from Ottosson, N. E. et al. Sci. Rep. 5, 13278 (2015). What happens if we combine an anchor modification with a stalk/effector modification? Wu181 combine both these two modifications (FIG. 10A). Wu181 has very large effects at 3 M on the 3R Shaker Kv channel (FIG. 10C); the shift of the G(V) curve was 21.32.9 mV (n=5). While the max shift was not significantly increased (53.64.0 mV for Wu181), the affinity was increased by a factor 6 (to 6.11.7 M for Wu181). Thus, a systematic modification of the mother compound DHAA with respect to the anchor motif and the effector motif increased the affinity at pH 7.4 by a factor of 16 and the efficacy by a factor of 2.0. Altogether this suggests an increased G(V) shift at low concentrations by a factor of 32.
EXAMPLE 8
[0199] DHAA Derivatives Open the Human M-Channel
[0200] Wu161 and Wu181 were shown to have large effects on the 3R Shaker Kv channel (FIG. 11B). Wu161 functionally reminds about the PUFA DHA (FIG. 9C), which is known to act on the human M-type (hKv7.2/7.3) channel at low micromolar concentrations. Therefore, we explored the effect of Wu161 and Wu181 on the hKv7.2/7.3 channel. 100 M Wu161 increased the M-current at negative voltages (FIG. 11A) by shifting the G(V) curve in negative direction along the voltage axis by 23.81.3 mV (n=4; FIG. 11B). As for the wt Shaker Kv channel a stalk length of three atoms had the largest effect on the M-channel (FIG. 11C) and the charge was also critical for the effect (FIG. 11D); the uncharged P3 compound (Wu110), had no effect, and a fully charged P3 compound (Wu161) had larger effect than a partially charged P3 compound (Wu179). 10 M Wu161 also significantly shifted the G(V) of the M channel by 6.30.9 mV (n=4; FIG. 11E). The half-maximal concentration, c.sub.1/2, was 4213 M and the maximum shift was 33.63.7 mV. However, Wu181, combining the effector of Wu161 with the anchor of Wu50 had a much larger effect on the M channel. 10 M Wu181 shifted the G(V) by 18.92.4 mV (n=4) along the voltage axis, and 1 M shifted the G(V) by 4.41.6 mV (n=4). The half-maximal concentration, c.sub.1/2, was 12.43.7 M and the maximum shift was 42.15.7 mV.
Summary of Examples 1-8
[0201] This investigation suggests that resin acids act on a voltage-gated K channel by having (i) an anchor, which bind the molecule close to the VSD, and (ii) an effector, which electrostatically exerts the effect on the voltage sensor S4.
[0202] The major findings are the following: [0203] (1) There is an optimal stalk length for the charged effector. For short stalks, the effect on voltage gating is increased when the stalk length is increased (FIG. 3E). A sudden drop in effect occurs at a certain cut-off length (FIG. 3B, 3E). These data fits with a simple electrostatic model where an increased stalk length allows the effector charge to come close to the charged voltage sensor S4 (FIG. 12, P1.fwdarw.P3), and that the effector charge finds a position far away from the voltage sensor when the stalk exceeds a certain cut-off length (FIG. 12, P4). [0204] (2) The charge of the effector is absolutely critical. An uncharged molecule has no effect. But, a double charged effector does not increase the effect, but rather decrease the effect. The electrostatic model suggests that the double charged effector tends to, for electrostatic reasons, choose a location further away from the voltage sensor. [0205] (3) The P3 molecule Wu161 affects the voltage sensor in a similar fashion as PUFAs (FIG. 9, FIG. 12). [0206] (4) The G(V) shifting effects of an improved anchor and effector are additive, making Wu181 very potent. [0207] (5) The human M-type Kv (hKv7.2/7.3) channel is clearly opened by 1 M of Wu181.
[0208] Knowledge of resin acids in detail can potentially lead to development of new drugs with high specificity, affinity and selectivity.
[0209] The family of resin acids includes many compounds acting on several types of ion channels a general theme on mechanism of their effects emerges. Most compounds open voltage-gated ion channels by shifting the G(V) curve along the voltage axes. Pimaric acid, isopimaric acid, DHAA, and abietic acids shifts the G(V) of the Shaker Kv channel, while podocarpic acid with a polar side chain in its anchor does not shift the G(V). Pimaric acid shifts the voltage dependence of activation of Kv1.1-2.1 channels, but not of Kv4.3 channels. Resin acids also open ion channels outside the Kv family. They open large-conductance voltage- and Ca2+-activated K+(BK) channels, also by shifting the G(V) curve along the voltage axis. But the effects are not limited K channels but also include voltage-gated Na and Ca channels; isopimaric acid acts on five of six explored voltage-gated ion channels in a mouse cardiac atrial cell line. However, in addition to the effects on the G(V) curves, also the steady-state inactivation curves were shifted in negative direction along the voltage axis. Thus, it is clear that several resin acids act on many types of voltage-gated ion channels.
[0210] Although, binding sites for the resin acids have not been assigned to most ion channels, the communalities in effects suggests that the different types of channels share a common binding site. We have suggested that resin acids electrostatically interact with the voltage sensor of the Shaker Kv channel by binding in a pocket between the transmembrane segments S3 and S4 of the VSD, and the lipid bilayer. Together with the effects reported in the present investigation on the M channel, all these data suggests that the resin acid pocket is conserved between different Kv channels. However, it is also known that subtle side chain alterations of the compounds can have large effects on the effects, and we know that the effect varies from channel to channel. Therefore, it is likely that compounds with high channel specificity can be developed.
[0211] In the present investigation, the G(V)-shifting effect of DHAA was improved with modifications at the carboxyl-site, up to five-fold for Wu161 with a permanent negative charge on a stalk three atoms away for the anchor. Thus, modifications at the carboxyl-site can help to increase the G(V)-shifting effect and possible help to tune the resin acid sensitivity between different ion channels, with different charge profile around the voltage sensor, suggesting that the carboxyl-site of resin acids is a powerful site to modify Kv channel opening activity in future drug design.
[0212] In conclusion, all flexibilities at the carboxyl-site described in the present investigation suggests that it is likely that lipoelectric compounds can be improved to increase the G(V) shift and developed into channel specific compounds to cause desired effects in different tissues.
TABLE-US-00001 TABLE 1 Summary of compound properties. G(V) Position Charged shift Name Anchor of charge group LogP pKa Channel pH n (mV) SEM p* Wu110 DHAA None None 4.48 3R 7.4 4 0.56 0.32 N.S M 7.4 3 1.50 0.40 N.S Wu111 DHAA None None 4.99 3R 7.4 3 1.83 1.25 N.S DHAA P1 COOH 5.57 4.55 3R 7.4 11 13.59 0.81 <0.0001 3R 10 9 24.73 1.34 <0.0001 wt 10 7 1.59 0.35 0.0019 M 10 3 11.63 0.97 0.0069 AA P1 COOH 4.95 4.59 3R 7.4 4 16.05 0.92 0.0004 Wu180 DHAA P2 COOH 5.68 4.79 3R 5.5 4 1.575 0.47 0.0431 3R 7.4 5 11.04 1.384 0.0013 3R 10 5 19.18 2.739 0.0022 wt 10 4 2.40 0.38 0.0078 Wu164 DHAA P2 S(O2)OH 4.99 0.49 3R 7.4 4 18.00 0.60 <0.0001 wt 7.4 4 4.10 0.59 0.0061 M 7.4 6 6.58 1.25 0.0033 Wu179 DHAA P3 COOH 6.13 4.91 3R 5.5 5 2.80 0.45 0.0033 3R 6.5 4 3.23 0.81 N.S 3R 7.4 3 10.43 0.58 0.0030 3R 9 4 16.63 0.03 <0.0001 3R 10 4 22.83 0.67 <0.0001 wt 10 4 3.83 0.43 0.0031 M 7.4 5 6.68 1.02 0.0028 Wu109 DHAA P3 S(O2)OH 4.60 0.51 3R 7.4 5 30.68 0.65 <0.0001 Wu161 DHAA P3 S(O2)OH 5.33 0.18 3R 5.5 4 28.10 1.57 0.0004 3R 6.5 4 27.53 1.61 0.0004 3R 7.5 6 33.38 2.77 <0.0001 3R 9 6 25.65 2.81 0.0003 3R 10 4 26.38 1.76 0.0006 wt 7.4 3 10.00 1.06 0.0110 M 7.4 4 23.75 1.33 0.0004 Wu181 Wu50 P3 S(O2)OH 5.90 2.04 3R 7.4 4 31.90 3.73 0.0004 10 M M 7.4 4 18.90 2.40 0.0043 Wu162 DHAA P3 P(O)(O.sup.).sub.2 5.15 1.87/6.89 3R 5.5 5 18.6 1.31 0.0001 3R 6.5 4 10.03 1.40 0.0055 3R 7.4 5 11.80 1.48 0.0013 3R 9 4 11.55 0.25 <0.0001 3R 10 3 8.93 0.65 0.0052 wt 7.4 4 1.18 0.45 N.S Wu176 DHAA P3 COOH 6.12 5.01 3R 5.5 3 2.80 0.52 0.0328 P1=P2 3R 7.4 7 15.16 1.26 <0.0001 3R 10 4 17.10 0.36 <0.0001 Wu117 DHAA P4 COOH 4.64 4.15 3R 7.4 6 7.27 0.36 <0.0001 3R 10 3 5.27 0.46 0.0247 wt 10 4 0.80 0.48 N.S Wu158 DHAA P4 P(O)(O.sup.).sub.2 4.11 1.57/8.08 3R 7.4 5 4.94 0.93 0.006 Wu152 DHAA P5 COOH 4.88 4.59 3R 7.4 5 6.2 1.29 0.0087 3R 10 3 5.20 0.83 0.0247 wt 10 3 1.17 0.26 0.0464 Wu154 DHAA P5 S(O2)OH 4.19 0.68 3R 7.4 4 9.85 1.352 0.0053 wt 7.4 5 2.48 0.20 0.0002 M 7.4 4 4.60 0.43 0.0017 Wu149 DHAA P6 COOH 5.17 4.32 3R 7.4 6 4.80 0.80 0.0018 3R 10 3 5.17 0.55 0.0111 wt 10 7 0.57 0.89 N.S Wu150 DHAA P6 S(O2)OH 4.25 0.87 3R 7.4 4 9.98 0.63 0.0005 wt 7.4 4 4.18 0.44 0.0024 Wu157 AA P6 COOH 4.32 4.37 3R 7.4 5 7.10 0.53 0.0002 Wu153 DHAA P9 COOH 4.01 4.06 3R 7.4 5 2.54 1.36 N.S Wu151 DHAA P11 COOH 4.59 4.48 3R 7.4 4 2.53 0.42 0.0093 Wu148 DHAA P4 and P5 COOH x2 6.24 3.88/5.71 3R 5.5 3 13.30 2.01 0.0220 3R 6.5 3 14.23 0.46 0.0011 3R 7.4 6 14.47 0.97 <0.0001 3R 9 4 11.20 0.89 0.0011 3R 10 3 9.53 0.35 0.0013 wt 7.4 3 0.73 0.09 0.0142 Concentration = 100 M if not stated otherwise. Name: Name of compound. Anchor: Three ring structure derived from dehydroabietic acid (DHAA), abietic acid (AA) or Wu50. Position of charge: Position of charged group as described in FIG. 2B. Charged group: Carboxyl group COOH, sulfonic acid group S(O2)OH, phosphorous-acid group (P(O)(O.sup.).sub.2). pK.sub.a: calculated value for the logarithmic acid dissociation constant (see Methods). Log P: calculated value for the logarithm of the partition coefficient (see Methods). Channel: Channel used, wt or 3R Shaker Kv channel. M: hKv7.2/7.3, M-channel. pH: pH used. Mean and SEM: average G(V) shift and standard error of mean. n: Number of cells.
