A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels

Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects. Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects. Atrial fibrillation (AF), a common cardiac arrhythmia, affects over two million Americans and is associated with increased morbidity, mortality, and healthcare costs. Antiarrhythmic drugs (AADs) used to treat AF aim to either maintain the normal sinus rhythm (SR) or control ventricular rate (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). However, conventional drugs such as sodium channel blockers (class I antiarrhythmic drugs) and potassium channel blockers (class III antiarrhythmic drugs) have limited efficacy, especially in persistent AF patients. In addition, these conventional AADs carry a risk of ventricular proarrhythmia, such as torsade de pointes (TdP) through excessive delay of ventricular repolarization (2Torp-Pedersen C. Moller M. Bloch-Thomsen P.E. Kober L. Sandoe E. Egstrup K. Agner E. Carlsen J. Videbaek J. Marchant B. Camm A.J. Dofetilide in patients with congestive heart failure and left ventricular dysfunction. Danish Investigations of Arrhythmia and Mortality on Dofetilide Study Group.N. Engl. J. Med. 1999; 341: 857-865Crossref PubMed Scopus (898) Google Scholar, 3Waldo A.L. Camm A.J. deRuyter H. Friedman P.L. MacNeil D.J. Pauls J.F. Pitt B. Pratt C.M. Schwartz P.J. Veltri E.P. Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. The SWORD Investigators. Survival with oral d-Sotalol.Lancet. 1996; 348: 7-12Abstract Full Text Full Text PDF PubMed Scopus (1176) Google Scholar). Thus, development of new antiarrhythmic agents with a more effective and safer profile is highly desirable (4Hashimoto N. Acetylcholine-activated potassium channel as a novel target for AF treatment.in: Atrial Fibrillation - Basic Research and Clinical Applications. IntechOpen Limited, London, UK2012: 321-338Crossref Google Scholar). The cardiac acetylcholine- (ACh-) activated inwardly rectifying K+ current (IKACh) consists of heterotetrameric ion channel subunits composed of Kir3.1 (GIRK1 or G1) and Kir3.4 (or GIRK4) subunits and is activated via the M2 muscarinic receptor to mediate vagal influences on heart rate and atrial repolarization. The Kir3.1/4 heterotetramers are the main contributors to the cardiac IKACh, while Kir3.4 homotetramers contribute considerably less activity. In contrast, Kir3.1 homotetramers are inactive and mostly trapped in the endoplasmic reticulum (ER) (5Ma D. Zerangue N. Raab-Graham K. Fried S.R. Jan Y.N. Jan L.Y. Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart.Neuron. 2002; 33: 715-729Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Heteromerization with the Kir3.4 traffics the heterotetramer efficiently to the plasma membrane to contribute to IKACh. Mutation of Kir3.1(F137) to the corresponding Ser residue found in Kir3.4 produces functional Kir3.1(F137S) channels although their trapping in the ER continues (6Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (98) Google Scholar, 7Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. Probing the G-protein regulation of and the two subunits of the KACh functional Full Text Full Text PDF PubMed Scopus Google Scholar). Kir3.1 an tool to the role of Kir3.1 subunits in the regulation of heteromeric channel but in addition, it found to a critical determinant of activity in the action of channel N. de E. K. the of inwardly rectifying K+ channels by the novel Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, L. D. S. A. Logothetis D.E. The channel and in Full Text Full Text PDF PubMed Scopus Google Scholar). channel activity is inwardly the IKACh is an to the repolarization and a role in the membrane potential U. E. of potassium in cardiac PubMed Scopus Google Scholar, N. 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Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). of IKACh channel have in recent However, selective IKACh channel have approved for to N. D. potassium channel Full Text Full Text PDF PubMed Scopus Google Scholar, S. for the treatment of atrial PubMed Scopus Google Scholar). is a of the from the of the that selectively inhibits and or as as potassium channels novel inhibitor for K+ PubMed Scopus Google Scholar, D.J. and K+ channels in a Pharmacol. PubMed Scopus Google Scholar). selectively inhibits IKACh channels in the cardiac with on channels of a of inhibitor for K+ 1999; PubMed Scopus Google Scholar). side of could from of in the central D.J. and K+ channels in a Pharmacol. PubMed Scopus Google as as and plasma in potassium as a target for atrial fibrillation Cardiovasc. Pharmacol. 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H. of a highly selective K+ channel on atrial PubMed Scopus Google Scholar, N. J. N. K. S. K. H. S. of the selective KACh channel on atrial fibrillation in a model of atrial with and III Cardiovasc. Pharmacol. PubMed Scopus Google Scholar). However, a to the to the AF in patients with is that was due to the was to central side (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). In addition, IKACh and not atrial repolarization in atrial patients H. A. L. S. M. L. the inwardly rectifying potassium current a antiarrhythmic of and in and 2015; PubMed Scopus Google Scholar). However, the of desirable of these compounds not the of IKACh channel for persistent AF (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). the to new drug selectively targeting IKACh channels. of benzopyran derivatives with was a on the atrial used for treatment of antiarrhythmic agents that as their main mechanism of action of the due to of the action potential in ventricular highly to such as de pointes In contrast, benzopyran compounds selectively the atrial on the ventricular K. as Scholar). However, the mechanism of their antiarrhythmic activity series a M. S. Novel of and for a of a of compound is to the N. J. A. J. N. H. of a highly selective K+ channel on atrial PubMed Scopus Google Scholar). We the M. S. Novel of and for benzopyran compounds and found BP-G1 to selectively and heteromeric Kir3.1/4 or channels over cardiac and channels. The compound used as a useful tool to the mechanism of action of benzopyran compounds through their with the as as their selectivity over ion channels. In we the molecular mechanism of BP-G1 action and its with the Kir3.1/4 a of and of benzopyran derivatives with was to have a on the of atrial on the and action potential of ventricular K. as Scholar). However, the mechanism of the antiarrhythmic activity of these drugs We benzopyran derivatives selectively channels that cardiac IKACh that is found in the atrial We the inhibitory activity of the benzopyran BP-G1 on inwardly K+ and Kir3.1/4 channels by two The compound inhibitory selectivity for heteromeric channels Kir3.1 and over channels and and of of Kir3.1/4 and channels to the of the BP-G1 the (or channel current was over as with the channel BP-G1 selectively heteromeric and Kir3.1/4 in a and with of and in and and and in G and The residue to a critical Kir3.1 determinant for current of heteromeric and for and current (6Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (98) Google Scholar, 7Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. Probing the G-protein regulation of and the two subunits of the KACh functional Full Text Full Text PDF PubMed Scopus Google Scholar, N. de E. K. the of inwardly rectifying K+ channels by the novel Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, L. D. S. A. Logothetis D.E. The channel and in Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the M2 residue D173 was to critical for and current Thus, we to Kir3.1(F137) and for BP-G1 in the of Kir3.4 channels. We to the and corresponding residues of the Kir3.4 to the F137 and D173 of the Kir3.1 channel and with Kir3.4 subunits (6Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (98) Google Scholar). while BP-G1 not the Kir3.4 channel or the channel it the channel in a and In the of the BP-G1 the a less by BP-G1 an residue in in the of the not contribute to the by the of that the residue in Kir3.4 corresponding to F137 in Kir3.1 is a critical determinant for selective by BP-G1. the molecular mechanism of the BP-G1 on we for the Kir3.4 and Kir3.1/4 channels on a of the channel and channels was by the The and Kir3.4 or and Kir3.1 are and the an to for the and Kir3.1/4 channels M. L. A. Logothetis D.E. channel molecular and of Scholar). We used the A. by of PubMed Scopus Google to for Kir3.4 and on the and the one with the best for the compound and channel on the model of Kir3.1/4 we molecular simulations to the potential in the channel. that the Kir3.1(F137) residue to the is critical for the inhibitory activity of we these residues with the residues and to the for The the central cavity of the channel. We and and by the the and The with the from the was as the predicted of the The the BP-G1 and the channel the to of PubMed Scopus Google Scholar). and predicted the BP-G1 and Kir3.1/4 channel. predicted two the of of the compound and a residue in channels E141 in the and one the of of the compound and D173 that is unique to Kir3.1 with residues found in the channels M. L. A. Logothetis D.E. channel molecular and of Scholar). the critical Kir3.1(F137) that is for BP-G1 and and was not predicted by the model to with the the model that F137 residue with the BP-G1 the of F137 to E141 to the The F137 side to the E141 and the the role of the residue in the BP-G1 we it to The a current with the to with the Kir3.1/4 and and In contrast, the in BP-G1 We the role of the Kir3.1 unique residue The a current by BP-G1 and the current by BP-G1 and we in although BP-G1 of Kir3.4 the as BP-G1 of the on the model of the Kir3.4 we the model and molecular simulations to the potential of BP-G1 in channel. with the Kir3.1/4 the the residue in the the residue in the with the residue for The with the from the was as the predicted of the The the BP-G1 and the channel the to of PubMed Scopus Google Scholar). and predicted the BP-G1 and the could in the Kir3.1/4 with the model with BP-G1. the of the subunit with the of to the corresponding and the with the of of the compound to the corresponding E141 of Kir3.1 or the corresponding of the subunit hydrogen with the of the the We to the of the with BP-G1. was a determinant for by the as the in the subunit by the compound and we pointed the in the subunit not in and it less blocking of BP-G1 to through the Kir3.1 subunit of the as Kir3.4 these such as the or on the of BP-G1 to heteromeric channel with Kir3.1 these validated the model that the BP-G1 compound inhibits the Kir3.1/4 channel through the central cavity by Kir3.1 residues E141 the F137 and simulations and their the model of BP-G1 as a and we molecular dynamics simulations We to simulations on Kir3.1/4 and the predicted in and The two after of and channels two and G of to to potassium to the from the simulations on the BP-G1 the central cavity of the channel and blocked from The of the in subunits of heterotetramers or Kir3.4 homotetramers or or less to D. D. M. Logothetis D.E. the mechanism of channel by and the Full Text Full Text PDF PubMed Scopus Google for Kir3.1/4 and the simulations The G after a in the that was that of Kir3.1/4 an that BP-G1 the it the of the of the Kir3.1/4 of channel activity. are two in the BP-G1 by we the BP-G1 was to effectively the or Kir3.1/4 channels in the Kir3.1 with its and could two hydrogen bonds with the of and with the of with not hydrogen bonds with the the of and with the of but hydrogen bonds with Kir3.1/4 channels and the with the of is not as its in not of Kir3.1/4 Thus, the proved with the as to the critical of the BP-G1 for its inhibitory activity on Kir3.1/4 channel compounds of BP-G1 and to and inhibitory activity to that the of was not a determinant of the but that to less BP-G1 with the and and critical predicted with the of but with the of and the of as as the of the the that by the BP-G1 on channel activity as by for compound to on a drug the to BP-G1 not inhibitory activity on current that was to by in in The inhibitory activity of BP-G1 was with for on cardiac ion channels and are on In addition, the inhibitory activity of BP-G1 and was on targets a and and the brain the and the receptor BP-G1 of BP-G1 for the receptor and the and The brain channels not BP-G1 and we in one of the sodium channels most in and found that BP-G1 not these We the in vivo of BP-G1 of on heart rate in was is the that after carbachol of was the heart with a after carbachol BP-G1 was via the of was a of on the heart is a control heart rate after carbachol control was through the the heart rate to and not In we the inactive The rate after carbachol However, of the inactive of was through the the heart rate not to as was the for BP-G1 in A. is a of in with BP-G1 with control and with In the was a of the of IKACh by after the to the due to of IKACh by the of carbachol on the carbachol and BP-G1. with In the was a of the after the to to carbachol and with In the was a of the after the not to as with BP-G1 and carbachol and with that BP-G1 is an in vivo of IKACh. derivatives with such as have to of the action potential selectively in atrial of these derivatives was on the and action potential in ventricular potential antiarrhythmic drug to treat AF with ventricular side K. as Scholar). However, the molecular mechanism of the action not We a benzopyran on ion channels in and We found that BP-G1 and selectively Kir3.1 heteromeric over inwardly rectifying K+ channels and cardiac channels such as and channels. BP-G1 could channel the Kir3.1 and but not channel to (or for Kir3.1-containing BP-G1 is a unique inhibitor of Kir3.1-containing channels. Previous that residues F137 and in the Kir3.1 channel critical for by and The could activated by and N. de E. K. the of inwardly rectifying K+ channels by the novel Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, L. D. S. A. Logothetis D.E. The channel and in Full Text Full Text PDF PubMed Scopus Google that these two residues of Kir3.1 are and for by these We the and with Kir3.4 and found that the Kir3.1 was and for the selective of heteromeric channels by the BP-G1 compound the molecular BP-G1 and we a model of Kir3.1/4 and BP-G1 the central cavity of the channel was identified by the BP-G1 not with F137 F137 the side of residue E141 to two hydrogen bonds with the the model we the predicted Kir3.1 residues E141 and D173 to of the a by while the the Thus, the Kir3.1 residues F137 and D173 contribute to the BP-G1 on Kir3.1-containing BP-G1 F137 through the minimal of BP-G1 for we or The compounds and not in Kir3.1/4 channel activity. In contrast, the channel with activity to BP-G1 the or is critical for the inhibitory activity of the The could used to the compound in to and The are in with the and residue with the of but residue with the and associated with C. BP-G1 good inhibitory activity for IKACh, it channels with channels are highly in H. A. K. S. rectifying potassium and PubMed Scopus Google blocking these channels could side effects. in the selectivity of BP-G1 for Kir3.1/4 channels over and highly the of Kir3.1/4 and channel for two residues are not and in Kir3.4 to residues and in the for the and The and Kir3.4 could to BP-G1 to the selectivity of for Kir3.1/4 over channels. by the Kir3.1/4 selective BP-G1 could The compound BP-G1 as a was is The predicted was are a good model for that BP-G1 have brain The BP-G1 inhibitory on as as cardiac ion channels such as and channels and rodent model that BP-G1 is an in vivo of IKACh In addition, in vivo and the the BP-G1 could a potential drug for AF the selectivity of the compound to approved by the and the of and as (6Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (98) Google Scholar). and Kir3.4 used for in the of for to to for a of to a in with a K+ to a for the current was in a potassium the the was with in the current was used for the inhibitory activity of the BP-G1 in was and the current was a with a potential from to was the of a in the the in G and with an and with Kir3.1 with or Kir3.4 The in with and in a and to after in K+ with K+ with was to BP-G1 in was in a response to in to and from the current The and the from to with with The of was to and the series was used in the on from to was used and in the the from the of The in and The channel after of was on the with the current activity of the channel. from in the in by and in and either in or in and for to for on and used for was by are or or and current as current and as current on repolarization to and as current on as current the of the the subunit of the cardiac channel was a with in the of either a of of compound or control or on a with of of a the was a a was and of is in and a of BP-G1 was in and and as of the in was in to in to the with a of to was the in in from and to drug and and a with a drug was through the the was the and BP-G1 or the inactive of was by through the of control the was are The of BP-G1 was in a of to to a of ion and of the and the compound for are In was to a of and and with a to and In was the and to The predicted channel was in an of and with molecular of E. of in a functional PubMed Scopus Google and a in by the in the selectivity as from the and the The of the The of was used to the for and BP-G1 the The for the BP-G1 potential by the C.M. from molecular for in Scopus Google Scholar, H. M. A. J. Scholar). The in was used to the The simulations with to the D. L. an for in Scopus Google with a to of the was the was from to dynamics with the of the the was an of and to and to the through a of was and for the in of the BP-G1 and its inactive through and not and and BP-G1 was and by proved with a on the BP-G1 the of future from as by or by with was on an from and used and for of compounds and their was by a of and in on a in million to or as the are as and are reported in compounds as by with a and The compound was and in a was in and for The was and with The with and over and The residue was by with the compound in to of The compound was with the of compound compound to by in and for the was to to the and in The was with sodium and through to the from the The after and over sodium and to compound was compound was by in acid and and was and for and to the and for The was to to in and with and sodium The was and the was over sodium and The residue was by to the of a for was in and and sodium was to the and for to the The was and with The with and over and The was The was in to by the of and for to of a for acid to a and in and to and for to of a for are the The that have of with the of The by the for and by the We for and M. L. S. C. M. H. K. M. L. D. A. S. J. and D. E. M. M. L. S. C. M. H. K. M. L. D. A. S. J. and D. E. M. L. S. C. M. H. K. M. L. D. A. S. J. and D. E. M. L. S. C. M. H. K. M. L. D. A. S. J. and D. E. M. L. S. C. M. H. K. M. L. D. A. S. J. and D. E. M. L. S. C. M. H. K. M. L. D. A. S. J. and D. E. M. and D. E. M. J. and D. E. M. A. S. J. and D. E. A. S. J. and D. E. A. S. J. and D. E. A. S. J. and D. E. M. and D. E. and The was by to D. E. and to S. N. The is the of the and not the of the of