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Title: Locating the Route of Entry and Binding Sites of Benzocaine and Phenytoin in a Bacterial Voltage Gated Sodium Channel}
Article:
Abstract:
Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.
Introduction:
Voltage-gated sodium channels( Navs) are transmembrane proteins that initiate action potentials in excitable cells by responding to small depolarizing signals to allow the rapid influx of into the cell[1]–[3]. Mutations and aberrant expression of sodium channels are responsible for a range of diseases related to nerve and muscular function including neuropathic pain, cardiac arrhythmia and epilepsy[4],[5]. These conditions often benefit from treatment with drugs that block the passage of and thereby rein in electrical activity. In clinical settings, the local anesthetic activity of sodium channel-blockers is also used to ease surgery or acute pain resulting from trauma. While there are many molecules capable of blocking sodium channels, most medically relevant drugs are small organic molecules which bind inside the pore to impede ion flow by either directly occluding the ion conduction pathway or by stabilizing a non-conductive channel conformation[3]. The result is reduced signal propagation, which manifests symptomatically as a reduction in seizure occurrence, pain sensation or cardiac excitability. Sodium channel inhibitors exhibit a wide range of chemical moieties, but common to most of them is the presence of a phenyl ring connected to a basic nitrogen, with some combination of aromatic and aliphatic decorations[6],[7]. Neutral local anesthetics are known to be able to block and leave resting sodium channels when applied from either side of the membrane, a process known as ‘tonic block’. In contrast highly polar or charged compounds can only block the pore after channel opening yielding ‘use dependent’ block that is the basis of anti-epileptic or anti-arrhythmic activity[8],[9]. The observation of multiple modes of drug block led to the suggestion that there are two access routes for drugs to enter the channel: through the activation gate from the cytosol or directly from the lipid bilayer through a ‘hydrophobic route’[8]. Humans have nine different types of voltage-gated sodium channels, which are preferentially expressed in different tissue[2]. Most sodium channel blocking drugs target all subtypes, but the development of selective channel blockers would allow a new range of clinical applications and a decrease in side effects[7]. For example, there is significant interest in increasing the specificity of drugs for the channel subtypes found in pain receptor neurons as a way to combat chronic pain[10],[11]. However, developing subtype-selective Nav channel blockers requires the generation of in-depth knowledge of the location of binding and the route of entry of existing drugs in order to assess how small sequence differences in the subtypes can be exploited. The presence of a common chemical moiety in sodium channel inhibitors also raises the question of how drugs with similar structure and binding sites, such as local anesthetics and anticonvulsants, have differing therapeutic effects[12]. A better understanding of the specific interactions of these compounds with residues in the channel and their access route may suggest strategies for altering drug kinetics and affinities. While there are no atomic resolution structures of eukaryotic voltage gated sodium channels( eNavs), the recent publication of a number of structures of homologous channels from bacteria( bNavs)[13]–[17] provides an avenue to understanding how sodium channel blockers work at the molecular level. The first crystal structure of a bNav from Arcobactor Butzleri, called NavAb, shows the protein is a tetramer of four identical subunits, with the pore axis running down the central axis[13]. Each subunit contains a pore forming domain consisting of two transmembrane helices( S5 and S6) joined by a linker that forms the narrow selectivity filter at the extracellular end of the pore. Beneath this is a central, water-filled cavity, walled by the S6 helices, where most channel-blocking drugs are known to bind in eNavs[18]–[22]. The pore tapers at the intracellular end to form the activation gate which is closed in this structure so that drugs cannot pass from the cytosol into the central cavity. Recent electrophysiological studies have shown that the local anesthetics lidocaine, ranozaline and benzocaine can block sodium flux in NaChBac, the most well characterised bNav, with comparable affinity to tonic block in eukaryotic channels[9],[23]. Supporting the similarity between the bacterial and eukaryotic channels, uncharged drugs such as benzocaine can block NaChBac when applied extracellularly indicating that the hydrophobic route for drug entry is still present[9]. The published bNav structures present a candidate for this drug access route as they show lateral fenestrations extending to the membrane from the center of the pore, which are large enough to fit small molecules[24],[25], and through which the general anesthetic isoflurane has been seen to pass in simulation studies of a NachBac homology model[26]. Despite the similarities between bacterial and eukaryotic voltage gated sodium channels that may make bNavs good models for studying tonic block, there are also functional and structural differences. NaChBac exhibits far slower gating kinetics than eNavs[27], precluding the study of use-dependent block in which recovery from inactivation must occur faster than drug dissociation. Structurally, bNavs are composed of four identical subunits, whereas eNavs are a single, heterotetrameric protein chain[27],[13]. Although there is common hydrophobic character in the pore, sequence alignments show that residues forming the putative drug receptor site on the internal S6 helix are not conserved between bacterial and eukaryotic Navs[28], and specifically NavAb lacks aromatic residues near the drug binding site that are suspected to be particularly important in use-dependent binding[21]. In order to better understand the mode of action of sodium channel inhibitors, here we use molecular dynamics simulations to examine the route of entry and mechanism of binding in NavAb of two channel-blocking drugs: the local anesthetic benzocaine, and the anti-epileptic phenytoin. Both are small, neutral drugs that bind inside the pore of eNavs[29],[20] and whose blocking effect tapers off after some time[30],[31], indicating they may leave the channel via the observed hydrophobic fenestrations. Benzocaine has been shown to illicit tonic block of NachBac[9]. Equilibrium and biased simulations are used to identify the potential binding sites of these two drugs in the pore, and to quantify the affinity of binding. Accompanying this is a demonstration of drug passage through the hydrophobic fenestration, showing that this pathway is a feasible route of access or escape for neutral channel-blocking drugs.
Discussion:
Using molecular dynamics simulations we have characterised the likely binding sites for benzocaine and phenytoin inside the central cavity of NavAb, with 3 independent simulation methods yielding consistent results. Both drugs are found to bind either in the hydrophobic pocket formed by the activation gate or at the entrance to the hydrophobic fenestrations. In both cases, the drugs interact with residues found to be important in binding in eNavs. But, we find this association is due to non-specific hydrophobic interactions meaning that binding occurs due to the overall architecture of the central cavity rather than interactions to specific residues. Although our simulations clearly identify the likely drug binding sites in the channel, it is not yet clear if the binding of more than one drug is necessary to occlude the pore or prevent channel opening. Our simulations also provide a plausible rationalisation for the differences seen between tonic and use dependent block, as interaction of cationic drugs with aromatic residues are essential in the use dependent case but not for resting state block[21],[32]. It is possible that the most likely binding position in the resting channel could be at the activation gate as found here, but this will be abolished upon channel activation allowing the drug to move close to the aromatic residues present in eNavs. If this hypothesis is correct, mutation of residues involved only in binding at the activation gate would be expected to change the affinity of tonic block, but not use-dependent block. Unfortunately, the lack of aromatic residues in the pore and the slow channel kinetics seen for NaChBac make these channels less ideal for modelling use-dependent block in either simulation or experiment- but they still may allow for modeling of eukaryotic tonic block. We note that the dissociation constant for tonic block by benzocaine in eukaryotic channels[41] closely matches that for bacterial channels[9]. Our results also show that the hydrophobic fenestrations seen in NavAb can serve as conduits for benzocaine and phenytoin to enter the central cavity and thus can form the long hypothesised hydrophobic entrance route[8]. There are three lines of evidence supporting this conclusion:( i) benzocaine exits through a fenestration during one of the equilibrium simulations,( ii) the energy barriers to reach the outside of the protein are small as found from metadynamics simulations, and( iii) the PMF plots indicate small barriers for drug entry, although larger barriers are present for penytoin than for benzocaine. The main barriers to passage through the fenestration appear to be physical constrictions in the fenestrations which is consistent with experiments in eukaryotic channels, in which analogs of benzocaine show slower rates of dissociation from sodium channels when they have bulkier or longer moieties attached[42]. It has been suggested that there may be scope to exploit any differences in the size of the fenestrations in different eNav subtypes or in different functional states to rationally design sub-type or state dependent channel blocker[16],[14], however, our previous simulation have suggested there is little state dependent difference in fenestration size[24]. In addition the concept of drug entry through the fenestration may be further tested using mutations in this area. Surprisingly, the potential entropic issues associated with the drug finding the small fenestration entrance appear to be small. The fact that lipids are seen to exchange in long simulations, that they are replaced by drug and water, and that polar residues on the surface of the protein can attract drugs from the bilayer may all help to overcome this potential barrier. Polar residue in this region( T138, Y142) are well conserved in bNavs and it would be interesting to see if their mutation altered the kinetics of drug entry. The large degree of sequence similarity in the pore forming regions of eNavs makes the design of subtype selective channel blockers a difficult goal. Elucidating the mode of entry and binding of existing channel blockers is an important step toward achieving this aim. Here we have been able to do this for benzocaine and phenytoin in a bacterial channel, but it is not clear how much about binding in eukaryotic channels can be inferred from this given the significant differences between these families of proteins. For example, the nature of binding seen here is likely to be less specific than that in the eukaryotic homologues. Additional functional studies of drug block in bacterial channels, increased availability of structural information about the different functional states of bacterial channels, reliable models of eukaryotic channels and detailed investigations of specific drug-residue interactions in tonic and use-dependent block will all aid progress toward the design of subtype selective channel blockers.
The treatment of cardiac arrhythmia , epilepsy and pain usually involves blocking the protein channels responsible for initiating electrical activity in nerves and muscles . Current drugs block all such channels , but improved medication requires compounds that can differentiate between the channels present in different parts of the body . Achieving this goal calls for a better understanding of the interactions of current drugs with the proteins . Here we use computer simulation to understand where a local anesthetic and an anti-epileptic bind to a bacterial sodium channel and how they find their way to this position , helping to uncover ways to selectively target different human channels .