View metadata,citationandsimilarpapersatcore.ac.uk The Journal of General Physiology www.jgp.org/cgi/doi/10.1085/jgp.200709946 399–405 J. Gen.Physiol.Vol. 131No.5 $30.00 The RockefellerUniversity Press © 2008Kaczorowski etal. Correspondence toOwenMcManus: o timize compoundactivity, evenifthenatureof signed tosimulatehumandisease states,wasusedtoop- approaches, inwhichprofi used classicalpharmacological channel drugdiscovery to resultfromtargetingthisproteinfamily. Earlyion nels, newgenerationsoftherapeuticagentsareexpected cium, orpotassiumchannels,ligand-gatedionchan- that modulatetheactivityofvoltage-gatedsodium,cal- for discoveringandcommercializingsuccessfuldrugs 2000 dysfunction canleadtopathophysiology( physiological processes( widespectrumof play acrucialroleincontrollingvery Ionchannelsareimportantdrugtargetsbecausethey Introduction is presentedtoillustratetheseprinciples. didates. Acasestudywithvoltage-gatedsodiumchannels mize theactivityofionchanneldrugdevelopmentcan- practices, provideanopportunitytodiscoverandopti- methodologies, alongwithclassicaldrugdevelopment leaddevelopment.Together,dicinal chemistry these assayseffectivelysupportme- andsecondary primary to provideinsightintomechanismofaction.Thesame and mine selectivityacrossionchannelsuperfamilies, fi screenstocon- clamp technologiesprovidesecondary density formatplatereaders.Newautomatedpatch membrane potentialorintracellularcalciumwithhigh fl ured formanydifferenttypesofionchannelsusing results. Automatedcell-basedHTSassayscanbeconfi small moleculelibraries,havealsoyieldedpromising ble leadstructures,whichtypicallyarenotabundantin screening (HTS)strategiesdevelopedtoidentifytracta- been successful.New, functional,highthroughput molecular biological/pharmacologicalstudies,have Proof ofconceptapproaches,focusingoncombined identifi includevalidationofnewchannel targetsand discovery eral challengesassociatedwithmolecular-based drug drugs targetingthisproteinclasshasbeendiffi physiologies. Historically, however, developmentof peutic targetsfortreatinganumberofdifferentpatho- Ionchannelsarewellrecognizedasimportantthera- IonChannelsasDrugTargets: TheNextGPCRs rm theactivityofhitsatchannellevel,todeter- methods tomonitoreitherchangesin uorescence ). Giventhestronghistoricalprecedentthatexists leads. cation ofacceptablemedicinalchemistry DepartmentofIonChannels,Merck Research Laboratories,Rahway, NJ07065 Gregory J.

Kaczorowski Hille, 2001 ling inanimalmodels,de- w e n , _ Owen B. m ), andbecausetheir c m a n u s @ m

McManus cult. Sev- e Ashcroft, r c k . c o m g- , Birgit T. discovery effortonvoltage-gatedsodiumchannels. discovery the remainderofthisarticle,usingexamplesfromadrug Each ofthesechallengesisaddressedinmoredetail cient todifferentiatenegativefromfailedclinicaltrails. egies tovalidatethatdrugexposureinpatientsissuffi and thelackofbiomarkertargetengagementstrat- fi species-specifi gets, whichmaybehinderedbycomplexandpotentially Signifi ion channeldrugcandidatesremainsanarduoustask. anddevelopmentofnew these advances,thediscovery andautomatedelectrophysiology.capabilities Despite technologies, includinghighthroughputscreening been signifi geting thisproteinclass.Themolecularapproachhas ing onamolecular-based strategytoidentifydrugstar- and controltheirfunction,researchersarenowfocus- lar componentsthatconstituteindividualchanneltypes of cellularphysiologyandidentifi Kaczo ( ent eraofionchanneldrugdiscovery gating mechanisms,allofwhichhassustainedthepres- ways, purifyingchannelproteins,andcharacterizing have beeninvaluableinmappingoutsignalingpath- ofresearchtoolsthat cessful therapiesanddiscovery resulted inanumberofnotablesuccessesincludingsuc- effortsand brute forceeffortdrovethesedrugdiscovery molecular targetwasunclear. Serendipity, insight,and biphenyl-4-yl]methyl Abbreviationsusedinthispaper: BPBTS, garding thepredictivenatureofanimalmodelsusedfor lection ofinappropriatetargetsduetouncertaintiesre- to lifethreateningarrhythmias; (e.g.,inhibitionofKv11.1,potentiallyleading opment candidatewithunrelatedchannelsorother failure includetoxicity, duetotheinteractionofadevel- with limitedsuccess.Issuesthatcontributetoprogram consuming processwhich,unfortunately, oftenmeets anddevelopmentisacostlytime Drugdiscovery Identifi tem; UHTS,ultraHTS. ing; Nav, sys- voltage-gated sodiumchannel;PNS,peripheralnervous fl uorescence resonanceenergytransfer;HTS,highthroughputscreen- culties indiscoveringacceptablesmallmoleculeleads, Withtheadventofamorecompleteunderstanding cant challengesexistinthevalidationofnewtar- cationandValidation ofIonChannelTargets rowski, 2005

Priest cantly strengthenedbytheadventofnew

c physiology( , and } - N ). ’ -(2,2 Maria L. ’ -bithien-5-ylmethyl)succinimide; FRET, Yu andCatterall,2004

Garcia Ashcroft, 2000 PERSPECTIVE cation ofthemolecu- N

- { [2 ’ -(aminosulfonyl)- Garcia and ), andse- brought toyouby provided byPubMedCentral ), dif- 399 - CORE preclinical testing when compared with human patho- antidepressants, such as amitriptyline, which are effi ca- physiology. An essential route to increased success in cious in treating neuropathic pain, possess a broad drug discovery is rigorous application of spectrum of pharmacological activities including in- traditional and novel in vitro and in vivo target valida- hibition of Nav1 channels. A comparison between ther- tion approaches, including genetic and pharmacologi- apeutic effi cacy and ability to inhibit Nav1.7 channels cal validation studies, expression profi ling, and altering for two classes of antidepressants, tricyclics and sero- channel expression in model systems. Improvements in tonin reuptake inhibitors, suggests a role of sodium the target identifi cation and validation stage can, argu- channel inhibition in the effi cacy of these compounds ably, have the greatest overall impact on ion channel in treating neuropathic pain (Dick et al., 2007). Tricyclic drug discovery efforts. antidepressants were potent inhibitors Human genetics and ablation studies in rodents and their potency in binding to the inactivated state have identifi ed a number of new ion channel targets of Nav1.7 was within the range of plasma concentra- (e.g., Nav1.7, Nav1.4, Cav2.2, KCNMA1, Kir1.1, Kir6.2- tions required for the treatment of neuropathic pain. SUR2, KCNQ, etc.) (Ashcroft, 2000; Lifton et al., 2001; In contrast, antidepressant serotonin reuptake inhibi- Yu and Catterall, 2004; Dib-Hajj et al., 2007). In addi- tors that are not effective in treating post-herpetic neu- tion to traditional knockout techniques, modulation of ralgia or diabetic neuropathy were weaker inhibitors of channel expression by regulation of promotor activity, Nav1.7, with in vitro potencies mostly above their thera- siRNA technology, or the employment of dominant- peutic concentration ranges. These data suggest that negative interference strategies can be used to aid in inhibition of voltage-gated sodium channels may con- the validation of novel targets. tribute to the anti-hyperalgesic effi cacy of tricyclic anti- As an example of human genetic validation, recent depressants and is further support for targeting sodium evidence has pointed to Nav1.7 as the most promising channels to treat chronic pain with more potent and sodium channel target for new analgesics (Dib-Hajj selective inhibitors. et al., 2007). The syndrome of Congenital Indifference Another example of pharmacological validation comes to Pain has been linked to nonsense mutations in Nav1.7 from the use of peptides, such as Ziconotide, a synthetic in individuals from 12 families representing 8 nationali- analogue of a peptide contained in cone snail venom, ties. These individuals have a complete inability to sense and a potent blocker of the N-type voltage-gated cal- pain, and yet they appear normal in all other respects, cium channel, Cav2.2 (Miljanich, 2004). Ziconotide was including intelligence, physical development, motor and developed clinically as a treatment for intractable pain autonomic refl exes, and sensation with the exception by intrathecal administration. In vivo pharmacological of the sense of smell. Additionally, several human gain results with Ziconotide strongly support the hypothesis of function mutations have been identified in Nav1.7 that a systemic small molecule inhibitor of Cav2.2 channels and shown to be linked to inherited pain dis- should be useful for treating pain. Similarly, in vitro stud- orders such as Inherited Erythromelalgia or Paroxysmal ies with the spider toxin peptide GxTX demonstrate Extreme Pain Disorder ( Dib-Hajj et al., 2007 ). Interest- that the pancreatic ␤ cell delayed rectifying potassium ingly, the human loss of Nav1.7 function was not repli- channel, Kv2.1 (KCNB1) is a potential target for en- cated in mice, where a conventional knockout is lethal, hancing glucose-dependent secretion, and thus and a nociceptor-specifi c knockout (Nassar et al., 2004 ) for the treatment of type II diabetes (Herrington et al., develops normally but displays a mild pain phenotype 2006). Since peptidyl modulators of ion channels are (resistance to infl ammatory pain). abundant in venoms, they are rich sources for reagents Given the possibility of compensatory changes in ge- useful in proof of concept studies. In addition, small netically derived disease models, the most convincing molecule natural product channel modulators, including target validation is derived from pharmacological proof indole diterpene blockers of high conductance, calcium- of concept in an animal model refl ecting human physi- activated potassium channels (KCNMA1), have been ology. Such validation can be obtained through use of used as probes in target validation studies ( Garcia and existing small molecule channel modulators, peptide Kaczorowski, 2001). neurotoxins, or antibodies specifi cally developed to inhibit channel function. Specifi c examples of target Lead Identifi cation and Characterization validation using existing drugs (lidocaine, tricyclic anti- The most challenging aspect of ion channel drug dis- depressants) or peptides (Ziconotide and GxTX) are covery may be the identification of an appropriate, discussed below. small molecule drug lead with desirable chemical prop- Systemic administration of the local anesthetic lido- erties that qualify it for exploration by medicinal chem- caine is approved for the treatment of neuropathic pain istry (MacCoss and Baillie, 2004). This is a key element ( Priest and Kaczorowski, 2007). At clinically used con- in successful ion channel preclinical drug development. centrations, block of Nav1 channels appears to be the Ion channels have traditionally been considered diffi - only mode of action of this agent. Similarly, tricyclic cult targets to engage in high throughput functional

400 The Next GPCRs screening formats, and large scale screening campaigns formats are amenable to application of ultrahigh have often yielded a paucity of potent, selective hits. throughput automation strategies. The scarceness of acceptable ion channel leads may Until recently, the characterization of screening hits derive from the long-standing emphasis within the using manual voltage clamp techniques was slow and te- pharmaceutical industry on programs directed at G dious, because of the low throughput inherent to this – coupled receptors, , and other enzymes, technique. However, secondary screening of initial hits leading to biased sample collections. Successful target- has now been revolutionized with the use of new auto- ing of ion channels critically requires robust and sensi- mated patch clamp technologies that can confi rm a tive, mechanism-based, HTS technologies that can compound’ s direct functional effects on the channel, detect a vanishingly small number of actives in large determine its selectivity across superfamilies of ion compound libraries, or in other words, can “ fi nd a nee- channels, and provide mechanistic insights, all accom- dle in a haystack” ( Herrington et al., 2005; Garcia and plished in a very rapid fashion. Several different plat- Kaczorowski, 2006). Implementation of reliable and in- forms are commercially available with specifi c features formative counter-screens for likely off-target activities is determining their optimal application, from specializ- essential for meaningful hit assessment and lead prioritiza- ing in high throughput analysis to more quantitative tion to ensure that resources are not wasted in pursuing measurements of channel activity using complex volt- nondevelopable chemical structures. Recent introduc- age protocols (Priest et al., 2007). Hopefully, automated tion of fl uorescent, cell-based screening technologies patch clamp technologies will soon be adapted to UHTS has enabled dependable HTS and ultra high through- requirements, enabling a new set of UHTS approaches put screening (UHTS) paradigms, allowing screening toward fi nding novel ion channel modulators. Pres- of libraries consisting of millions of compounds in ently, the combination of biochemical and biophysical a timely, cost effective manner, and thereby allow de- approaches is needed to identify useful lead structures. tection of true lead structures with adequate initial po- Together, these strategies, along with more classical drug tency, selectivity across ion channel superfamilies, and development techniques, provide a means for discovering defi ned mechanisms of action. Early mechanistic stud- and optimizing the activity of potential ion channel ies are also important, since a particular mechanism of drug development candidates for almost any member action may lead to functional selectivity for the target of the various ion channel super families. channel through state dependence and/or use-dependent channel inhibition. Case Study: Discovering Inhibitors of Voltage-gated In one general confi guration, HTS assays can be insti- Sodium Channels tuted for many different types of ion channels by estab- As a way of illustrating the issues related to ion channel lishing cell lines heterologously expressing the target in drug discovery outlined above, the remainder of this ar- a context where changes in the activity of the channel ticle will describe a case study focusing on the identifi ca- of interest can affect the cellular plasma membrane po- tion of voltage-gated sodium channel inhibitors to treat tential. Potential sensitive fl uorescent dyes can then be chronic neuropathic pain. Treatment of pain is a seri- used to monitor changes in of ous medical issue and there is a major effort in the phar- such cells grown in high density, multiwell format plates maceutical industry to develop new therapies for this during screening procedures (Gonzalez and Maher, condition. In particular, the treatment of neuropathic 2002). Detection of an active compound is achieved if pain, defi ned as “ chronic pain that results from a pri- addition of test compound causes a corresponding mary lesion or dysfunction of the peripheral nervous change in membrane potential. This strategy works well system” by the International Association for the Study of to identify sodium or modulators, Pain (IASP), remains a major unmet medical need. and such paradigms can be used to screen for ligand- It is clear that voltage-gated sodium (Nav1) channels gated ion channel modulators, as well. Identifi cation of play a key role in the origination and propagation both channel inhibitors and channel openers can be of sensory nerve action potentials necessary for pain accomplished using this general screening method signaling. Local applications of nonsubtype-selective (Garcia et al., 2007). For detecting voltage-gated calcium sodium channel blockers, such as novocaine, provide channel effectors, a similar approach can be adopted, complete pain relief through conduction block. How- except that fl uorescent dye indicators are employed ever, this approach to pain relief is limited to very few to monitor the concentration of intracellular calcium. applications, such as dental procedures, since sodium Cotransfection with an inwardly rectifying potassium chan- channels are also vital to conduction in the , CNS, nel, together with the controlled variation in extracellu- skeletal muscle, and nonnociceptive sensory neurons. lar potassium concentration, has been used to control The Nav1 super family is comprised of 10 members (Yu cellular membrane potential, in order to establish the and Catterall, 2004 ). Seven of these subtypes, Nav1.1, most sensitive and mechanistically meaningful assay Nav1.3, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Nav1.9, are confi guration (Xia et al., 2004 ). All of these screening present in the peripheral nervous system (PNS). Of these,

Kaczorowski et al. 401 Figure 1. A functional, membrane potential FRET-based assay for Nav1.7 channels. In the absence of other ionic conductances that can hyperpolarize the cell, heterologous expression of Nav1.7 channels provides a system where at the cell resting membrane potential most chan- nels will reside in the nonconducting inactivated state. Removal of fast inactivation by the addition of veratridine shifts the channel’ s equilibrium to the conductive, open state that allows sodium entry leading to cell . The changes in voltage can be monitored with a pair of FRET voltage-sensing dyes, coumarin and oxonol. Cell depolarization alters the distribution of oxonol across the membrane, causing a change in the FRET signal. In the presence of a Nav1.7 inhibi- tor, channel equilibrium shifts toward the inac- tivated, drug-bound conformation, reducing the number of channels that will be available for veratridine modifi cation, and preventing the agonist-induced FRET signal. The dose– re- sponse curve for the veratridine-induced change in FRET signal is steep, suggesting that modifi ca- tion of a small number of Nav1.7 channels is suf- fi cient to cause cell depolarization.

Nav1.7, Nav1.8, and Nav1.9 are expressed predomi- testing compounds of interest on Nav1.7, Nav1.5 (the nantly in nociceptive neurons, and Nav1.3 is predomi- primary cardiac sodium channel) and Nav1.8 in parallel. nantly embryonic, but is up-regulated in the adult PNS A membrane potential– based assay was used to screen -compounds on Nav1.8 stably expressed in a re 200,000 ف after injury. This limited expression pattern makes these subtypes attractive targets for the development of novel combinant cell line. This HTS assay was based on fl uores- analgesic agents. However, their relative contribution to cence resonance energy transfer (FRET) between two pain signaling, and specifi cally to neuropathic pain sig- members of a membrane potential– sensitive dye pair de- naling, is unclear and may vary with different etiologies veloped by Aurora Biosciences (Priest et al., 2004). Nav1.8 and sensory qualities of pain. channels were preincubated with test compound and the In the absence of molecular selectivity for one Nav1 chemical agonist deltamethrine in the absence of extracel- subtype, it is possible to specifi cally target Nav1 chan- lular sodium. Subsequent addition of sodium resulted in nels in a given conformational state while preserving membrane depolarization and Nav1 block was quantifi ed sodium channel– dependent impulse conduction. This as interference with that cellular depolarization process. type of state-dependent inhibition is the basis for the Although the initial screen on Nav1.8 yielded a variety therapeutic window seen with sodium channel blocking of hits, only a single compound was considered a viable anticonvulsants and antiarrhythmics, such as lamotrig- lead for medicinal chemistry efforts. Before committing ine and lidocaine. These drugs have higher affi nity for resources to this lead, the compound, a disubstituted channels in the open and/or inactivated states than for succinimide termed BPBTS (N - { [2’ -(aminosulfonyl) resting, closed channels. This mechanism of inhibition biphenyl-4-yl] methyl} - N ’ -(2,2’ -bithien-5-yl methyl) favors binding in rapidly fi ring or partially depolarized succinimide), was examined in detail by manual whole tissues. Neuropathic pain should be sensitive to this in- cell voltage clamp. BPBTS was found to inhibit all Nav1 hibitory mechanism, since it is thought to arise from in- subtypes with similar potency, and inhibition was depen- jury-induced areas of , a hypothesis that dent on membrane potential and stimulation frequency. is supported by the clinical effi cacy of lidocaine admin- This inhibitory mechanism was consistent with higher istered systemically at subanesthetic doses. Moreover, affi nity of the compound for channels in the open and nonsubtype-selective, state-dependent block may afford inactivated state, compared with channels in the resting the greatest effi cacy, since individual knockout of Nav1.3, state. In addition, BPBTS was two orders of magnitude Nav1.7, Nav1.8, or Nav1.9 did not provide convincing more potent than the clinically used anticonvulsant and evidence for a dominant role of any of these channels antiarrhythmic Nav1 blockers, inhibiting the inactivated in neuropathic pain signaling. state of Nav1.8, Nav1.7, Nav1.5, and Nav1.2, with Ki val- Based on this rationale, a decision was made to ini- ues of 0.09, 0.15, 0.08, and 0.14 μ M and the resting state tially pursue nonsubtype selective, state-dependent Nav1 with Kr values of 1.5, 1.3, 0.3, and 1.2 μ M, respectively inhibitors, while monitoring molecular selectivity by ( Priest et al., 2004).

402 The Next GPCRs Figure 2. 1-Benzazepin-2-one Nav1 inhibitors. The structures of two 1-benzazepin-2-one Nav1 inhibitors are illustrated together with their po- tencies for hNav1.5, hNav1.7, and hNav1.8 chan- nels as determined in functional membrane potential, FRET-based assays. The estimated po- tencies of these compounds for the inactivated state of hNav1.5 and hNav1.7 channels, as de- termined from electrophysiological recordings, are also presented. Note that only compound 2 displays selectivity for the hNav1.7 channel. Both compounds are weaker inhibitors of the hNav1.8 channel.

As such, BPBTS was an attractive lead for medicinal with a brain to plasma ratio greater than 10 for mexi- chemistry; its main liabilities being a poor pharmacoki- letine. These data obtained with CDA54 strongly sug- netic profi le. Over the course of profi ling analogues of gested that inhibition of PNS sodium channels alone BPBTS, as well as published Nav1 inhibitors, using the is effi cacious in animal models of neuropathic pain, membrane potential– based fl uorescent screening assay, and that limiting CNS exposures of Nav1 inhibitors is structure-based discrepancies between potencies deter- a viable approach to developing Nav1 inhibitors with mined in the fl uorescent assay and by electrophysiology an improved therapeutic index. were noted for a few compounds. These discrepancies A UHTS campaign, using the membrane potential– were traced to an interaction between these compounds based assay described to screen for inhibitors of Nav1.7, and the agonist veratridine used to open Nav1.7 chan- discovered the novel 1-benzazepin-2-one channel inhibi- nels. Subsequently, the fl uorescent assay was modifi ed tors (Hoyt et al., 2007; Williams et al., 2007). This class of such that Nav1 channels were preincubated with test com- inhibitors demonstrated a defi ned structure–activity rela- pound in physiological extracellular sodium concen- tionship and, when evaluated in vivo, members of this se- trations and Nav1-dependent depolarization was initiated ries were orally effi cacious in rodent neuropathic pain by agonist addition (Fig. 1). Channel inhibitory poten- and epilepsy models. Importantly, some members of this cies measured in this modifi ed assay correlated very class displayed molecular selectivity for Nav1.7 channels well with the inactivated state inhibition determined (Williams et al., 2007). For example, compound 2 of Fig. 2 fold selective for-10 ف by electrophysiology across many structural classes of was highly state dependent and Nav1 inhibitors (Felix et al., 2004; Liu et al., 2006). Nav1.7 over Nav1.8 and Nav1.5. The most potent, albeit Although analogues of BPBTS failed to surpass the not subtype-selective, member of this class of Nav1.7 in- initial lead in potency, medicinal chemistry succeeded hibitors (compound 1, BNZA; Fig. 2) was tritiated. at improving the pharmacokinetic profile, eventu- [3 H]BNZA binds with high affi nity (Kd of 1.6 nM) to re- ally generating trans-N - { [2 ’ -(aminosulfonyl)biphenyl- combinant Nav1.7 channels. This is the fi rst demon- 4-yl]methyl} - N -methyl-N ’-[4-(trifl uoromethoxy)benzyl] stration of high-affi nity ligand binding to Nav1.7 and cyclopentane-1,2-dicarboxamide (CDA54) with 44% oral provides a valuable screening tool with which to search bioavailability, one hour half life, and a clearance rate for Nav1.7-selective compounds. Data obtained with the of 14 ml/min/kg, which was profi led extensively in vivo 1-benzazepin-2-one structural series suggest that Nav1.7- (Brochu et al., 2006). In two rat models of neuropathic selective analogues can be identifi ed, and with the pain, CDA54 (10 mg/kg, given orally) signifi cantly re- appropriate pharmacokinetic and drug metabolism duced nerve injury– induced behavioral hypersensitiv- properties, such compounds could be developed as anal- ity by 44– 67%. The same dose/plasma concentration gesic agents, potentially displaying improved tolerability of CDA54 did not affect acute nociception (rat hot over existing drugs used to treat neuropathic pain. Sup- plate assay), motor coordination (rat rotorod assay), or port for the feasibility of developing subtype-selective so- cardiac conduction (electrophysiological parameters dium channel inhibitors as novel analgesics comes from measured in the cardiovascular dog). These properties the recent report of a high affi nity Nav1.8 selective agent, are in contrast to those of current sodium channel block- which given intraperitoneally was effi cacious in a wide ers used in the clinic, which cause impaired motor co- range of rodent pain models (Jarvis et al., 2007). ordination in rats and CNS side effects in man at all A potential alternative approach to searching for sub- effi cacious doses. Interestingly, upon oral dosing, the type-selective sodium channel inhibitors would be to brain to plasma ratio for CDA54 was 0.03. In contrast, screen for compounds that target channel gating mech- clinically used Nav1 blockers accumulate in the CNS, anisms. Several peptides have previously been shown to

Kaczorowski et al. 403 modify gating of sodium channels, but few small mole- 2006 . Block of peripheral nerve sodium channels selectively cules, especially inhibitors, have been described to inhibits features of neuropathic pain in rats. Mol. Pharmacol. function in this fashion. One such agent is ProTx-II, a 69 : 823 – 832 . Dib-Hajj , S.D. , T.R. Cummins , J.A. Black , and S.G. Waxman. 2007 . 30– peptide purifi ed from tarantula venom; From to pain: Na(v)1.7 and human pain disorders. Trends this peptide blocks sodium channels and shows selectiv- Neurosci. 30 : 555 – 563 . ity for Nav1.7 (Smith et al., 2007). ProTx-II binds to the Dick , I.E. , R.M. Brochu , Y. Purohit , G.J. Kaczorowski , W.J. Martin , resting state of sodium channels and shifts the voltage and B.T. Priest . 2007 . Sodium channel blockade may contribute dependence of channel activation to more depolarized to the analgesic effi cacy of antidepressants. J. Pain . 8 :315 – 324. potentials. Strong depolarizations overcome channel Felix , J.P. , B.S. Williams , B.T. Priest , R.M. Brochu , I.E. Dick , V.A. Warren , L. Yan , R.S. Slaughter , G.J. Kaczorowski , M.M. Smith , inhibition, which is a hallmark of this type of gating and M.L. Garcia . 2004 . Functional assay of voltage-gated sodium modifi er peptide. One possible strategy to identify small channels using membrane potential-sensitive dyes. Assay Drug molecule mimetics of a gating modifi er peptide is to ra- Dev. Technol. 2 :260 – 268 . diolabel ProTx-II in biologically active form, and to Garcia , M.L. , and G.J. Kaczorowski . 2001 . Pharmacology of high- develop a binding assay with Nav1.7 channels heterolo- conductance, Ca2+ -activated potassium channels. In Potassium gously expressed in a cell line. Screening for small mol- Channels in Cardiovascular Biology. A.A. Rusch, editor. Academic/ Plenum Publishers, New York . 219–234. ecules that modulate ProTx-II binding could reveal new Garcia , M.L. , and G.J. Kaczorowski . 2005 . Potassium channels as tar- classes of channel inhibitors that partition into the gets for therapeutic intervention. Sci. STKE . 2005 : pe46 . membrane and interfere with movement of the gating Garcia , M.L. , and G. Kaczorowski . 2006 . Critical success. European paddle, thereby preventing channel opening. An added Pharmaceutical Review . 11 : 42 – 45 . advantage of this type of UHTS is that high concentra- Garcia , M.L. , D.-M. Shen , and G.J. Kaczorowski . 2007 . High-con- tions of test compounds could be employed, a situation ductance calcium-activated potassium channels: validated tar- that is precluded in dye-based screening due to fl uores- gets for smooth muscle relaxants? Expert Opin. Ther. Patents . 17 : 832 – 842 . cence interference that typically occurs with high con- Gonzalez , J.E. , and M.P. Maher . 2002 . Cellular fl uorescent indica- centrations of many small organic molecules. Given that tors and voltage/ion probe reader (VIPR) tools for ion channel some gating modifi er peptides bind to regions that and receptor drug discovery. Receptors Channels . 8 :283 – 295 . are unique to specifi c channels within a super family, Herrington , J. , O. McManus , and L. Kiss . 2005 . The road ahead and subtype-selective inhibitors might be identifi ed using how to get there. European Pharmaceutical Review . 10 : 79 – 84 . such a strategy. Herrington , J. , Y.P. Zhou , R.M. Bugianesi , P.M. Dulski , Y. Feng, V.A. Warren , M.M. Smith , M.G. Kohler , V.M. Garsky , M. Sanchez , et al . 2006 . Blockers of the delayed-rectifi er potassium current in Conclusions pancreatic ␤ -cells enhance glucose-dependent insulin secretion. Although identifi cation of novel sodium channel Diabetes . 55 : 1034 – 1042 . inhibitors was used to illustrate current molecular Hille , B. 2001 . Ion Channels of Excitable Membranes. Third edi- approaches to ion channel drug discovery, these prin- tion. Sinauer Associates, Inc., Sunderland, MA . 814 pp. Hoyt , S.B. , C. London , D. Gorin , M.J. Wyvratt , M.H. Fisher , C. ciples can be generalized to any ion channel target. Abbadie , J.P. Felix , M.L. Garcia , X. Li , K.A. Lyons , et al . 2007 . It would appear that establishment and orchestration Discovery of a novel class of benzazepinone Na(v)1.7 blockers: of functional UHTS is no longer the rate-determin- potential treatments for neuropathic pain. Bioorg Med Chem Lett . ing step in ion channel drug development. Rather, 17:4630–4634. the synthesis of ion channel friendly small molecule Jarvis , M.F., P. Honore , C.-C. Shieh , M. Chapman , S. Joshi , X.-F. libraries to facilitate lead discovery, and the establish- Zhang , M. Kort , W. Carroll , B. Marron , R. Atkinson , et al . 2007 . 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