Effect of Conformational Diversity on the Bioactivity of Μ-Conotoxin PIIIA Disulfide Isomers

marine drugs

Article Eﬀect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulﬁde Isomers

1, 1, 2, 1 Ajay Abisheck Paul George †, Pascal Heimer †, Enrico Leipold †, Thomas Schmitz , Desiree Kaufmann 3, Daniel Tietze 3 , Stefan H. Heinemann 4 and Diana Imhof 1,*

1 Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany 2 Department of Anesthesiology and Intensive Care, University of Lübeck, Ratzeburger Allee 160, D-23562 Lübeck, Germany 3 Eduard Zintl Institute of Inorganic and Physical Chemistry, Darmstadt University of Technology, Alarich-Weiss-Str. 4, D-64287 Darmstadt, Germany 4 Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, D-07745 Jena, Germany * Correspondence: [email protected]; Tel.: +49-228-73-5254 These authors contributed equally to this work. † Received: 5 June 2019; Accepted: 25 June 2019; Published: 2 July 2019

Abstract: Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer’s activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.

Keywords: µ-conotoxin; PIIIA; voltage-gated sodium channel; disulﬁde connectivity; peptide folding; electrophysiology; molecular docking; molecular dynamics

1. Introduction Covalent linkage of cysteine residues by disulfide bonds is fundamental for the folding, stability, and function of many peptides and proteins [1–4]. The formation of correct disulfide linkages is particularly important for peptides and proteins with higher-order disulfide networks (>2 disulfide bonds) because incorrectly assembled disulfide bonds may directly interfere with the biological functions of these molecules. The venom of marine cone snails is a rich source of small- to medium-sized disulfide-stabilized peptides, so-called conotoxins, most of which specifically target ion channels and receptors in the membranes of excitable cells [5]. Conotoxins are recognized for their pharmacological and therapeutic

Mar. Drugs 2019, 17, 390; doi:10.3390/md17070390 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 390 2 of 18

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Whilebind to some pharmacologicalthe extracellularsodiumµ-conotoxins, channels porelead such structuresstructures(NaV channels) [13].[13]. µ-Conotoxins µ-Conotoxinsmaking them areare attractive soso-called-called research porepore blockersblockers tools asbecausebecause well astheythey potential bindbind toto pharmacologicalthethe extracellularextracellular poreporelead (Navestibule(NaVV channels)channels) and, hence, makingmaking obstruct themthem theattractiveattractive permeation researchresearch of sodium toolstools asasions wellwell (Na asas+) throughpotentialpotential the pharmacologicalpharmacological channels [14]. While leadlead as µ-GIIIA(NavestibulestructuresV arechannels) veryand, [13]. specifichence, µ-Conotoxinsmaking obstruct for them skeletal aretheattractive sopermeation muscle-called research pore sodium of blockerssodium tools channels asions because well (Na (Na as+ )they throughpotential1.4), bindµ-PIIIA—originally theto pharmacological the channels extracellular [14]. While isolatedleadpore vestibulestructuresstructures and, [13].[13]. hence, µ-Conotoxinsµ-Conotoxins obstruct arearethe sosopermeation-called-called porepore of blockersblockerssodium ions becausebecause (Na + they)they) Vthroughthrough bindbind thetotheto thethe channelschannels extracellularextracellular [14].[14]. WhileWhile porepore structuressomestructures µ-conotoxins, [13].[13]. µ-Conotoxinsµ-Conotoxins such as µ-GIIIA areare soso-called-called are very porepore spec blockersblockersific for becausebecauseskeletal theymuscle+they bindbind sodium toto thethe channels extracellularextracellular (Na V porepore1.4), structuressomevestibule µ-conotoxins, and,[13]. hence,µ-Conotoxins such obstruct as µ-GIIIA are the so permeation-called are very pore specof blockers sodiumific for ionsbecauseskeletal (Na +theymuscle) through bind sodium tothe the channels channelsextracellular [14]. (Na While Vpore1.4), + from Conusvestibulesome µ-conotoxins, purpurascens and, hence, such—also obstruct as aµ-GIIIAff theects permeation neuronal are very specof Na sodiumVificchannels for ions skeletal (Na [10+ +muscle]) through and even sodium the some channels channels voltage-gated [14]. (Na WhileV1.4), K vestibulesomevestibule µ-conotoxins, and,and, hence,hence, such obstructobstruct as µ-GIIIA thethe permeationpermeation are very specofof sodiumsodiumific for ions ionsskeletal (Na(Na +muscle)) throughthrough sodium thethe channelschannels channels [14].[14]. (Na WhileWhileV1.4), µ-PIIIAvestibulesome µ-conotoxins,— and,originally hence, isolatedsuch obstruct as µ-GIIIAfrom the permeationConus are verypurpurascens ofspec sodiumific for— alsoions skeletal affects(Na+ muscle) through neuronal sodium the Na channelsV channelschannels [14]. (Na[10] WhileV and1.4), vestibuleµ-PIIIA— and,originally hence, isolated obstruct from the permeationConus purpurascens of sodium— alsoions affects(Na ) through neuronal the Na channelsV channels [14]. [10] WhileV and channelssomeµ-PIIIA [15 µ-conotoxins,].— Recently,originally we suchisolated synthesized as µ-GIIIAfrom Conus allare 15 verypurpurascens possible specific disulfide for—also skeletal affects isomers muscle neuronal (denoted sodium NaV channels channels in the current (Na [10]V 1.4),and study someµ-PIIIAsome µ-conotoxins,µ-conotoxins,—originally suchisolatedsuch asas+ µ-GIIIAµ-GIIIAfrom Conus areare veryverypurpurascens specspecificific forfor—also skeletalskeletal affects musclemuscle neuronal sodiumsodium NaV channels channelschannels (Na (Na[10]V 1.4),1.4),and evensomeµ-PIIIA some µ-conotoxins,—originally voltage-gated suchisolated Kas+ channelsµ-GIIIA from Conus are [15]. very purpurascens Recently, specific we for— synthesi alsoskeletal affectszed muscle allneuronal 15 sodiumpossible Na Vchannels disulfidechannels (Na [10]isomersV1.4), and someeven someµ-conotoxins, voltage-gated such Kas+ µ-GIIIAchannels are [15]. very Recently, specific we for synthesi skeletalzed muscle all 15 sodium possibleV channels disulfide (Na isomersV1.4), µ-PIIIAeven some—originally voltage-gated isolated K+ channelsfrom Conus [15]. purpurascens Recently, we— synthesialso affectszed neuronalall 15 possible Na V channelsdisulfide [10]isomers and as 1–15µ-PIIIAevenµ-PIIIA) of some the——originally conotoxinoriginally voltage-gated isolatedisolatedµ-PIIIA K + channels fromfrom (sequence: ConusConus [15]. purpurascenspurpurascens Recently, ZRLCCGFOKSCRSRQCKOHRCC-NH we—— synthesialsoalso affectsaffectszed neuronalallneuronal 15 possible NaNaV channelschannelsdisulfide2), determined [10][10]isomers andand (denotedµ-PIIIAeven some— originally voltage-gatedin the isolatedcurrent K+ channelsfrom study Conus [15]. purpurascensas Recently, 1–15) we—of synthesialso the affects zedconotoxin neuronalall 15 possible Naµ-PIIIAV channels disulfide (sequence: [10] isomers and µ-PIIIAeven(denoted some— originally voltage-gatedin the isolatedcurrent K + channelsfrom study Conus [15]. purpurascensas Recently, 1–15) we—of synthesialso the affects zedconotoxin neuronalall 15 possible Naµ-PIIIA channelsdisulfide (sequence: [10]isomers and their structure(denotedeven some and voltage-gatedin verifiedthe theircurrent K++ disulfidechannels study [15]. bondas Recently, patterns1–15) we byof synthesi applyingthe zedconotoxin aall combination 15 possible µ-PIIIA disulfide of MS(sequence:/MS isomers analysis evenZRLCCGFOKSCRSRQCKOHRCC-NHeven(denoted somesome voltage-gatedvoltage-gatedin the current KK+ channelschannels study [15].[15].2 ), determinedasRecently,Recently, 1–15 ) wewe their ofsynthesisynthesi thestructure zedzedconotoxin allall 15and15 possiblepossible verified µ-PIIIA disulfidedisulfide their (sequence:disulfide isomersisomers even(denotedZRLCCGFOKSCRSRQCKOHRCC-NH some voltage-gatedin the current K channels study [15]. 2), as determinedRecently, 1–15) we theirof synthesi thestructure zedconotoxin all and15 possible verifiedµ-PIIIA disulfide their (sequence: disulfide isomers (denotedZRLCCGFOKSCRSRQCKOHRCC-NH(denoted inin thethe currentcurrent studystudy 22 ),), asasdetermineddetermined 1–151–15)) theiroftheirof thethestructurestructure conotoxinconotoxin andand verified verifiedµ-PIIIAµ-PIIIA theirtheir (sequence:(sequence: disulfidedisulfide and 2Dbond(denotedZRLCCGFOKSCRSRQCKOHRCC-NH NMR patterns spectroscopy in bythe applying current [16]. a Incombination thestudy present 2), asofdetermined study,MS/MS1–15 these) analysis oftheir isomers the structureand 2Dconotoxin and NMR and further spectroscopy verifiedµ-PIIIA analogs their (Table[16].(sequence: disulfide In1 )the were (denotedZRLCCGFOKSCRSRQCKOHRCC-NHbond patterns in bythe applying current a combination study 2), asdeterminedof MS/MS1–15) analysis theirof thestructure and 2Dconotoxin NMR and spectroscopy verifiedµ-PIIIA their [16].(sequence: disulfide In the bondZRLCCGFOKSCRSRQCKOHRCC-NH patterns by applying a combination2), ofdetermined MS/MS analysis their structureand 2D NMR and spectroscopyverified their [16]. disulfide In the analyzedZRLCCGFOKSCRSRQCKOHRCC-NHZRLCCGFOKSCRSRQCKOHRCC-NH in electrophysiological experiments22),), determineddetermined for their potencytheirtheir structurestructure to block andand the verifiedverified human theirtheir skeletal disulfidedisulfide muscle ZRLCCGFOKSCRSRQCKOHRCC-NHpresentbondbond patternspatterns study, by bythese applyingapplying isomers aa combination combinationand further2), determinedofofanalogs MS/MSMS/MS (T analysisanalysisable their 1) structure were andand 2D 2Danalyzed NMRNMR and spectroscopyspectroscopyverified in electrophysiological their [16].[16]. disulfide InIn thethe bondpresentbond patternspatterns study, by bythese applyingapplying isomers aa combination combinationand further ofofanalogs MS/MSMS/MS (T analysisanalysisable 1) wereandand 2D 2Danalyzed NMRNMR spectroscopyspectroscopy in electrophysiological [16].[16]. InIn thethe voltage-gatedbondexperimentspresent patterns sodiumstudy, for by these their channelapplying isomerspotency Na a combination V and1.4.to block further Interactions the of analogs humanMS/MS between (T skeletalanalysisable 1) the muscleandwere isomers 2D analyzed voltage-gatedNMR and spectroscopy thein electrophysiological channel sodium [16]. protein channel In the were experimentspresent study, for these their isomers potency and to blockfurther the analogs human (T skeletalskeletalable 1) musclemusclewere analyzed voltage-gatedvoltage-gated in electrophysiological sodiumsodium channelchannel presentNapresentexperimentsV1.4. study,Interactionsstudy, for thesethese their betweenisomersisomers potency andandthe to isomersfurtherblockfurther the analogsanalogsand human the (T (T channelskeletalableable 1)1) proteinwereweremuscle analyzedanalyzed werevoltage-gated examined inin electrophysiologicalelectrophysiological sodium via molecular channel examinedpresentexperimentsNaV1.4. via study,Interactions molecular for these their dockingbetweenisomers potency and the simulations.to blockisomersfurther the analogsand human All the the (T channelskeletalable 21 PIIIA1) proteinmusclewere analogs analyzed voltage-gatedwere (Table examined in electrophysiological1) weresodium via subjectedmolecular channel to experimentsNaexperimentsVV1.4. Interactions forfor theirtheir between potencypotency the toto blockblockisomers thethe and humanhuman the channelskeletalskeletal proteinmusclemuscle voltage-gatedwerevoltage-gated examined sodiumsodium via molecular channelchannel dockingexperimentsNaV1.4. Interactionssimulations. for their between potencyAll the the21to blockPIIIAisomers theanalogs and human the (Table channelskeletal 1) weremuscleprotein subjected voltage-gatedwere examined to all-atom sodium via molecularmolecular channel all-atomexperimentsNadocking molecularV1.4. Interactionssimulations. for dynamics their between potencyAll (MD) the the 21to simulations blockisomersPIIIA theanalogs and human in solutionthe (Table channelskeletal while1) wereproteinmuscle 3 selectedsubjected voltage-gatedwere examined isomer-channel to all-atom sodium via molecular channel complexes dockingNaV1.4. Interactionssimulations. between All the the21 PIIIAisomers analogs and the (Table channel 1) wereprotein subjected were examined to all-atom via molecularmolecular NaNaVV1.4.1.4. InteractionsInteractions betweenbetween thethe isomersisomers andand thethe channelchannel proteinprotein werewere examinedexamined viavia molecularmolecular NadynamicsdockingdockingV1.4. Interactions simulations.simulations. (MD) simulations between AllAll thethe in the 2121solution isomersPIIIAPIIIA whileanalogsanalogs and 3the selected (Table(Table channel 1)isomer-channel1) wereproteinwere subjectedsubjected were complexes examined toto all-atomall-atom obtainedvia molecularmolecular from obtaineddockingdynamicsdocking from simulations. thesimulations. (MD) docking simulations All simulationsAll thethe in 2121solution PIIIAPIIIA were whileanalogsanalogs subjected 3 selected (Table(Table to further 1)1)isomer-channel werewere MD-based subjectedsubjected complexes refinementtoto all-atomall-atom obtained formolecularmolecular additional from dockingthedynamicsdynamics docking simulations. (MD)(MD) simulations simulationssimulations All were the inin 21subjected solutionsolution PIIIA whilewhileanalogsto further 33 selectedselected(Table MD-based 1)isomer-channelisomer-channel were refinement subjected complexescomplexes for to additionalall-atom obtainedobtained moleculardetails fromfrom of detailsdynamicsthethedynamics of dockingdocking how the(MD)(MD) simulationssimulations isomer-channel simulationssimulations werewere inin subjected subjectedsolutionsolution interaction while whiletoto furtherfurther stabilizes 33 selectedselected MD-based over isomer-channelisomer-channel time. refinement The complexes analysiscomplexes for additional of obtainedobtained the details 21 peptidesfromfrom of dynamicshowthethe dockingdocking the isomer-channel (MD) simulationssimulations simulations interactionwerewere in subjectedsubjectedsolution stabilizes while toto furtherfurther over3 selected time.MD-basedMD-based isomer-channelThe analysis refinementrefinement of complexesthe forfor 21 additionaladditional peptides obtained detailsdetails(Table from of1)of (Table1thehowthe) revealed dockingdocking the isomer-channel simulations thatsimulations the channel-blocking interactionwerewere subjectedsubjected stabilizes toto ability furtherfurther over is MD-basedtime.time.MD-based determined TheThe analysisanalysis refinementrefinement by a ofof complex the the forfor 2121 additionaladditional peptidespeptides interplay detailsdetails(Table(Table between of1)of1) therevealedhowhow docking thethe isomer-channelisomer-channelthat simulations the channel-blocking interactionwereinteraction subjected stabilizesstabilizes ability to further overisover determined time.MD-basedtime. TheThe analysis analysisrefinementby a complex ofof the thefor 2121 additional interplay peptidespeptides details (Table(Tablebetween of1)1) conformationalhowrevealedrevealedhow thethe isomer-channelisomer-channel stability,thatthat thethe orientationschannel-blockingchannel-blocking interactioninteraction of the stabilizesstabilizes abilityability functionally overisoveris determineddetermined time.time. significant TheThe analysisanalysisbyby side aa chainscomplex complex ofof thethe [ 172121 interplay],interplay peptidespeptides and the (Table(Table electrostaticbetweenbetween 1)1) howconformationalrevealedrevealed the isomer-channelthatthat thethestability, channel-blockingchannel-blocking interactionorientations stabilizes abilityabilityof the overisfunctionallyis determineddetermined time. The significant analysisbyby aa complex complex ofside the chains21 interplayinterplaypeptides [17], (Tablebetweenandbetween the 1) propertiesrevealedconformationalrevealed of the thatthat molecules. thethestability, channel-blockingchannel-blocking This orientations study provides abilityabilityof the aisisfunctionally conceptual determineddetermined frameworksignificant byby aa complexcomplex side to understand chains interplayinterplay [17], how betweenandbetween di thefferent revealedelectrostaticconformationalconformational that properties thestability,stability, channel-blocking of theorientationsorientations molecules. abilityof ofThis thethe study isfunctionallyfunctionally determined provides asignificantsignificant conceptualby a complex sideside framework chainschains interplay to[17],[17], understand betweenandand thethe conformationalelectrostaticconformational properties stability,stability, of theorientationsorientations molecules.lecules. of of ThisThis thethe studystudy functionallyfunctionally providesprovides significantaasignificant conceptualconceptual sideside frameworkframework chainschains to[17],to[17], understandunderstand andand thethe disulfideconformationalhowelectrostatic connectivities different properties disulfide stability, affect ofconnectivities theorientations the mo structurelecules. affectof This ofthe theµ study -PIIIAfunctionally structure provides and of ultimately significantaµ-PIIIA conceptual and itsside frameworkultimately interaction chains its[17],to withunderstandinteraction and the the pore howelectrostatic different properties disulfide of connectivities the molecules.lecules. affect ThisThis the studystudy structureructure providesprovides ofof aaµ-PIIIAµ-PIIIA conceptualconceptual andand frameworkframeworkultimatelyultimately toitstoits understand understandinteractioninteraction electrostaticwithelectrostatichow thedifferent pore propertiesproperties of disulfide NaV1.4. ofof connectivities thethe momolecules.lecules. affect ThisThis studystudythe st ructure providesprovides of aa µ-PIIIA conceptualconceptual and frameworkframework ultimately totoits understandunderstand interaction of NaVelectrostatichowwith1.4. differentthe pore properties ofdisulfide NaV1.4. of connectivities the molecules. affect This thestudy structure provides of aµ-PIIIA conceptual and frameworkultimately toits understandinteraction howwithhow differentthedifferent pore of disulfidedisulfide NaVV1.4. connectivitiesconnectivities affectaffect thethe ststructureructure ofof µ-PIIIAµ-PIIIA andand ultimatelyultimately itsits interactioninteraction howwith differentthe pore ofdisulfide NaV1.4. connectivities affect the structure of µ-PIIIA and ultimately its interaction how different disulfideV connectivities affect the structure of µ-PIIIA and ultimately its interaction Tablewith 1.theNomenclature, pore of Na V1.4. disulfide connectivity, type of structure, and source references for the peptides withwith Tablethethe porepore 1. Nomenclature,ofof NaNaV1.4.1.4. disulfide connectivity, type of structure, and source references for the with Tablethe pore 1. Nomenclature,of NaV1.4. disulfide connectivity, type of structure, and source references for the used inTable this study.1. Nomenclature, disulfide connectivity, type of structure, and source references for the peptidesTable 1. used Nomenclature, in this study. disulfide connectivity, type of structure, and source references for the peptidesTable 1. usedNomenclature, in this study. disulfide connectivity, type of structure, and source references for the TableTablepeptides 1.1. usedNomenclature,Nomenclature, in this study. disulfidedisulfide connectivity,connectivity, typetype ofof structure,structure, andand sourcesource referencesreferences forfor thethe IsomerTablepeptides 1. usedNomenclature, inNumber this study. of disulfide connectivity, type of structure, andType source of referencesSource for the peptidespeptidesIsomer usedused ininNumber thisthis study.study. of Disulfide Connectivity Type of Source peptidesIsomerIsomer used inNumber this study. of Disulfide connectivity Type of Source NomenclaturenomenclatureIsomer DisulfidesNumberdisulfides of Disulfide connectivity structureStructureType of reference(s)Reference(s)Source nomenclatureIsomer Numberdisulfides of Disulfide connectivity structureType of reference(s)Source nomenclatureIsomerIsomer NumberNumberdisulfides ofof DisulfideDisulfide connectivityconnectivity structureTypeType ofof reference(s)SourceSource nomenclatureIsomer Numberdisulfides of DisulfideDisulfide connectivityconnectivity structureType of HeimerHeimerreference(s)Source et et al. al. [16], [16 ], nomenclature1 1 disulfides33 Disulfide connectivity structureNMRNMR Heimerreference(s)reference(s) et al. [16], nomenclaturenomenclature1 disulfidesdisulfides3 structurestructureNMR HeimerTietzereference(s)reference(s) et al. [[16],10 ]. nomenclature1 disulfides3 structureNMR HeimerHeimerTietzereference(s) et etet al. al.al. [10]. [16],[16], 1 3 NMR HeimerHeimerTietze et etet al. al.al. [10]. [16],[16], 1 3 NMR HeimerTietze etet al.al. [10].[16], 121 33 NMRNMR HeimerTietzeHeimer et et et al. al. al. [10]. [16], [16], 2 12 33 NMRNMR HeimerTietzeTietze etet et al.al. al. [10].[10]. [16], 2 3 NMR HeimerTietzeTietze et etet al. al.al. [10]. [10[16],]. 2 3 NMR HeimerTietze et et al. al. [10]. [16], 2 3 NMR HeimerHeimerTietze etet al.al. [10].[16],[16], 22 33 NMRNMR HeimerTietze et et al. al. [10]. [16], 23 3 NMR HeimerTietzeTietzeHeimer et etet et al. al.al. al. [10]. [10].[16], [16], 3 3 33 NMRNMR HeimerTietze et et al. al. [10]. [16], 3 3 NMR HeimerTietzeTietze et et al. al.al. [10]. [[16],10]. 3 3 NMR HeimerHeimerTietze et etet al. al.al. [10]. [16],[16], 33 33 NMRNMR HeimerTietzeTietze et etet al. al.al. [10]. [10].[16], 34 3 NMR HeimerTietzeTietze et etet al. al.al. [10].[10]. [16]. 4 3 NMR HeimerTietze et et al. al. [10]. [16]. 4 4 3 3 NMR Heimer Heimer et et al. [16]. [16]. 4 3 NMR Heimer et al. [16]. 454 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 45 3 NMR Heimer et al. [16]. 5 3 NMR Heimer et al. [16]. 5 55 333 NMRNMR Heimer Heimer Heimer etet et al.al. [16].[16]. [16]. 565 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 56 3 NMR Heimer et al. [16]. 6 3 NMR Heimer et al. [16]. 6 66 333 InNMRNMR silico Heimer Heimer Heimer etet et al.al. [16].[16]. [16]. 676 33 InNMRNMR silico Heimer Heimer etet al.al. [16].[16]. 67 3 InNMR silico Heimer et al. [16]. 7 3 InInmodel silicosilico Heimer et al. [16]. 7 3 InmodelIn silico silico Heimer et al. [16]. 7 7 33 InInmodel silicosilico HeimerHeimer et et al. al. [16]. [16]. 77 33 Inmodelmodel silico HeimerHeimer etet al.al. [16].[16]. 78 3 modelmodelNMR Heimer et al. [16]. 8 3 modelNMR Heimer et al. [16]. 8 3 NMR Heimer et al. [16]. 8 3 NMR Heimer et al. [16]. 8 88 333 NMRNMRNMR Heimer Heimer Heimer etet et al.al. al. [16].[16]. [16]. 89 3 NMR Heimer et al. [16]. 9 3 NMR Heimer et al. [16]. 9 3 NMR Heimer et al. [16]. 9 3 NMR Heimer et al. [16]. 9 99 333 NMRNMRNMR Heimer Heimer Heimer etet et al.al. al. [16].[16]. [16]. 109 3 NMR Heimer et al. [16]. 10 3 NMR Heimer et al. [16]. 10 3 NMR Heimer et al. [16]. 10 3 NMR Heimer et al. [16]. 1010 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 10 10 33 NMRNMR Heimer Heimer et et al. al. [16]. [16].

Mar. Drugs 2019, 17, 390 3 of 18

Table 1. Cont.

Mar.Isomer Drugs 2019, 17, x FORNumber PEER ofREVIEW Type of Source 3 of 19 Mar.Mar. Drugs Drugs 2019 2019, ,17 17, ,x x FOR FOR PEER PEER REVIEW REVIEW Disulfide Connectivity 33 of of 19 19 NomenclatureMar.Mar. DrugsDrugs 20192019,, 1717,, xx FORFORDisulfides PEERPEER REVIEWREVIEW Structure Reference(s)33 ofof 1919 Mar. Drugs 2019,, 17,, xx FORFOR PEERPEER REVIEWREVIEW 3 of 19 Mar.Mar. DrugsDrugs 20192019,,, 1717,,, xxx FORFORFOR PEERPEERPEER REVIEWREVIEWREVIEW 33 ofof 1919 11 11 33 NMRNMR Heimer Heimer et et al. al. [16]. [16]. 1111 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 1111 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 11 3 NMR Heimer et al. [16]. 1111 33 InInNMRNMR silicosilico Heimer Heimer etet al.al. [16].[16]. 12 3 InIn silico silico Heimer et al. [16]. 12 1212 333 InInmodel silicosilico HeimerHeimerHeimer etet et al.al. al. [16].[16]. [16]. 1212 33 Inmodelmodelmodel silico HeimerHeimer etet al.al. [16].[16]. 12 3 InInmodelmodel silicosilico Heimer et al. [16]. 1212 33 InInmodel silicosilico HeimerHeimer etet al.al. [16].[16]. 13 3 InmodelInmodel silico silico Heimer et al. [16]. 13 1313 333 InInmodel silicosilico HeimerHeimerHeimer etet et al.al. al. [16].[16]. [16]. 1313 33 Inmodelmodelmodel silico HeimerHeimer etet al.al. [16].[16]. 13 3 InInmodel silicosilico Heimer et al. [16]. 1313 33 model HeimerHeimer etet al.al. [16].[16]. 14 3 modelNMR Heimer et al. [16]. 1414 33 modelmodelNMRNMR Heimer Heimer etet al.al. [16].[16]. 14 1414 333 NMRNMRNMR Heimer Heimer Heimer etet et al.al. al. [16].[16]. [16]. 14 3 NMR Heimer et al. [16]. 1414 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 15 3 NMR Heimer et al. [16]. 1515 33 NMRNMR Heimer Heimer etet al.al. [16].[16]. 15 1515 333 NMRNMRNMR Heimer Heimer Heimer etet et al.al. al. [16].[16]. [16]. 15 3 NMR Heimer et al. [16]. 1515 33 InInNMRNMR silicosilico Heimer Heimer etet al.al. [16].[16]. 16* 2 In silico Current study 1616** 22 InInmodel silicosilico CurrentCurrent studystudy 1616** 22 InmodelInmodel silico silico CurrentCurrent studystudy 16*16* 22 InInmodel silicosilico CurrentCurrent study study 1616** 22 Inmodelmodel silico CurrentCurrent studystudy 17* 2 InInmodel silicosilico Current study 17* 2 InInmodelmodel silicosilico Current study 17* 2 Inmodelmodel silico Current study 17** 2 InInmodel silico silico Current study 17*17* 22 InInmodel silicosilico CurrentCurrent study study 1717** 22 InInmodelmodel silicosilico CurrentCurrent studystudy 1818** 22 Inmodelmodel silico CurrentCurrent studystudy 18* 2 InInmodelmodel silicosilico Current study 18* 2 Inmodel silico Current study 18* 2 InInmodelInmodel silicosilico silico Current study 18*1818** # 222 Inmodel silico CurrentCurrentCurrent studystudy study ΔΔ(C5-C21)2# 2 Inmodelmodel silico Current study Δ(C5-C21)2 # 2 Inmodelmodel silico Current study Δ(C5-C21)2# 2 Inmodel silico Current study Δ(C5-C21)2 # 2 Inmodelmodel silico Current study Δ(C5-C21)2## 2 InInInmodel silicosilico silico Current study Δ(C5-C21)# 2## 2 Inmodel silico Current study ∆(C5-C21)ΔΔ(C5-C21)(C5-C21)2 22 # 222 Inmodel silico CurrentCurrentCurrent studystudy study ΔΔ(C11-C21)4# 2 Inmodelmodel silico Current study Δ(C11-C21)4 # 2 Inmodelmodel silico Current study Δ(C11-C21)4# 2 Inmodel silico Current study Δ(C11-C21)4 # 2 Inmodelmodel silico Current study Δ(C11-C21)4## 2 InInInmodel silicosilico silico Current study Δ(C11-C21)# 4## 2 Inmodel silico Current study ∆(C11-C21)ΔΔ(C11-C21)(C11-C21)4 44# 222 Inmodel silico CurrentCurrentCurrent studystudy study ΔΔ(C5-C22)10# 2 Inmodelmodel silico Current study Δ(C5-C22)10 # 2 Inmodelmodel silico Current study Δ(C5-C22)10# 2 Inmodel silico Current study Δ(C5-C22)10 # 2 Inmodelmodel silico Current study Δ(C5-C22)10## 2 InInInmodel silicosilico silico Current study Δ(C5-C22)# 10## 2 ## model Current study ∆(C5-C22)ΔΔ(C5-C22)(C5-C22)* Mutated10 1010 disulfide 2 22bonds replaced by serine residues. # Isomers withmodel in silico disulfideCurrentCurrentCurrent studystudybond study (C5-C22)** MutatedMutated10 disulfide disulfide 2bondsbonds replacedreplaced byby serineserine residues.residues. # IsomersIsomers withwithmodel inin silico silico disulfideCurrentdisulfide studybondbond * Mutated disulfide bonds replaced by serine residues. # Isomers withmodelmodel in silico disulfide bond * Mutated disulfide bonds replaced by serine residues. # Isomers withmodelmodel in silico disulfide bond *opening Mutated yielding disulfide two bondsSH-groups. replaced by serine residues. # Isomers with in silico disulfide bond opening*opening Mutated yieldingyielding disulfide twotwo bonds SH-groups.SH-groups. replaced by serine residues. ## Isomers with in silico disulfide bond **opening MutatedMutated yielding disulfidedisulfide two bonds bondsSH-groups. replacedreplaced byby serineserine #residues.residues. ## IsomersIsomersIsomers withwithwith ininin silicosilicosilico disulfidedisulfidedisulfide bondbondbond * Mutatedopening disulfide yielding bonds two replaced SH-groups. by serine residues. Isomers with in silico disulfide bond opening yielding two SH-groups.opening yielding two SH-groups. 2.2. ResultsResultsopeningopening yieldingyielding twotwo SH-groups.SH-groups. 2. Results 2. Results 2.2. ResultsResults 2. Results2.1.2.1. BioactivityBioactivity ofof 3-3- andand 2-Disulfide-bonded2-Disulfide-bonded µ-PIIIAµ-PIIIA AnalogsAnalogs atat NaNavv1.41.4 2.1. Bioactivity of 3- and 2-Disulfide-bonded µ-PIIIA Analogs at Nav1.4 2.1. Bioactivity of 3- and 2-Disulfide-bonded µ-PIIIA Analogs at Nav 1.4 2.1. Bioactivity of 3- and 2-Disulfide-bonded µ-PIIIA Analogs at Na v1.4 2.1. BioactivityPreviously, of we 3- andsynthesized 2-Disulfide-bonded all 15 possible µ-PIIIA 3-disulfide-bonded Analogs at Nav1.4 isomers of the conopeptide 2.1.2.1. BioactivityBioactivityPreviously, ofofwe 3-3- andsynthesizedand 2-Disulfide-bonded2-Disulfide-bonded all 15 possible µ-PIIIAµ-PIIIA 3-disulfide-bonded AnalogsAnalogs atat NaNavv1.41.4 isomers of the conopeptide 2.1. BioactivityPreviously, of 3- and we 2-Disulfide-bonded synthesized all 15µ-PIIIA possible Analogs 3-disulfide-bonded at Nav1.4 isomers of the conopeptide µ-PIIIAµ-PIIIAPreviously, (here(here termedtermed we synthesized1–151–15,, TableTable 1) 1)all andand 15 determined determinedpossible 3- theirdisulfide-bondedtheir 3-dime3-dimensionalnsional isomers structurestructure of [1 [1the6].6]. BasedBasedconopeptide onon thethe µ-PIIIAPreviously,Previously, (here termed wewe synthesized synthesized1–15, Table 1)allall and 1515 determined possiblepossible 3-3- disulfide-bondedtheirdisulfide-bonded 3-dimensional isomersisomers structure ofof [1thethe6]. conopeptideBasedconopeptide on the Previously,nativeµ-PIIIAnative disulfidedisulfide (here we termed synthesized connectivityconnectivity 1–15,, TableTable all ofof 15 isomer1)isomer1)possible andand determineddetermined22,, wewe 3-disulfide-bonded nownow theirtheirproducedproduced 3-dime3-dime threethreensional isomers additionaladditional structure of the 2-disulfide-bonded2-disulfide-bonded conopeptide[16]. Based onµ the-PIIIA µ-PIIIAµ-PIIIAnative disulfide (here(here termedtermed connectivity 1–151–15,,, TableTableTable of 1)isomer1)1) andandand determined determineddetermined2, we now theirtheirtheirproduced 3-dime3-dime3-dime threensionalnsional additional structurestructure 2-disulfide-bonded [1[16].6]. BasedBased onon thethe mutantsnativemutants disulfide ofof µ-PIIIAµ-PIIIA connectivity (termed(termed 16–1816–18 of isomer),), eacheach 2lackinglacking,, wewe nownow oneone produced producedofof thethe disulfidedisulfide threethree bondsadditionalbondsadditional ofof 22 (Table2-disulfide-bonded(Table2-disulfide-bonded 1,1, FigureFigure S1,S1, (here termednativemutants disulfide1 of–15 µ-PIIIA, Table connectivity (termed1) and determined16–18 of isomer), each 2 lacking, their, wewe nownow 3-dimensional one producedproduced of the disulfide threethree structure additionaladditionalbonds [ of16 2]. 2-disulfide-bonded2-disulfide-bonded(Table Based 1, onFigure the nativeS1, mutantsnativeTablesnativemutants disulfidedisulfideS1 ofof and µ-PIIIAµ-PIIIA S2). connectivityconnectivity (termedPrevious(termed 16–18 16–18 reports ofof isomerisomer),), eacheach have 2 lacking2lacking, , shownwewe nownow oneone that produced producedofof µ-PIIIA thethe disulfidedisulfide threehasthree the bondsadditionalbondsadditional highest ofof 22 potency (Table2-disulfide-bonded(Table2-disulfide-bonded 1,1, to FigureFigure interfere S1,S1, disulfideTablesmutantsTables connectivity S1S1 of andand µ-PIIIA S2).S2). of Previous(termedPrevious isomer 16–18 2reportsreports, we), now each havehave producedlacking shownshown one thatthat three of µ-PIIIA µ-PIIIAthe additional disulfide hashas thethe bonds 2-disulfide-bonded highesthighest of 2 potency potency(Table(Table 1,1, toto FigureFigure interfereinterfere mutants S1,S1, of TablesmutantsmutantsTables S1S1 ofof andand µ-PIIIAµ-PIIIA S2).S2). Previous(termedPrevious(termed 16–18 16–18reportsreports),), each each havehave lackinglacking shownshown oneone thatthat ofof µ-PIIIA µ-PIIIA thethe disulfidedisulfide hashas thethe bondsbonds highesthighest ofof 2 2 potency potency (Table(Table 1, 1, toto FigureFigure interfereinterfere S1,S1, withTableswith skeletalskeletal S1 and musclemuscle S2). Previous channelchannel reports NaNaVV1.41.4 havealthoughalthough shown alsoalso that beingbeing µ-PIIIA activeactive has onon theneuronalneuronal highest NaNa potencyVV channels,channels, to interfere suchsuch asas µ-PIIIATableswith (termed skeletal S1 and16 muscle– S2).18), Previous each channel lacking reports NaV one1.4 havealthough of the shown disulfide also thatthat being µ-PIIIAµ-PIIIA bonds active ofhashas on2 thethe(Tableneuronal highesthighest1, FigureNa potencypotencyV channels, S1, to Tablesto interfereinterfere such S1 as and withTablesTableswith skeletalskeletal S1S1 andand musclemuscle S2).S2). PreviousPrevious channelchannel reports reports NaNaV1.41.4 havealthoughhavealthough shownshown alsoalso thatthat beingbeing µ-PIIIAµ-PIIIA activeactive+ hashas onon theneuronaltheneuronal highesthighest NaNa potencypotencyV channels,channels, toto interfere interfere suchsuch asas withNaVV1.6 skeletal and Na muscleVV1.8 [18], channel and NaevenV1.4 on although select voltage-gated also being active K+ (K onVV) neuronalchannels Na[15,19].V channels, We therefore such as NawithV1.6 skeletal and Na muscleV1.8 [18], channel and NaevenV1.4 on although select voltage-gated also being active K + (K onV) neuronalchannels Na[15,19].V channels, We therefore such as S2). PreviouswithNaV1.6 skeletal and reports Na muscleV have1.8 [18], channel shown and Na thatevenVV1.4µ on-PIIIA although select has voltage-gated also the being highest active potencyK+ (KonV )neuronal channels to interfere Na [15,19].VV withchannels,channels, We skeletal therefore suchsuch muscle asas NawithwithV1.6 skeletalskeletal and Na musclemuscleV1.8 [18], channelchannel and NaevenNaV1.41.4 on althoughalthough select voltage-gated alsoalso beingbeing activeactive K + (K ononV) neuronalneuronalchannels NaNa[15,19].V channels,channels, We therefore suchsuch asas appliedNaappliedV1.6 andallall 3-disulfide-bonded3-disulfide-bonded NaV1.8 [18], and even asas wellwell on as asselect thethe 2-disulfide-bonded2-disulfide-bonded voltage-gated K++ (K isomersisomersV) channels toto HEK293HEK293 [15,19]. cellscells We expressingexpressing therefore NaappliedV1.6 and all 3-disulfide-bondedNaV1.8 [18], and even as well on asselect the 2-disulfide-bondedvoltage-gated K++ (K(K isomersV) channels to HEK293 [15,19]. cells We expressing therefore channelNaappliedNaapplied NaVV1.61.6V 1.4 and andallall although 3-disulfide-bonded 3-disulfide-bonded NaNaVV1.81.8 [18],[18], also andand being even even asas active wellwell onon asas selectselect on thethe neuronal 2-disulfide-bonded 2-disulfide-bonded voltage-gatedvoltage-gated NaV channels, KK (K (K isomersisomersVV)) channelschannels such toto asHEK293HEK293 Na[15,19].[15,19].V1.6 cellscells We andWe expressingexpressing thereforetherefore NaV1.8+ [ 18], appliedhuman NaallV V3-disulfide-bonded1.4 channels and measured as well as the the impact 2-disulfide-bonded of the toxin isomers isomers on todepo HEK293larization-elicited cells expressing Na+ humanapplied Na all V3-disulfide-bonded1.4 channels and measured as well asthe the impact 2-disulfide-bonded of the toxin isomers isomers on todepo HEK293larization-elicited cells expressing Na + appliedhuman Naall V3-disulfide-bonded1.4 channels and measured +as well as the the impact 2-disulfide-bonded of the toxin isomers isomers on to depo HEK293larization-elicited cells expressing Na+ and evenhumanappliedapplied on select Na allall V 3-disulfide-bonded3-disulfide-bonded1.4 voltage-gated channels and measured K asas(K wellwellV) channelsasasthe thethe impact 2-disulfide-bonded2-disulfide-bonded [15 of, 19the]. toxin We thereforeisomers isomersisomers on applied todepoto HEK293HEK293larization-elicited all 3-disulfide-bonded cellscells expressingexpressing Na + inwardhumaninward Nacurrents,currents,V1.4 channels expressedexpressed and as asmeasured thethe timetime the constantconstant impact forfor of currentcurrentthe toxin blockblock isomers andand on thethe depo maximalmaximallarization-elicited blockblock afterafter longlong Na++ humaninward Na currents,V1.4 channels expressed and asmeasured the time the constant impact for of thcurrente toxin block isomers and on the depo maximallarization-elicitedlarization-elicited block after longNaNa++ as wellhumaninwardtoxinhumaninward as the exposure Na currents,Nacurrents, 2-disulfide-bondedVV1.41.4 channels channels(Figure expressedexpressed 1, andand Figure as as measuredmeasured thethe isomers S2 timetime). Figure the the constantconstant to impactimpact HEK293 1 summarizes forfor ofof currentthcurrentth cellsee toxintoxin expressingthe blockblock isomersisomers inhibitory andand on on thethe human depo depoactivity maximalmaximallarization-elicitedlarization-elicited Na of block blockthe1.4 most channels afterafter active long long NaNa and toxininwardinwardtoxin exposureexposure currents,currents, (Figure(Figure expressedexpressed 1,1, FigureFigure asas thethe S2S2 timetime).). FigureFigure constantconstant 11 summarizessummarizes for current thethe block inhibitoryinhibitory and the activity activitymaximal ofof Vblock thethe mostmost after activeactive long toxininwardinwardtoxin exposureexposure currents,currents, (Figure(Figure expressedexpressed 1,1, FigureFigure asas thethe S2S2 timetime).). FigureFigure constantconstant 11 summarizessummarizes forfor currentcurrent thethe blockblock inhibitoryinhibitory andand+ thethe activity activity maximalmaximal ofof block blockthethe mostmost afterafter activeactive longlong measuredisomerstoxinisomers the exposure 1 impact1,, 22,, 77,, 11 11(Figure of,, 1212 the,, 1414 toxin1,,, andand Figure 15 isomers15.. As AsS2 ).demonstrateddemonstrated Figure on depolarization-elicited 1 summarizes inin FigureFigure the 1A,1A, inhibitory currentscurrents Na inward mediatedmediated activity currents, of byby the humanhuman most expressed NaNa activeVV1.41.4 as toxintoxinisomers exposureexposure 1, 2, 7, 11(Figure(Figure, 12, 14 1,1,, and FigureFigure 15. AsS2S2). demonstrated Figure 1 summarizes in Figure the 1A, inhibitory currents mediated activity of by the human most NaactiveV1.4 isomerstoxintoxinisomers exposureexposure 11,, 22,, 77,, 11 11 (Figure(Figure,, 1212,, 1414 1,,1,, and and FigureFigure 1515.. As AsS2S2 ). ).demonstrateddemonstrated FigureFigure 11 summarizessummarizes inin FigureFigure the the1A,1A, inhibitory inhibitory currentscurrents mediatedmediated activityactivity of of byby the the humanhuman mostmost Na Na activeactiveV1.41.4 the timewereisomerswere constant diminisheddiminished 1, 2, 7 for, 11 current ,by by12 ,all all14 ,isomers,isomers, and block 15. and As albeitalbeit demonstrated the toto maximal differedifferentnt in blockdegrees.degrees. Figure after 1A, AtAt longacurrentsa concentrationconcentration toxin mediated exposure ofof by1010 humanµM, (FigureµM, isomerisomer Na1,V Figure1.4 22,, isomersisomerswere diminished 1,, 2,, 7,, 11,, by12,, all14, , isomers,andand 15.. AsAs albeit demonstrateddemonstrated to different inin degrees. FigureFigure 1A,1A, At currentscurrentsa concentration mediatedmediated of by by10 humanhumanµM, isomer NaNaV1.4 2, wereisomerswhichisomerswere diminisheddiminished has 11,, 22the,, 77,, native 1111 , , by by1212 , ,all all disulfide1414 , ,isomers, isomers, andand 1515 co.. As Asnnectivityalbeitalbeit demonstrateddemonstrated toto differediffere of µ-PIIIAntnt inin degrees.degrees. FigureFigure (C4-C16/C5-C 1A, 1A, AtAt currentsacurrentsa concentrationconcentration21/C11-C22), mediatedmediated ofof was by10by10 human µM,humanµM,most isomerisomer effective NaNaVV1.41.4 22,, S2). Figurewhichwerewhich 1diminished hassummarizeshas thethe nativenative by the alldisulfidedisulfide isomers, inhibitory coco nnectivitynnectivityalbeit activity to differe ofof of µ-PIIIAµ-PIIIA thent mostdegrees. (C4-C16/C5-C(C4-C16/C5-C active At a isomers concentration21/C11-C22),21/C11-C22),1, 2, 7 of, was11was 10, 12 µM, mostmost, 14 isomer ,effectiveeffective and 15 2,,. As werewhichwerewhich diminisheddiminished hashas thethe nativenative byby alldisulfidealldisulfide isomers,isomers, coco nnectivity nnectivityalbeitalbeit toto differediffere ofof µ-PIIIAµ-PIIIAntnt degrees.degrees. (C4-C16/C5-C(C4-C16/C5-C AtAt aa concentrationconcentration21/C11-C22),21/C11-C22), ofof waswas 1010 µM, mostµM,most isomer isomer effectiveeffective 22,, inwhichin inhibitinginhibiting has the NanativeNaVV1.41.4 disulfide channels,channels, co followednnectivityfollowed bybyof µ-PIIIAisomersisomers (C4-C16/C5-C 1515,, 1111,, 1414,, 21/C11-C22),11,, 77,, andand 1212 was.. AnalysisAnalysis most effective ofof thethe demonstratedwhichin inhibiting has in the Figure nativeNaV11.4A, disulfide channels, currents co mediatednnectivityfollowed byofby µ-PIIIA humanisomers (C4-C16/C5-C Na 15V, 1.411, were14, 21/C11-C22),1 diminished, 7, and 12 was. by Analysis allmost isomers, effective of the albeit whichwhichin inhibiting hashas thethe nativeNanativeV 1.4 disulfide disulfidechannels, coco nnectivitynnectivityfollowed ofbyof µ-PIIIA µ-PIIIAisomers (C4-C16/C5-C(C4-C16/C5-C 15, 11, 14, 21/C11-C22),21/C11-C22),1, 7, and 12 waswas. Analysis mostmost effectiveeffective of the inconcentration inhibiting NadependenceV1.4 channels, revealed followed that isomer by isomers 2 blocked 15, human11, 14, Na1, V71.4, and with 12 an. Analysisapparent ofIC 50the of concentrationin inhibiting dependenceNaV1.4 channels, revealed followed that isomer by isomers 2 blocked 15, human11, 14 , Na1, V1.47, andwith 12an. apparentAnalysis ICof 50the of to differentininconcentration inhibitinginhibiting degrees. NaNadependence AtV1.4 a concentration channels, revealed followed ofthat 10 isomer µbyM, isomers isomer 2 blocked 215, which,, human11,, 14 has,, Na1 the,, V71.4,, nativeandand with 12 an disulfide.. AnalysisAnalysisapparent connectivity ofofIC 50thethe of ininconcentration inhibitinginhibiting± NaNadependenceVV1.41.4 channels,channels, revealed followedfollowed that isomer byby isomersisomers 2 blocked 1515,, human1111,, 1414,, Na11,, V 771.4,, and andwith 1212 an.. AnalysisAnalysisapparent ofICof± 50 thethe of concentration105.3 ± 29.9 nM, dependence which is comparablerevealed that to isomerthe value 2 blocked for the humanparalog Na ratV 1.4Na Vwith1.4 channels an apparent (103.2 IC ±50 9.9of 105.3concentration ± 29.9 nM, dependence which is comparable revealed that to isomerthe value 2 blocked for the paraloghuman NaratV Na1.4V with1.4 channels an apparent (103.2 IC ±50 9.9 of µ concentration105.3 ± 29.9 nM, dependence which is comparablerevealed that to isomer the value 2 blockedblocked for the humanhumanparalog Na NaratV 1.4Na Vwith1.4 channels an apparent (103.2 IC ±5050 9.9ofof of -PIIIAconcentrationconcentration105.3 (C4-C16± 29.9 nM, /dependencedependenceC5-C21 which/ C11-C22),is comparablerevealedrevealed was thatthat to most isomer isomerthe value eff 22ective blockedblocked for the in human inhibitingparaloghuman Na NaratVV Na1.41.4Na V withwith1.41.4 channels channels,anan apparentapparent (103.2 followed ICIC ±5050 9.9 ofof by 105.3nM) [10].± 29.929.9 As nM,nM, observed whichwhich for isis comparablecomparablesome µ-conotoxins, toto thethe valuevalue even forforat athethe saturating paralogparalog concentrationratrat NaNaV1.4 channels of 100 (103.2µM a small± 9.99.9 nM)105.3nM) [10]. [10].± 29.929.9 AsAs nM,nM, observedobserved whichwhich forfor isis somecomparablecomparablesome µ-conotoxins,µ-conotoxins, toto thethe valuevalue eveneven for foratat a athethe saturatingsaturating paralogparalog concentrationconcentrationratrat NaNaV1.4 channels ofof 100100 µM(103.2µM aa smallsmall± 9.99.9 isomers105.3105.3nM)15 [10]., ±±11 29.929.9, As14 nM,nM, ,observed1, which7which, and for isis12 ± comparablesomecomparable. Analysis µ-conotoxins, toto of thethe the valuevalue even concentration for forat a thethe saturating paralogparalog dependence concentrationratrat NaNaVV1.41.4 revealedchannelschannels of 100 (103.2(103.2µM that a isomersmall±± 9.99.9 2 currentnM)current [10]. componentcomponent As observed ofof 7.27.2for ± some 2.2%2.2% µ-conotoxins, remained,remained, indicatingindicating even at thatathat saturating channelchannel concentrationoccupancyoccupancy byby ofisomerisomer 100 µM 22 doesdoes a small notnot nM)current [10]. component As observed of 7.2for ±some 2.2% µ-conotoxins, remained, indicating even at athat saturating channel concentrationoccupancy by of isomer 100 µM 2 does a small not blockedcurrentnM)nM)current human [10].[10]. componentcomponent AsAs Na observed+observed1.4 with ofof 7.2 7.2forfor an ± somesome 2.2% apparent2.2% µ-conotoxins, µ-conotoxins,remained,remained, IC ofindicatingindicating 105.3eveneven atat thata29.9thata saturatingsaturating channelchannel nM, which concentrationoccupancyconcentrationoccupancy is comparable byby of isomerofisomer 100100 to µMµM 22 the doesdoes aa small valuesmall notnot for currenteliminate component Na+ Vconduction of 7.2 ±(Figure 2.2% remained, 1A,B). 50The indicating remaining that isomers channel (15 occupancy, 11, 14, 1, 7by, 12 isomer) blocked 2 does NaV Vnot1.4 eliminatecurrent component Na + conduction of 7.2 ±(Figure 2.2% remained, 1A,B). The indicating remaining that isomers channel (15 occupancy, 11, 14, 1, 7by, 12 isomer) blocked 2 does Na Vnot1.4 currenteliminate component Na+ conduction of 7.2 ± ±(Figure 2.2%2.2% remained,remained, 1A,B). The indicatingindicating remaining± thatthat isomers channelchannel (15 occupancyoccupancy, 11, 14, 1, 7byby, 12 isomerisomer) blocked 2 doesdoes Na Vnotnot1.4 the paralogcurrenteliminatecurrent rat componentcomponent Na Na+ conduction1.4 channels ofof 7.27.2 (Figure 2.2%2.2% (103.2 remained,remained, 1A,B).9.9 The nM)indicatingindicating remaining [10]. that Asthat isomers observed channelchannel (15 occupancy,occupancy 11 for, 14 some, 1, 7 byby, µ12 -conotoxins, isomerisomer) blocked 22 doesdoes Na V even notnot1.4 at channelseliminatechannels Nalessless++ V conductionpotentlypotently than than(Figure isomerisomer 1A,B). 22 The.. InIn remaining addition,addition, isomersthethe onsetonset (15 , 11ofof , 14blockblock, 1, 7 ,as as12 )estimated estimatedblocked Na withwithV1.4 eliminatechannels Naless++ conduction conductionpotently than(Figure(Figure isomer 1A,B).1A,B).± 2TheThe. In remainingremaining addition, isomersisomers the onset ((15,, 11of,, 14block,, 1,, 7 ,, as12 ) estimatedblocked Na withV1.4 a saturatingchannelseliminatesingle-exponentialeliminatechannels concentration NaNalessless conduction conductionpotentlypotently functions of than than 100(Figure(Figure was µisomerisomerM 1A,B).1A,B).substantially a small 2 2 The.The. In currentIn remaining remaining addition, addition,slower component than isomers isomersthethe foronsetonset ((isomer1515 of, , 7.21111ofof, , 14142blockblock, ,2.2%, thus 11,, 7 7 ,, as as12 remained,precluding12 )) estimated estimatedblockedblocked faithfulindicatingNaNa withwithVV1.41.4 single-exponentialchannelssingle-exponential less potently functionsfunctions than waswas isomer substantiallysubstantially 2.. InIn addition,addition, slowerslower thanthan thethe for foronsetonset isomerisomer ofof 22blockblock,, thusthus asasprecludingprecluding estimatedestimated faithfulfaithful withwith single-exponentialchannelschannelssingle-exponential lessless potentlypotently functionsfunctions thanthan waswas isomerisomer substantiallysubstantially 22.. InIn addition, addition, slowerslower thanthan +thethe for foronsetonset isomerisomer ofof ±22blockblock,, thusthus asasprecludingprecluding estimatedestimated faithfulfaithful withwith that channelassessmentsingle-exponentialassessment occupancy ofof channelchannel functions by blockblock isomer atat was lowerlower2 does substantially concentrationsconcentrations not eliminate slower thanthan Na than 1010conduction µMµM for and andisomer toto determinedetermine (Figure2, thus 1precluding A,B). thethe associatedassociated The remainingfaithful ICIC5050 single-exponentialassessment of channel functions block atwas lower substantially concentrations slower than than 10 µMfor andisomer to determine 2,, thusthus precludingprecluding the associated faithfulfaithful IC50 single-exponentialsingle-exponentialassessment of channel functionsfunctions block at waswas lower substantiallysubstantially concentrations slowerslower than thanthan 10 µM forfor andisomerisomer to determine 22,, thusthus precludingprecluding the associated faithfulfaithful IC50 assessment of channel block at lower concentrations than 10 µM and to determine the associated IC 50 assessment of channel block at lower concentrations than 10 µM and to determine the associated IC5050 assessment assessment ofof channelchannel blockblock atat lowerlower concentrationsconcentrations thanthan 1010 µMµM andand toto determinedetermine thethe associatedassociated ICIC5050

Mar. Drugs 2019, 17, 390 4 of 18

isomers (15, 11, 14, 1, 7, 12) blocked NaV1.4 channels less potently than isomer 2. In addition, the onset of block as estimated with single-exponential functions was substantially slower than for isomer 2, thus µ precludingMar. Drugs faithful2019, 17, x assessmentFOR PEER REVIEW of channel block at lower concentrations than 10 M and to determine4 of 19 the associated IC50 values (Figure1C). Assessment of higher concentrations revealed that particularly isomersvalues15 (Figureand 71C).are Assessment interesting: of total higher current concen blocktrations at revealed 10 and 100 thatµ particularlyM was virtually isomers identical 15 and 7 (15 : 48.2are interesting:5.9% and 51.1total current2.2%, block respectively; at 10 and 7100: 32.3 µM was4.3% virtually and 35.1 identical4.6%, (15: respectively), 48.2 ± 5.9% and while 51.1 ± the ± ± ± ± ± ± time2.2%, constant respectively; characterizing 7: 32.3 the 4.3% kinetics and of 35.1 onset of4.6%, block, respectively),τblock, roughly while scaled the linearlytime constant with the τ concentrationcharacterizing (15 the: 10 kineticsµM: 160.0 of onset34.9 of block, ms; 100 blockµ,M: roughly 4.7 1scaled ms; 7linearly: 10 µM: with 853.0 the concentration195.0 ms; 100 (15µ: M: ± ± ± ± ± ±± 59.010 µM:18.1 160.0 ms; Figure 34.9 ms;1C). 100 This µM: result4.7 1indicates ms; 7: 10 µM: saturated 853.0 association 195.0 ms; 100 of µM: isomers 59.0 1518.1and ms;7 Figureand the ± channel,1C). This with result imperfect indicates (about saturated 50% andassociation 35%, respectively) of isomers 15 occlusion and 7 and of thethe Nachannel,+ permeation with imperfect pathway. + In(about contrast, 50% for and isomers 35%, respectively)11, 14, 1, and occlusion12 saturation of the Na of channel permeation block pathway. was not In apparent contrast, for as increasingisomers the11 concentration, 14, 1, and 12 saturation of the peptides of channel from block 10 µ Mwas to not 100 apparentµM also as increased increasing channel the concentration block (Figure of the1C). peptides from 10 µM to 100 µM also increased channel block (Figure 1C). The channel block of The channel block of isomers with even lower potency (3–6, 8, 10, 13 and 16–18) were analyzed at isomers with even lower potency (3–6, 8, 10, 13 and 16–18) were analyzed at 10 µM. As shown in 10 µM. As shown in Figure S2, sample 13 was most active among this group, as it inhibited NaV1.4 Figure S2, sample 13 was most active among this group, as it inhibited NaV1.4 by 24.6 ± 3.0%, by 24.6 3.0%, followed by samples 4 (21.7 3.5%), 9 (17.7 1.7%), 3 (17.6 3.6%), 17 (16.5 0.5%), followed± by samples 4 (21.7 ± 3.5%), 9 (17.7± ± 1.7%), 3 (17.6 ±± 3.6%), 17 (16.5± ± 0.5%), and 18 (16.5± ± and 18 (16.5 0.3%). Isomer samples 6, 8, 16, 5, and 10 diminished NaV1.4-mediated currents by less 0.3%). Isomer± samples 6, 8, 16, 5, and 10 diminished NaV1.4-mediated currents by less than 15% (6: than 15% (6: 12.6 1.3%, 8: 11.4 2.2%, 16: 10.5 1.3%, 5: 6.7 2.1%, 10: 1.0 3.0%), and thus were 12.6 ± 1.3%, 8: 11.4± ± 2.2%, 16: 10.5± ± 1.3%, 5: 6.7± ± 2.1%, 10: 1.0± ± 3.0%), and thus± were considered consideredinactive under inactive these under conditions. these conditions.

Figure 1. Disulfide isomers of µ-PIIIA differentially inhibit NaV1.4-mediated currents. Figure 1. Disulfide isomers of µ-PIIIA differentially inhibit NaV1.4-mediated currents. A) (A) Representative current traces of transiently expressed NaV1.4 channels evoked at a test potential Representative current traces of transiently expressed NaV1.4 channels evoked at a test potential of of 20 mV before (black, ctrl) and after (red) application of 10 µM of the indicated µ-PIIIA isomers. −20 mV before (black, ctrl) and after (red) application of 10 µM of the indicated µ-PIIIA isomers. B) B ( )Normalized Normalized peak peak current current amplitudes amplitudes obtained obtained from from repetitively repetitively evoked evoked current current responses responses were were plottedplotted as aas function a function of time of totime follow to thefollow time the course time of course current of block current mediated block by mediated various concentrationsby various of µconcentrations-PIIIA isomers 2of(circles) µ-PIIIA and isomers15 (triangles). 2 (circles) Continuous and lines15 (triangles). are single-exponential Continuous data lines fits usedare to characterizesingle-exponential the onset data of channel fits used block. to characterize The arrowhead the onset marks of cha thennel start block. of peptide The arrowhead application. marks The the time axisstart was of split peptide to illustrate application. that channel The time block axis by was isomer split15 tosaturates illustrate atthat about channel 50% suggestingblock by isomer that isomer 15 15 saturatesseals the channelat about pore50% onlysuggesting partially. that In isomer contrast, 15 seals isomer the 2-mediated channel pore channel only partially. inhibition In saturated contrast, at aboutisomer 95%. 2-mediated (C) Steady-state channel block inhibi estimatedtion saturated from at single-exponential about 95%. C) Steady-state fits of the block time estimated course of currentfrom inhibitionsingle-exponential (bottom) as fits well of as the the time associated course single-exponentialof current inhibition time (bottom) constant as (top), well describingas the associated the onset ofsingle-exponential channel block, for time the indicated constant isomers(top), describing and concentrations. the onset of Lines channel connect block, data for points the indicated for clarity. Symbolsisomers and and color-coding concentrations. of data Lines obtained connectwith data isomerspoints for2 andclarity.15 areSymbols as in (andB). Numbers color-coding of individual of data experimentsobtained with (n) areisomers provided 2 andin 15 parentheses. are as in B). Numbers of individual experiments (n) are provided in parentheses.

2.2. In silico Toxin Binding Studies of the 3-Disulfide-Bonded µ-PIIIA Analogs at Nav1.4

Mar. Drugs 2019, 17, 390 5 of 18

2.2. In Silico Toxin Binding Studies of the 3-Disulﬁde-Bonded µ-PIIIA Analogs at Nav1.4

Interactions of the µ-PIIIA isomers with the NaV1.4 channel were further investigated by docking isomers 1, 2, 4, 7, 11–15 (Table1) to the channel (pdb ID 6AGF [ 20]) using the HADDOCK easy web interface (Figure2, Figures S15 and S16). The remaining isomers 3, 5, 6, 8, 9, 10, and 16–18 were not further analyzed as they were considered to be inactive with respect to their poor pore blockage (FigureMar.1 Drugs). 2019, 17, x FOR PEER REVIEW 6 of 19

Figure 2. µ Visualization of -PIIIA–NaV1.4 complex conformations obtained from docking experiments for (A) the native isomer 2,(B) isomer 15, and (C) isomer 7. Left panel – top view of the toxin-channel Figure 2. Visualization of μ‐PIIIA–NaV1.4 complex conformations obtained from docking complex. Middle panel – side view of the toxin-channel complex. The four NaV1.4 domains are experiments for A) the native isomer 2, B) isomer 15, and C) isomer 7. Left panel – top view of the indicated and hydrogen bonds between the toxin and the channel are given as yellow dashed lines, toxin‐channel complex. Middle panel – side view of the toxin‐channel complex. The four NaV1.4 speciﬁed in the right panel. The Na 1.4 channel surface (molecular surface) is given in gray, the domains are indicated and hydrogenV bonds between the toxin and the channel are given as yellow selectivity ﬁlter motif (DEKA) is highlighted in red. The toxin is shown with side chain atoms present dashed lines, specified in the right panel. The NaV1.4 channel surface (molecular surface) is given in (coloring scheme: carbon – cyan, nitrogen – blue, oxygen – red, sulfur – green, backbone – isomer 2: gray, the selectivity filter motif (DEKA) is highlighted in red. The toxin is shown with side chain purple, 15: pink, 7: red). atoms present (coloring scheme: carbon – cyan, nitrogen – blue, oxygen – red, sulfur – green, Thebackbone docking – results isomer were2: purple, clustered 15: pink, according 7: red). to the interface-ligand root mean square deviations (RMSDs) and scored according to the HADDOCK scoring function (Table S7). Multiple poses of the 2.3. MD‐ and Molecular Electrostatic Potential (MEP)‐Based Analysis toxin isomers bound to the channel were observed and described in each case. The observations from 2.3.1. Analysis of 3‐Disulfide‐bonded μ‐PIIIA Isomers 1–15 With the biological activity of the 3‐disulfide‐bonded μ‐PIIIA isomers 1–15 and the 2‐disulfide‐bonded analogs 16–18 determined via electrophysiological assessment, and their probable binding modes estimated by molecular docking, we conducted MD simulations to determine the impact of the distinct disulfide bonding on conformational stability and dynamics. From the electrophysiological investigation, it was evident that the native isomer 2

Mar. Drugs 2019, 17, 390 6 of 18 the docking experiments were in line with the experimental observations indicating that the native and most active isomer 2 (Figure1, Figure2A) preferred a binding conformation that covered the central pore, while the moderately active ones chose conformations that left large portions of the pore exposed (isomers 11, 12, 14, 1 and 7) and/or did not insert as deeply into the pore (isomers 13, 14 and 15) as the native isomer 2 (Figure1, Figure2B,C, Figures S15 and S16). Isomer 2 was observed to reside closest to the selectivity filter residues located at the center of the pore in a similar position as recently found for the structurally related µ-conotoxin KIIIA occluding the pore with its central R14, while tightly binding to three out of the four channel subunits (Figure2A). The moderately active isomers, were found to bind away from the center of the pore at the interface of the subunits I, III, and IV leaving only a single toxin residue close to the pore (except for isomers 15 and 4, Figure1, Figure2B,C, Supplementary Figures S14 and S15). Another general observation from our docking experiments was that the C- or N-terminus of the moderately active isomers was located closest to the selectivity filter region, again only allowing for an insufficient pore blockage by a single toxin residue. However, at this point it shall be noted that the degree of pore blockage, which corresponds to the remaining current as revealed by the electrophysiological experiments, can only partly be rationalized from the docked pose of the isomers since the absolute degree of pore block could only be determined for isomers 2, 15 and 7 (Figures1 and2). Nevertheless, the binding poses of isomers 2, 15 and 7 might explain their decreasing ability to block the pore (Figure2) unveiling a smaller number of hydrogen bonds with residues close to the selectivity filter for 15 and 7 compared to the native isomer 2 (Figure2). Moreover, 15 and 7 were found to only block the pore by their flexible N-terminal residues Z1 and R2 (Figure2). In contrast to isomer 2 and 15, the center of isomer 7 is not located above the pore leaving enough space for sodium ions to get close to the selectivity filter region, which might explain the even lower degree of pore block compared to 2 and 15 (Figure2.).

2.3. MD- and Molecular Electrostatic Potential (MEP)-Based Analysis

2.3.1. Analysis of 3-Disulfide-bonded µ-PIIIA Isomers 1–15 With the biological activity of the 3-disulfide-bonded µ-PIIIA isomers 1–15 and the 2-disulfide-bonded analogs 16–18 determined via electrophysiological assessment, and their probable binding modes estimated by molecular docking, we conducted MD simulations to determine the impact of the distinct disulfide bonding on conformational stability and dynamics. From the electrophysiological investigation, it was evident that the native isomer 2 (C4-C16/C5-C21/C11-C22) of µ-PIIIA showed the highest activity by blocking >90% of channel current (Figure1). The biological activity dropped to ~50% block for isomer 15 (C4-C22/C5-C21/C11-C16) and even below for all other active analogs 11, 14, 1, 7, and 12. Based on this premise, two independent 400-ns MD simulations were conducted on each investigated disulfide isomer. The most obvious finding from the MD simulations was that isomer 2 was the most thermodynamically stable among the 3-disulfide-bonded analogs studied herein. All the structural and energetic properties computed from the two independent simulations (Figures S3–S14) were almost identical for the trajectories of isomer 2, while the other 14 analogs, despite converging to similar conformations and measured average properties, took diverging paths to equilibration. The ∆Gsolv estimated from atomic hydrophobicity and surface solvent accessibility [21,22] was the lowest for 2 among all the 3-disulfide isomers (Figures S3 and S4). This is an indicator that the folded conformation of 2 is energetically slightly preferred over e.g., isomers 15, 11, and 14. From Figure3, Figures S5 and S6 it is evident that the structure of isomer 2 exhibits good backbone stability with the average root mean square deviation (RMSD) value between the two trajectories of 2.55 0.47 Å (Table S3). From the structural superimpositions (Figure3A) and root mean ± square fluctuation (RMSF) plot (Figure3E, Figure S7) it is evident that the leading three N-terminal residues possess greater flexibility than the rest of the structure as this part of the sequence is not constrained by disulfide bonds. Mar. Drugs 2019, 17, x FOR PEER REVIEW 8 of 19

(C4-C16/C5-C11/C21-C22), arise from their disulfide bonding architecture of pairing between adjacent cysteines with no crosslinking disulfides to stabilize the segment. Hence they all fall into the inactive category. Visual inspection of the MD trajectory of these isomers revealed excessive rotational and translational motion which can be quantified by their RMSDs averaging at ~ 6 Å from their initial conformations (Table S3). In terms of channel binding this means that these isomers are never found in a conformation to form stable contacts with the channel residues and leave the pore Mar. Drugsarea2019 constantly, 17, 390 exposed. For the isomers in the active category (1, 7, 11, 12, 14 and 15), a combination 7 of 18 of moderate to good backbone stability and favorable orientations of at least some of the key residues result in conformations that could partially but stably block the channel pore.

Figure 3.FigureMD-based 3. MD-based structural structural comparison comparison of of isomers isomers 22,, 77, ,and and 1010. A.(–CA) –20C cartoon) 20 cartoon styled styledsimulation simulation snapshotssnapshots of isomer of isomer2 (blue), 2 (blue),7 (orange), 7 (orange) and, and10 10 (red)(red) superimposed superimposed on their on their respective respective starting starting NMR NMR structuresstructures (gray). (gr Theay). disulﬁde The disulfide connectivity connectivity of each of each isomer isomer is given is given below below the the individual individual isomer isomer images.

(D) Overlay of backbone RMSD progression plots for isomers 2 (blue), 7 (orange), and 10 (red) from a 400-ns MD simulation. (E) Overlay of per-residue root mean square fluctuation (RMSF) plots for isomers 2 (blue), 7 (orange), and 10 (red) from a 400-ns MD simulation. (F) Cartoon style structural superimposition of the representative structure of isomer 7 (orange) from MD simulation over the representative structure of isomer 2 (blue). The lysine and arginine residues significant for channel blocking are shown as sticks and labelled for both isomers. (G) Cartoon style structural superimposition of the representative structure of isomer 10 (orange) from MD simulation over the same of isomer 2 (blue). The lysine and arginine residues significant for channel blocking are shown as sticks and labelled for both isomers.

Comparing the structure and dynamics of 7 with 2 reveals that in terms of backbone mobility, the key difference again lies in the flexibility of the N-terminal residues (Figure3). From the superimposed structures in Figure3B, we noticed that the C4-C11 disulfide bond renders its bracketed residues to adopt an anti-parallel β-sheet conformation (Figure3B). This secondary structural adoption reorients Mar. Drugs 2019, 17, 390 8 of 18 the R2 residue towards the core of the peptide where it forms intramolecular H-bonds with C11 at the backbone in 40% of the trajectory, thus keeping it away from possible contacts with the channel. Added to this, 7 has the largest Molecular Electrostatic Potential (MEP) among all the studied isomers at 11.49 kJ/mol keeping the peptide bound to the channel by electrostatic attraction. The reduced channel-blocking ability of 7 primarily results from the orientations of its basic side chain residues, which are significantly different from the native 2 (Figure2F). Moreover, the key residue R12 was involved in intramolecular H-bonds with Q15 for ~25% of the simulation taking it out of play for making contacts with the channel. 10 (C4-C16/C5-C22/C11-C21), whose disulfide architecture is the closest to that of 2 (Figure2C), is surprisingly the isomer with the lowest measured bioactivity (Suppl. Figure1). The isomer’s backbone undergoes an average change of 3.3 0.6 Å RMSD to reach an equilibrated state ± (Figure3D) with the average fluctuations of all its residues being much higher than those of 2 (Figure3E). Structural superimpositions of 10 on 2 reveals clear misalignments between their key arginine and lysine residues. Furthermore, a series of intramolecular H-bonds by these key residues R2, R12, R14 and K17 for up to ~60% of total simulation time render them unavailable for interacting with the channel residues. In addition, 10 has the lowest MEP value at 6.76 kJ/mol leaving it at a disadvantage in terms of favorable electrostatics to retain it near the channel pore. Despite the stable backbone conformations observed for 8, it still falls in the inactive set for the same reason as 10, the formation of intramolecular H-bonds between the side chains of key residues that take them out of play from channel binding. In 8, H-bond interactions between R12 and Z1 for ~65% of the simulation, K9 and Q15 for ~8% of the simulation, R20 and K17 for ~10% of the simulation, K17 and C21 for ~24% of the simulation and the side chain of R2 to its own main chain for ~5% of the simulation ensure that these residues are almost never available to make contacts with the channel. Structures from the MD trajectories were clustered based on a backbone RMSD threshold of 1 Å using the “single-linkage” algorithm to facilitate the selection of representatives out of isomers that sample diverse conformations. From Table S4 it is evident that some of the less active isomers (e.g., 3, 4, 5, and 9) result in many clusters and their largest clusters represent only a small percentage of the trajectory. The corresponding RMSD plots (Figures S5 and S6) also convey the same message. It can be deduced that the extreme conformational instability in isomers 3 (C4-C5/C11-C16/C21-C22), 4 (C4-C5/C11-C21/C16-C22), 5 (C4-C5/C11-C22/C16-C21), 6 (C4-C11/C5-C16/C21-C22), and 9 (C4-C16/C5-C11/C21-C22), arise from their disulfide bonding architecture of pairing between adjacent cysteines with no crosslinking disulfides to stabilize the segment. Hence they all fall into the inactive category. Visual inspection of the MD trajectory of these isomers revealed excessive rotational and translational motion which can be quantified by their RMSDs averaging at ~ 6 Å from their initial conformations (Table S3). In terms of channel binding this means that these isomers are never found in a conformation to form stable contacts with the channel residues and leave the pore area constantly exposed. For the isomers in the active category (1, 7, 11, 12, 14 and 15), a combination of moderate to good backbone stability and favorable orientations of at least some of the key residues result in conformations that could partially but stably block the channel pore. Additionally, 400-ns long refinement MD simulations of the isomer-channel complexes were conducted with a membrane system embedding the channel for the isomers 2, 7, and 15. All of the simulation parameters between the these isomer-channel complex MD simulations and the isomer in solution simulations were kept identical to maintain uniformity of the systems and comparability of results. All the 3 isomer-channel systems had attained equilibration providing molecular level insights into the toxin-channel interactions and how this related to the extent of pore block obtained from the electrophysiological studies. Differences in the dynamic behavior of these three isomers between their unbound and channel-bound forms were measured in terms of average RMSD, radius of gyration (Rg), and solvent accessible surface area (SASA) values (Table S8). As expected, the mean SASA values of the channel-bound forms of the isomer were comparatively lower than their unbound counterparts (Table S8) owing to the fact that some part of the peptide’s solvent accessible surface is not available to the solvent but to the channel to interact with. An estimate of the binding energy (Ebind) was computed from the simulation and the values (Table S9) showed a clear correlation with the binding potency Mar. Drugs 2019, 17, 390 9 of 18 quantified by the electrophysiological results. Isomer 2, the best binder among all the isomers had an Ebind of -99.56 kJ/mol, while isomer 15 had -144.71 kJ/mol and finally isomer 7 with an Ebind of -332.83 kJ/mol (Table S9), preserving the trend observed from the electrophysiological experiments. As observed in the docking simulations, isomer 2 was found to occupy the central region of the channel with its R14 residue occluding the pore facilitating channel block at the beginning of the simulation. Over the course of the 400-ns simulation, it was observed that the isomer underwent a conformational change accounting to an average backbone RMSD of 3.2 Å. ~95% of conformations sampled by the isomer had the R14 residue placed directly above the channel pore persistently forming H-bonding interactions with the channel residue E184 making sure that the pore was covered. Additional isomer-channel H-bonding interactions namely Z1-D472, R12-L475, and R12-N474, stabilized the residence of the isomer 2 over the channel pore. For ~5% of the simulation, the isomer adopted a conformation where the sidechain of R14 reoriented away from its original position covering the pore and formed intramolecular H-bonds with the S13 residue on its own backbone. During this event it was observed that the channel pore was clearly exposed in a way that allows the passage of ions. This could be a reason as to why the channel was not blocked to 100% by this isomer (Figure1). The docked conformation of isomer 15 had its N-terminal oriented towards the interior with Z1 and R2 residues occupying the area over the pore. A H-bond between the residues R2 of the isomer and D500 of the channel added to the initial stability of the configuration. As the simulation progressed, the isomer adopted an altered conformation in which the channel pore was only partly covered, and no single residue occluded the pore. The isomer underwent an average conformational change of 4.36 Å backbone RMSD during the simulation. For isomer 7, the starting structure (docked complex conformation) in simulation had the isomer oriented over the channel pore with the Z1 residue found occluding the pore forming a H-bond with the channel residue G773. This conformation was different from what was observed in the MD simulation of this isomer in solution wherein the N-terminal residues formed an anti-parallel ß-sheet (Figure3B). During the simulation it was observed that isomer 7 quickly (under 5 ns) adopted a similar conformation to that in solution forming anti-parallel ß-sheets between residues C5-O8 and K9-C11, pulling the Z1 residue away from the channel pore. Hence the channel pore remained exposed for large parts of the simulation. A small fraction of the conformations (~15%) sampled by the isomer involved the R2 residue stretching out over the pore providing the partial block. For the rest of the simulation this residue was found H-bonded to T772 which is a part of the loop structure of segment II of the channel, leaving the pore constantly exposed. The isomer had an overall RMSD of 1.69 Å during the simulation indicating that it was stable in its bound state although it did not block the pore sufficiently. It was also observed that this isomer’s most preferred conformation, where the pore remains exposed, is different by 5.13 Å from its less preferred conformation where pore block was observed. This could further relate to the large time constant of block observed for this isomer because a large conformational change was required for channel block to be initiated. Finally, the per-residue contribution to the solvent accessible surface area (SASAres) was computed for the three isomers and compared against their unbound forms (Table S10). Residues found close to the channel pore (e.g., R14 for isomer 2, Z1 and R2 for isomer 15 and Z1 for isomer 7) had lower SASAres values compared to their unbound versions, serving as a passive indicator to their involvement in and the extent of pore blockage (Table S10). The observations from these simulations serve as a guide to understanding the molecular details of how the difference in disulfide connectivity between these three selected isomers influence the way they bind to the channel, thereby defining the function. 2.3.2. Analysis of 2-Disulfide-bonded µ-PIIIA Isomers 16–18 We studied the structural and functional contribution of the individual disulfide bonds to the native isomer 2 of µ-PIIIA. From the electrophysiological experiments, significant loss of channel-blocking activity among all the three 2-disulfide analogs (16, 17, and 18) was demonstrated. MD analysis revealed that all the three 2-disulfide analogs underwent a reasonable amount of backbone conformational change (Figure4). Among the three, the backbone of isomer 18 lacking the C5-C21 disulfide bond evolved to be the most stable as observed from the RMSD profiles in Figure4. 18 also had the least Mar. Drugs 2019, 17, 390 10 of 18 increase of per-residue fluctuations as shown in the RMSF comparison plots in Figure4. This results in the marginally higher activity seen for 18 over 16 in Figure S2. In all the three 2-disulfide-bonded analogs, clear misalignment of the key basic residues was observed in comparison to the native 3-disulfide-bonded isomer 2 and hence the loss of bioactivity (Figure4J, K, L). A recent report using a modified version of µ-PIIIA produced similar results with respect to assessing the role of individual disulfide bonds on the structure and bioactivity of µ-PIIIA [23], although in this study the N-terminal pyroglutamate residue was replaced by proline and respective cysteines were substituted by alanineMar. residues. Drugs 2019, 17, x FOR PEER REVIEW 11 of 19

Figure 4. MD-basedFigure 4. MD- structuralbased structural comparison comparison of of the the 2-disulfide 2-disulﬁde-bonded-bonded µ-PIIIA analogsµ-PIIIA 16, analogs17, and 18. 16, 17, and 18.(A–C) 20A–C cartoon) 20 cartoon styled styled simulation simulation snapshots snapshots of isomer of isomer 16 (maroon),16 (maroon), 17 (green) 17, and(green), 18 (cyan) and 18 (cyan) superimposed on the starting NMR structure of µ-PIIIA isomer 2 (blue). The disulfide connectivity superimposed on the starting NMR structure of µ-PIIIA isomer 2 (blue). The disulﬁde connectivity and the C-S mutations of each isomer are given below the individual isomer images. D) Overlay of and the C-Sbackbone mutations RMSD of progression each isomer plots of are isomer given 16 below(maroon) the on the individual native isomer isomer 2 (blue). images. E) Overlay (D of) Overlay of backbone RMSDbackbone progression RMSD progression plots plots of isomer of isomers16 17(maroon) (green) on on the the native native isomer isomer 2 (blue).2 F(blue).) Overlay ( Eof) Overlay of backbone RMSDbackbone progression RMSD progression plots plots of isomers of isomers17 18(green) (cyan) on on the thenative native isomer isomer 2 (blue).2 G(blue).) Overlay ( Fof) Overlay of per-residue RMSF plots of isomer 16 (maroon) on the native isomer 2 (blue). E) Overlay of backbone RMSD progression plots of isomers 18 (cyan) on the native isomer 2 (blue). (G) Overlay of per-residue RMSF plots of isomer 17 (green) on the native isomer 2 (blue). F) Overlay of per-residue per-residueRMSF RMSF plots plots of isomers of isomer 18 (cyan)16 (maroon) on the native on isomer the native 2 (blue). isomer J–L) Cartoon2 (blue). styled (E) representative Overlay of per-residue RMSF plotsstructures of isomer from17 MD(green) for isomers on the 16 (maroon), native isomer 17 (green)2 (blue). and 18 (cyan) (F) Overlay superimposed of per-residue on the structure RMSF plots of isomers 18 (cyan)of the native on the isomer native 2 (blue). isomer The functionally2 (blue). ( Jsignificant–L) Cartoon lysine styled and arginine representative residues arestructures shown as from MD sticks and labelled. for isomers 16 (maroon), 17 (green) and 18 (cyan) superimposed on the structure of the native isomer 2

(blue). The functionally signiﬁcant lysine and arginine residues are shown as sticks and labelled. Mar. Drugs 2019, 17, 390 11 of 18

2.3.3. Analysis of 2-Disulfide-bonded µ-PIIIA Isomers ∆(C5-C21)2, ∆(C11-C21)4, and ∆(C5-C22)10 The concept of disulfide bonds holding a “structurally frustrated” peptide in its bioactive form is known from the literature [24]. A means to measure this inherent frustration induced by the disulfide Mar. Drugs 2019, 17, x FOR PEER REVIEW 12 of 19 bonding pattern can be studied by observing the dynamics of partially folded states of the peptide. Previous studies2.3.3. haveAnalysis also of 2- shownDisulfide that-bonded this µ- observationPIIIA Isomers Δ(C could5-C21) give2, Δ(C hints11-C21 into)4, and the Δ(C folding5-C22)10 pathways that the peptide prefersThe concept to take of disulfide as it folds bonds [8 holding]. Isomer a “structurally2, 4, and frustrated”10 have peptide been in selected its bioactive as form representatives of structurallyis known well-defined from the literature (2), moderately [24]. A means flexible to measure (10 this), and inherent highly frustration flexible induced (4) classes by the among the disulfide bonding pattern can be studied by observing the dynamics of partially folded states of the 15 µ-PIIIA isomerspeptide. [Previous16]. We studies created have partially also shown folded that this analogs observation of could these give isomers hints into by the arbitrarily folding removing the second disulfidepathways that bond the inpeptide these prefers structures to take as creating it folds [8] the. Isomer∆(C5-C21) 2, 4, and2 ,10∆ have(C11-C21) been selected4, and as ∆(C5-C22)10 to observe therepresentatives refolding preference of structurally of well these-defined partially (2), moderately folded flexible states ( (Figure10), and 5 highly). Isomer flexible ∆(4(C5-C21)) 2 did classes among the 15 µ-PIIIA isomers [16]. We created partially folded analogs of these isomers by not prefer toarbitrarily refold maintaining removing the second an average disulfide distance bond in these of 15 structures Å between creating the the S γΔ(C5atoms-C21)2 of, the opened C5-C21 disulfideΔ(C11 bond,-C21)4, and while Δ(C5 in-C22) analog10 to observe∆(C5-C22) the refolding10 at several preference time of these points partially the Sfoldedγ atoms states of the opened C5-C22 disulfide(Figure bond 5). Isomer came Δ(C5 up-C21) to 52 Ådid close not prefer but eventually to refold maintaining sampled an unfoldedaverage distance conformations of 15 Å towards between the Sγ atoms of the opened C5-C21 disulfide bond, while in analog Δ(C5-C22)10 at several the end of thetime simulation points the S (Figureγ atoms of5 ).the The opened analog C5-C22∆ disulfide(C11-C21) bond4 camedisplayed up to 5 Å an close equal but eventually mix of both unfolded and folded statessampled with unfolded respect conformations to its parent towards isomer the 4 endstructure of the simulation with the (distancesFigure 5). The between analog the unbound γ Δ(C11-C21)4 displayed an equal mix of both unfolded and folded states with respect to its parent S atoms ofisomer the opened 4 structure C11-C21 with the disulfide distances between bond fluctuating the unbound Sheavilyγ atoms ofbetween the opened 3 Å C11 to-C21 30 Å (Figure5). This analysisdisulfide confirms bond that fluctuating the folding heavily between preferences 3 Å to 30 between Å (Figure di5).ff Thiserent analysis disulfide confirm isomerss that the of the same sequence canfolding be highly preferences diverse, between further different supporting disulfide isomers that of µthe-PIIIA same sequence does require can be highly all three diverse of, its disulfide further supporting that µ-PIIIA does require all three of its disulfide bonds to remain structurally bonds to remainstable structurallyand bioactive. stable and bioactive.

Figure 5. MD-basedFigure 5. MD disulfide-based disulfide distance distance analysis analysis of of partially partially folded folded µ-PIIIA µ analogs-PIIIA Δ(C5 analogs-C21)2, ∆(C5-C21)2, ∆(C11-C21)4Δ(C11and-C21)∆(C5-C22)4 and Δ(C510-C22).(A10–. CA)–C Disulfide) Disulfide connectivity connectivity (black (black solid lines) solid of lines)the isomers. of the The isomers. The colored dotted lines (blue – Δ(C5-C21)2, red – Δ(C11-C21)4, and green – Δ(C5-C22)10) represent the coloreddotted lines (blue – ∆(C5-C21)2, red – ∆(C11-C21)4, and green – ∆(C5-C22)10) represent the disulfide bonds opened in silico.(D) Overlay of distance plots between the in silico opened disulfide bonds (blue – ∆(C5-C21)2, red – ∆(C11-C21)4, and green – ∆(C5-C22)10). (E–G) The cyan cartoon structures represent the NMR structures of the isomers 2, 4, and 10 with their disulfide bonds shown as yellow sticks. The overlaid gray cartoon is a single MD snapshot of the partially folded 2-disulfide-bonded versions of the isomers with 100 conformations of the opened disulfide bond versions (blue – ∆(C5-C21)2, red – ∆(C11-C21)4, and green – ∆(C5-C22)10) to represent the distances sampled between them during simulation. Mar. Drugs 2019, 17, 390 12 of 18

3. Discussion The significance of disulfide bonds in the stability and folding of peptides and proteins is well-established and accepted [25]. Beyond their involvement in proteins for thermodynamic and kinetic stability [26], disulfide bonds also play multifaceted roles in peptides with therapeutic potential [27]. While the so-called “native fold” is often what is studied with much rigor, we have in recent studies ventured into exploring all alternative folds using the 3-disulfide-bonded µ-PIIIA as target [17]. In the present study, we have clarified using a combination of electrophysiological and computational approaches that despite the native fold (isomer 2) still carrying the highest bioactivity, 6 of the remaining 14 isomers are still bioactive in a range between 30%–50% of maximum current block compared to 2. Due to its globular conformation, homogenous distribution of functionally significant basic arginine and lysine residues and their stable orientations [17,28,29] indicated by the lowest RMSF values among the 15 isomers studied, isomer 2 is both structurally and energetically the most favorable to optimally block the channel (Figure3). It is also the largest populated isomer and the most easily formed with ∆Gsolv computation [23] serving as an indicator of stability. Our current findings help to clarify questions about ambiguities regarding the potencies of the different isomers. In an earlier study (Tietze et al., 2012) [10], isomer 3 was active (~50% as active as the native isomer), while it was inactive in the present study. However, taken into account the recent study by Heimer et al., 2018 [16], we are now able to state that 3, when obtained from the oxidative self-folding approach, obviously contained other bioactive isomers, such as 2 or 15, as confirmed by corresponding HPLC elution profiles of the individual mixtures [16]. In addition, these impurities explain why NMR analysis could not be obtained for this isomer in our first study (Tietze et al., 2012) [10] but could be elucidated upon targeted synthesis and purification of isomer 3 by Heimer et al. [16]. In the same study by Tietze et al., 2012 [10], isomer 1 was more active than the natively folded isomer 2. Considering our recent report [16], this discrepancy can also be explained by the HPLC analysis of the isomer mixtures. In this recent report, it was demonstrated that isomer 1 cannot unambiguously be separated from 2 [16], which was also confirmed by impurities (unassigned) found in the NMR spectra, although the analysis of the structure of isomer 1 was still feasible at the time. The negligible to no activity of 8 and 10 represents an interesting case of how structural stability does not always relate to bioactivity and adds evidence to the notion that folding proceeds in the direction that preserves function [29], and that even slight alterations of the disulfide architecture can at times be functionally inefficient. The current study also provides experimental proof for an earlier in silico study which suggested that native µ-PIIIA does certainly need all its 3 disulfide bonds to remain functional [8] compared to other conotoxins such as µ-KIIIA [7] and α-conotoxin Vc1.1 [30]. In summary, using electrophysiology, molecular docking, MD simulations, and MEP analysis, we have established a molecular, structural and electrostatic basis for the impact of disulfide connectivity on the bioactivity of µ-PIIIA isomers. We have additionally used simulations to explain the loss of bioactivity in the 2-disulfide-bonded µ-PIIIA analogs.

4. Materials and Methods

4.1. Peptide Synthesis and Purification Synthesis, purification, and chemical and structural analysis of the 15 3-disulfide-bonded PIIIA isomers 1–15 (sequence: ZRLCCGFOKSCRSRQCKOHRCC-NH2, Table S1) has been described previously [16]. Freeze-dried aliquots of known concentrations for each isomer, as determined by amino acid analysis and HPLC, were submitted to electrophysiological experiments. In addition, three 2-disulfide-bonded isomers 16–18 were prepared and subjected to biological activity testing as well. In brief, these µ-PIIIA-mutants were automatically assembled using 9-fluorenyl-methyloxycarbonyl (Fmoc) chemistry and couplings employing 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-methylmorpholine/DMF (1:1) on a Rink-amide MBHA resin (0.53 mmol/g) and an EPS221 peptide synthesizer (Intavis Bioanalytical Instruments AG, Cologne, Mar. Drugs 2019, 17, 390 13 of 18

Germany). The different cysteine pairs were protected with Trt- and Acm-groups, or exchanged with Ser according to the desired disulfide connectivity (Table S2). Peptide cleavage and concomitant side chain deprotection was accomplished with reagent K (phenol/thioanisole/ethanedithiol 1:1:0.5, 150 µl/100 mg resin) and TFA (95%, 1 mL/100 mg resin) for 3 h. The crude peptides were precipitated with diethyl ether, centrifuged, and washed several times with diethyl ether. Linear precursor peptides were purified as previously described [16] and chemically characterized (Table S3). The oxidation of the 2-disulfide bonded peptides 16–18 was performed in a mixture of isopropanol/water/1 M HCl (31:62.5:7.5) with a final peptide concentration of 0.05 mM. Then, 1.1 equiv. iodine (0.1 M in MeOH) was added to the peptide solutions, and the reaction was stirred for 1 h at room temperature. After the first disulfide bond was closed, further 13.9 equiv. iodine (0.1 M in MeOH) was added to deprotect the Acm-group and to form the second disulfide bond. After the reaction was stirred for up to 1 h, the oxidation was stopped by the addition of an excess of ascorbic acid (1 M in water). Reaction progress was monitored via RP-HPLC and LC-ESI-MS. The products were freeze-dried and stored at 20 C. The purification of − ◦ the crude peptides was performed by semi-preparative RP-HPLC as reported [16]. Peptide purity and constitution was confirmed by analytical RP-HPLC, MALDI-TOF mass spectrometry and amino acid analysis (Figure S1, Table S3) using methods and instruments as described previously [16]. Amino acid analysis was used for the determination of peptide concentrations in solution prior to the further experiments to ensure equal concentrations in analysis and biological testing. In general, µ-PIIIA derivatives were dissolved in double distilled water at a stock concentration of 10 µM and further diluted as described in the electrophysiological experiments.

4.2. Electrophysiological Experiments

Human SCN4A (encoding the NaV1.4 channel α subunit, UniProt ID P35499) on a plasmid with Cytomegalovirus (CMV) promoter was transiently expressed in HEK 293 cells as shown previously [10]. Co-transfection of a plasmid encoding the CD8 antigen ensured visual detection of transfected cells with CD8-speciﬁc Dynabeads (Deutsche Dynal, Hamburg, Germany). Currents were measured with the whole-cell patch clamp method 24–48 h after transfection [10]. The patch pipettes contained (in mM): 35 NaCl, 105 CsF, 10 EGTA (ethylene glycol bis(2-amino-ethylether)tetraacetic acid), 10 HEPES (pH 7.4 with CsOH). The bath solution contained (in mM): 150 NaCl, 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH). Holding potential was –120 mV, Na+ currents were elicited with depolarizing steps to 20 mV. Series resistance was corrected electronically up to 80%. Peptides, − diluted in the bath solution, were applied focally to cells under consideration with a ﬁne-tipped glass capillary. Time course of peak current decrease after peptide application was described with single-exponential functions.

4.3. Molecular Modeling and Docking Simulations The toxin-channel binding was predicted by docking the lowest energy conformer of the NMR structures of the µ-PIIIA isomers 1, 2, 4, 7, 11-15 [16] to the recently solved structure of the human NaV1.4 voltage-gated sodium channel (pdb ID 6AGF [20]) using the Easy Interface of the HADDOCK online platform [31,32](https://haddock.science.uu.nl/services/HADDOCK2.2/haddockserver-easy. html), a web service known to be suitable for handling more complex peptide ligand structures [32]. The receptor structure was energy-minimized in explicit solvent (TIP3 water) before the docking runs. For the docking process, all toxin residues and the channel residues that are part of the channel’s upper surface were deﬁned as “active”, because they were assumed to be able to form contacts with the toxin. As “passive” channel residues we deﬁned all residues within a radius of 6.5 Å from the “active” residues. From the docking results, the best scoring structure from the highest scoring complex cluster was selected for further analysis (Table S7). As the HADDOCK web interface cannot handle γ-pyroglutamic acid, glutamic acid was used instead, and was re-converted to γ-pyroglutamic acid, followed by a subsequent energy minimization step for further analysis. Mar. Drugs 2019, 17, 390 14 of 18

Analysis and energy minimizations were performed using the YASARA molecular modeling software (YASARA structure, Vers. 18.3.23, YASARA Biosciences GmbH, Vienna, Austria) [33,34]. The energy minimizations of the toxin-channel complex were performed in explicit water (TIP3P) and the Particle Mesh Ewald (PME) method [35] in order to describe long-range electrostatics at a cut-oﬀ distance of 8 Å in physiological conditions (0.9% NaCl, pH 7.4), at a constant temperature (298 K) using a Berendsen thermostat, and with constant pressure (1 bar). The charged amino acids were assigned according to the predicted pKa of the amino acid side chains from the Ewald summation, and were neutralized by adding counter ions (NaCl). The YASARA2 force ﬁeld was used for energy minimization by simulated annealing, including the optimization of the hydrogen bond network and the equilibration of the water shell, until system convergence was achieved. The molecular graphics were created using YASARA (YASARA structure, Vers. 18.3.23, YASARA Biosciences GmbH, Vienna, Austria, www.yasara.org) and POVRay (Persistence of Vision Raytracer Pty. Ltd., Williamstown, Australia www.povray.org).

4.4. MD Simulations of the µ-PIIIA Isomers and Analogs All-atom unbiased MD simulations of the 15 3-disulfide-bonded isomers (1–15), three 2-disulfide bonded analogs (16–18) and three partially folded 2-disulfide bonded models 2-2S, 4-2S and 10-2S were conducted with the GROMACS 2018 program [36,37]. The 3D coordinates for isomers 1–15 were obtained from the NMR structural ensembles reported [16]. The first out of the 20 structures of the ensemble was taken as the starting conformation for MD simulations. Analogs 16–18 were modeled in YASARA [34] by replacing the respective cysteine residues by serine. These models were energy-minimized using the AMBER99SB-ILDN [38] force field (Amber 11 in YASARA) before being used for simulations. The partially folded conformations were created in GROMACS via the pdb2gmx module as earlier described [8]. All steps and parameters involved in preparing the MD system were taken from the previous work [8] that involved the simulation of the native isomer of µ-PIIIA, except that the simulation run times increased herein. Solvent equilibration for 50 ns in constant temperature and constant-volume NVT ensemble, and 50 ns in the constant temperature and constant-pressure NPT ensemble conditions were carried out with position restraints on the energy-minimized peptide prior to the production MD run. Production runs at 300 K were conducted for 400 ns with a 2 fs time step. Two independent simulations were conducted per peptide using the random seed method that assigns different initial velocities (from the Boltzmann distribution) to the system thereby ensuring adequate sampling of the conformational space. 10,000 frames from each trajectory were used for analysis. The RMSD of the protein backbone atoms, per-residue RMSF of all peptide atoms, the radius of gyration (Rg) of all peptide atoms, and the SASA were computed using analysis tools within GROMACS. The double cubic lattice method [39] with the default probe radius of 1.4 Å was used to compute the SASA as well as the per-residue contribution to the solvent accessible surface (SASAres). An estimate of solvation free energies (∆Gsolv) from the atomic solvation energies per exposed surface area as implemented by Eisenberg et al. [21] were computed and plotted as a function of simulation time. Single-linkage clustering analysis was done on each trajectory using the backbone RMSD cut-off of 1 Å as the metric for the clustering. The centroid of the largest cluster from each trajectory was used as a representative for further analysis. Visualizations of the MD trajectories, molecular graphics and movies from the simulations were created in Visual Molecular Dynamics (VMD) (version 1.9.3) [40]. Plots were created using the Grace program (version 5.1.25).

4.5. MD Simulations of the µ-PIIIA Isomer-Channel Complexes Owing to the ease of setting up a membrane simulation, YASARA was used in conducting 400-ns long MD simulations of isomers 2, 7, and 15 bound to the recently resolved cryo-electron microscopy structure (PDB 6AGF [20]) of the human voltage-gated sodium channel NaV1.4. The 3 isomers were selected based on a consensus obtained from the electrophysiological experiments. The preparation of the membrane embedded system was achieved by providing the docked Mar. Drugs 2019, 17, 390 15 of 18 isomer-channel complex as input to the md_runmembrane.mcr macro in YASARA. The program first identified the transmembrane helices by scanning the channel structure for secondary structure elements with hydrophobic surface residues to orient and embed a membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) fatty acid residues. The protein was oriented such that the axis through the transmembrane helices is perpendicular to the membrane. A cubic simulation cell large enough to enclose the entire membrane was then drawn. The cell was extended by 15 Å on either side of the membrane so that the membrane was 30 Å larger than the protein ensuring that during simulation, the protein never sees its periodic image. The simulation cell was filled with the + water molecules including enough number of Na and Cl− ions to achieve a physiological concentration of 0.9% as a mass fraction. Next, successive steps of steepest descents energy minimizations followed by simulated annealing minimizations were done to remove bumps between the lipid molecules by deleting those lipids resulting in unfavorable geometries. The minimization steps were done iteratively until the potential energy of the system was optimized. Any solvent molecule found within the lipids was removed and the membrane was slowly packed closer by iteratively reducing the size of the simulation cell. As the final step, the membrane was artificially stabilized by position restraints and a pre-production simulation for 250 ps was done to equilibrate the solvent around the membrane and protein. The equilibrated system was composed of a total of ~163000 atoms including ~300 POPC molecules making up the membrane, within a cubic simulation cell with periodic boundaries of side ~120 Å was ready for the production MD simulation. A 2 fs time step was used to run the 400-ns long production MD simulations. Temperature was maintained at 300 K by a velocity rescaling algorithm and the pressure maintained at 1 bar by rescaling the volume. The cut-off for non-bonded forces was set at 8 Å. Analysis of the resulting trajectories were done via the standard macros within YASARA (md_analzye.mcr and md_analyzeres.mcr). An estimate of the binding energy Ebind was obtained using the Poisson-Boltzmann method [41,42] via the md_analyzebindenergy.mcr macro in YASARA by analyzing each frame in the 400-ns MD simulation. The Ebind was given by Ebind = EpotRecept + EsolvRecept + EpotLigand+ E Ligand EpotComplex E Complex where the subscripted “pot” and “solv” represent solv − − solv potential energy and solvation energy, respectively. Positive values from this result indicate better association between the ligand and receptor although negative values do not indicate no binding. The values obtained from the 3 simulations were compared against each other to estimate and compare the nature of the toxin-channel interactions for the 3 isomers (2, 7, and 15) in this study.

4.6. MEP Calculations MEP calculations were made for multiple conformations for all peptides used in this study to help quantify the diﬀerences in the surface electrostatic properties. MEP calculations were done using the Poisson-Boltzmann method [41,42] via the Adaptive Poisson-Boltzmann Solver (ABPS) program [43] built into YASARA. The method uses an implicit solvation model wherein the solvent is treated as a high dielectric (ε = 80) continuum. To maintain consistency with the MD simulations, the AMBER99SB-ILDN force ﬁeld was used. Frames from the last 300 ns of the MD trajectory were used as inputs for the MEP calculation.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/7/390/s1, Figures S1–S16 and Tables S1–S10. Author Contributions: P.H., T.S., and D.I. planned and performed synthesis and characterization of the peptides. E.L. and S.H.H. planned and conducted electrophysiological experiments. Molecular modeling, docking and simulation studies have been planned and carried out by A.A.P.G., D.K., D.T., and D.I. The manuscript was written through contributions of all authors. All authors have given approval to the ﬁnal version of the manuscript. Funding: Financial support by the University of Bonn (to D.I.) is gratefully acknowledged. Acknowledgments: We are thankful to Tim Wermund (University of Bonn) for technical assistance in peptide synthesis and puriﬁcation. Mar. Drugs 2019, 17, 390 16 of 18

Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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