DTIC ADA583921: Evaluation and Computational Characterization of the Faciliated Transport of Glc Carbon C-1 Oxime Reactivators Across a Blood Brain Barrier Model

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Evaluation and computational characterization of the facilitated transport of Glc carbon C-1
oxime reactivators across a blood brain barrier model 5b. GRANT NUMBER
1.E005.08.WR
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6. AUTHOR(S) 5d. PROJECT NUMBER
Bhonsle, JB, Causey, R, Oyler, BL, Bartochucci, C, Lamba, D, Pesaresi, A, Bhamare, NK,
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Published in Chemico-Biological Interactions 203 (2013) 129–134. This work was supported by Defense Threat Reduction Agency (Grant number
1.E005.08.WR).
14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Facilitated transport, Sugar-oximes, Sox, Blood brain barrier, Reactivation, Acetylcholinesterase
16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON
OF ABSTRACT OF PAGES Gregory Garcia
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Chemico-BiologicalInteractions203(2013)129–134
ContentslistsavailableatSciVerseScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Evaluation and computational characterization of the facilitated transport
of Glc carbon C-1 oxime reactivators across a blood brain barrier model
Jayendra B. Bhonslea, Robert Causeyb, Benjamin L. Oylerc, Cecilia Bartoluccid, Doriano Lambae,
Alessandro Pesaresie, Nanaji K. Bhamareb, Iswarduth Soojhawonf, Gregory E. Garciab,⇑
aAsthaDrugDiscovery&Research,5284RandolphRoad#262,Rockville,MD20852,USA
bResearchDivision,USArmyMedicalResearchInstituteofChemicalDefense,3100Ricketts,PointRoad,AberdeenProvingGround,MD21010-5400,USA
cAnalyticalToxicologyDivision,USArmyMedicalResearchInstituteofChemicalDefense,3100RickettsPointRoad,AberdeenProvingGround,MD21010-5400,USA
dIstitutodiCristallografia,ConsiglioNazionaledelleRicerche,AreadellaRicercadiRoma,ViaSalariaKm.29.300,I-00015MonterotondoScalo,Roma,Italy
eIstitutodiCristallografia,ConsiglioNazionaledelleRicerche,AreaSciencePark,Basovizza,S.S.14,Km.163.5,I-34149Trieste,Italy
fDivisionofBiochemistry,WalterReedArmyInstituteofResearch,503RobertGrantRoad,SilverSpring,MD20910-7500,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Weareevaluatingafacilitativetransportstrategytomoveoximesacrossthebloodbrainbarrier(BBB)to
Availableonline13October2012 reactivateinhibitedbrainacetylcholinesterase(AChE).Weselectedglucose(Glc)transporters(GLUT)for
thispurposeasthesetransportersarehighlyrepresentedintheBBB.Glcconjugateshavesuccessfully
Keywords: moveddrugsacrosstheBBBandpreviousworkhasshownthatGlc-oximes(sugar-oximes,SOxs)can
Facilitatedtransport reducetheorganophosphonateinducedhypothermiaresponse.Wepreviouslyevaluatedthereactivation
Sugar-oximes potentialofGlccarbonC-1SOxs.HerewereportthereactivationparametersforVX-andGB-inhibited
SOx
human(Hu)AChEofthebestSOx(13c)andourfindingsthatthekineticsaresimilartothoseofthepar-
Bloodbrainbarrier
entoxime.AlthoughcrystalsofTorpedocalifornicaAChEwereproduced,neithersoakedorco-crystallized
Reactivation
experimentsweresuccessfulatconcentrationsbelow20mM13c,andhigherconcentrationscrackedthe
Acetylcholinesterase
crystals.13cwasnon-toxictoneuroblastomaandkidneycelllinesat12–18mM,allowinghighconcen-
trationstobeusedinaBBBkidneycellmodel.Thetransferof13cfromthedonorsidewasasymmetric
withthegreatestlossof13cfromtheapical-orluminal-treatedside.Therewasnoapparenttransfer
from the basolateral side. The 13c P results indicate a ‘low’ transport efficiency; however, mass
app
accountingrevealedonlya20%recoveryfromtheapicaldoseinwhichhighconcentrationswerefound
inthecelllysatefraction.Molecularmodelingof13cthroughtheGLUT-1channeldemonstratedthat
transportof13cwasmorerestrictedthanGlc.Selectedsiteswerecomparedandthe13cbindingenergies
weregreaterthantwotimesthoseofGlc.
PublishedbyElsevierIrelandLtd.
1.Introduction theirbreakdownproductsmaybetootoxicforuse[6,7].Otherstrat-
egiestodeliveroximesacrosstheBBB(reviewedbyMerceyetal.
Currently fielded oxime reactivators 2-pralidoxime (2-PAM), [8])includefacilitativetransportwheresugar-oxime(SOx)conju-
obidoxime and potential replacements 1,10-methylenebis[4- gates could be transferred by glucose (Glc) transporters (GLUT)
[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate (MMB- [9].GLUTsareabundantintheBBBwithGLUT-1capillarydensity
4) and [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxy- of 80–90pmol/mg membranes [10]. Glc-conjugate transport has
methyl]pyridin-2-ylidene]methyl]-oxoazaniumdichloride (HI-6), beenreported[11–13].GLUTshavenotyetbeencrystallized,but
donotappreciablycrossthebloodbrainbarrier(BBB)anddonot ahomologymodelbaseduponthebacterialglycerol-3-phosphate
preventOP-inducedbrainseizureactivity[1–3].TheBBBiscom- transporter (PDB ID 1PW4 [14]) was developed and reported by
prised of an endothelial cell layer with tighter intercellular junc- Salas-Burgosetal.(PDBID1SUK[15])andformedthebasisforstud-
tions as compared to normal capillaries [4]. BBB-penetrating iestoexploreGlctransportersasafacilitativetransportmechanism
oximes such as monoisonitrosoacetone (MINA) and dihydropyri- toimprovereactivatorpenetrationacrosstheBBB.
dine 2-pralidoxime (pro-2-PAM) can act centrally and abrogate ExpandingontheworkofHeldmanetal.[16]wecomparedtheir
brain OP-induced seizure activity [5,6], but these compounds or best SOx (N-[(3-b-D-glucopyranosyloxy)propyl]-4-pyr-
idiniumaldoximechloride(8b))andanovelSOxofourowndesign
[9] (Fig. 1). Our SOx, N-[(3-b-D-glucopyranosyloxy)octyl]-2-pyr-
⇑ Correspondingauthor.Tel.:[phone];fax:[phone]. idiniumaldoxime iodide (13c), reactivated 133% and 113% better
E-mailaddress:[email protected](G.E.Garcia). than 8b for diisopropylfluorophosphate- and paraoxon-inhibited
0009-2797/$-seefrontmatterPublishedbyElsevierIrelandLtd.
http://dx.doi.org/10.1016/j.cbi.2012.09.012
130 J.B.Bhonsleetal./Chemico-BiologicalInteractions203(2013)129–134
wherek isestimatedastheslopeofthebest-fitline.Asecondary
obs
plotwasmadeofk vs.[SOx].Theequationk =k =SOx/(K +-
obs obs r D
SOx)andnonlinearregressionwasusedtoestimateK andk.The
D r
specific reactivity, k , was calculated using equation k =k/K .
r2 r2 r D
CurvefittingwasdoneusingPrismsoftwareVer.4.0(GraphpadInc.).
2.3.Toxicity
NeuroblastomaSHSY-5Ycellsweretreatedwithincreasingcon-
Fig. 1. Structures for Glc-1-position ether sugar-oxime series of compounds.
centrations(0.1–12mM)of13c.The24hpost-treatmentviability
Specificcompounds(n,parentoxime):8b(3,4PAM);8c(3,2-PAM);13c(7,2-PAM).
was determined using the AqueousOne Cell Titer kit (Promega
Corp.,Madison,WI).
20
2.4.Bloodbrainbarrier(BBB)modeling
15
se S Themultidrugresistance-1Madin-Darbycaninekidney(MDR1-
on P MDCK)cellmodelwasused(AbsorptiveSystemsInc.,Exton,PA).
sp4C 10 Compoundswerepreparedinwater.2-PAM(poorBBBpenetrant)
3c re x 10 5 w50aslMuseadndat6,51l2M, a,nadnd18thmeMte.sStamcopmlepsouwnedre1c3ocllewcatesduaseftderat2h5.,
1
ForthemMoximetestscelllysateswereprepared.Luciferyellow
andtransepithelialelectricresistance(TEER)measurementswere
0
usedascultureintegritychecks.TEERvaluesrangedfrom1640to
0 2 4 1942Ohm-cm2.Theapparentpermeabilites,P ,werecalculated
app
Time (min)
according to Wang et al. [19] for the receiver compartments fol-
lowing apical and basolateral treatment. In these cultures, apical
Fig. 2. Multiple reactionmonitoring (MRM) chromatogramof 13cincelllysate referstotheluminalorexteriorsideandbasolateralreferstothe
sample from 18mM apical side treated MDCK BBB model test. 13c Retention
Time=3.20min.Thetransitionmonitoredwas413?251m/z.Theconcentration abluminal or tissuecompartmentside. Thebrain penetration po-
determinedfromthistestwas175lMasshowninTable3. tentialof a test compoundis classifiedas low, moderate, or high
using the following criteria: a. Low: P A?B<3.0(cid:3)10(cid:2)6cm/s,
app
b. Low: P A?BP3.0(cid:3)10(cid:2)6cm/s, EffluxP10, c. Moderate:
app
humanredbloodcell(HuRBC)AChE,andwegeneratedpharmaco- P A?BP3.0(cid:3)10(cid:2)6cm/s, 10>EffluxP3.0, d. High: P
app app
phore models from quantitative structure–activity relationships, A?BP3.0(cid:3)10(cid:2)6cm/s,Efflux<3.0.
and2Dand3Dcomparativemolecularfieldanalysismodels[17].
Thepharmacophorecomputationadjusted(PCA)predictedactivity 2.5.AnalyticalSOxassay
for13chadanexcellentcorrelationwiththeobservedactivity.We
havecontinuedthisworkandnowreportthereactivationparame- WedevelopedamoresensitiveLC–MS/MSassaythantheCrea-
tersoftheseSOxsandtheanalysisofanadditionalnovelSOxN-[(3- seyandGreenassay[20]toanalyzebloodbrainbarrier(BBB)sam-
b-D-glucopyranosyloxy)propyl]-2-pyridiniumaldoxime chloride ples. Samples were acidified to pH 2 and then deproteinized by
(8c)predictedtobeactive.Togaininsightsofimportantmolecular ultrafiltration.Sampleswerestoredat(cid:2)80(cid:2)Cuntiluse.LCsepara-
contactsbetweenSOxandAChEweexploredX-raycrystallographic tion was performed using a 1260 series binary pump equipped
studies of 8b and 13c. In addition, we developed computational witha1290seriesautosampler(Agilent,SantaClara,CA)operated
modelstopredictthetransportpotentialofGLUT-1fornovelcom- ataflowrateof300lLmin(cid:2)1isocraticconditions(44%SolventA:
poundsthatcouldbeextremelyvaluablefordesigningBBB-pene- acetonitrile, 56% Solvent B: 40mM ammonium acetate) at 24(cid:2)C.
tratingSOxsandcorrelatethismodelwithexperimentalresults. Separations were over a PolyCAT A column (100mm(cid:3)2.1mm
i.d.,3lm)andaJavelin(10mm(cid:3)2.1mm)guardcartridge(PolyLC
l
Inc.,Columbia,MD)of5 Linjections.Oximesweredetectedwith
2.Materialsandmethods
a 4000 QTrap mass spectrometer (AB Sciex, Foster City, CA)
equippedwithanelectrosprayionsourceoperatedinpositiveion-
2.1.Sugar-oximesynthesis
izationmodeat5kV.Thesourceconditionswerecurtaingasflow
of 20psi, temperature of 50(cid:2)C, GS1 (cone gas) at 60psi, GS2
MaterialsandreagentswerepurchasedfromSigma–Aldrich(St.
(desolvation gas) at 25psi, and interface heater on. Collision gas
Louis,MO).8cwassynthesizedasdescribedearlier[9].Structures
(nitrogen) was set on ‘‘high’’. Multiple reaction monitoring
were confirmed by NMR utilizing a Varian Unity INOVA at
(MRM)modewas used,and theconditionsfor eachMRMtransi-
600MHz using 5mm Penta (H,C,N,P,D) pulse field Z-gradient
tionwereoptimizedandareasfollows:
(PFG) probe at 25(cid:2)C and liquid chromatography–mass spectros-
copy(LC–MS)(datanotshown). Transition Q1 Q3 Dwell DP EP CE CXP
name m/z m/z time
2.2.Reactivation (ms)
13cQuantifier 413.3251.2150 193.57.5 35.0915.91
Humanwholebloodwasobtainedfromvolunteersinaccordance 13cQualifier 413.3122.9150 193.57.5 43.076.2
withtheUSAMRICDdonorprogram(USAMRICDM-10088).HuRBCs 2-PAM 137.193.1 150 60.2913.1831.127.58
were treated with OPs to yield 80–95% inhibited AChE [9]. AChE
reactivationwasdeterminedatseveralSOxconcentrationsandas-
sayed by the discontinuous method [18] to determine the initial DP=declustering potential, EP=entrance potential, CE=collision
(v),final(v ),andintermediate(v)AChEactivitylevels.Thedata energy,CXP=collisioncellexitpotential;allpotentialsarereported
i 0 t
were plotted according to equation ln((v (cid:2)v)/(v (cid:2) v))=(cid:2)k t inV.
0 t 0 i obs
J.B.Bhonsleetal./Chemico-BiologicalInteractions203(2013)129–134 131
Fig.3. ReactivationofVX-inhibitedhuRBCAChEby2-PAMand13c,8contheleft,and8bontheright.
DatawereprocessedusingAnalystversion1.5.2(ABSciex,Fos- Table2
terCity,CA).Thelowerlimitofquantitation(LLOQ)for13cand2- SOx13ctransportinMD-MDCK.
PAMwas(cid:4)20ng/mLandtherewasnegligibleion-suppression.A Sample SOxConcentration
LC-MS/MSrepresentativechromatogramfromacelllysateanalysis TreatmentSOx(mM) MeasuredSOx(lM)
isshowninFig.2.Matrix-matchedstandardswereusedforstan-
mM Mean SD
dardcurvegeneration.
Apical
Receiver 6 2.65 0.09
12 3.56 0.51
2.6.Molecularmodeling
18 7.08 0.22
Cell 6 37.8 6.51
ThetranslocationofGlcand13cthroughtheGLUT-1receptor 12 66.1 4.79
was studied using two computational techniques, viz, molecular 18 175.4 4.28
Donor 6 1169 53.0
dynamics (MD) simulation and Glc–GLUT-1 docking studies. The
12 2153 283.5
MD simulations were performed using Discovery Studio version
18 3722 234.2
3.1(DS3.1)molecularmodelingsoftware,andthedockingstudies
Basolateral
wereperformedwithSybyl-Xversion1.3molecularmodelingsoft-
Receiver 6 0.82 0.02
ware. All MD simulations with DS3.1 used CHARMm forcefield, 12 1.46 0.29
Momany–Ronechargesandimplicitwatersolvation(Generalized 18 1.71 0.17
Bornapproach).AlldockingstudiesusedAmber7FF02forcefield Cell 6 0.77 0.1
12 1.93 1.04
with Amber charges. PDB ID 1SUK (homology modeling derived
18 3.15 0.62
structure)was cleaned,solvated in water,and equilibratedwhile Donor 6 6041 247
constrainingthebackboneatomstopreservethebioactiveconfor- 12 11883 135
mationandoptimalside-chainlocations.TheFlexiDockalgorithm 18 19554 135
based on Genetic algorithm was used for docking. Compounds
werepositionedmanuallyatthevariouslocationsintheGlcchan-
nel. Flexidock experiments were performed with the specified
numberofgenerationsasshowninTable6.For13cdockingexper-
Table1 iments 13c was aligned with Glc from the Glc–GLUT1 docked
Reactivationparametersofsugar-oximes. poses.ForallMDexperimentsthestandarddynamicscascadepro-
Agent Oxime kr(min) KD(lM) kr2(min(cid:2)1mM(cid:2)1) kr2ratio2-PAM tocol as devised in DS3.1 was used. The GLUT-1 backbone atoms
wereconstrainedbyemployingtheFixedAtomconstraintsmeth-
VX 2-PAM 0.116 28.67 4.060 1.000
od.Inthestandarddynamicscascadeprotocol,theNVTapproach
8b 0.089 629 0.141 0.035
8c 0.085 138.5 0.616 0.152 was employed with the following settings in the minimization
13c 0.101 15.08 6.680 1.645 (steepest descent 500 steps followed by conjugate descent 500
GB 2-PAM 0.131 23.59 5.556 1.000 steps),heatingandequilibration(5000stepseach)andproduction
8b 0.447 2669 0.168 0.030 (10,000steps)steps.Thetargettemperaturewassetto310Ktore-
8c 0.063 156.60 0.401 0.072 flectthephysiologicaltemperatureratherthanthestandardroom
13c 0.056 15.45 3.637 0.654
temperature.
132 J.B.Bhonsleetal./Chemico-BiologicalInteractions203(2013)129–134
Table3 welltoleratedbyMDR1-MDCKcellstreatedfor2hinlowGlcmed-
ListofaminoacidsinvolvedinthePDBstructure1SUK-definedhelicesofGLUT-1. ia(5mM)with13cconcentrationsashighas18mM(thehighest
1SUKPDBhelices#/aminoacidresidue# concentrationtested-datanotshown).Theseresultsindicatethat
toxicityof13cappearssimilartothatof8b[17].
PDB-H# Residue# PDB-H# Residue# PDB-H# Residue#
H1 S5-G31 H8 L169-N182 H15 N360-F375
H2 I33-I40 H9 L185-P187 H16 V376-F379 3.3.Bloodbrainbarriermodel
H3 T47-R51 H10 L188-C207 H17 G384-A392
H4 T60-F90 H11 R253-E261 H18 L394-N397
The MDR1-MDCK cell line is a well documented BBB model
H5 G91-S113 H12 R264-Y292 H19 G398-F422
[20]. This cell line expresses several GLUTs (GLUT-1, 2, 3 and 5)
H6 M121-S148 H13 N304-R330 H20 G430-G471
l
H7 A151-L169 H14 T335-L357 H21 H484-S490 withasymmetricdistribution[21].ThePappwaslowfor5–50 M
concentrationsof2-PAMand13c(datanotshown).Astheseexper-
imentswereperformedinGlcconcentrationsof15–20mM(typi-
cal culture media) the experiments were repeated at the
Table4
ListofaminoacidsinvolvedintheMuecklerdefinedhelicesofGLUT-1. physiologicallyrelevantGlcconcentrationof5mM.13cconcentra-
tionsupto18mMrevealedadose-dependentasymmetrictrans-
Mueckler2008helices#/aminoacidresidue#
ference with the apical treatment yielding higher levels in the
M-H# Residue# M-H# Residue# M-H# Residue# receiverwellandcelllysates.The13cP waslowforallconcen-
app
MH1 S5-I40 MH5 A151-N182 MH9 T335-L357 trationstestedwiththe apicaltreatmentyielding>4(cid:3)therecei-
MH2 T60-F90 MH6 L185-C207 MH10 N360-A392 ver side concentration than the basolateral side as shown in
MH3 G91-S113 MH7 R264-Y292 MH11 L394-F422
Table 2. The summation of compartment concentrations reveals
MH4 M121-S148 MH8 N304-R330 MH12 G430-G471
that18mMapicaldosingis20%ofthatfromthebasolateralside
((3904.48lM/19558.86lM)(cid:5)100).
Table5 3.4.Modeling
ListofaminoacidsinvolvedintheGLUT-1GlucosechannelalongwiththeMueckler
andPDBdefinedhelicesnumbers.
The homology model structure of GLUT-1 (PDB ID 1SUK) was
Muecklerhelices/PDBhelices/aminoacidresidue# defined to comprise 21 alpha-helices by Salas-Burgos et al. [15],
M-H# PDB-H# Residue# M(cid:2)H# PDB-H# Residue# andtheaminoacidscomprisingthehelicesareshowninTable3.
Theaminoacidscomprisingthe12alpha-helicesdefinedbyMeuc-
MH1 H2 S23-I40 MH7 H12 R264-Y292
MH2 H4 T60-F90 MH8 H13 N304-R330 kleretal.[22]areshowninTable4,whichalsoshowsthattheGlc
MH4 H6 M121-S148 MH10 H15-H18 N360-N397 channelisflankedbyhelices1,2,4,5,7,8,10and11.InthePDB
MH5 H7-H8 A151-N182 MH11 H19-H20 G398-G471 1SUKstructurethesameGlcchanneltranslatestohelicesnumber-
ing2,4,6,7,8,12,13,15,16,17,18,19and20.Theexactamino
acids defining the Glc channel of the Meuckler et al. helices and
3.Results PDB1SUKhelicesareshowninTable5.Theexactaminoacidsfrom
theGlctranslocationchannelhavebeenidentifiedasGlu-254and
3.1.Reactivation Lys-256[23],Trp-388[15],andTrp-412[24].Sevendockingloca-
tions in the Glc channelwere identifiedand are listed in Table 6
The reactivation of VX-inhibited Hu RBC AChE by 2-PAM and andnamedaccordingtotheirpositionsinthechannel.Theouter-
SOxs is shown in Fig. 3 (GB data not shown). The reactivation most docking location is termed extra cellular mouth-1 (ECM-1).
parameters are shown in Table 1. The k ratio of 13c to 2-PAM The next location, deeper into the mouth, is ECM-2, while the
r2
was1.67,while8cyielded0.152and8b0.035.Thisindicatesthat deepest location is ECM-3. The docking location closest to Trp-
13cisthebestSOxreactivatortodateandthata2-PAMderivative 412 in the central region is termed central Glc channel-1 (CGC-
isbetterforVXandGBthanthe4-PAMderivative.Wehaveprevi- 1), and the next location deeper toward the cytoplasm is CGC-2,
ously reported that 13c is a better reactivator than 13a (3-PAM whilethedeepestisCGC-3.Thedockinglocationattheintracellu-
derivative)and13b(4-PAMderivative)[9],soitappearsthepre- lar mouth is termed ICM. A GLUT-1 ribbon model is shown in
ferredparentoximeis2-PAM. Fig.4A,andthemovementofGlcisshownfortheindividualsites
inFig.4B.ThedockingparametersandresultsareshowninTable6.
3.2.Toxicity The binding energies of Glc and 13c varied from (cid:2)477 to
(cid:2)166Kcals/mol, and (cid:2)868 to (cid:2)269Kcals/mol, respectively. For
13cwasfoundtobenontoxicupto12mM(highestconcentra- boththerewasamaximumaffinityatECM-2.MDsimulationswith
tiontested)fortheneuroblastomacelllineSHSY-5Y.13cwasalso Glcand13cplacedatthevariouspositionsfortimeperiodsof2ps
Table6
DockingexperimentsparametersandresultsforGlcand13c.
Glc ReportedGlc Bindingsite# FlexiDock# Obsdbinding GLUT-1overlay Bindingsite# FlexiDock# Obsdbindingenergy
location bindingAA bondsforGlc generationsfor energyKcals/mol 1SUKRMSDfor bondsfor13c generationsfor Kcals/molfor13c
Glc forGlc Glc 13c
ECM-1 – 91 110500 (cid:2)241.7 0.25 339 181000 (cid:2)581.9
ECM-2 – 232 122000 (cid:2)476.7 0.28 444 240000 (cid:2)867.5
ECM-3 W412 199 105500 (cid:2)382.8 0.27 487 240000 (cid:2)750.9
CGC-1 W412 187 122000 (cid:2)334.7 0.34 453 238000 (cid:2)743.2
CGC-2 – 112 122000 (cid:2)166.0 0.27 220 240000 (cid:2)288.9
CGC-3 W388 107 122000 (cid:2)168.4 0.27 254 240000 (cid:2)395.9
ICM E254/K256 137 122000 (cid:2)288.6 0.23 137 80000 (cid:2)268.5
J.B.Bhonsleetal./Chemico-BiologicalInteractions203(2013)129–134 133
ime derivatives [9,16,17,25], and 13c is the most effective SOx
reactivatortodate(Table1).Wedevelopedandvalidatedmolecu-
lar models with reactivation improvement (8c vs. 8b, Table 1).
SOxs also appear to be relatively non-toxic with guinea pig LD
50
of1590mgkg(cid:2)1[9].Assumingaonecompartmentmodel,theesti-
matedbloodlevelofSOxattheLD wouldbe42mM.TheLD is
50 12.5
10.5mMandgreaterthantwotimesthenormalGlcbloodlevelof
5mM.8bpharmacokineticsshowsthatSOxcanattainT quickly
max
(10min, similar to 2-PAM) from an IM administration but has
longer mean residence time (2h vs. less than 80min for 2-PAM)
[17].Thesearealldesirablecharacteristicsofareactivator,butit
was the finding of the amelioration of the body temperature
depressionresponsetopesticidesthatmadethesecompounds of
interest to cross the BBB [16]. The first experiments to test the
transport of 13c in a BBB model were done with 0.005 and
0.05mMSOx.Nomeasurable13cwasfoundinthereceiverwells
(Table2).Theseexperiments,however,wereconductedatthetyp-
icalcellularmaintenanceGlcconcentrationof15–20mM,soitis
possible that at low concentrations 13c would not compete well
forthetransporter.Wethentested13cupto18mMatthephysi-
ologically relevant 5mM Glc concentration and found that 13c
transport appears to be asymmetric with greater efficiency from
theapical(luminal)side. The18mM13ctreatmentyieldedafter
2h(cid:4)7and1.7lM13conthereceiversideofapical-andbasolat-
eral-treatedsides.Thissidednesswasalsoapparentforcelllysates
l
with175vs.3.15 M13cfoundfromapicalandbasolateraldosing
respectively.Theseresultsindicatethatthereappearstobesignif-
icantremovalof13cfromtheapicalsideandnearlynonefromthe
basolateral side. We hypothesize that GLUT transporters on the
apicalsidearemoreefficientthanthoseonthebasolatereralside
for13csuchthat13cbecomestrappedinsidethecell.Inaddition,
since not all the 13c mass could be accounted for ((cid:4)80% unac-
counted)whichsuggeststhatonceinsidethecell13cismetabo-
lized or catabolized and that the products not are detectable as
13c(413m/z).Furtheranalysisisneededtodeterminewhatthese
might be and whether a functional oxime moiety is transferred
across the BBB, though no longer as parent SOx, and explain the
apparentCNSeffectsofSOx;or,thereissomeotherasyetuniden-
tifiedmechanismforSOxBBBpenetrationthatmaynotprimarily
involveGLUTs.Regardlessofmechanism,efficacystudiesareinor-
dertoevaluatetheutilityoftheSOxs.
Attempts at soaking of Torpedo californica (TcAChE) crystals
with8bor13cwereunsuccessful(datanotshown),TcAChEcrys-
talsweresoakedwithvariousconcentrations(5–80mM)ofeither
8bor13cSOx.TheseresultsdemonstratethatSOxshaveanappar-
ent low affinity for the apo active TcAChE. The weak molecular
interactionsarelikelytheoutcomeofentropy–enthalpycompen-
sation [26]. A stronger protein–ligand interaction will also result
in a reduction of the configurational freedom of the system and
thusareductionoftheentropy.Correspondingly,bytakingintoac-
Fig.4. GLUT-1structureandGlcmovementthroughchannel.(A)Cartoonrepre- counteffectssuchasthedesolvationenthalpyandentropyandthe
sentationoftheGLUT-1theoreticalmodel(PDBID1SUK)[15]createdusingPyMOL conformational restriction that accompanies binding, weaker
[27];(B)PictureofbindinglocationsalongtheGlcchannelinGLUT-1shownwith molecularinteractionswillproducealoosermolecularassociation
Glc: (1) extra cellular mouth-1 (ECM-1); deeper into the mouth, is (2) ECM-2;
andanincreaseinentropy.Studiesareinprogresstoelucidatethe
deepestlocationis(3)ECM-3;thedockinglocationclosesttoTrp-412inthecentral
region is (4) central Glc channel-1 (CGC-1); next location deeper toward the structures of VX and GB nonaged and aged conjugated forms of
cytoplasmis(5)CGC-2;thedeepestlocationis(6)CGC-3.Thedockinglocationat TcAChEwith8bor13c.
theintracellularmouth(ICM)is(7). The modeling results lay the ground work to examine the
molecular dynamics of GLUT-1 transport of SOxs. The docked
GLUT-1 molecule and the original PDB molecule 1SUK had an
showedthemtravelingin the intracellular mouthdirection(data
excellent overlay with RMSD values varying from 0.2 to 0.3Å as
notshown).
showninTable6.Table6showsthat13chadlowerbindingenergy
valuesateachlocationexaminedthanGlcindicatingthatGLUT-1
4.Discussion would be a relatively poor transporter for 13c. The ECM2 site
hadthelowestvaluesforGlcand13candwethereforepostulate
SOxs have now been shown by us and others to be relatively thattheGlcchannelhasabottleneck,wherethetransportedmol-
good reactivators compared to monoamine quaternary pralidox- eculeiscapturedwithmoderatebindingaffinityof(cid:2)242Kcals/mol
134 J.B.Bhonsleetal./Chemico-BiologicalInteractions203(2013)129–134
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