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Facilitatedantibiotictranslocationthrough and porins

K.R.Mahendran,T.Mach,A.Bessonov,H.Weingart,M.Winterhalter JacobsUniversity,Bremen 2008

Outline

• ArtificialBilayermeasurements
• Antibioticentrythroughbacterialporins

OmpF(Cephalosporinsandfluroquinolones) OmpC(Cephalosporinsandfluroquinolones) Omp36(Betalactams)

• Effectoftemperatureonampicillin passage

throughOmpF

• Prospects0 newstructuredevelopment

Motivation

Antibiotic Target

Mainresistancemechanismstodrugs

Yim 2007,modified

Gramnegativebacteria

BacterialPorins

• Specificandnon0specificchannels
• 3majorporins(OmpF,OmpCandPhoE)
• OmpFplaysimportantroleinantibiotictranslocation

OmpF2OMPF OmpC2J1N

Artificiallipidbilayermeasurements

• Electrophysiology

Ionchannels,bacterialporins,toxinsetc

• Currentmeasurement0 Ionflow
• BLMtechniqueallowstoseeinteractionsbetween

antibioticandporinastimeresolvedfluctuationsofion current

• SubstrateinteractingwithPore

canproduce

• Artificiallipidbilayermeasurements

Antibioticinteractswithporinresulting intransientblockageofioniccurrent

CephalosporinspathwaythroughOmpF

• Target:thebacterialcellwall
• Fourthgeneration

Cefpirome+/0 Cefepime+/0

TimeresolvedioncurrentblockagesthroughOmpF

50 100 150 200

• 1

2 3 4 5

OmpFnoantibiotic

time,s current,pA

1 2 3 4 5 50 100 150 200

• time,s

current,pA

OmpF010mMcefepime

1 2 3 4 5 50 100 150 200

time,s current,pA

OmpF05mMcefepime

1 2 3 4 5 50 100 150 200

• time,s

current,pA

OmpF10mMcefpirome

Conditions:1MKCl,pH6,antibioticaddedattransside,V=+50mV

BindingkineticsOmpF0 cefepime

• cisside+50mV

cisside050mV transside+50mV transside050mV IMKClpH6,10mMCefepime

Cefepime(mM) Numberofevents

Singleexponentialfittingofblockagetim ehistogram DwellTim e(m s)

0.5 1 1.5

Count(N)

500 1000

Twoquantitiesaremeasured 1)Numberofblockages 2)Averagetimeofblockage

10mMcefepime Tau=0.12± 0.02ms

At10mMcefepime

Averageresidencetime,Tau=0.12± 0.02ms AssociationrateconstantKon=1868M01s01 DissociationrateconstantKoff=9523.8s01

Antibiotictranslocationisfacilitatedbybindingsiteinthe channel

BindingkineticsOmpF0 cefepime

Microbiologicalassays

Lipidbilayermeasurementscorrelateswithmicrobiologicalassays

meanlysis diameter(mm)

Bredin etal,2002;Vidaletal,2005

PathwayoffluroquinolonesthroughOmpF

• Fluroquinolones0 targetDNA
• Enrofloxacin
• zwitterionic

• Conditions:150mMKCl,pH6,antibioticaddedatcisside,V=0 50mV
• enrofloxacin

Anexampleofstronginteraction0 OmpFand Enrofloxacin

Τau050mV=2.98±0.56msec Τau50mV=1.82±0.4msec

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 300 600 900 1200

50mV 050mV C[Enro],mM

ν,ev/sec

BindingKinetics

Nestorovichetal,2002

Conditions:150mMKCl,pH6 Conditions:1MKCl,pH6

≈10ev/secat3mM,V=50mV ≈20ev/secat3mM,V=050mV ≈65ev/secat3mM,V=50mV ≈720ev/secat3mM,V=050mV

Enrofloxacinblockingevents

Ampicillinblockingevents

AntibiotictranslocationthroughOmpC

Trimerformssmallestfunctionalunit OmpC 60%identityinaminoacidsequencetoOmpF Keyresiduesconserved,differentpositions (Basle2006) Moreacidicresiduesandcation selectivecomparedtoOmpF Liposomeswellingassays(NikaidoandRosenburg1985)

Porin Singlechannel Conductance(nS) 1MKCLpH6 Criticalvoltagefor Channelclosure(mV) Ionicselectivity PK/Cl 204 7030 4 2.5 1000150 2000250

Cefepime0 Fourthgeneration+/0 Ceftriaxone0 Thirdgeneration02 Norfloxacin– Fluroquinolone+/0

AntibiotictranslocationthroughOmpC

OmpF OmpC

TimeresolvedioncurrentblockagesthroughOmpC

OmpCnoantibiotic

1s 50pA

OmpC10mMCefepime

1s 50pA

OMPC10mMCEFTRIOXONE

1s 50pA

OmpC2mMNORFLOXACIN

1s 50pA

Conditions:1MKCl,pH6,antibioticaddedatcis side,V=+100mV

PowerSpectrumOmpC0Norfloxacin

Frequency(Hz)

100 1000 10000

Amplitude(pA²/Hz)

1e04 0.001 0.01 0.1

0mMnorfloxacin 1mMnorfloxacin 2mMnorfloxacin 3mMnorfloxacin

• Determinationofbindingparameters

Determinationofbindingparameters

Residencetime

CefepimeTau0.1± 0.02ms CeftriaxoneTau0.14± 0.02ms Norfloxacin Tau0.2±0.02 Ampicilin nobindingevent

StrengthantibioticinteractionwithOmpC Norfloxacin>ceftriaxone>cefepime>ampicillin

Antibiotictranslocationthrough Omp36

• possessesthreeknownporins
• Omp35( OmpF,OmpK35homolog)
• Omp36(OmpC,OmpK36homolog)
• Omp37

Antibiotics

• Ertapenem,cefepime,ceftazidimeandampicillin

2 4 6 8 10 50 100 150 200 250

• cisside050m

V cisside+50m V transside050m V transside+50m V

Ertapenem concentration,(m M ) Numberofeventspersecond

BindingkineticsOmp36

5 10 15 20 25 5 10 15 20 25 30

• trans0side;

V=050mV cis0side; V=050mV trans0side; V=+50mV cis0side; V=+50mV

ν,s01

cefepime,mM Numberofeventspersecond

BindingkineticsofOmp36

Frequency(Hz)

100 1000 10000

A m p litu d e ( p A ² /H z )

0.001 0.01

10mMertapenem 25mMcefepime Noantibiotic

• cisertapenem

transertapenem cisandtransertapenem

Appliedvoltage,m V Averageresidencetime,ms

Bilayermeasurementscorrelatedwithantibioticactivitiesinbacteria (Chloë E.James) Tauertapenem0.14±0.02ms Taucefepime0.1±0.02ms

Temperatureeffectonantibiotic translocationthroughOmpF

• OmpFhighlystable.
• Newparameter
• OmpFconductancestronglydependsontemperature
• AmpicillinandNorfloxacin(Catalin)
• Temperature (50550C)

2 5 m s 2 5 p A

t i m e , m s current,pA

t i m e , m s current,pA t i m e , m s current,pA

50c 150c 250c

Ioncurrentblockagesatdifferenttemperatures

Opencurrent Closedporecurrent 10mMampicillin,1MKClpH6,+50mV

t i m e , m s currentpA

350c

t i m e , m s current,pA

450c 550c

2 5 m s 2 5 p A

t i m e , m s current,pA

Determinationofbindingparameters

10 20 30 40 50 60 2 4 6 8 10 12

• Temperature(c)

Numberofeventspersecond

10mMAmpicillincisside IMKClpH6+50mV

DependenceofAmpicillin0OmpFbindingeventsontemperature

10 20 30 40 50 60 0.0 0.2 0.4 0.6 0.8 1.0 1.2

• cisside+50mV

transside+50mV 10mMampicillinpH6

Dependenceofresidencetimeontemperature

TemperatureC Residencetime,ms

Chemicalrateconstants(konandkoff)weredetermined Dependsontemperature

Prospects

• Wehavebeenabletomeasuretime0resolvedsingle0 molecule

eventsofantibioticentryintothepore,andobtaindetailedkinetic information. Highresolutionconductancemeasurementscorrelatedwith antibioticactivitiesinbacteria(MICassays) Experimentalresultsfromlipidbilayermeasurements andmicrobiologicalassayscomparedwithmolecular dynamicssimulations. Miniaturation(Nanion) Developmentofnewantibioticswithhightranslocation efficiencyinfuture.

Nestorovichetal,2002

Acknowledgements

GroupofProf.Winterhalter Prof.Jean0MariePagès Chloë E.James GroupofProf.Ceccarelli GroupofProf.PaulaGameiro Dr.MalcolmPage(Basilea)