The target evaluation platform revealed an AdeR …c1940652.r52.cf0.rackcdn.com/57639c74b8d39a469d0034d9/...SWITCHING OFF BACTERIAL RESISTANCE The target evaluation platform revealed

SWITCHING OFF BACTERIAL RESISTANCE

ThetargetevaluationplatformrevealedanAdeRunrelatedtigecycline resistancemechanismin

XDRAcinetobacterbaumannii

VincentTrebosc1,2,SarahGartenmann1,KevinRoyet1,PabloManfredi2,BirgitSchellhorn1,MichelPieren1,SergioLociuro1,PeterC.Sennhenn1,MarcGitzinger1,MarcelTigges1,DirkBumann2,andChristianKemmer1

1 BioVersysAG,Hochbergerstrasse60c,CH-4057Basel,Switzerland2 Biozentrum,UniversityofBasel,Klingenbergstrasse70,CH-4056Basel,Switzerland

6/17/16 ECCMID2016-BioVersys_Proprietary_Material

1


Acinetobacterbaumannii– anincreasingthreatinhealthcareunits

2

2011

2016

2007

Photo:JaniceCarr

- Mainly affects patients inthe ICU- Increasing emergence of A.baumannii outbreaks inhospitals- MDR/XDRstrains are emerging worldwide- Remaining treatment options:a)polymyxins,b)minocycline,c)tigeycycline



3

Unknown

AdeRS TCSscontrolstigecycline effluxAdeR asdrugtargetforadjuvants?

AdeR

AdeS

Tigecycline

Tigecycline resistance:1. Tigecycline triggersAdeABC effluxpumpoverexpression2. AdeABC effluxestigecycline toreduceintracellularconcentration

EffluxbyAdeABC

AdeABC



4

0 . 4 0 . 8 1 . 6 3 . 1 6 . 31 2 . 5 2 5

0

2

4

6

T i g e c y c l i n e M I C d i s t r i b u t i o n

M I C ( m g / m l )

nu

mb

er

of

iso

late

s

w i l d t y p e

Da d e R

• adeRdeletionreducedMIC90 from25µg/mlto3.1µg/ml

• Clinicallyacceptedsusceptibility breakpoint:1ug/mL

à 60%of∆adeRmutantsremainedtigecyclinenon-susceptible

WT:wildtypeMIC:minimal inhibitory concentration

StrainTigecyclineMIC

WT ΔadeR

ATCC-17978 0.4 0.4

BV26 6.3 0.4

BV94 25 3.1

BV173 6.3 0.8

BV175 3.1 0.8

BV185 6.3 3.1

BV186 3.1 3.1

BV187 3.1 3.1

BV189 6.3 3.1

BV190 6.3 0.8

BV191 25 1.6

AdeR playsamajorroleintigecycline resistancebutpresenceofalternativemechanism

ECCMID2016-BioVersys_Proprietary_Material



AdeRSmutations(aminoacidsubstitutions)WT ΔadeR

ATCC-17978 0.4 0.4 reference

BV94 25 3.1 F170SinAdeS

BV173 6.3 0.8 D21VandD26NinAdeR

BV186 3.1 3.1 Nomutation

BV189 6.3 3.1 Nomutation

BV191 25 1.6 G79SinAdeS

AdeR playsamajorroleintigecycline resistancebutpresenceofalternativemechanism

5

AT

CC

- 17 9 7 8

BV

9 4

BV

1 7 3

BV

1 8 6

BV

1 8 9

BV

1 9 1

0

5 0

1 0 0

1 5 0

2 0 0

8 0 0

1 0 0 0

1 2 0 0

a d e B e x p r e s s i o n l e v e l

fold

ex

pre

ss

ion

(re

lati

ve

to

AT

CC

-17

97

8)

w i l d t y p e

Da d e R

• adeR is essentialforAdeABCexpression• Tigecyline resistance is mediated byadeABC drug efflux• Sequencing ofTCSidentified nomutationinstrains notresponding toloss ofadeR

à Alternativeresistancemechanism(s),independentofAdeR


WT ΔadeR

ATCC-17978 0.4 0.4

BV94 25 3.1

BV173 6.3 0.8

BV186 3.1 3.1

BV189 6.3 3.1

BV191 25 1.6

WT:wildtypeMIC:minimal inhibitory concentration

ECCMID2016-BioVersys_Proprietary_Material


Artificalresistancedevelopmentandwholegenomesequencing

6

MutatedgenesorregionsMutations

GenefunctionATCC-17978(TGC) ATCC-17978ΔadeR(TGC)

trm (A1S_2858)IS-17liketransposondisruption

Lossofadenineatposition311leadstoprematurestopcodon

MethyltransferasepotentiallyinvolvedinrRNAs methylation

RNaseE(A1S_0403) 58bp deletion - RibonucleaseinvolvedinrRNAsprocessingandRNAdecay

IntergenicregionbetweenRNaseE(A1S_0403)and23SrRNA pseudouridylatesynthase (A1S_0404)

ISaba11elementinsertedatposition439,336 -

RNaseEseeabove.23SrRNA pseudouridylate synthaseinvolvedinmodificationof23SrRNA

adeN (A1S_1979) - IStransposondisruption TranscriptionalrepressoroftheAdeIJKeffluxpump

abeM (A1S_0395) -C->Tmutation(position429,259)inthepromoterregionofabeM

MATEfamilyeffluxpump

MIC (µg/ml) Tigecycline

WT

ATCC-17978 0.4

ATCC-17978(TGC) 25

∆adeR ATCC-17978ΔadeR 0.4

ATCC-17978ΔadeR(TGC) 6.3

• ∆adeRmutantwereabletodeveloptigecyclineresistance• trmwastheonlymutatedgeneinbothstrainsà Istrm disruptionageneraltigecyclineresistancemechanism?



trm disruptionisnottheonlytriggerforthealternativeresistancemechanism

7

• Correlationbetweentrm disruptionandtigecyclineexposure• However,lossofadeR inrenders3strainstigecycline susceptiblewhiletrmwasdisruptedà trm disruptioncannotbetheonlydriverofresistance

à isolatedbeforetigecyclineapproval

Strain

IsolationTigecyclineMIC trmmutations

(nucleotide)Location Date WT ΔadeR

ATCC-17978 France 1951 0.4 0.4 NomutationBV26 Switzerland 1979 6.3 0.4 NomutationBV94 USA 2011 25 3.1 G1065BV173 Greece 2012 6.3 0.8 A240BV175 Turkey 2012 3.1 0.8 A240BV185 Mexico 2013 6.3 3.1 A240BV186 USA 2013 3.1 3.1 G1065BV187 USA 2013 3.1 3.1 G1065BV189 Spain 2013 6.3 3.1 A240BV190 Greece 2012 6.3 0.8 T1050BV191 China 2013 25 1.6 A240

trm disruptioncannotbetheonlydriverofresistance



Factsthatdevalidated AdeR asadrugtarget

1. AdeABC-mediatedtigecyclineeffluxisthemajorresistancemechanism• lossofAdeR reducestigecyclineMIC90from25µg/mlto3.1µg/ml• theadeABC effluxpumpexpressionisabolishedin∆adeRmutants• wefoundacorrelationbetweentigecyclineMICs,AdeRS mutationsandAdeABC

expressionlevel

2. 60%of∆adeRmutantsremainedresistanttotigecycline

3. Wesuccessfullydevelopedtigecyclineresistanceina∆adeR strain

4. Weidentifiedacandidateproteininvolvedinanalternativeresistancemechanism• wholegenomesequencingidentifiedtrm astheonlygenemutatedinthetwo

independentlyevolvedtigecyclineresistantstrains• trm genewasdisruptedin9of10primarilytestedtigecyclineresistantclinicalisolates• lossofadeR renders3strainstigecycline susceptiblewhiletrm isdisruptedà trm disruptioncannotbetheonlydriverforanalternativeresistancemechanism

8

AdeR inhibitionbysmallmoleculedrugwillnotre-sensitizeA.baumannii totigecycline



Acknowledgements

9

*Funded by the Marie Curie Initial Training ITN-2013-607694-Translocation

VincentTrebosc*SarahGartenmannKevinRoyetBirgitSchellhornMichelPieren,PhDSergioLociuro,PhDPeterC.Sennhenn,PhDMarcGitzinger,PhDMarcelTigges,PhDChristianKemmer,PhD

PabloManfrediProf. Dr. DirkBumann

Forfurtherinformation,[email protected]



backup



OverviewDrugDiscovery

6/17/16 11ECCMID2016- BioVersys_Proprietary_Material

Genetic,cellularandinvivoexperimentalmodelstoidentifyandvalidatetargets

Highthroughput(HTP)primaryscreenings

Constant,iterativecompoundsynthesistoimprovecompoundproperties

Invitroandexvivosecondaryassays(modeofaction,selectivity)

Invitroassessment ofpharmacologicalpropertiesofthecompounds

Invivoassessment ofpharmacologicalproperties

Infectionefficacymodel

Earlysafetyandtoxicitystudies

TargetIdentificationandValidation CompoundScreening

SecondaryAssayInvivo

AdaptedfromHughesetal.BJP,2011


12

UrgentunmetmedicalneedinGram-negativebacteria



Drugtargetevaluationwithknockoutinrelevantclinicalstrains

13ECCMID2016-BioVersys_Proprietary_Material

• Non-antibioticresistancemarkerenablesmanipulationofdrugresistantisolates

• Induciblecounterselectioncassette

Straindesignation

isolationMLST

Resistance profileforXDR(non-susceptibleto≥1antibioticinallbut≤2categories)location date GENT MERO CIP T/C TZP CTX SXT SAM COL TET

ATCC17978 France 1951 77 S S S S S I R S S SBV26 Switzerland 1979 1 R S R R S I R R S RBV94 USA 2011 2 R R R R R R R I R RBV173 Greece 2012 2 R R R R R R R R R RBV175 Turkey 2012 2 R R R R R R R R R RBV185 Mexico 2013 2 R R R R R R R R R RBV186 USA 2013 2 R R R R R R R R R IBV187 USA 2013 2 R R R R R R R I R IBV189 Spain 2013 2 R R R X R R R R R RBV190 Greece 2012 1 R R R X R R R R R RBV191 China 2013 2 R R R X R R R R R R

Straindesignation

isolationMLST

location dateATCC17978 France 1951 77

BV26 Switzerland 1979 1BV94 USA 2011 2BV173 Greece 2012 2BV175 Turkey 2012 2BV185 Mexico 2013 2BV186 USA 2013 2BV187 USA 2013 2BV189 Spain 2013 2BV190 Greece 2012 1BV191 China 2013 2

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The target evaluation platform revealed an AdeR …c1940652.r52.cf0.rackcdn.com/57639c74b8d39a469d0034d9/...SWITCHING OFF BACTERIAL RESISTANCE The target evaluation platform revealed

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