Visualising the molecular drivers behind drug resistance Thehugeexpenseofdevelopinganewdrugcanbewastedifnaturalmuta7onsofaminoacidresiduesinthetargetedproteinleadtoalossofdrugbindingaffinity.Ra5onaldrugdesignisacon5nualstruggle,withevolu5ondrivingmuta5onsthatdevelopemergingdrugresistanceintowidespreaddruginefficacy.WehavedevelopedanewfreeenergymethodcallledProteinSwap,basedonWaterSwap,thatcanprovidedrugdesignerswiththeinsightneededtounderstandhowproteinmuta5onsaffectdrugbinding.Thismethodallowsthechangeinbindingfreeenergyofthedrugassociatedwiththemuta7onoftheproteintobecalculated.Inaddi5on,asforotherSwap-Basedmethods,thisfreeenergychangecanbedecomposedintocomponentsthatcanbeusedforvisualis7on.Thesecomponentsallowthechangeinbindingfreeenergytobeunderstoodintermsofchangesinspecificligand-proteininterac7ons,orchangesinthesolva5ngwaternetworkintheac5vesite.Thismethodallowsdrugdesignerstopro-ac5velyscreenanewdrugcomputa7onallyagainstarangeoflikelyproteinmutants,therebyenablingthemtogetonestepaheadofnature.Alterna5vely,itallowschemiststoinves5gatethemolecularbasisforreducedbindingaffinityinknowndrug-resistantmutantsofaprotein.

MaturosMalaisreeandChristopherJ.Woods,SchoolofChemistry,UniversityofBristol,Bristol,BS81TS,UnitedKingdom

ProteinSwapisanewfreeenergymethodthatcanbeusedtoexploredrugresistance. Residue-based decomposi5ons provide a useful graphicalvisualisa5on.Theseenabletheeffectofproteinmuta5onondrugbindingtobera5onalisedatthemolecularlevel.

ProteinSwapwillsoonbeavailableasanopensourcecommand-linetoolas part of Sire (hLps://siremol.org). A graphical interfacewill bemadeavailablebyCressetviaFlare(hLps://www.cresset-group.com/flare).

References(1)  Woods,CJ,Malaisree,M,Hannongbua,S&Mulholland,AJ,“Awater-swapreac5oncoordinateforthecalcula5onof

absoluteprotein-ligandbindingfreeenergies”,JournalofChemicalPhysics,vol134,2011(2)  Woods,CJ,Malaisree,M,Michel,J,Long,B,McIntosh-Smith,S&Mulholland,AJ,“Rapiddecomposi5onand

visualisa5onofprotein-ligandbindingfreeenergiesbyresidueandbywater”,FaradayDiscussions,vol169,2014(3)  Baek,YH,etal,“ProfilingandCharacteriza5onofInfluenzaVirusN1StrainsPoten5allyResistanttoMul5ple

NeuraminidaseInhibitors”,JournalofVirology,vol89,2015

AcknowledgementsWethanktheEPSRCandCressetforfundingviaanEPSRCImpactAccelera5onAward.CWisanEPSRCResearchSocwareEngineeringFellow(EP/N018591/1).WethanktheAdvancedCompu5ngResearchCentreatUoB forprovidingearly access to thenewBlueCrystalPhase4cluster(hfp://www.acrc.bris.ac.uk)

λ=0.0 λ=0.2 λ=0.5 λ=0.8 λ=1.0

Wildtype

Mutant

E(�) = (1� �)

Eligand:wildtype + Ewater:mutant

�+ �

Ewater:wildtype + Eligand:mutant

�

ProteinSwap, based onWaterSwap1,2, calculates the change in protein-ligand binding free energycaused by protein muta5on. A λ-coordinate swaps the ligand from the wildtype protein to the mutant.Simultaneously, a cluster of water molecules is swapped from the mutant to the wildtype. Monte Carlosimula5onsateachvalueofλareusedtoevaluatethefreeenergychangeusingthermodynamicintegra5on.Theresultisthefreeenergypreferenceoftheligandforeitherthewildtypeorthemutantprotein.

Valida7on,wasperformedbycalcula5ngtheProteinSwapfreeenergiesofoseltamivirandperamivirligandsinwildtypeandmutant formsof influenzaneuraminidase (H1N1-2009). Logical tests, such as swapping the ligandbetween two iden5calproteins, showed themethodwas sound.Reverse tests, comparingwildtypeàmutant againstmutantàwildtype, showed theProteinSwap free energies were consistent. Closure tests showed free energies closed. Finally, comparison against knownexperimentalbindingaffini7esshowedthatProteinSwapcouldcorerctlyiden5fyknowndrug-resistantmuta5ons.

-8

-6

-4

-2

0

2

4

6

8

0 0.2 0.4 0.6 0.8 1

FreeEne

rgy/kcalm

ol-1

-0.1±0.6kcalmol-1Peramivir:WildtypeàWildtype

LogicalTest-shouldbezero

-16

-12

-8

-4

0

4

8

12

16

0 0.2 0.4 0.6 0.8 1

FreeEnergy/kcalm

ol-1

λ-16

-12

-8

-4

0

4

8

12

16

0 0.2 0.4 0.6 0.8 1

FreeEnergy/kcalm

ol-1

λ

8.3±0.8kcalmol-1 -7.7±1.2kcalmol-1Peramivir:WildtypeàR292K R292KàWildtype

ReverseTest-shouldbeequalandopposite ClosureTest–pathsequal

Wildtype R292K

R152K

E119G

8.3±0.8kcalmol-1

Peramivir:Wildtype,R292K,R152K,E119G

ComparisontoExpt.GreenShowsAgreement Oseltamivir PeramivirExperimentalResistance3 Wildtype≈E119G<R152K<R292K Wildtype<R152K<R292K<<E119G

ProteinSwapFreeEnergy/kcalmol-1

0.0≈0.6<5.1<8.4 0.0<3.0<8.3!!3.0

R e s i d u e - b a s e ddecomposi7on, allowsProteinSwap free energies to bebroken down to residue-basedcomponents. Each residue can becolored as preferring bindingligand (blue) or preferring bindingwater (red). The difference ofthese between the mutant andwildtype shows how muta5onweakens b ind ing for someresidues(red),whilestrengtheningbindingtoothers(blue).

-30

-25

-20

-15

-10

-5

0

5

10

R118

E119

D151

R152

R156

S179

D198

I222

R274

E277

S246

E276

E277

R292

N294

G344

K3

46

R371

FreeEne

rgy/kcalm

ol-1

-30

-25

-20

-15

-10

-5

0

5

10

R118

E119

D151

R152

R156

S179

D198

I222

R274

E277

S246

E276

E277

K292

N294

G344

K3

46

R371

FreeEne

rgy/kcalm

ol-1

-30

-25

-20

-15

-10

-5

0

5

10

R118

E119

D151

R152

R156

S179

D198

I222

R274

E277

S246

E276

E277

R292K

N294

G344

K3

46

R371

FreeEne

rgy/kcalm

ol-1

- =

Oseltamivir:Wildtype Oseltamivir:R292K Difference

R292

R371

R118 R118

R371

K292 R292K

R371

R118

Eresiduewildtype(�) = (1� �)

Eresidue

ligand:wildtype

�+ �

Eresidue

water:wildtype

�

Eresiduemutant(�) = (1� �)

Eresidue

water:mutant

�+ �

Eresidue

ligand:mutant

�

Conclusion