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•  LaurieB.Schenkel•  RibonTherapeutics

APotentandSelectivePARP14InhibitorDecreasesPro-TumorMacrophageFunctionandElicitsInflammatoryResponsesinTumorExplants

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Disclosure Statement

•  IamanemployeeandshareholderofRibonTherapeutics

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PARP14 Is Overexpressed in Cancer and Implicated in Macrophage and T Cell Biology •  ThePARPfamilyconsistsof17proteins(16catalyticallyactive)thatuseNAD+topost-translationally

ADP-ribosylatesubstrates•  PolyPARPsarewell-studied,andPARP1/PARP2inhibitorshavebeenapproved•  MonoPARPsofferamechanisticallydistinctanduntappedopportunity

•  PARP14isamemberofthemonoPARPsub-family•  DownstreamregulatorofIFN-γandIL-4signaling•  Immuno-oncologyfunctionsincludingregulationofM1/M2macrophagedifferentiation•  Highexpressioninmultipletumortypes

Overexpressedinseveralcancertypescomparedtonormaltissue

Affectsthedifferentiationofimmunecellsubsetsassociatedwithhumandisease

Interferon-stimulatedgene

Vehicle IFN-γ

SU-DHL-8 lymphoma cells

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Screening Ribon’s Compound Collection and Profiling Against a Panel of PARP Assays Identified a PARP14 Hit RibonPlatformenabledthe

monoPARPfamilyfordrugdiscoveryPARP14wasscreenedagainstRibon’s

proprietarycompoundcollectionAmoderatelypotenthitwas

identifiedandprofiledacrossthePARPenzymes

Compound1

*

*

*

Biochemical-DELFIA

Biophysical-SPR

X-Raycrystalstructure

Biophysical-TR-FRET

Cellbiophysical-NanoBRET

Potentandselectiveinhibitor*Literaturetoolinhibitor

CellMARylation/biochemicalassay

PARPAssay IC50(µM)

PARP14 1

PARP1 0.1

PARP2 0.3

PARP3 1

PARP4 1

PARP5a/5b 0.09/0.03

PARP6 1

PARP7 10

PARP8 4

PARP9 >100

PARP10 1

PARP11 4

PARP12 4

PARP15 6

PARP16 7

polyPARPsmonoPARPs

≤0.01 0.3 ≥10Wigle,et.al.SLASDiscovery,2019

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Asp1685Ser1688

I

IIIII

IVTyr1727

Tyr1714

Leu1782

Tyr1721

His1682

Compound 1 Provides Vectors to Access Areas of the NAD+ Binding Pocket that Were Hypothesized to Increase PARP14 Potency and Selectivity

•  HittoleadeffortstargetedinteractionsinkeyareasoftheNAD+bindingpocket

•  RegionIV:theAsp/SermotifisuniquetoPARP14andPARP15

•  RegionI:themonoPARPscontainhydrophobicresidues,whilethepolyPARPscontainpolarresiduesthatformaconservedsaltbridge

•  Nicotinamidepocket•  I:MAR/PARdifference•  II:D-Loop•  III:Adenosineregion•  IV:Asp/Serregion

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Modification of the Thioether Led to High Selectivity for PARP14 over the Other MonoPARPs

Compound2

Compound1

OptimizationofthearomaticthioetherimprovedPARP14potencyandselectivityoverthe

monoPARPs

Changesinthebindingconformationofthethioetherledtothealcoholofcompound2interactingwith

Asp1685andthePARP14D-loopadoptinga“closed”,orderedconformationPARP14IC50(µM): 1 0.3

Compound: 21

polyPA

RPs

mon

oPAR

Ps

≤1 10 ≥100

PARP14selectivity(fold):

PARP1

PARP2

PARP5a

PARP5b

PARP3

PARP4

PARP6

PARP7

PARP8

PARP9

PARP10

PARP11

PARP12

PARP15

PARP16

V

Ser1688

I

II III

Asp1685

IV

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RBN012759 Is a Potent PARP14 Inhibitor that Is Highly Selective over All Mono- and PolyPARPs

polyPA

RPs PARP1

PARP2

PARP5a

PARP5b

PARP3

PARP4

PARP6

PARP7

PARP8

PARP9

PARP10

PARP11

PARP12

PARP15

PARP16

mon

oPAR

Ps

RBN012759

PARP14IC50(µM): 1 0.3 <0.003Compound: 21

≤1 10 ≥100

PARP14selectivity(fold):

RBN012759is>300-foldselectiveoverthemonoPARPsand>1000-foldselectiveoverthepolyPARPs

OptimizationforPARP14potency,selectivityandPKpropertiesledto

RBN012759

Compound2

RBN012759

RBN012759makesnewinteractionswithPARP14intheAsp/Ser(IV)andMAR/PAR

difference(I)regions

Ser1688

I

II III

IVAsp1685

Tyr1727

Tyr1714

Leu1782

Tyr1721

His1682

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The Profile of RBN012759 Supports Its Use as an In Vitro and In Vivo PARP14 Chemical Probe

PARP14Activity/AffinityIC50humancatalyticdomain(µM) <0.003IC50mousecatalyticdomain(µM) 0.005IC50humanfulllength(µM) <0.005

CellularTargetEngagementPARP14NanoBRETcellprobedisplacement(µM) 0.003

PARPFamilyActivityPARP1(µM) >100PARP2(µM) >100PARP3(µM) >100PARP4(µM) 10PARP5a(µM) 8PARP5b(µM) 10PARP6(µM) 4PARP7(µM) 4PARP8(µM) 20PARP9(µM) >100PARP10(µM) 1PARP11(µM) 1PARP12(µM) 5PARP15(µM) 3PARP16(µM) 6

InVitroADMEandPhysicochemicalPropertiesKineticsolubilityatpH7.4(µM) 198Caco-2Papp(A-B)(x10-6cm/s)/effluxratio 19/1MousemicrosomesClint(mL/min/kg,scaled) 469Mouseppb(Fu) 0.13MW/TPSA/XLogP 379/71/2.9

InVivoPKMouseCl(mL/min/kg)/t1/2(h)/Vss(L/kg) 54/0.4/1.4Mouse%Fat100mg/kg 30

ThechemicalprobeRBN012759:•  Isapotentinhibitorofhumanandmouse

PARP14withhighPARPfamilyselectivity•  Issufficientlysoluble,highlypermeable,low

efflux•  Hasmoderateclearanceandoral

bioavailabilityinmice•  Iswell-toleratedinmicewithrepeatdosing

upto500mg/kgBID•  75-foldcoverageofthePARP14mouse

freeEC50observedatCtrough

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RBN012759 Demonstrates Intracellular Engagement of PARP14

RBN012759treatmentofhumanprimarymacrophagesdecreasedtheMAR/PARsignalcorrespondingtoPARP14self-

MARylationandstabilizedPARP14proteininadose-dependentmanner

NAD+

nicotinamide

Substratemodifiedwithmono(ADP-ribose)(MAR)

Substrate

Substratemodifiedwithpoly(ADP-ribose)(PAR)

PARPsutilizeNAD+topost-translationallymodifysubstratesviaMAR

(monoPARPs,includingPARP14)orPAR(polyPARPs)

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(M2)

PARP14 Inhibition Reverses IL-4-Driven Gene Expression in Primary Human Macrophages

Experimentaldesign

PARP14ireversesIL-4stimulatedgeneexpression

PARP14ireducesIL-4stimulatedcytokinesecretion

1 1 1 1 16 0

7 0

8 0

9 0

1 0 0

1 1 0

IL 1 0 p a p e r (1 :2 )

IL-1

0 (

pg

/mL

) D M S O

D M S O

R B N 0 1 2 7 5 9 1 0 u M

R B N 0 1 2 7 5 9 1 .0 u M

R B N 0 1 2 7 5 9 0 .1 u M

N a iv e

IL -4 +IL -1 3

1 1 1 1 10

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

C C 2 4 p a p e r (1 :2 )

CC

L2

4 (

pg

/mL

)

D M S O

D M S O

R B N 0 1 2 7 5 9 1 0 u M

R B N 0 1 2 7 5 9 1 .0 u M

R B N 0 1 2 7 5 9 0 .1 u M

1 1 1 1 10

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

IL 1 - ra 1 :4 p a p e r

IL1

-ra

(p

g/m

L)

D M S O

D M S O

R B N 0 1 2 7 5 9 1 0 u M

R B N 0 1 2 7 5 9 1 .0 u M

R B N 0 1 2 7 5 9 0 .1 u M

1 1 1 1 16 0

7 0

8 0

9 0

1 0 0

1 1 0

IL 1 0 p a p e r (1 :2 )

IL-1

0 (

pg

/mL

) D M S O

D M S O

R B N 0 1 2 7 5 9 1 0 mM

R B N 0 1 2 7 5 9 1 .0 mM

R B N 0 1 2 7 5 9 0 .1 mM

N a iv e

IL -4 +IL -1 3

Datasimilarity(0=100%similar)

•  TreatmentofprimaryhumanmacrophageswithRBN012759ledtodecreasedIL-4drivenM2-likegeneexpression,suggestingthatPARP14inhibitionresultsinalessimmunosuppressivephenotype

M2

Naive

(M2)

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PARP14 Inhibition Induces Inflammatory Gene Expression in an Unbiased Set of Kidney Cancer Tumor Explants

Experimentaldesign:kidneyrenalclearcellcarcinoma(KIRC)explants

RBN012759inducesgeneexpressionchangessimilartoimmunecheckpointinhibitorcombinationinsometumors

• RBN012759treatmentinducedgeneexpressionchangesintumors5,9and2thatclusterwithimmunecheckpointinhibitor(ICI)hyper-respondertumor5

Expressionfoldchange(log2)

ICIhyper-responder

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Conclusions

•  DetailedknowledgeofthePARPfamilyNAD+bindingpocketsenabledstructure-basedoptimizationofmoderatelypotent,unselectivePARP14hitCompound1intoRBN012759

•  RBN012759(<0.003µMPARP14,>300-foldselectiveovermonoPARPs,>1000-foldselectiveoverpolyPARPs)isthemostpotentandselectiveinhibitorofPARP14reportedtodate

•  ThepropertiesofRBN012759enableitsuseasaninvitroandinvivoPARP14chemicalprobe

•  DatageneratedinhumanprimarymacrophagesandKIRChumantumorexplantswithRBN012759treatmentlinksPARP14withsuppressionoftheanti-tumorimmuneresponse

•  RBN012759canserveasausefultoolforfurtherexplorationofPARP14functionsincellbiologyandasadrugtarget

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Acknowledgements

VictoriaRichonAndySantospagoLaurieSchenkelRichardSchroederPrashantShambharkarJeffSongTadStewartKerrenSwingerLukeUtleyZachareniaVarsamisMelissaVasbinderTimWigleJodieWong

RyanAboEllenBambergDanielleBlackwellRichardBushellAnneCheungW.DavidChurchLisaClearyDavidCordoBryanDorseyJenniferDowningJosephGozgitLinetteGrayBinGuiHeikeKeilhackPeterKimDanielleKnightKaikoKunii

TeamRibon:KevinKuntzKristyKuplast-BarrJenkinsLemeraChangLiuAlvinLuAhmedMadyChristinaMajerKristenMcEachernMaeganMikulaElenaMinissaleJasonMoJenniferMolinaSunainaNayakMarioNiepelSudhaParasuramanNicholasPerlYueRen

JamesAudiaMarkBoothbyPaulChang

LeeKrausLarryLaskyTimothyMitchison

FoundersandAdvisors: