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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:
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