increase protein levels and complement other CF ... · Organoid Model 103% 134% 160% 0% 20% 40% 60%...
1
In order to identify novel modulators of mutant CFTR function, a high-throughput screening strategy that enriches for small molecules that act on the translated sequence of CFTR protein was used. This approach resulted in the identification of a new class of modulator, a CFTR amplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide more CFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon. Patients homozygous for the F508del realize a modest benefit in lung function from a combination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftor provided no significant clinical benefit in patients with F508del compound heterozygous genotypes. Clinical benefit for patients with two copies, but not for patients with a single copy of F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for corrector and potentiator efficacy. In vitro , amplifiers nearly double the functional rescue conferred by these compounds. In addition, amplifiers are selective in increasing the total amount of CFTR protein without an increase in the levels of a second mutant ABC transporter protein. Amplifiers have activity both in cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, and do not possess corrector or potentiator activity, instead complementing the action of these other modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs with demonstrated activity across models derived from CF donors with different genotypes, and across different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiers show activity in primary cells from non-lung tissues, and when orally dosed in animals. The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNA stabilization. This is consistent with a model in which the novel amplifier class of CFTR modulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTR synthesis to provide more protein, amplifiers can be used in combinations to boost the activity of additional CFTR modulators. Funded in part by a Therapeutics Development Award from Cystic Fibrosis Foundation Therapeutics, Inc. CONCLUSIONS > Amplifiers are a new class of CFTR modulator > Amplifiers increase CFTR immature protein and stabilize CFTR mRNA > Amplifiers increase substrate for additional CFTR modulators > Amplifiers work across CFTR genotypes > Amplifiers are active in non-lung tissues and in vivo ACKNOWLEDGEMENTS We would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago Medical School for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of Sharp Edge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB for organoid experiments. CFTR Modulators with Distinct Mechanisms of Action Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator Model for Amplifier Effect on CFTR Translational Efficiency Amplifier Increases F508del-CFTR Protein Amplifier Stabilizes CFTR mRNA Amplifier Increases F508del-CFTR Function Amplifier Provides More Substrate for Other CFTR Modulators Physiological Response to Amplifier in Forskolin-Induced Swelling Assay Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo » The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows that PTI-CH selectively increases F508del-CFTR total expression and does not have this effect on G268V-PgP » HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR more than mature F508del-CFTR, distinguishing it from correctors which preferentially increase maturation of CFTR » In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in the presence of amplifier » Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH is increased by nearly 2-fold » Total mRNA levels are not impacted by PTI-CH » Using chamber current measurements of F508del homozygous HBEs treated for 24 hours with PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor » PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or in combination with corrector and/or potentiator » CFBE cell western blot of the PTI-CH increase in immature F508del-CFTR that can then be converted into mature F508del-CFTR by lumacaftor » PTI-CH triple combination with PTI modulators provides greater in vitro efficacy in G85E/F508del HBEs than a two corrector triple combination » PTI-CH works in genotypes other than F508del/F508del » PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftor alone » PTI-CH enhances ivacaftor activity in R117H/F508del HBEs » PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB) » Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-type CFTR Characterization of CFTR amplifiers, mutation-agnostic modulators that increase protein levels and complement other CF therapeutic modalities John Preston Miller, Lawrence Drew, Olivia Green, Danijela Dukovski, Adriana Villella, Nipul Patel, Cecilia Bastos, David Kombo, Daniel Parks, Matthew Cullen, Hoang Danh, Shinichiro Wachi, Kenneth A Giuliano, Soheil Aghamohammadzadeh, Ken Longo, Akhil Bhalla, Daniel Qiu, Chaosheng Zou, Magnus Ivarsson, Benito Munoz, Huseyin Mehmet Proteostasis Therapeutics, Inc., Cambridge, Massachusetts, United States -1 4 9 14 19 600 800 1000 1200 Isc (uAmp/cm^2) Time (seconds) R117H/F508del HBEs fsk IVA CFTRinh172 71% 100% 128% 201% 0% 50% 100% 150% 200% 250% R117H/F508del CFTR Chloride Transport Activity (% of ivacaftor) ivacaftor + PTI-CH PTI-CH ivacaftor DMSO • PTI-CH works in genotypes other than F508del/F508del DMSO + lumacaftor ivacaftor + + PTI-CH + + 0 min 100 min F508del/ F508del Intestinal Organoids A455E/ F508del Intestinal Organoids lumacaftor lumacaftor + PTI-CH DMSO PTI-CH Experiments performed at HUB 30% 50% 100% 145% 0% 20% 40% 60% 80% 100% 120% 140% 160% Forskolin-Induced Swelling (Relative to lumacaftor + ivacaftor) F508del/F508del Human Intestinal Organoid Model 103% 134% 160% 0% 20% 40% 60% 80% 100% 120% 140% 160% 180% CFTR mRNA Levels (Relative to vehicle) Non-CF Rat Lung vehicle PTI-CH lumacaftor + + ivacaftor + + + + PTI-CH + + 0% 100% 200% 300% DMSO VRT-325 lumacaftor PTI-CH Expression Level (Relative to DMSO) Surface F508del CFTR Protein Total F508del CFTR Protein CFTR mRNA 0% 100% 200% 300% 400% Expression Level (Relative to DMSO) Surface G268V PgP Protein Total G268V PgP Protein G268V PgP mRNA 0% 50% 100% 150% 200% 250% F508del CFTR Protein Levels (Relative to DMSO) Band C (Mature CFTR) Band B (immature CFTR) HEK Cell Reporter System F508del/F508del HBE Western DMSO PTI-CH 1.5 2 2.5 3 3.5 4 4.5 5 5.5 Half-life (hours) DMSO PTI-CH Approach to steady-state in CFBE Cells Tet-Off CFTR transcriptional shut-off in HEKs 0 5 10 15 20 25 0 0.5 1 1.5 2 2.5 3 3.5 Time (h) EU incorporation (DMSO standardized) DMSO 0 10 20 0 1 2 3 Total mRNA Levels Treatment Time (hours) PolyA RNA ( m grams) 0 2 4 6 hours post DOX t 1/2 = 1 hour t 1/2 = 3 hours PTI-CH • In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in th -5 0 5 10 15 20 900 1400 1900 Isc (uAmp/cm^2) Time (seconds) 23% 38% 35% 65% 64% 113% 100% 180% 0% 20% 40% 60% 80% 100% 120% 140% 160% 180% 200% F508del CFTR Inhibitable Current (% ivacaftor + lumacaftor) fsk IVA CFTRinh172 PTI-CH + lumacaftor lumacaftor DMSO • Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hours DMSO + lumacaftor + + + + ivacaftor + + + + PTI-CH + + + + Tubulin Band C Band B 150 100 250 DMSO lumacaftor PTI-CH PTI-CH + lumacaftor 100% 211% 239% 413% 100% 229% 338% 461% 0% 100% 200% 300% 400% 500% CFTR Chloride Transport Activity (%lumacaftor + ivacaftor) F508del/F508del G85E/F508del lumacaftor + VX-661 + + PTI-C1811 (Corrector) + + + ivacaftor + + PTI-P271 (Potentiator) + + PTI-CH + + Action POTENTIATORS CORRECTORS AMPLIFIERS Potentiators, such as ivacaftor, act by increasingthe openingtime of the CFTR channel resultingin higher ion flow. Correctors, such as lumacaftor, are thought to facilitate the processingof mutated CFTR protein leadingto improved delivery of CFTR protein to the cell membrane. Amplifiers selectively increase the amount of immature CFTR protein in the cell providingadditional substrate for correctors and potentiators to act upon. Construct mRNA ATG STOP ATG STOP CFTR 5’UTR 3’ UTR exons/ introns ATG STOP CFTR amplifiers targeting CFTR translation independent of endogenous CFTR transcriptional control Endogenous CFTR Gene CFTR translation enhancement Endogenous CFTR transcriptional modulation Molecular Targets Enriched Molecular Targets Minimized CFTR translation enhancement CMV and exogenous transcriptional regulation PTI Curated Compound Library Phenotypic HTS Lead Optimization in HBE cells Disease-Relevant Translation TM in HBE CFBE HBE CFTR CFTR IREs PUROMYCIN 5’UTR 3’ UTR CMV CFTR mRNA binds to the ribosome Signal sequence emerges and recognized by the signal recognition particle (SRP) SRP binds mRNA-ribosome complex to form the Ribosome-Nascent Chain Complex (RNC) SRP shuttles the RNC to the ER surface and immature CFTR protein is available Improved efficiency of translation leads to increased levels and stability of CFTR mRNA INTRODUCTION RESULTS Amplifier Can Exceed Ivacaftor in vitro Efficacy and Doubles It in Combination
Transcript of increase protein levels and complement other CF ... · Organoid Model 103% 134% 160% 0% 20% 40% 60%...
In order to identify novel modulators of mutant CFTR function, a high-throughput screening strategy that enriches for small molecules that act on the translated sequence of CFTR protein was used. This approach resulted in the identification of a new class of modulator, a CFTR amplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide more CFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon. Patients homozygous for the F508del realize a modest benefit in lung function from a combination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftor provided no significant clinical benefit in patients with F508del compound heterozygous genotypes. Clinical benefit for patients with two copies, but not for patients with a single copy of F508del-CFTR, suggests the amount
of F508del-CFTR protein may be limiting for corrector and potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. In addition, amplifiers are selective in increasing the total amount of CFTR protein without an increase in the levels of a second mutant ABC transporter protein. Amplifiers have activity both in cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, and do not possess corrector or potentiator activity, instead complementing the action of these other modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs with demonstrated activity across models derived from CF donors with different genotypes, and across different classes of mutant CFTR. In addition to lung-derived cellular models,
amplifiers show activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNA stabilization. This is consistent with a model in which the novel amplifier class of CFTR modulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTR synthesis to provide more protein, amplifiers can be used in combinations to boost the activity of additional CFTR modulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis Foundation Therapeutics, Inc.
CONCLUSIONS> Amplifiers are a new class of CFTR modulator
> Amplifiers increase CFTR immature protein and stabilize
CFTR mRNA
> Amplifiers increase substrate for additional CFTR modulators
> Amplifiers work across CFTR genotypes
> Amplifiers are active in non-lung tissues and in vivo
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt
Green at Chicago Medical School for TECC24 functional profiling;
Scott Sneddon, Qi Yan and Junriz Delos Santos of Sharp Edge Labs
for Surface Expression Assays; and Sylvia Fernandez Boj and Rob
Vries of HUB for organoid experiments.
CFTR Modulators with Distinct Mechanisms of Action
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Model for Amplifier Effect on CFTR Translational Efficiency
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
» The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows that PTI-CH selectively increases F508del-CFTR total expression and does not have this effect on G268V-PgP
» HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR more than mature F508del-CFTR, distinguishing it from correctors which preferentially increase maturation of CFTR
» In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in the presence of amplifier
» Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH is increased by nearly 2-fold
» Total mRNA levels are not impacted by PTI-CH
» Using chamber current measurements of F508del homozygous HBEs treated for 24 hours with PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
» PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or in combination with corrector and/or potentiator
» CFBE cell western blot of the PTI-CH increase in immature F508del-CFTR that can then be converted into mature F508del-CFTR by lumacaftor
» PTI-CH triple combination with PTI modulators provides greater in vitro efficacy in G85E/F508del HBEs than a two corrector triple combination
» PTI-CH works in genotypes other than F508del/F508del
» PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftor alone
» PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
» PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
» Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-type CFTR
Characterization of CFTR amplifiers, mutation-agnostic modulators that increase protein levels and complement other CF therapeutic modalitiesJohn Preston Miller, Lawrence Drew, Olivia Green, Danijela Dukovski, Adriana Villella, Nipul Patel, Cecilia Bastos, David Kombo, Daniel Parks, Matthew Cullen, Hoang Danh, Shinichiro Wachi, Kenneth A Giuliano, Soheil Aghamohammadzadeh, Ken Longo, Akhil Bhalla, Daniel Qiu, Chaosheng Zou, Magnus Ivarsson, Benito Munoz, Huseyin MehmetProteostasis Therapeutics, Inc., Cambridge, Massachusetts, United States
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)EU
inco
rpor
atio
n (D
MSO
st
anda
rdiz
ed)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)EU
inco
rpor
atio
n (D
MSO
st
anda
rdiz
ed)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3CF
TR m
RNA
Leve
ls(R
elat
ive
to v
ehic
le)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3CF
TR m
RNA
Leve
ls(R
elat
ive
to v
ehic
le)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
0%
100%
200%
300%
DMSO VRT-325 lumacaftor PTI-CH
Expr
essi
on L
evel
(Rel
ativ
e to
DM
SO) Surface F508del CFTR Protein
Total F508del CFTR ProteinCFTR mRNA
-1
4
9
14
19
600 800 1000 1200
Isc(uA
mp/cm
^2)
Time(seconds)
R117H/F508delHBEs
CharacterizationofCFTRamplifiers,mutation-agnosticmodulatorsthatincreaseproteinlevelsandcomplementotherCFtherapeuticmodalities
JohnPrestonMiller,LawrenceDrew,OliviaGreen,DanijelaDukovski,AdrianaVillella,NipulPatel,CeciliaBastos,DavidKombo,DanielParks,MatthewCullen,HoangDanh,ShinichiroWachi,KennethAGiuliano,SoheilAghamohammadzadeh, KenLongo,AkhilBhalla,DanielQiu,ChaoshengZou,MagnusIvarsson,BenitoMunoz,HuseyinMehmetProteostasisTherapeutics, Inc.,Cambridge,Massachusetts, UnitedStates
In order to identify novel modulators of mutant CFTR function, a high-throughput screeningstrategy that enriches for small molecules that act on the translated sequence of CFTR proteinwas used. This approach resulted in the identification of a new class of modulator, a CFTRamplifier, which increases the levels of immature CFTR protein. Amplifiers thus provide moreCFTR protein for other CFTR modulators, for example, correctors and potentiators, to act upon.Patients homozygous for the F508del realize a modest benefit in lung function from acombination treatment of lumacaftor and ivacaftor. In contrast, lumacaftor and ivacaftorprovided no significant clinical benefit in patients with F508del compound heterozygousgenotypes. Clinical benefit for patients with two copies, but not for patients with a single copyof F508del-CFTR, suggests the amount of F508del-CFTR protein may be limiting for correctorand potentiator efficacy.
In vitro, amplifiers nearly double the functional rescue conferred by these compounds. Inaddition, amplifiers are selective in increasing the total amount of CFTR protein without anincrease in the levels of a second mutant ABC transporter protein. Amplifiers have activity bothin cell lines and in primary human bronchial epithelial (HBE) cells across a variety of assays, anddo not possess corrector or potentiator activity, instead complementing the action of theseother modulators. Amplifiers increase CFTR protein levels in cell lines and HBEs withdemonstrated activity across models derived from CF donors with different genotypes, andacross different classes of mutant CFTR. In addition to lung-derived cellular models, amplifiersshow activity in primary cells from non-lung tissues, and when orally dosed in animals.
The increase in immature CFTR protein levels elicited by amplifiers also results in CFTR mRNAstabilization. This is consistent with a model in which the novel amplifier class of CFTRmodulators work by enhancing CFTR translation efficiency. Acting at an early step in CFTRsynthesis to provide more protein, amplifiers can be used in combinations to boost the activityof additional CFTRmodulators.
Funded in part by a Therapeutics Development Award from Cystic Fibrosis FoundationTherapeutics, Inc.
INTRODUCTION RESULTS
ConstructmRNA
ATG STOP ATG STOP
CFTR5’UTR 3’UTR
exons/introns
ATG STOP
CFTRamplifierstargetingCFTRtranslationindependentofendogenousCFTRtranscriptionalcontrol
EndogenousCFTRGene
CFTRtranslationenhancement
Endogenous CFTRtranscriptionalmodulation
MolecularTargets
Enriched
MolecularTargets
Minimized
CFTRtranslationenhancement
CMVandexogenous
transcriptionalregulation
PTICuratedCompound
Library
PhenotypicHTS
LeadOptimizationinHBEcells
Disease-RelevantTranslationTM in
HBE
CFBE
HBE
CFTR
CFTR IREs PUROMYCIN5’UTR 3’UTRCMV
Cell Line Reporter Strategy Identifies A New Class of CFTR Modulator
Amplifier Increases F508del-CFTR Protein
Amplifier Stabilizes CFTR mRNA
Amplifier Increases F508del-CFTR Function
Amplifier Provides More Substrate for Other CFTR Modulators
• The Mutant Surface and Total Protein Expression Assay (Sharp Edge Labs, Inc.) shows thatPTI-CH selectively increases F508del-CFTR total expression and does not have this effect onG268V-PgP
• HBE western blot analysis shows PTI-CH increases the levels of immature F508del-CFTR morethan mature F508del-CFTR, distinguishing it from correctors which preferentially increasematuration of CFTR
0%
100%
200%
300%
400%
DMSO VRT-325 lumacaftor PTI-CHEx
pres
sion
Lev
el(R
elat
ive
to D
MSO
)
Surface G268V PgP ProteinTotal G268V PgP ProteinG268V PgP mRNA
0%
50%
100%
150%
200%
250%
DMSO lumacaftor PTI-CH PTI-CH+
lumacaftor
F508
delC
FTRProteinLevels
(RelativetoDMSO
)
BandC(MatureCFTR)
BandB(immatureCFTR)
HEK Cell Reporter System F508del/F508del HBE Western
DMSO
PTI-CH
1.52
2.53
3.54
4.55
5.5
Hal
f-life
(hou
rs)
DMSO PTI-CH
Approach to steady-state in CFBE CellsTet-Off CFTR transcriptional shut-off in
HEKs
0 5 10 15 20 2500.5
11.5
22.5
33.5
Time (h)
EU in
corp
orat
ion
(DM
SO
stan
dard
ized
)
DMSO
0 1 0 2 00
1
2
3
T o ta l m R N A L e v e ls
T re a tm e n t T im e (h o u rs )
Pol
yA R
NA
(m
gram
s)0 2 4 6 hours post DOXt1/2 = 1 hourt1/2 = 3 hours
PTI-CH
• In inducible cells with transcription shut-off, F508del-CFTR mRNA is stabilized 3-fold in thepresence of amplifier
• Approach to steady state analysis shows CFTR mRNA half-life in response to PTI-CH isincreased by nearly 2-fold
• Total mRNA levels are not impacted by PTI-CH
-5
0
5
10
15
20
900 1400 1900
Isc(u
Amp/cm
^2)
Time(seconds)
23%38% 35%
65% 64%
113%
100%
180%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
F508
del C
FTR
Inhi
bita
ble
Curr
ent
(% iv
acaf
tor +
lum
acaf
tor)
fsk
IVA CFTRinh172
PTI-CH+
lumacaftor
lumacaftor
DMSO
• Ussing chamber current measurements of F508del homozygous HBEs treated for 24 hourswith PTI-CH and lumacaftor show enhanced chloride transport relative to lumacaftor
• PTI-CH confers a 1.5 to 2-fold increase to F508del-CFTR function as a stand-alone or incombination with corrector and/or potentiator
fsk
IVACFTRinh172
71%
100%128%
201%
0%
50%
100%
150%
200%
250%
R117
H/F508
delC
FTRCh
lorid
eTran
sportA
ctivity
(%ofivacafto
r)
ivacaftor+
PTI-CH
PTI-CHivacaftor
DMSO
• PTI-CH works in genotypes other than F508del/F508del
• PTI-CH enhances R117H/F508del CFTR function to levels greater than or equal to ivacaftoralone
• PTI-CH enhances ivacaftor activity in R117H/F508del HBEs
Tubulin
Band CBand B
150
100
250
DM
SO
lum
acaf
tor
PTI-C
H
PTI-C
H +
lum
acaf
tor
• PTI-CH works in F508del/F508del intestinal organoids (experiments performed at HUB)
• Increasing doses of PTI-CH show in vivo activity in non-CF rats with endogenous wild-typeCFTR
Amplifier Exhibits Activity in an Intestinal Organoid Model and in vivo
30%
50%
100%
145%
0%
20%
40%
60%
80%
100%
120%
140%
160%
Forskolin-In
ducedSw
elling
(Relativetolumacaftor+
ivacaftor)
F508del/F508del Human Intestinal Organoid Model
103%
134%
160%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
1 2 3
CFTR
mRN
A Le
vels
(Rel
ativ
e to
veh
icle
)
Non-CF Rat Lung
vehicle PTI-CH
Amplifier Can Exceed Ivacaftor in in vitro Efficacy and Doubles It in Combination
Model for Amplifier Effect on CFTR Translational Efficiency
CFTRmRNAbindstotheribosome
Signalsequenceemergesandrecognizedbythesignalrecognitionparticle(SRP)
SRPbinds mRNA-ribosomecomplextoformtheRibosome-NascentChainComplex(RNC)
SRPshuttles theRNCtotheERsurfaceandimmatureCFTRproteinisavailable
ImprovedefficiencyoftranslationleadstoincreasedlevelsandstabilityofCFTRmRNA
• CFBE cell western blot of the PTI-CH-driven increase in immature F508del-CFTR that can thenbe converted into mature F508del-CFTR by lumacaftor
• PTI-CH triple combination with other PTI modulators provides greater in vitro efficacy inG85E/F508del HBEs than a two corrector triple combination
100%
211%239%
413%
100%
229%
338%
461%
0%
100%
200%
300%
400%
500%
CFTR
Chl
orid
e Tr
ansp
ort A
ctiv
ity(%
lum
acaf
tor +
ivac
afto
r)
F508del/F508del
G85E/F508del
lumacaftor +VX-661 + +PTI-C1811(Corrector) + + +ivacaftor + +PTI-P271(Potentiator) + +PTI-CH + +
ACKNOWLEDGEMENTSWe would like to thank Robert Bridges, Amita Thakerar and Matt Green at Chicago MedicalSchool for TECC24 functional profiling; Scott Sneddon, Qi Yan and Junriz Delos Santos of SharpEdge Labs for Surface Expression Assays; and Sylvia Fernandez Boj and Rob Vries of HUB fororganoid experiments
CONCLUSIONS• Amplifiers are a new class of CFTR modulator• Amplifiers increase CFTR immature protein and stabilize CFTR mRNA• Amplifiers increase substrate for additional CFTR modulators• Amplifiers work across CFTR genotypes• Amplifiers are active in non-lung tissues and in vivo
DMSO +lumacaftor + + + +ivacaftor + + + +PTI-CH + + + +
DMSO +lumacaftorivacaftor + +PTI-CH + +
lumacaftor + +ivacaftor + + + +PTI-CH + +
Physiological Response to Amplifier in Forskolin-Induced Swelling Assay
0 min
100 min
F508del/ F508del
Intestinal Organoids
A455E/ F508del
Intestinal Organoids
lumacaftor lumacaftor
+
PTI-CH
DMSO PTI-CH
Experiments performed at HUB
CFTR Modulators with Distinct Mechanisms of Action
POTENTIATORS
CORRECTORS
AMPLIFIERS
Potentiators,suchasivacaftor,actbyincreasingtheopeningtimeoftheCFTRchannelresultinginhigherionflow.
Correctors,suchaslumacaftor,arethoughttofacilitatetheprocessingofmutatedCFTRproteinleadingtoimproveddeliveryofCFTRproteintothecellmembrane.
AmplifiersselectivelyincreasetheamountofimmatureCFTRproteininthecellprovidingadditionalsubstrateforcorrectorsandpotentiators toactupon.
INTRODUCTION
RESULTS
Amplifier Can Exceed Ivacaftor in vitro Efficacy and Doubles It in Combination
