V avg = Δd/Δt a avg = Δv/Δt Δd = v i Δt +.5aΔt 2 v f = v i + aΔt v f 2 = v i 2 + 2aΔd.
V 6 V 1 inhibitor PLN-74809 blocks multiple TGF- 2 A J. Wu ......PLN-74809 is a more potent...
1
Dual α V β 6 /α V β 1 inhibitor PLN - 74809 blocks multiple TGF - β activation pathways associated with IPF M. Decaris, J. Schaub, C. Chen, J. Cha, M. Reilly, S. Ho, G. Lee, M. Rexhepaj, V. Rao, M. Marlow, P. Kotak, L. Hooi, J. Wu, S. Martin, T. Chen, M. Munoz, N. Cooper, T. Hom, F. Rock, K. Leftheris, P. Andre, E. Gorina, E. Lefebvre, S. Turner Pliant Therapeutics, South San Francisco, CA, USA Rationale Integrin α V β 6 - and α V β 1 -mediated activation of TGF-β has been shown to promote fibrosis in the bleomycin (bleo) lung injury model, and elevated levels of α V β 6 are present in human IPF lung tissue. 1-4 We have developed PLN-74809, a dual selective α V β 6 /α V β 1 small-molecule inhibitor (SMi), and compared its antifibrotic properties with single/pan-α V integrin inhibitors and standard-of-care molecules (nintedanib and pirfenidone) across multiple IPF model systems. Methods Inhibitor potency and selectivity were characterized using ligand-binding, primary cell adhesion, and TGF-β reporter cell-based assays. A novel ELISA- based assay was developed to assess α V β 1 tissue levels. Gene expression analysis, hydroxyproline (OHP) quantitation, and/or second harmonic generation imaging of collagen fiber deposition were used to evaluate the antifibrotic properties of inhibitors in precision-cut lung slices (PCLS; generated from IPF patients and bleo-injured mouse explants) as well as in the standard murine bleo model of IPF. Results 1) Integrin α V β 6 and α V β 1 were present at elevated levels in fibrotic human and mouse lung tissue 2) Dual α V β 6 /α V β 1 inhibitor PLN-74809 blocked lung epithelial cell (α V β 6 ) and lung fibroblast (α V β 1 ) adhesion to latency-associated peptide (LAP)/latent TGF-β 3) Collagen gene expression levels in PCLS showed: ▪ Dual inhibition of α V β 6 /α V β 1 is more effective than single inhibition of either integrin alone at reducing collagen gene expression ▪ Dual α V β 6 /α V β 1 inhibition is equipotent to pan-α V inhibition ▪ PLN-74809 is a more potent inhibitor of collagen gene expression than nintedanib or pirfenidone 4) Dual inhibition of α V β 6 /α V β 1 significantly reduced pulmonary collagen deposition in the murine bleo model of IPF Conclusions Dual α V β 6 /α V β 1 inhibitor PLN-74809 offers a targeted approach to block multiple avenues of TGF-β activation in the fibrotic lung. Integrins α V β 6 and α V β 1 Activate Latent TGF-β Resulting in Profibrotic Gene Expression TGF-β-Activating Integrins α V β 6 and α V β 1 Are Present at Elevated Levels in Fibrotic Lung Tissue Dual α V β 6 /α V β 1 Inhibitor PLN-74809 Blocks Lung Epithelial Cell and Fibroblast Adhesion to LAP Dual α V β 6 /α V β 1 Inhibition Reduces Collagen Expression in Precision-Cut Lung Slices (PCLS) Dual α V β 6 /α V β 1 Inhibition Reduces Pulmonary Collagen Deposition in the Murine Bleo Model Conclusions DMSO V 1 SMi V 6 mAb V 1 SMi + V 6 mAb PLN-74809 1.8 M Alk5 SMi 1 M 0.0 0.5 1.0 1.5 Relative Expression (COL1A1 mRNA) ** **** **** DMSO PLN-74809 200 nM Nin 75 nM (Cmax) Pirf 50 M (Cmax) PLN-74809 + Nin PLN-74809 + Pirf Alk5 SMi 1 M 0.0 0.5 1.0 1.5 Relative Expression (COL1A1 mRNA) *** **** *** **** Normal IPF 0 1 2 3 4 5 Lung V 1 Levels IPF Explants V 1 (pg/g total protein) * -9 -8 -7 -6 -5 -4 0 2 4 6 [Cpd] log M [Ab] log g/mL Cell Adhesion Index V 6 Ab PLN-74809 V 1 SMi -10 -9 -8 -7 -6 -5 0.0 0.5 1.0 1.5 [Cpd] log M [Ab] log g/mL Cell Adhesion Index V 1 SMi V 6 Ab PLN-74809 No Bleo Vehicle PBS 100 mg/kg 250 mg/kg 500 mg/kg 0.05 0.10 0.15 0.20 Lung p-Smad3 (Normalized to total Smad3) * *** *** Healthy Lung IPF Lung IPF IPF + Unilateral Lung Transplant IHC staining and PET imaging show elevated α V β 6 levels in IPF lung tissue ELISA-based assay shows elevated α V β 1 levels in IPF lung tissue Horan et al. 2008 1 ▪ Dual inhibition of α V β 6 (on epithelial cells) and α V β 1 (on fibroblasts) offers a targeted approach for blocking TGF-β signaling in the fibrotic lung α V β 6 levels are elevated in bleo-injured mouse lungs; Itgb6 -/- mice are protected from fibrosis α V β 1 levels are elevated in bleo-injured mouse lungs; SMi blocks fibrosis PCLS from IPF patient lung explants (n=5-7 patients) showed: ▪ Dual inhibition of α V β 6 /α V β 1 with PLN-74809, or a combination of inhibitors, is more effective than single inhibition of either integrin at reducing collagen expression ▪ PLN-74809 inhibits collagen gene expression in the presence or absence of nintedanib or pirfenidone Anti-α V β 6 antibody – blocks epithelial cell adhesion only α V β 1 SMi – blocks fibroblast adhesion only PLN-74809 – blocks adhesion by both cell types ▪ Lung epithelial cells increase expression of α V β 6 upon injury, leading to elevated TGF-β activation ▪ PLN-74809 potently blocks epithelial cell adhesion to LAP, thereby preventing activation of TGF-β Epithelial Cell Adhesion to LAP Fibroblast Adhesion to LAP ▪ Fibroblasts also activate latent TGF-β via α V β 1 binding to LAP ▪ PLN-74809 potently blocks fibroblast adhesion to LAP, thereby preventing activation of TGF-β Adapted from: Chen et al. 2016 5 Second harmonic generation imaging showed a dose-dependent reduction in fibrotic collagen deposition with PLN-74809 Interstitial Collagen Area (%) pSmad3/Smad3 Ratio Collagen (OHP) Fractional Synthesis Total Lung Collagen (OHP) Dual α V β 6 /α V β 1 inhibition with PLN-74809 significantly and dose-dependently reduced pSmad3/Smad3 ratio and OHP deposition in bleo-injured mouse lungs A clear PK/PD relationship between plasma levels of PLN-74809 (oral 250 mg/kg) and pSmad3/Smad3 ratio in mouse lung tissue/bronchoalveolar lavage cells was observed ▪ Levels of α V β 6 and α V β 1 , two integrins that activate latent TGF-β through binding to LAP, are increased in fibrotic lung tissue ▪ PLN-74809 blocks lung epithelial cell (α V β 6 ) and lung fibroblast (α V β 1 ) adhesion to LAP, thereby preventing TGF-β activation ▪ Dual inhibition of α V β 6 /α V β 1 in PCLS prepared from IPF patient explants or bleo-injured mouse lungs was more effective at reducing collagen gene expression than single integrin inhibition alone or standard-of-care drugs nintedanib and pirfenidone ▪ Dual inhibition of α V β 6 /α V β 1 with PLN-74809 significantly reduced pulmonary collagen deposition in the bleo mouse model of IPF v 1 SMi V 6 Ab PLN-74809 200 nM pan- V SMi #1 1 M pan- V SMi #2 1 M Alk5 SMi 1 M 0.00 0.25 0.50 0.75 1.00 1.25 Col1a1 expression vs vehicle control ** ** ** **** Vehicle PLN-74809 200 nM Nin 75 nM Pirf 50 uM PLN-74809 + Nin PLN-74809 + Pirf ALK5 SMi 0.0 0.5 1.0 1.5 2.0 Col1a1 expression vs DMSO control ** ** ** **** PLN-74809 Nintedanib Pirfenidone 0 200 400 600 800 1000 500000 600000 700000 800000 [c] resulting in 50% inhibition of Col1a1 expression (nM) 172 nM 897 nM 678 M 1 2 3 4 COL1A1 Expression in Human IPF PCLS COL1A1 Expression in Human IPF PCLS PCLS from bleomycin-injured mouse lungs showed: ▪ Dual inhibition of α V β 6 /α V β 1 with PLN-74809, or a combination of inhibitors, is more effective than single inhibition of either integrin at reducing collagen expression ▪ Dual α V β 6 /α V β 1 inhibition is equipotent to pan-α V inhibition ▪ PLN-74809 is more potent than nintedanib or pirfenidone at blocking collagen gene expression in fibrotic lung tissue COL1A1 Expression in Bleo-injured Mouse PCLS COL1A1 Expression in Bleo-injured Mouse PCLS Concentration Required to Inhibit PCLS Collagen Expression by 50% References: 1. Horan et al. Am J Respir Crit Care Med 2008;177:56-65; 2. Munger et al. Cell 1999;96:319-328; 3. Henderson et al. Nat Med 2013;19:1617-24; 4. Reed et al. Sci Transl Med 2015;7:288ra79; 5. Chen et al. J Mol Cell Cardiol 2016;93:162-174 Key abbreviations: Bleo, bleomycin; LAP, latency-associated peptide; OHP, hydroxyproline; PCLS, precision-cut lung tissue slices; SMi, small-molecule inhibitor Disclosures: All authors were employed by Pliant Therapeutics Inc. at the time of their contribution to the studies reported here. p < 0.05 **p<0.01 ***p<0.001 ****p<0.0001 **p < 0.01 ****p < 0.0001 *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 β6+/+ β6-/- Normal Bleomycin 0 20 40 60 Murine V 6 (pg/ug total protein) Lung v 6 Levels Murine Tissue Normal Bleomycin 0.0 0.1 0.2 0.3 Murine V 1 (pg/ug total protein) Lung v 1 Levels Murine Tissue Munger et al. 1999 2 Reed et al. 2015 4 Lung OHP p < 0.01 p < 0.01
Transcript of V 6 V 1 inhibitor PLN-74809 blocks multiple TGF- 2 A J. Wu ......PLN-74809 is a more potent...
Dual αVβ6/αVβ1 inhibitor PLN-74809 blocks multiple TGF-β activation pathways associated with IPFM. Decaris, J. Schaub, C. Chen, J. Cha, M. Reilly, S. Ho, G. Lee, M. Rexhepaj, V. Rao, M. Marlow, P. Kotak, L. Hooi,
J. Wu, S. Martin, T. Chen, M. Munoz, N. Cooper, T. Hom, F. Rock, K. Leftheris, P. Andre, E. Gorina, E. Lefebvre, S. TurnerPliant Therapeutics, South San Francisco, CA, USA
Rationale
Integrin αVβ6- and αVβ1-mediated activation of TGF-β has been shown topromote fibrosis in the bleomycin (bleo) lung injury model, and elevatedlevels of αVβ6 are present in human IPF lung tissue.1-4 We have developedPLN-74809, a dual selective αVβ6/αVβ1 small-molecule inhibitor (SMi), andcompared its antifibrotic properties with single/pan-αV integrin inhibitorsand standard-of-care molecules (nintedanib and pirfenidone) acrossmultiple IPF model systems.
Methods
Inhibitor potency and selectivity were characterized using ligand-binding,primary cell adhesion, and TGF-β reporter cell-based assays. A novel ELISA-based assay was developed to assess αVβ1 tissue levels. Gene expressionanalysis, hydroxyproline (OHP) quantitation, and/or second harmonicgeneration imaging of collagen fiber deposition were used to evaluate theantifibrotic properties of inhibitors in precision-cut lung slices (PCLS;generated from IPF patients and bleo-injured mouse explants) as well as inthe standard murine bleo model of IPF.
Results
1) Integrin αVβ6 and αVβ1 were present at elevated levels in fibrotic humanand mouse lung tissue
2) Dual αVβ6/αVβ1 inhibitor PLN-74809 blocked lung epithelial cell (αVβ6)and lung fibroblast (αVβ1) adhesion to latency-associated peptide(LAP)/latent TGF-β
3) Collagen gene expression levels in PCLS showed:
▪ Dual inhibition of αVβ6/αVβ1 is more effective than single inhibition ofeither integrin alone at reducing collagen gene expression
▪ Dual αVβ6/αVβ1 inhibition is equipotent to pan-αV inhibition
▪ PLN-74809 is a more potent inhibitor of collagen gene expression thannintedanib or pirfenidone
4) Dual inhibition of αVβ6/αVβ1 significantly reduced pulmonary collagendeposition in the murine bleo model of IPF
Conclusions
Dual αVβ6/αVβ1 inhibitor PLN-74809 offers a targeted approach to blockmultiple avenues of TGF-β activation in the fibrotic lung.
Integrins αVβ6 and αVβ1 Activate Latent TGF-β Resulting in Profibrotic Gene Expression
TGF-β-Activating Integrins αVβ6 and αVβ1 Are Present at Elevated Levels in Fibrotic Lung Tissue
Dual αVβ6/αVβ1 Inhibitor PLN-74809 Blocks Lung Epithelial Cell and Fibroblast Adhesion to LAP
Dual αVβ6/αVβ1 Inhibition Reduces Collagen Expression in Precision-Cut Lung Slices (PCLS)
Dual αVβ6/αVβ1 Inhibition Reduces Pulmonary Collagen Deposition in the Murine Bleo Model
Conclusions
DM
SO
V 1
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V 6
mAb
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mAb
PLN-7
4809
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M
Alk
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Rela
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xp
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(CO
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RN
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******
****
DM
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PLN-7
4809
200
nM
Nin
75
nM (C
max
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Pirf 5
0 M
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ax)
PLN-7
4809
+ N
in
PLN-7
4809
+ P
irf
Alk
5 SM
i 1
M
0.0
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1.0
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xp
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A)
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Normal IPF
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Lung V1 Levels
IPF Explants
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pg
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2
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[Cpd] log M[Ab] log g/mL
Cell A
dh
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V6 Ab
PLN-74809
V1 SMi
-10 -9 -8 -7 -6 -50.0
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[Cpd] log M[Ab] log g/mL
Cell A
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V1 SMi
V6 Ab
PLN-74809
No B
leo
Vehic
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mg/k
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g
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0.10
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Lu
ng
p-S
mad
3
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rmalize
d t
o t
ota
l S
mad
3) *
***
***
Healthy Lung
IPF Lung
IPF
IPF + Unilateral Lung
Transplant
IHC staining and PET imaging showelevated αVβ6 levels in IPF lung tissue
ELISA-based assay shows elevated αVβ1 levels in IPF lung tissue
Horan et al. 20081
▪ Dual inhibition of αVβ6 (on epithelial cells) and αVβ1 (on fibroblasts) offers a targeted approach for blocking TGF-β signaling in the fibrotic lung
αVβ6 levels are elevated in bleo-injured mouse lungs; Itgb6-/- mice are protected
from fibrosis
αVβ1 levels are elevated in bleo-injured mouse lungs; SMi blocks fibrosis
PCLS from IPF patient lung explants (n=5-7 patients) showed:
▪ Dual inhibition of αVβ6/αVβ1 with PLN-74809, or a combination of inhibitors, is more effective than single inhibition of either integrin at reducing collagen expression
▪ PLN-74809 inhibits collagen gene expression in the presence or absence of nintedanib or pirfenidone
Anti-αVβ6 antibody – blocks epithelial cell adhesion onlyαVβ1 SMi – blocks fibroblast adhesion only
PLN-74809 – blocks adhesion by both cell types
▪ Lung epithelial cells increase expression of αVβ6 upon injury, leading to elevated TGF-β activation
▪ PLN-74809 potently blocks epithelial cell adhesion to LAP, thereby preventing activation of TGF-β
Epithelial Cell Adhesion to LAP
Fibroblast Adhesion to LAP
▪ Fibroblasts also activate latent TGF-β via αVβ1 binding to LAP
▪ PLN-74809 potently blocks fibroblast adhesion to LAP, thereby preventing activation of TGF-β
Adapted from: Chen et al. 20165
Second harmonic generation imaging showed a
dose-dependent reduction in fibrotic collagen
deposition with PLN-74809
Interstitial Collagen Area (%)
pSmad3/Smad3 Ratio Collagen (OHP) Fractional Synthesis
Total Lung Collagen (OHP)
Dual αVβ6/αVβ1 inhibition with PLN-74809 significantly and dose-dependently reduced pSmad3/Smad3 ratio and OHP deposition in bleo-injured mouse lungs
A clear PK/PD relationship between plasma levels of PLN-74809 (oral 250 mg/kg) and pSmad3/Smad3 ratio in mouse lung tissue/bronchoalveolar lavage cells was observed
▪ Levels of αVβ6 and αVβ1, two integrins that activate latent TGF-βthrough binding to LAP, are increased in fibrotic lung tissue
▪ PLN-74809 blocks lung epithelial cell (αVβ6) and lung fibroblast (αVβ1) adhesion to LAP, thereby preventing TGF-β activation
▪ Dual inhibition of αVβ6/αVβ1 in PCLS prepared from IPF patient explants or bleo-injured mouse lungs was more effective at reducing collagen gene expression than single integrin inhibition alone or standard-of-care drugs nintedanib and pirfenidone
▪ Dual inhibition of αVβ6/αVβ1 with PLN-74809 significantly reduced pulmonary collagen deposition in the bleo mouse model of IPF
v 1
SM
i
V 6
Ab
PLN-7
4809
200
nM
pan-
V S
Mi #
1 1 M
pan-
V S
Mi #
2 1 M
Alk
5 SM
i 1
M
0.00
0.25
0.50
0.75
1.00
1.25
Co
l1a1 e
xp
ressio
n v
s
veh
icle
co
ntr
ol
**
** **
****
Vehicle
PLN-7
4809
200
nM
Nin
75
nM
Pirf 5
0 uM
PLN-7
4809
+ N
in
PLN-7
4809
+ P
irf
ALK
5 SM
i
0.0
0.5
1.0
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2.0
Co
l1a1 e
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n v
s
DM
SO
co
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ol
**** **
****
PLN-7
4809
Nin
tedan
ib
Pirfe
nidone
0
200
400
600
800
1000500000
600000
700000
800000
[c]
res
ult
ing
in
50%
in
hib
itio
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of
Co
l1a1 e
xp
ressio
n (
nM
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172 nM
897 nM
678 M
1
2
3 4
COL1A1 Expressionin Human IPF PCLS
COL1A1 Expressionin Human IPF PCLS
PCLS from bleomycin-injured mouse lungs showed:
▪ Dual inhibition of αVβ6/αVβ1 with PLN-74809, or a combination of inhibitors, is more effective than single inhibition of either integrin at reducing collagen expression
▪ Dual αVβ6/αVβ1 inhibition is equipotent to pan-αV inhibition
▪ PLN-74809 is more potent than nintedanib or pirfenidone at blocking collagen gene expression in fibrotic lung tissue
COL1A1 Expressionin Bleo-injured Mouse PCLS
COL1A1 Expressionin Bleo-injured Mouse PCLS
Concentration Required to Inhibit PCLS Collagen Expression by 50%
References: 1. Horan et al. Am J Respir Crit Care Med 2008;177:56-65; 2. Munger et al. Cell 1999;96:319-328; 3. Henderson et al. Nat Med 2013;19:1617-24; 4. Reed et al. Sci Transl Med 2015;7:288ra79; 5. Chen et al. J Mol Cell Cardiol 2016;93:162-174
Key abbreviations: Bleo, bleomycin; LAP, latency-associated peptide; OHP, hydroxyproline; PCLS, precision-cut lung tissue slices; SMi, small-molecule inhibitor
Disclosures: All authors were employed by Pliant Therapeutics Inc. at the time of their contribution to the studies reported here.
p < 0.05
**p<0.01***p<0.001
****p<0.0001
**p < 0.01****p < 0.0001
*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
β6+/+ β6-/-
Norm
al
Ble
omyc
in
0
20
40
60
Mu
rin
e
V
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(pg
/ug
to
tal p
rote
in) **
Lung v6 Levels
Murine Tissue
Norm
al
Ble
omyc
in
0.0
0.1
0.2
0.3
Mu
rin
e
V
1
(pg
/ug
to
tal p
rote
in)
**
Lung v1 Levels
Murine Tissue
Munger et al. 19992
Reed et al. 20154
Lung OHP
p < 0.01p < 0.01
