Small molecule inhibitors of PI4 Kinase
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Potent, selective small molecule inhibitors of type III phosphatidylinositol-4-kinase α- and β- and their effects on phosphatidylinositol signalling D Mik W i Dr Mike Waring Principal Scientist – Medicinal Chemistry Oncology Innovative Medicines AstraZeneca Chemical Biology Meets Drug Discovery 2014 12 th June 2014 Windlesham
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Potent, selective small molecule inhibitors of type III phosphatidylinositol-4-kinase α- and β- and their effects on phosphatidylinositol signalling. The importance of small molecule probes to help us understand cellular pathways in cancer
Transcript of Small molecule inhibitors of PI4 Kinase
Potent, selective small molecule inhibitors of type III phosphatidylinositol-4-kinase α-yp p p yand β- and their effects on phosphatidylinositol signallingp p y g g
D Mik W iDr Mike WaringPrincipal Scientist – Medicinal ChemistryOncology Innovative MedicinesAstraZeneca
Chemical Biology Meets Drug Discovery 201412th June 2014Windlesham
The PI3Kinase cascade in cell signalling
2
Type III PI4K and the PIP3 cascade• Phosphatidylinositol (PI) 4-phosphate (PI4P) is generated by multiple PI 4-kinases which phosphorylate the D4
Class III PI3K PI4Ks PIKFYVE
kinases which phosphorylate the D4 position of PI
• Type II PI 4-kinases α- and β- isoformsll i l bl h
PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
are small insoluble enzymes, whereas type III PI 4-kinases α- and β- isoformsare soluble enzymes which are t t ll l t d t PI 3 kistructurally related to PI 3-kinases
• PI4P is generated in many cellular membranes where it plays a role in
PTENClass I PI3K
SHIP1/2
p yvesicular trafficking from the Golgi to the plasma membrane or endosomes
•PI4P is also the precursor of the key OP
OOR1O
R2
OO
OH
OH
Chiral
OH
OO
O
OO
R2
OR1OH
Chiral
R1 >0 R2 >0
PI4K6•PI4P is also the precursor of the key signaling molecule phoshatidylinositol-4,5-bisphosphate (PIP2)
PO O
OH
OH
OH OP
O
O
OOH
OH
OP
O
O
O
OH
OH POO
OOOO
R2 ChiralOH
O
PO
O O
OO
P
OO
OO
R2
OR1
Chiral
12
PI4K
PIP5K
PI3K
34
65
3
OO
OP
O
O
O
OH
OH
OP
O
O
OO
O
R1
OP
O
O
O
OP
OO
O
OH
P
OO
n-C1 7 H3 5
PI4K in cancer• Strategy to target tumours with INPP4B
loss
( )
Class III PI3K PI4Ks PIKFYVE
• PI4K inhibition (siRNA) has been shown to result in inhibition of AKT signalling and cell growth (3 cell lines, AZ data)
PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
• It is unknown whether inhibition of PI4K will result in efficacy in pre-clinical models with INPP4B loss PTEN
Class I PI3KSHIP1/2
• INPP4B loss has been reported in a number of tumour types:
Breast (~ 50% loss of INPP4B reported in TN BrCa), Prostate (~40% loss of INPP4B reported in metastatic disease), O
POOR1
O
O
R2
O
O
OOH
OH
Chiral
OH
OO
P
O
O
O
OO
R2
O
O
R1OH
Chiral
R1 >0 R2 >0
12
PI4K3
4
65
melanoma, ovarian
• Key challenge is to confirm linkage between INPP4B loss and PI4K inhibition
OH
OH
OH OP
OO
OH
OHOO
OH
O
POO
OOO
O
OO
R2
OR1
ChiralOH
O
PO
O O
OO
P
OO
OO
R2
O
O
R1
ChiralPIP5K
PI3K
3
4
between INPP4B loss and PI4K inhibition O
OP
O
O
O
OH
OH
OP
O
O
OOP
OO
OP
OO
O
OHOO
n-C1 7 H3 5
Known probe compounds
O
O
N
O
H
O
O
O
O
O
LY294002O
OO
Wortmannin
pIC50 pIC50p 50
PI4Kα <4.3
PI4Kβ 4.4
p 50
PI4Kα 5.9
PI4Kβ 5.8
PI3Kα 6.2 PI3Kα 8.1
• siRNA mediated depletion of type II and type III PI 4-kinases identifiedsiRNA mediated depletion of type II and type III PI 4 kinases identifiedexpression of type III PI 4-kinase β as critical for proliferation of multiplebreast cancer cell line models (BT474c and MDA MB 468)
A hi h th h t (HTS) f ki b t t t d i t• A high throughput screen (HTS) of a kinase subset was tested againstrecombinant type III PI 4-kinase β using an ADP-Glo assay format
• Follow-up screening against type III PI 4-kinase α and other related
5
p g g ypenzymes on the PI pathway also took place
PI4Kβ Series – Amide SAR
pIC50 pIC50 pIC50 pIC50 pIC50
PI4Kα 5.3
PI4Kβ 7.2
PI3K 6 2
PI4Kα 5.0
PI4Kβ 7.7
PI3K 4 8
PI4Kα 5.5
PI4Kβ 8.0
PI3K 5 4
PI4Kα 6.0
PI4Kβ 8.2
PI3K 5 9
PI4Kα 5.0
PI4Kβ 8.0
PI3K 4 5PI3Kα 6.2
PIP5Kγ 4.9
LogD 2.5
PI3Kα 4.8
PIP5Kγ <4.0
LogD -
PI3Kα 5.4
PIP5Kγ <4.4
LogD 3.6
PI3Kα 5.9
PIP5Kγ 4.7
LogD 1.6
PI3Kα 4.5
PIP5Kγ 4.9
LogD 3.0g g g g g
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Effect of PI4Kβ inhibitors on PI3K pathway
Class III PI3K PI4Ks PIKFYVE
PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
• 10 nM PI4Kβ inhibitor• No effect on phospho-Akt up to 30 μM
PTENClass I PI3K
SHIP1/2
PDPK1
7
Akt Akt Ser308 P
PI3Kγ +
8
PI4Kβ +
9
PI4Kβ +
10
PI4Kα +S
O
O
NH N
OS
NHN
11
PI4Kα Series – initial SAR
pIC50
PI4Kα 6.8
pIC50
PI4Kα 6.2
pIC50
PI4Kα 5.8PI4Kα 6.8
PI4Kβ 6.4
PI3Kα 5.8
PI4Kα 6.2
PI4Kβ 6.0
PI3Kα 6.1
PI4Kα 5.8
PI4Kβ 6.2
PI3Kα 5.8
PIP5Kγ <4.0
LogD 3.9
PIP5Kγ 4.8
LogD -
PIP5Kγ 4.5
LogD 2.7
12
PI4Kα Series – core changes
pIC50
PI4Kα 5.8
pIC50
PI4Kα 5.6
pIC50
PI4Kα 5.0
pIC50
PI4Kα 6.5PI4Kα 5.8
PI4Kβ 6.2
PI3Kα 5.8
PI4Kα 5.6
PI4Kβ 6.1
PI3Kα 5.6
PI4Kα 5.0
PI4Kβ 5.6
PI3Kα 5.1
PI4Kα 6.5
PI4Kβ 6.2
PI3Kα 5.4
PIP5Kγ 4.5
LogD 2.7
PIP5Kγ 5.6
LogD 2.5*
PIP5Kγ <4.0
LogD 2.3
PIP5Kγ 4.8
LogD 1.9
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PI4Kα Series – core changes
pIC50 pIC50 pIC50 pIC50
PI4Kα 6.5
PI4Kβ 6.2
PI3Kα 5 4
PI4Kα 6.4
PI4Kβ 6.5
PI3Kα 5 6
PI4Kα 6.5
PI4Kβ 6.5
PI3Kα 5 2
PI4Kα 8.2
PI4Kβ 5.9
PI3Kα 5 2PI3Kα 5.4
PIP5Kγ 4.8
LogD 1.9
PI3Kα 5.6
PIP5Kγ <4.0
LogD 1.9*
PI3Kα 5.2
PIP5Kγ <4.0
LogD 2.4*
PI3Kα 5.2
PIP5Kγ <4.0
LogD 3.0
14
PI4Kα +S
O
O
NH N
OS
NHN
15
PI4Kα +
16
PI4Kβ +
17
Effect of PI4Kβ inhibitors on PI3K pathway
S
O
O
NH
O
Class III PI3K PI4Ks PIKFYVE
H NO
N
NHN
S
PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
• 10 nM PI4Kβ inhibitor• No effect on phospho-Akt(BT474) up to 30 μM
N
O
N H
S
PTENClass I PI3K
SHIP1/2
N H 2
N
• 6.3 nM PI4Kα inhibitor• Inhibits phospho-Akt with IC50 = 21 μM
PDPK1
p p 50 μ
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Akt Akt Ser308 P
Kinase selectivity of inhibitors
N
O
N H 2
S
N
@ 1
0μM
60
70
80
90
100
60
70
80
90
100
@ 1
0μM
FGR 98%
ZIPK 72%
STK17A 68%
inhi
bitio
n @
20
30
40
50
20
30
40
50
inhi
bitio
n @
%
Millipore 125 kinase panel
0
10
0 50 100 150 200 250 3000
10
0 20 40 60 80 100 120 140
%
Millipore 259 kinase panel
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Inhibition of type III PI 4-kinase prevents l ti f i it l 1 h h t (IP )accumulation of inositol-1-phosphate (IP1)
IP1 accumulates when NIH3T3-PDGFRβ cells
Class III PI3K PI4Ks PIKFYVE
are stimulated with PDGF
PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
PLCγ
IP3
PTENClass I PI3K
SHIP1/2 IP1
20
Inhibition of type III PI 4-kinase prevents l ti f i it l 1 h h t (IP )accumulation of inositol-1-phosphate (IP1)
PLCγ
IP3 IP1
N
O
IP3
N
N H 2
S
N
21
IC50 = 0.50 µM IC50 > 30 µM
Inhibition of type III PI 4-kinaseα modulates levels of PIP and PIP2levels of PIP and PIP2
• Cellular PIP, PIP2 and PIP3 using chemical derivatisationClass III PI3K PI4Ks PIKFYVE
, 2 3 gand tandem mass spectrometry
• NIH3T3-PDGFRβ cells were treated with 30μM inhibitorprior to PDGF stimulation then lipid extraction
PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
prior to PDGF stimulation then lipid extraction
-1 -1 -2.4
PIP PIP2 PIP3
PTENClass I PI3K
SHIP1/2
-1.4
-1.3
-1.2
-1.1
1
-1.2
-1.1
1
-3.2
-3
-2.8
-2.6
2.4
-1.9
-1.8
-1.7
-1.6
-1.5
-1.5
-1.4
-1.3
-4
-3.8
-3.6
-3.4
N
O
-2Basal PDGF stim
-1.6Basal PDGF stim
-4.2Basal PDGF stim
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N
NH
N
N
S
N
N
SO
O
Live cell imaging
•The PH domain of PLCδ1 binds specifically to PI(4,5)P2
• U2OS cells overexpressing PH-PLCδ1 pre-incubated with inhibitors for 60 minp g pat 37ºC before reading fluorescence
• Treatment of cells with the PI4Kα selective inhibitor reduces basal PI(4,5)P2 tolevels similar to Wortmanninlevels similar to Wortmannin
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Cancer cell growth inhibition
Add INPP3 +/- dataClass III PI3K PI4Ks PIKFYVE
PIKFYVE PIP4KsPIP5KsPIKFYVEINPP4 Class II PI3K
PTENClass I PI3K
SHIP1/2
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Literature on other PI4K work
PI4Kβ 24 nM
Antimicrob. Agents Chemother. 2012, 56, 5149
F
F3C
F
NHS
O
O
O
NN
O PI4Kα 5.0 nM
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J. Biol. Chem. 2014, 289, 6120J. Med. Chem. 2014, 57, 2091
N H 2N
Acknowledgements
AstraZeneca:AstraZeneca: CRTCRTAstraZeneca:AstraZeneca:David AndrewsDavid AndrewsVikki FlemingtonVikki FlemingtonR b t G iR b t G i
CRT:CRT:Jennifer McKelvieJennifer McKelvieSarita MamanSarita Maman
Robert GarciaRobert GarciaCarol LenaghanCarol LenaghanMarian PrestonMarian Preston
James SmithJames SmithMartin SwarbrickMartin SwarbrickIris TreiniesIris Treinies
Piotr RauboPiotr RauboGraeme RobbGraeme RobbKaren RobertsKaren Roberts
Robert WoodRobert Wood
Paterson:Paterson:Rachel RowlinsonRachel RowlinsonJonathan G. WinterJonathan G. Winter
Paterson:Paterson:Nullin DivechaNullin Divecha
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