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

1. Potent, selective small molecule inhibitors
of type III phosphatidylinositol-4-kinase α-yp p p y
and β- and their effects on
phosphatidylinositol signallingp p y g g
D Mik W iDr Mike Waring
Principal Scientist – Medicinal Chemistry
Oncology Innovative Medicines
AstraZeneca
Chemical Biology Meets Drug Discovery 2014
12th June 2014
Windlesham

2. The PI3Kinase cascade in cell signalling
2

3. 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- isoforms
ll i l bl h
PIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
are small insoluble enzymes, whereas
type III PI 4-kinases α- and β- isoforms
are 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
PTEN
Class I PI3K
SHIP1/2
p y
vesicular trafficking from the Golgi to the
plasma membrane or endosomes
•PI4P is also the precursor of the key O
P
OOR1
O
R2
O
O
OH
OH
Chiral
OH
O
O
O
OO
R2
OR1
OH
Chiral
R1 >0
R2 >0
PI4K6
•PI4P is also the precursor of the key
signaling molecule phoshatidylinositol-
4,5-bisphosphate (PIP2)
P
O O
OH
OH
OH
O
P
O
O
O
OH
OH
O
P
O
O
O
OH
OH P
OO
OO
OO
R2 Chiral
OH
O
P
O
O O
O
O
P
O
O
OO
R2
OR1
Chiral
1
2
PI4K
PIP5K
PI3K
3
4
6
5
3
O
O
O
P
O
O
O
OH
OH
O
P
O
O
OO
O
R1
O
P
O
O
O
O
P
O
O
O
OH
P
OO
n-C1 7 H3 5

4. 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)
PIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
• It is unknown whether inhibition of PI4K
will result in efficacy in pre-clinical models
with INPP4B loss PTEN
Class I PI3K
SHIP1/2
• 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
P
OOR1
O
O
R2
O
O
O
OH
OH
Chiral
OH
O
O
P
O
O
O
OO
R2
O
O
R1
OH
Chiral
R1 >0
R2 >0
1
2
PI4K
3
4
6
5
melanoma, ovarian
• Key challenge is to confirm linkage
between INPP4B loss and PI4K inhibition
OH
OH
OH
O
P
O
O
OH
OH
OO
OH
O
P
OO
O
O
O
O
OO
R2
OR1
Chiral
OH
O
P
O
O O
O
O
P
O
O
OO
R2
O
O
R1
Chiral
PIP5K
PI3K
3
4
between INPP4B loss and PI4K inhibition O
O
P
O
O
O
OH
OH
O
P
O
O
OO
P
O
O
O
P
O
O
O
OH
OO
n-C1 7 H3 5

5. Known probe compounds
O
O
N
O
H
O
O
O
O
O
LY294002
O
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 identified
expression of type III PI 4-kinase β as critical for proliferation of multiple
breast 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 against
recombinant 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 yp
enzymes on the PI pathway also took place

6. 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
6

7. Effect of PI4Kβ inhibitors on PI3K pathway
Class III PI3K PI4Ks PIKFYVE
PIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
• 10 nM PI4Kβ inhibitor
• No effect on phospho-Akt up to 30 μM
PTEN
Class I PI3K
SHIP1/2
PDPK1
7
Akt Akt Ser308 P

8. PI3Kγ +
8

9. PI4Kβ +
9

10. PI4Kβ +
10

11. PI4Kα +
S
O
O
N
H
N
O
S
N
HN
11

12. 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

13. 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
13

14. 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

15. PI4Kα +
S
O
O
N
H
N
O
S
N
HN
15

16. PI4Kα +
16

17. PI4Kβ +
17

18. Effect of PI4Kβ inhibitors on PI3K pathway
S
O
O
N
H
O
Class III PI3K PI4Ks PIKFYVE
H
N
O
N
N
HN
S
PIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
• 10 nM PI4Kβ inhibitor
• No effect on phospho-Akt
(BT474) up to 30 μM
N
O
N H
S
PTEN
Class 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
18
Akt Akt Ser308 P

19. Kinase selectivity of inhibitors
N
O
N H 2
S
N
@10μM
60
70
80
90
100
60
70
80
90
100
@10μM
FGR 98%
ZIPK 72%
STK17A 68%
inhibition@
20
30
40
50
20
30
40
50
inhibition@
%
Millipore 125 kinase panel
0
10
0 50 100 150 200 250 300
0
10
0 20 40 60 80 100 120 140
%
Millipore 259 kinase panel
19

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)
IP1 accumulates when NIH3T3-PDGFRβ cells
Class III PI3K PI4Ks PIKFYVE
are stimulated with PDGF
PIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
PLCγ
IP3
PTEN
Class I PI3K
SHIP1/2
IP1
20

21. 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

22. Inhibition of type III PI 4-kinaseα modulates
levels of PIP and PIP2levels of PIP and PIP2
• Cellular PIP, PIP2 and PIP3 using chemical derivatisation
Class III PI3K PI4Ks PIKFYVE
, 2 3 g
and tandem mass spectrometry
• NIH3T3-PDGFRβ cells were treated with 30μM inhibitor
prior to PDGF stimulation then lipid extractionPIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
prior to PDGF stimulation then lipid extraction
-1 -1 -2.4
PIP PIP2 PIP3
PTEN
Class 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
-2
Basal PDGF stim
-1.6
Basal PDGF stim
-4.2
Basal PDGF stim
22
N
NH
N
N
S
N
N
S
O
O

23. 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 p
at 37ºC before reading fluorescence
• Treatment of cells with the PI4Kα selective inhibitor reduces basal PI(4,5)P2 to
levels similar to Wortmanninlevels similar to Wortmannin
23

24. Cancer cell growth inhibition
Add INPP3 +/- dataClass III PI3K PI4Ks PIKFYVE
PIKFYVE PIP4KsPIP5Ks
PIKFYVE
INPP4 Class II PI3K
PTEN
Class I PI3K
SHIP1/2
24

25. Literature on other PI4K work
PI4Kβ 24 nM
Antimicrob. Agents Chemother. 2012, 56, 5149
F
F3C
F
NH
S
O
O
O
N
N
O PI4Kα 5.0 nM
25
J. Biol. Chem. 2014, 289, 6120
J. Med. Chem. 2014, 57, 2091
N H2N

26. Acknowledgements
AstraZeneca:AstraZeneca: CRTCRTAstraZeneca:AstraZeneca:
David AndrewsDavid Andrews
Vikki FlemingtonVikki Flemington
R b t G iR b t G i
CRT:CRT:
Jennifer McKelvieJennifer McKelvie
Sarita MamanSarita Maman
Robert GarciaRobert Garcia
Carol LenaghanCarol Lenaghan
Marian PrestonMarian Preston
James SmithJames Smith
Martin SwarbrickMartin Swarbrick
Iris TreiniesIris Treinies
Piotr RauboPiotr Raubo
Graeme RobbGraeme Robb
Karen RobertsKaren Roberts
Robert WoodRobert Wood
Paterson:Paterson:
Rachel RowlinsonRachel Rowlinson
Jonathan G. WinterJonathan G. Winter
Paterson:Paterson:
Nullin DivechaNullin Divecha
26