Modulation of PIP2 Levels through small molecule inhibition of PIP5K David M. Andrews1, Sabina Cosulich1, Nullin Divecha4, Daniel Fitzgerald4, Vikki Flemington1, Cliff Jones1, David R. Jones1, Oliver T. Kern3, Sylvie Lachmann2, Ellen MacDonald3, Sarita

Maman3, Jenny McKelvie2, Kurt Pike1, Rachel Rowlinson1, Michelle C. Riddick3, Graeme Robb1, Karen Roberts1, Martin L. Stockley3, Martin E. Swarbrick3, James M. Smith1, Iris Treinies2,

Mike J. Waring1, Robert J. Wood3

1 AstraZeneca, Oncology iMed, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG

2 CRT Discovery Laboratories, Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, UK 3 CRT Discovery Laboratories, Jonas Webb Building, Babraham Research Campus, Cambridge CB22 3AT, UK 4 Centre for Biological Sciences, University of Southampton, Life Sciences Building (85), Highfield Campus, Southampton SO17 1BJ, UK

HeLa Cells incubated with DMSO 1hr prior to treatment

Introduction Phosphatidyl inositol (4,5)-bisphosphate (PIP2) is a key phospholipid signalling molecule, involved

in cellular processes such as: actin cytoskeletal organisation, cell proliferation and survival1. As

the physiological substrate for both PI3K and PLC, modulation of PIP2 is expected to have an

effect on the AKT and PLC / DAG / IP3 pathways.

Medicinal Chemistry Two separate HTS campaigns of 50K and a focussed set of 100K compounds identified a number

of hit series. SAR was built around three chemotypes, (A, B & C) with biochemical assays

routinely run against all three isoforms of PIP5K, two isoforms of PI4K and PI3Kα. Compounds

were also routinely assayed in pAKT and IP1 cell assays, as surrogate markers for cellular PIP2

levels. MOI studies suggested these compounds were ATP competitive.

References 1. (a) Kisseleva et al., 2005. Mol Cell Biol. 10:3956-66; (b) Emoto et al., 2005. JBC. 280:37901–07

2. Weermink et al., 2004, Eur J Pharm., 500, 87-99

3. DeWald et al., 2005, Cancer Res., 65:713-7

4. Clark et al., 2011, Nature Methods, 8: 267-272

T=1s T=97s T=211s

PH-RFP

Basal conditions Ionomycin EGTA

Basal conditions Ionomycin EGTA

T=1s T=100s T=200s

HeLa Cells incubated with compound 1hr prior to treatment

Series A Series B Series C

PIP5Kα (pIC50) 5.8 7.8 9.3

PIP5Kβ (pIC50) 7.2 8.6 8.8

PIP5Kγ (pIC50) 7.2 8.9 8.9

PI3Kα (pIC50) 5.3 4.5 <4.0

PI4Kα (pIC50) 5.1 4.8 4.0

PI4Kβ (pIC50) 5.1 5.4 4.0

PIP2 inhib @ 3µM 3.7% 21% 17%

Compounds from series B and C emerged as

leading series, with good primary biochemical

inhibition of PIP5K, and improved potency

(pIC50 ~7) in one down-stream biomarker

assay (pAKT). We paid particular attention to

LipE as an efficiency metric and found that we

could design compounds with improved

efficiency. i.e. lower LogD & higher cellular

potency.

Live cell imaging: Ionomycin/Ca2+ activates

PLCs, depleting PIP2 levels; EGTA sequesters

calcium, leads to PIP5K-dependent re-

synthesis of PIP2

PIP5K inhibition slows rate of PIP2 re-

synthesis

Lipid Kinase Selectivity of Each Series Lead

Kinase Panel Selectivity of Each Series

Seri

es B

S

eri

es

A

Se

rie

s

C

PI4

K

Conclusions

1.Three series of potent,

chemically-distinct

series of PIP5K

inhibitors

2.High selectivity is

achievable within the

lipid kinase family

3.Selective tool

compounds

exemplifying PI3K,

PI4K and PIP5K

inhibition offer the

prospect of detailed

pathway deconvolution

4. Initial evidence of cell

potency

Hinge substitution tolerates ranges

of motifs, modest effect on

potency of phys chem props

Hinge binder required

3’-Aniline: required

for potency 5’-substitution:

• Range of amides and other

substituents tolerated

• Opportunity to modulate some phys

chem props (e.g. LogD)

Aryl ring required

Introduction of certain hetero atoms tolerated in both aryl rings

NH

N NH

O

NH

O

O

Thorough SAR evaluation

demonstrated the importance of the

hinge binding motif coupled with an

essential 3’ aniline. The hinge

substituent, along with the 5’ position

and core were extensively modified

to fully explore physicochemical

space, potency and selectivity

Series B SAR Summary

PTEN

Class III PI3K PI4K PIKFYVE

PIP4Ks PIP5Ks PIKFYVE

Class I PI3K

SHIP1/2

INPP4

Downstream pAkt

cell assays

Cellular PI(4,5)P2 is synthesised by phosphorylation of

PI(4)P on the D-5 position of the inositol head group by

phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks).

The family of PIP5K is comprised of 3 isoforms α, β and γ regulated by membrane receptors,

phosphorylation and small GTPases of the Rho and ARF family2. Activation of this pathway is

known to promote growth and invasion of cancer cells, rendering PIP5K an attractive therapeutic

target for antitumour therapies3.

Improved LipE Improved cell

potency HTS output

• Use of published methodology3 to extract and quantify multiple fatty acyl species of PI and PIPs

• Cell model: NIH3T3-PDGFRβ cells. Stimulation with PDGF causes PIP2 depletion

• IP3 and DAG are modulated by PLC; PIP3 via PI3K

• PIP5K inhibition reduces PIP2 and PIP3 in response to RTK activation

Mass Spectrometry Shows that PIP5K Inhibition Prevents Resynthesis of PIP2

• Stimulation with PDGF (green) ↑PIP3

• PI3K inhibitor (blue) ↓PIP3

• PIP5K inhibitor (red) ↓PIP3

•Stimulation with PDGF (green) ↓PIP2

prior to resynthesis

•PIP5K inhibitor (red) ↓PIP2

•PI3K inhibitor (blue) no effect

+ PDGF timecourse PIP

PIP3

PI3K

↑PLCγ

PIP5K

DAG

IP3

pY pY pY

pY

RTK (PDGFRβ)

Ligand (PDGF)

PLCγ

pY

PI3K

+

PIP2

Class II PI3K