Characterisation of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2Joanne Munck, Valerio Berdini, Luke Bevan, Hannah Braithwaite, Ildiko Buck, Megan Cassidy, Juan Castro, Aurelie Courtin, James Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte Griffiths-Jones, Tom Heightman, Chris Hindley, Birikiti Kidane, Justyna Kucia-Tran,

John Lyons, Vanessa Martins, Sandra Muench, Chris Murray, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, David Rees, Sharna Rich, Caroline Richardson, Harpreet Saini, Lukas Stanczuk, Neil Thompson, Hugh Walton, Nicola Wilsher, Alison Woolford, Nicola WallisAstex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, United Kingdom

Poster presented at AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics Conference, Philadelphia. October 26 - 30, 2017. (Poster available to download from Astex’s website at www.astx.com)

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• The MAPK pathway is commonly hyper-activated in human cancers due to the occurrence of oncogenic mutations in RAF and RAS, and multiple studies have demonstrated that MAPK pathway inhibition suppresses the growth of such cancer cells.

• MAPK inhibition has been clinically validated by BRAF and MEK inhibitors, which are approved for the treatment of BRAFV600E-mutant melanoma and NSCLC. However, response to such agents is often short-lived due to the onset of resistance mechanisms that result in re-activation of ERK1/2 (ERK) signalling1,2.

• RAF and MEK inhibitors have also been clinically tested in other cancers, including BRAF-mutant colorectal (CRC) and KRAS-mutant Non-small cell lung cancer (NSCLC), where they had limited clinical activity3,4.

• As ERK is the primary downstream effector of the MAPK pathway, it is hypothesized that ERK inhibitors may prove to be less susceptible to oncogenic bypass than RAF and MEK inhibitors and therefore have the potential to overcome the limitations of RAF and MEK inhibitors.

• Using fragment-based drug discovery we have developed a novel, potent and selective ERK inhibitor, which inhibits in vitro ERK catalytic activity with a low nM IC50 value and has strong anti-proliferative effects in a wide range of MAPK-activated cell lines.

• In addition to inhibiting ERK catalytic activity, the compound also inhibits the phosphorylation of ERK by MEK and confers a decrease in cellular pERK levels in both BRAF-mutant and KRAS-mutant cell lines (in vitro and in in vivo pharmacodynamic [PD] studies).

• Once daily oral dosing of the lead compound (50 mg/kg) conferred significant anti-tumor activity in a range of in vivo models.• These data support the further optimisation of this series of compounds for clinical development.

1. A novel, potent and selective ERK inhibitor

2. Inhibition of ERK catalytic activity and the proliferation of MAPK-activated cells

INTRODUCTION

• The lead compound inhibits ERK1/2catalytic activity with an IC50 of 3 nM (asdetermined in an ERK TRF Kinase assay).

• It binds to the active site of ERK2 (wherethe adenine of ATP binds) and thenexpands in an elongated shape, exploitinga pocket which is created by an unusualmovement of the P-Loop Tyr36 residue.

• The lead compound was highly selectivefor ERK1/2 in a screen of 465 kinases.

• The lead compound potently inhibited ERK catalytic activity in A375 (BRAF-mutant melanoma) and HCT116 (KRAS-mutant CRC) cells, with an IC50 value of 7.2 nM and 5.2 nM, respectively.

• The lead compound potently inhibited the proliferation of cell lines harbouring a range of MAPK aberrations. EC50 values ranged from 3.4 nMin HT29 cells (BRAF-mutant melanoma) to 210 nM in Ma-mel-28 cells (NRAS-mutant melanoma).

i) A375 cells (BRAFV600E melanoma) were treated for 2 hours with compound and pRSK levels determined using aMeso Scale Discovery (MSD) detection assay. Mean ± SEM shown. ii) 96-hr Alamar Blue Proliferation Assay

• A MEK/ERK cascade assay was used to evaluate the effects of the lead compound on the phosphorylation of ERK by MEK.• Assay: Active, phosphorylated MEK incubated with non-phosphorylated ERK in the presence of ATP and compound, and the

degree of phosphorylation of ERK determined. Compounds were also tested in a MEK binding assay in order to distinguishbetween a) ERK inhibitors that prevent the phosphorylation of ERK by MEK and b) MEK inhibitors.

MEK inhibition ERK Phosphorylation modulation

No inhibition

ERK

pMEK PP

ATPP

ERK

ERK

pMEKP

ATP ERK

ERK

pMEKP

ATP ERK

= Phosphorylation

= ERK inhibitor that prevents the phosphorylation of ERK by MEK

= MEK inhibitor

P

MEK/ERK cascade assay. pERK2 detected using anti-pERK2 Europium and d2 labelled antibodies (Cisbio).

• The lead compound prevents thephosphorylation of ERK by MEK, but doesnot inhibit MEK activity.

4. Modulation of pERK levels in BRAF- and KRAS-mutant cell linesi) Cells were treated for 2 hours with

compound and pERK levelsdetermined by ELISA

ii) A375 and HCT116 cells were treated for up to 48 hours with 24 nM and 290 nM, compound respectively (equivalent of 5 x proliferation assay EC50 value). Cell lysates analysed by western blotting.

iii) ERK directly phosphorylates and inhibits wild-type RAF activity. In KRAS-mutant cell lines (e.gHCT116), ERK inhibitors therefore confer an increase in RAF activity and increase in phosphorylation of MEK and ERK. As levels of wild-type RAF signalling are low in BRAFV600E cells (e.g A375), ERK inhibitors are not expected to confer such a strong increase in phosphorylation of MEK and ERK.

• The lead compound inhibited ERK catalytic activity in both A375 and HCT116 cell lines (indicated by the decrease in phosphorylation of the ERK substrates RSK and CRAF).

• Furthermore, it conferred a decrease in the phosphorylation of ERK itself in both cell lines (confirmed by ELISA and western blotting).

• An increase in pMEK was observed in HCT116 cells only. This is consistent with previous observations that the inhibition of ERK-dependent negative feedback has a greater impact in KRAS-mutant than BRAF-mutant cell lines5.

RAF

MEK

ERK

RASmut

RAF

MEK

ERK

RASmut

X

BRAFV600E

MEK

ERK

RASRAS Mutant BRAF MutantpRSK

RSK

pERK 1/2

ERK 1/2

pMEK

pCRAF

β-actin

0 2 4 8 16 24 48 Time (h) 0 2 4 8 16 24 48

MEK

A375 (BRAFmut melanoma)

CRAF

HCT116 (KRASmut CRC)

5. Anti-tumor activity in BRAF- and KRAS-mutant tumor xenografts

6. Inhibition of ERK catalytic activity and the phosphorylation of ERK in xenograft tumor tissue

• Once daily oral administration of 50 mg/kg lead compound conferred significant anti-tumor activity inA375 and Calu-6 xenograft models (p < 0.0001).

The lead compound was administered orally at 50 mg/kg once daily (qd) to BALB/c nude mice bearing either sub-cutaneous A375 or Calu-6 tumor xenografts. Each data point represents mean ± SEM. n = 8 mice per treatment group. Compound was well tolerated.

pERK

tERK

pRSK

Ctrl 2 hr 6 hr 16 hr 24 hr

Calu-6

tRSK

α-tubulin

A single dose of 50 mg/kg lead compound was administered orally to BALB/c mice bearing subcutaneous Calu-6 tumor xenografts. Animals were sacrificed and tumors removed at

indicated time-points. Tumor lysates were analysed by western blotting.

• A single dose of 50 mg/kg inhibited ERK catalytic activity in Calu-6 tumor xenografts (indicated by the decrease in phosphorylation of the ERK substrate RSK).

• Furthermore, the compound conferred a decrease in the phosphorylation of ERK itself.

• The effects on ERK catalytic activity and ERK phosphorylation were sustained for up to 24 hours.

• The direct targeting of ERK is an attractive therapeutic approach to overcoming the limitations of RAF or MEK inhibitors.

• Using fragment-based drug discovery we have developed a novel, potent and selective ERK inhibitor, which in addition to inhibiting ERK catalytic activity also inhibits the phosphorylation of ERK by MEK.

• The compound has in vivo activity, conferring significant anti-tumor activity in A375 (BRAF-mutmelanoma) and Calu-6 (KRAS-mut lung) xenograft models.

• These data support the further optimisation of this compound series for future clinical development. • For more information on the discovery of this compound series, please visit Poster Board Number 161

SUMMARY AND CONCLUSIONS

i)

NO

HNO

N

N

NH

Cl

OH

O

References: 1. Hauschild et al., (2012) Lancet, 380, 358–365 2. Solit and Rosen (2011) N Engl J Med. 364(8), 772-774 3. Kopetz et al. (2015) J Clin Oncol. 33(34), 4032-8 4. Jänne et al., (2017) JAMA. 317(18),1844-1853 5. Hatzivassiliou et al., 2013 Nature12; 501 (7466): 232-6

Heightman et al., Fragment-based discovery of a highly potent orally bioavailable ERK1/2 inhibitor which modulates the phosphorylation and catalytic activity of ERK1/2 (Abstract B161).

ii)

i) ii)

i)

ii) iii)

i) Structure of lead compoundii) Crystal structure of lead compound bound to human ERK2

iii) Activity of lead compound in a panel of 465 kinases

iii) >50% inhibitionat 100 nM

© Astex Pharmaceuticals

3. Modulation of the in vitro phosphorylation of ERK by MEK

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