NOVEL SMALL MOLECULE DUAL ANTAGONISTS OF XIAP AND CIAP1 GENERATED BY

FRAGMENT-BASED DRUG DISCOVERY (FBDD) ARE EFFECTIVE IN PRE-CLINICAL MODELS

Sandra Muench on behalf of the IAP project team

Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, United Kingdom.

INTRODUCTION

The inhibitor of apoptosis proteins (IAPs) are widely de-regulated in many tumours and contribute to cancer drug resistance. The targeted inhibition of IAPs can switch TNF-alpha signaling in cancer cells from pro-survival to pro-apoptotic. Therefore, IAPs represent an attractive target for cancer therapy.

The IAP family member cellular IAP1 (cIAP1) is involved in the regulation of TNF-alpha signaling and X-linked IAP (XIAP) directly interacts with and inhibits caspases. IAP family members are characterized by BIR (baculoviral IAP repeat) domains, to which the endogenous inhibitor of IAPs SMAC (second mitochondria derived activator of caspases) binds. Peptidomimetic compounds based on the SMAC sequence have been developed, but they show high selectivity for cIAP1. We used our fragment based-drug discovery approach to generate a non-alanine, non-peptidomimetic IAP antagonist, which has dual potency for XIAP and cIAP1. Here we describe the characterization of this compound in in vitro and in vivo models of melanoma and breast cancer.

Regulation of death receptor

signaling by Smac mimetic (). Fulda S Clin Cancer Res 2014;20:3915-3920

OPTIMISATION OF FRAGMENTS CELLULAR ACTIVITY OF AT-IAP

Poster presented at “Cancer Pharmacogenomics and Targeted Therapies 2014” 17-19 September 2014, Cambridge, UK © Astex Pharmaceuticals Inc.

MELANOMA AND BREAST CANCER XENOGRAFT EFFICACY MODEL

• AT-IAP was well tolerated up to 50 mg/kg. p.o. q.d. and shows significant in vivo activity in xenograft models.

MELANOMA AND BREAST CANCER XENOGRAFT PK/PD DATA

• Degradation of cIAP1 and induction of apoptosis markers observed in both tumour types after single doses of AT-IAP

SUMMARY AND CONCLUSIONS

• AT-IAP represents a novel IAP antagonist with a potent dual cIAP1 and XIAP antagonist profile.

• In vitro cell line testing suggests significant activity against a panel of melanoma and primary tumor cell lines, which is enhanced on addition of exogenous TNF-α (1ng/ml).

• In vitro biomarker evaluation shows robust inhibition of cIAP1 and XIAP-Caspase-9 interaction and up-regulation of apoptosis marker.

AT-IAP ACTIVITY IN MELANOMA CELL LINES AND PRIMARY TUMORS

APOPTOSIS EVALUATION OF MELANOMA CELL LINES BY FLOW CYTOMETRY

• Increased caspase-3-substrate staining after 48h treatment with AT-IAP + 1ng/ml TNF-α

A375 melanoma cell line

BIOMARKER EVALUATION IN VITRO BY WESTERN BLOTTING

• cIAP1/2 degradation and apoptosis marker 24h after AT-IAP addition in sensitive cancer cell lines

• Cell viability assay screen of cell lines for AT-IAP sensitivity +/- TNF-α

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Hs

294

T

Mal

me-

3M

RP

MI-

79

51

LB1

319

-MEL

SK-M

EL-2

8

C8

1 6

1

BB

74

-MEL

UZG

4-M

EL

A-3

75

SK-M

EL-2

SK-M

EL-5

KU

L58

-MEL

SK-M

EL-2

4

CM

EL-5

Act

ivit

y A

rea

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

PD

X-2

PD

X-1

5

PD

X-1

PD

X-3

PD

X-1

2

PD

X-5

PD

X-4

PD

X-6

PD

X-7

PD

X-1

0

PD

X-8

PD

X-9

PD

X-1

1

PD

X-1

3

PD

X-1

4

AT-IAPAT-IAP + 1ng/ml TNF-a

AT-IAPAT-IAP + 1ng TNF-a

pIC

50

Cell lines(proliferation)

Ex Vivo PDX Cells(colony formation)

A375Sensitive

SK-MEL-24Insensitive

cIAP1

cIAP2

Cl. PARP

Cl. Casp.-3

1 ng/ml TNF-a

1 mM AT-IAP

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Hs

294

T

Mal

me-

3M

RP

MI-

79

51

LB1

319

-MEL

SK-M

EL-2

8

C8

1 6

1

BB

74

-MEL

UZG

4-M

EL

A-3

75

SK-M

EL-2

SK-M

EL-5

KU

L58

-MEL

SK-M

EL-2

4

CM

EL-5

Act

ivit

y A

rea

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

PD

X-2

PD

X-1

5

PD

X-1

PD

X-3

PD

X-1

2

PD

X-5

PD

X-4

PD

X-6

PD

X-7

PD

X-1

0

PD

X-8

PD

X-9

PD

X-1

1

PD

X-1

3

PD

X-1

4

AT-IAPAT-IAP + 1ng/ml TNF-a

AT-IAPAT-IAP + 1ng TNF-a

pIC

50

Cell lines(proliferation)

Ex Vivo PDX Cells(colony formation)

A375Sensitive

SK-MEL-24Insensitive

cIAP1

cIAP2

Cl. PARP

Cl. Casp.-3

1 ng/ml TNF-a

1 mM AT-IAP

• Balanced cIAP1/XIAP profile

• Non peptidomimetic

• Non alanine warhead

• Potent cellular activity

• Oral activity in in vivo models

Assay Description EC50 (nM)

XIAP Cell Assay HEK293-XIAP-Caspase-9 (I.P) 5.1

ML-IAP Cell Assay HEK293-ML-IAP-SMAC (I.P) 11.0

cIAP1 Cell Assay MDA-MB-231 (cIAP1 degradation) 0.32

Cell Proliferation Assays EVSA-T

MDA-MB-231 HCT-116 (insensitive control)

0.83 4.4

>10,000

• AT-IAP induces cIAP1 inhibition and up-regulation of apoptosis marker in vivo.

• In vivo single agent efficacy can be demonstrated in the A375 melanoma and MDA-MB-231 breast cancer xenograft models.

SK-MEL28

A375

DMSO +TNFa

10nM AT-IAP + TNFa

100nM AT-IAP + TNFa

6 hVeh. Ctr.

AT-IAP 50 mg/kg p.o.

24 h

cIAP1

CleavedPARP

CleavedCaspase-3

48 h

0

1

2

3

4

0

20

40

60

80

100

120

CTR 6 h 24 h 48 h

Tum

ou

r [A

T-IA

P]

µM

cIA

P1

% V

eh. C

TR

cIAP1 level [AT-IAP] µM

PK/PD Data

A375 melanoma xenograft

MDA-MB-231 breast cancer xenograft HEK293 transfected with FLAG-XIAP MDA-MB-231 breast

cancer cell line

• XIAP-Caspase-9 interaction disrupted 2h after AT-IAP addition