Overview of oncology drug discovery and development projects

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Overview of oncology drug discovery and
development projects Burnham Institute for
Medical Research Kristiina Vuori, M.D.,
Ph.D. Inaugural Chemical Biology Consortium
Meeting August 10, 2009 NCI/NIH, Bethesda
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Burnham Institute for Medical Research

• 1 of 7 NCI-designated basic research cancer
  centers
• Research Programs
• Tumor Microenvironment
• Tumor Development
• Signal Transduction
• Apoptosis and Cell Death Research

• 1 of 4 Comprehensive Screening Centers in MLPCN
• Assay development HCS screens and algorithm
  development
• HTS uHTS compound libraries
• Cheminformatics
• NMR-based drug discovery
• Medicinal chemistry
• Exploratory pharmacology
• Mouse tumor models for efficacy studies
• Functional genomics (RNAi screens libraries)

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Burnham Molecular Target Projects

• Apoptosis targets (Bcl-2, IAP, DRs, Caspases)
• Autophagy targets (Atg4, ULK)
• Chaperones (Hsp 70 family)
• Cell growth survival (PI3K/Akt/mTOR)
• Cell proliferation (Erk/JNK/p38, ATF2)
• Cell adhesion, invasion angiogenesis
  (integrins, FAK, MT1-MMP, Eph, CCR6, invadopodia)
• NF-kB pathway activation (Ubc13)
• Hypoxia (HIF1a pathway) (Siah-2)
• Cancer cell metabolism (FAS, 3-D spheroid
  screens)
• Tumor suppressors (PML pathway)

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Burnham Molecular Target Projects

• Apoptosis targets (Bcl-2, IAP, DRs, Caspases)
• Autophagy targets (Atg4, ULK)
• Chaperones (Hsp 70 family)
• Cell growth survival (PI3K/Akt/mTOR)
• Cell proliferation (Erk/JNK/p38, ATF2)
• Cell adhesion, invasion angiogenesis
  (integrins, FAK, MT1-MMP, Eph, CCR6, invadopodia)
• NF-kB pathway activation (Ubc13)
• Hypoxia (HIF1a pathway) (Siah-2)
• Cancer cell metabolism (FAS, 3-D spheroid
  screens)
• Tumor suppressors (PML pathway)

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Apoptotic signaling pathways
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Broad Spectrum Bcl-2 antagonists for cancer
chemotherapy
Chemotherapy/Radiation
Chemotherapy/Radiation
DNA
Anti-apoptotic Bcl-2 proteins
Pro-apoptotic Bcl-2 proteins
n gt 15 Bax Bak Bad Bid etc.
n 6 Bcl-2 Bcl-XL Mcl-1 Bcl-W Bfl-1 Bcl-B
Caspase activation
Cancer progression proliferation
Cancer Cell Apoptosis
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Burnham Molecular Target Projects

• Apoptosis Targets
• Bcl-2 family proteins
• FPA-, TR-FRET-based assays (BH3-peptide ligands)
• NMR-based drug discovery and optimization
• Cell-based assays
• TR3/Nur77 conversion assay for Bcl-2
• GFP-reporter assays for Bcl-2 expression
• Lead candidates broad spectrum Bcl-2 antagonists

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Current Bcl-2-targeted therapies rely on drugs
specific for Bcl-2 and Bcl-XL, leaving other
family members unaddressed
Genasense (anti Bcl-2)
ABT-737 (anti Bcl-XL/Bcl-2)
Mar 6, 2009 - Genta Incorporated announced that
the Food and Drug Administration's (FDA) Center
for Drug Evaluation and Research (CDER) has
decided that available data are not adequate to
support approval of Genasense (oblimersen sodium)
Injection for treatment of patients with relapsed
or refractory chronic lymphocytic leukemia
(CLL).
An inhibitor of Bcl-2 family proteins induces
regression of solid tumours. Nature 2005, 435,
677-681 ABT-737 and ABT-263 in late preclinical
/early clinical studies against CLL. Very potent
against Bcl-XL and Bcl-2 but lacks activity
against Mcl-1, Bfl-1 and Bcl-B
http//www.jco.org/cgi/content/abstract/JCO.2006.0
6.0483v1
AT101 -Currently in Phase IB/II trials by Ascenta
Therapeutics
Concurrent upregulation of Bcl-XL and Bfl-1
induces 1000-fold resistance to ABT-737 in
chronic lymphocytic leukemia Vogler et al. Blood
2008
Concurrent upregulation of Bcl-2 and Mcl-1
renders AML cells insensitive to
ABT-737 Konopleva et al. Cancer Cell 2006
Kitada, S. Leone, M. Reed, J. C., Pellecchia, M
J. Med. Chem (2003)
Multiple cellular targets (e.g. sperm LDH male
contraceptive) Presence of two aldehydes makes it
highly reactive (GI toxicity) (Kitata et al.
Blood 2008)
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ApoGossypol and Derivatives as Drug Candidates

• Retain full activity against Bcl-2 family
  proteins, inhibiting 5 to 6 anti-apoptotic
  members
• Lack activity against LDH (more selective)
• Lack aldehydes (non-reactive)
• Stable formulations identified
• Superior stability in plasma
• Superior PK (AUC after oral administration)
• Much less toxic to normal tissues (allows higher
  doses)
• Display single-agent anti-tumor activity in mouse
  transgenic cancer model
• Reduced off-target effects compared to Gossypol
• Support obtained from the NCI-RAID program

Compound 8r (BI79H5)
Arg143
Tyr105
P1
Becattini et al., Reed Pellecchia, Chem Biol
2004 Wei et al., Reed Pellecchia, Mol. Cancer
Ther. 2009 Wei et al., Reed Pellecchia, J.
Med. Chem. 2009
Kitada, S, et al, Pellecchia Reed Blood 111
3211, 2008 Jia, L, et al, Reed Pellecchia
Cancer Chemothr Pharm 61 63, 2008
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NCI-CBC Development Opportunities for ApoGossypol
and its derivatives

• Broad oncology indications Prostate, Breast,
  Lung, Lymphoma, Leukemia
• GMP grade ApoGossypol available
• Enantiomers/atropoisomers separation established
  for BI79H5 GMP production required
• IND enabling Formulation/PK/TOX required
• IP patents pending on compounds shown

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Small Molecule Inhibitors of IAP proteins
3D Structure of IAP / Caspase complex (BIR2 /
caspase3)
(Adapted from Mufti et. al Arch Biochem Biophys.
2007 463168-74)
Riedl et al. Cell 104791, 2001
Riedl, et al. Cell 104791, 2001
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Burnham Molecular Target Projects

• Apoptosis Targets
• IAP family proteins
• FPA-based assays (SMAC-peptide ligands) Enzyme
  derepression assays
• NMR-based drug discovery and optimization
• yeast-based functional assay for XIAP/caspase-3
  interaction
• Structure-based drug design of novel inhibitors

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Small Molecule Inhibitors of IAP proteins
Small-molecule IAP antagonists (in Phase I
clinical trials) reported to date generally have
high affinities for the BIR3 domain of XIAP as
well as the BIR3 domains of c-IAP1 and 2.
BIR2

• Structure-based design and synthesis of new small
  molecule inhibitors at Burnham
• bind to both BIR2 and BIR3 domain
• are highly selective for BIR2 vs. BIR3
• CBC discovery and development opportunity Lead
  optimization in progress

BIR2 active Smac Mimetic
SMAC binding Groove (crystal contact)
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Autophagy as a cell survival mechanism
Autophagy is an evolutionarily conserved process
whereby cells catabolize proteins and organelles
for purposes of generating substrates for
sustaining ATP production during times of
nutrient deprivation.
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Autophagy and Cancer

• Recent evidence has implicated autophagy in the
  survival of cancer cells in the context of
  nutrient deprivation and other stressful
  circumstances.  
• Chemical modulators of autophagy are essentially
  non-existent, with the only available agent,
  3-methyladenine, requiring millimolar
  concentrations to inhibit class III phosphatidyl
  inositol kinases involved in autophagy.

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Burnham Molecular Target Projects

• Autophagy Targets
• Autophagins (ATG4 family cysteine proteases)
• In vitro biochemical and yeast-based assays
• HCS assays monitoring GFP-p62 and GFP-LC3
  localization

• Ulk1/Atg1 serine threonine kinase
• In vitro kinase assay
• GFP-LC3 HCS assay
• Lead candidate in development

Cys74
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Autophagy Machinery Reveals Several Potential
Drug Discovery Targets
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1º HTS Assay for ATG4 (Autophagin) Developed
based on Cleavable Fusion Protein Enzyme, PLA2
PLA2-based HTS assay for recombinant ATG4B. A,
ATG4B cleaves Glycine site of LC3 to release
catalytically active PLA2, which is able to turn
over NBD-C6-HPC, librating fluorescence. B,
Serial diluted recombinant ATG4B as indicated was
mixed with 100 nM LC3-PLA2 and 20 mM NBD C6-HPC
to final volume of 100 uL in a well in a
black-walled 384-well-plate. C, Serial diluted
LC3-PLA2 as indicated was incubated with or
without 0.1 nM recombinant ATG4B in the PLA2
assay buffer (20 mM Tris-HCl, pH 8.0, 2 mM CaCl2,
and 1mM DTT). D, 0.1 nM ATG4B was incubated with
20 nM LC3-PLA2 in PLA2 assay buffer at RT for 1
h. Fluorescence was measured with excitation and
emission filters of 465 nm and 533 nm,
respectively.
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Secondary ATG4 (Autophagin) Assay Developed Based
on Fluorigenic Peptide Substrates
D
Synthetic peptide-based secondary assay for
ATG4B. A, Recombinant ATG4B cleaves
AFC-conjugated peptide to release fluorescence.
B, Positional scanning strategy using
combinatorial peptide library to define optimal
substrate sequence for ATG4B. C, Summary of
cleavage of fluorogenic tetrapeptide is
presented. The enzyme concentration was 6-8 ?M.
ACC production was monitored using an fmax
multi-well fluorescence plate reader (Molecular
Devices) at excitation wavelength of 355 nm and
an emission wavelength of 460 nm. Assay time
varied from 15-60 min. Standard deviation for
each measurement shown is lt20. The x-axis
indicates the amino-acid tested at the P2, P3, or
P4 positions (standard single letter code for
natural L-amino acids O - nor-leucine, hC
cyclohexylalanine, hP homo-phenylalanine). The
y-axis represents the average relative activity
expressed as a percent of the best amino acid. D,
Recombinant ATG4B (5 ?m) was incubated with
synthetic peptide substrates (100 mM) at 37ºC.
ATG4B activity was measured by AFC production,
which was kinetically monitored at excitation
wavelength 400 nm and emission wavelength 505 nm.

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1º HTS Assay for ATG4B (Autophagin) Protease
Created in Yeast Using Cleavable Transcription
Factor and Reporter Genes
ATG4B WT / LC3 ATG4B C/A / LC3 Caspase-1 / WEHD
A
B
Cell membrane
ATG4B WT Caspase-1
72 55 36 28
LC3
ATG4B
TF
FasDD-LC3B-TF
SD/Leu
FasDD-LC3B
Nucleus
LacZ
8op
LEU2
6op
SD/X-gal
C
Yeast-based HTS assay for ATG4B. A, The basis for
the yeast-based cleavable reporter assay for
monitoring ATG4B activity is depicted. The
membrane tethered transcription factor consists
of the DNA-binding domain of the LexA protein and
the transactivation domain of the B42 protein,
fused to LC3 and the extracellular and
transmembrane domains of Fas. ATG4B cleaves LC3
to release the chimeric LexA/B42 transcription
factor, leaving the membrane and enter the
nucleus, where it induces expression of LEU2 and
LacZ genes. B, ATG4B wild type, catalytic mutant,
and caspase-1 expressed EGY48 yeast cell were
cultured on the selection medium SD/leucine or
induction medium SD/X-gal. C, For HTS
implementation, yeast cells ( 100 ml/well at
1x104/ml) expressing ATG4B/LC3 (red) or ATG4B
C/A/LC3 (blue) were plated in 384 well plates in
media containing 1 galactose/2raffinose, with
X-gal substrate solution. Plates were incubated
for 48 hrs at 30?C and the OD620 values were
recorded.
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High Content Screening (HCS) Assay for
Autophagy Using GTP-p62 Marker Protein
Knockdown of ATG4B causes GFP-p62 punctation and
reduces p62 degradation under autophagy-inducing
conditions. siRNA against ATG4B was transfected
into GFP-p62 expressed HeLa cells for 48 h. The
cells were cultured in rich medium (DMEM 10
serum) or nutrient poor medium (HBSS 0.1
serum) HBSS for 6 h. GFP-p62 was monitored by
confocal microscopy.
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Workflow for ATG4 (Autophagin) Protease
Inhibitors Program
Primary screening
Secondary assays
Optimization
Yeast-based cleavable Reporter assay
Counter-screen for PLA2 inhibitors
In vitro ADME Optimization
Primary assay LC3-PLA2 assay
Dose dependent IC50 lt 10 mM
Confirmatory Fluorogenic peptide assay
Compound Optimization (SAR)
Counter-screen for GAPDH inhibitors
Mammalian HCS assay (GFP-p62)
Cysteine Protease Selectivity Panel (Caspases
Calpains)
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ULK1 An Autophagy Regulator Downstream from mTOR

• Atg1 serine/threonine protein kinase is a
  critical autophagy regulator in yeast
• Mammalian homologue Unc (uncoordinated)-51-like
  kinase 1 (ULK1)

Hypothesis Inhibit ? tumour cell death
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ULK1 Inhibitor Hit-to-Lead
Small Molecule Hit
Kinase profiling
Novel Leads
Focused library of analogues
e.g. BIM-0206953 Low nanomolarpotency
(Ambit KinomeScan panel of 402 kinases)
BIM-0061859 ULK1 Kd 98 nM

• BIM-0206953
• Highly potent lead, novel structure
• Selectivity profile under evaluation
• Synthesis and testing of additional analogues in
  in vitro kinase assays in progress
• Test best inhibitors in cell culture-based models
  that respond with classic autophagic morphology
• Advance best compounds through ADME/T and PK
  studies to in vivo efficacy models

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Burnham Molecular Target Projects

• Tumor Suppressors
• PML pathway
• High-content cell imaging assays for
  identification of compounds that induce PML
  Oncogenic Domain (POD) activation
• Counter-screens and secondary screens have also
  been developed

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PODs A Tumor Suppressor Pathway
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HCS Assay Development
Assay Implementation Z-Factor 0.6
Algorithm Development
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HCS Hits from Combi-Chem Libraries
Z-Factor 0.6
4 µg/mL Compounds
1422-9
1422-62
1422-10
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Workflow and Secondary Assays
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Burnham Molecular Target Projects

• Apoptosis targets (Bcl-2, IAP, DRs, Caspases)
• Autophagy targets (Atg4, ULK)
• Chaperones (Hsp 70 family)
• Cell growth survival (PI3K/Akt/mTOR)
• Cell proliferation (Erk/JNK/p38, ATF2)
• Cell adhesion, invasion angiogenesis
  (integrins, FAK, MT1-MMP, Eph, CCR6, invadopodia)
• NF-kB pathway activation (Ubc13)
• Hypoxia (HIF1a pathway) (Siah-2)
• Cancer cell metabolism (FAS, 3-D spheroid
  screens)
• Tumor suppressors (PML pathway)