Transcript Lead Generation Through Scientific Collaborations

Carolina Innovations Seminar
Center for Integrative Chemical
Biology and Drug Discovery
Stephen V. Frye, Ph.D.
Jian Jin, Ph.D.
nd
Center for Integrative
Chemical Biology
& Drug Discovery
University of North Carolina
Chapel Hill
http://www.pharmacy.unc.edu/labs/center-for-integrative-chemical-biology-and-drug-discovery
September 2 , 2010
Genetic Medicine Building
Center For Integrative Chemical Biology & Drug Discovery
Drug Discovery –vs- Development
Drug Discovery:
Target identification,
Hit to Lead Chemistry,
Target Validation,
Candidate Selection
Hypothesis
generation, revision &
exploration at the
chemistry/biology
interface
>10 years
$1 billion
Drug Development:
Scale-up, Formulation,
Toxicology,
Phase I-III Clinical trials
Regulated process to
establish efficacy &
safety of a potential
new medicine
3
Center For Integrative Chemical Biology & Drug Discovery
Why Academic Drug Discovery?
• Global
• Biotech
– Many diseases are not
adequately addressed
– Understanding of both
molecular targets and disease
pathways is accelerating
• Pharmaceutical Drug
Discovery
– Industry’s reputation and
sustainability in question
– Trend to rely on ‘external’
discovery
– Organizational challenges as
big as the scientific ones
– no more successful per $
spent than majors
– less able to tackle ‘big
problems’
• predictive toxicology
• refinement of HTS –
compounds and technology
• Academic Drug Discovery
– a growing enterprise but…
• fit with academic mission?
• skill sets missing
• resources limited
– where to focus?
CICBDD July 2009
Center For Integrative Chemical Biology & Drug Discovery
Drug Discovery – what can be done better in
academia?
Target selection –
the disease connection
hypothesis
Current industrial paradigm for drug
discovery = A linear process with little
patience for deviation,
“FAIL FAST” mentality
When the target’s role in human
disease is unprecedented,
this rarely works (<5% success rate)
Efficacy
in treating
disease in
Humans
CICBDD July 2009
Center For Integrative Chemical Biology & Drug Discovery
Drug Discovery – what we are trying to do better
at UNC.
biology
Small molecule tool
to enhance target
understanding & validation
Target selection –
the disease connection
hypothesis
UNC can excel
at small molecule
driven elucidation of
biology relevant to
Drug Discovery
chemistry
RESEARCH:
hypothesis generation,
revision & exploration at
the chemistry/biology
interface
Efficacy
in treating
disease in
Humans
Center For Integrative Chemical Biology & Drug Discovery
Center for Integrative Chemical
Biology and Drug Discovery
Vision: The Center will be an
innovative and sustainable scientific
force in the creation of new small
molecule therapeutics to improve
public health, to transform the drug
discovery paradigm and to place UNC
at the forefront of translational
medicine.
Center For Integrative Chemical Biology & Drug Discovery
CENTER Capabilities
• Assay Development (Bill
• Medicinal Chemistry (Jian
Janzen)
Jin & Xiaodong Wang)
– 6 scientists
– 8 internal chemists
– protein expression &
– can support 3-5 projects
purification
– 4 external chemistry
– cell-based or in vitro assays
FTEs to support projects
and tool compound
– multiple platforms & readouts
synthesis
possible
• Computational Chemistry
• Compound Screening
(Dmitri Kireev)
– screens up to 100K
– design of focused
compounds possible
screening sets, virtual
– Three HTS run since 4Q09
screening
– Small tube store installed and
– structure-based design
now loaded
– IT infrastructure
Center For Integrative Chemical Biology & Drug Discovery
CENTER Mission
Center
Initiated
Projects in
Chemical
Biology
Target Proposals
from
UNC Faculty
“responsive
collaborations”
“prospective
science”
small molecule ‘probes’
target validation
drug leads
Center For Integrative Chemical Biology & Drug Discovery
Envisioned process at UNC for
responsive projects
Target I.D.
&
Validation
Investigator
driven target
proposals
with validation
data from
HT-genomics,
HT – siRNA,
In vivo
models
1 year,
50%
success
Hit I.D.
Assay
development
MTS,
focused
sets,
knowledge
-based
design,
HTS
1 year,
80%
Lead
I.D.
SAR dev.,
compd
optimiz.,
Cellular in vivo
efficacy,
initial
dmpk,
selectivity
profiling,
target
validation
2-3 yrs,
50%
Candidate
I.D.
lead opt.,
In vivo
efficacy &
safety,
7 day tox.,
scale-up,
dmpk in
3 species,
target
validation
Center For Integrative Chemical Biology & Drug Discovery
Portfolio Model
Number
of
items
Preproject
Assay
Develop
ment
Chemistry(H2L)
5 projects
Work in progress
10 projects
15-20 projects
Effort
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Project Progress
Center For Integrative Chemical Biology & Drug Discovery
Portfolio Goals
Stage
Assay
Developed
2010
12
Goals –
Eppin
projects that GRK2/3
will complete CCR7
RGS
the indicated G9a
stage during SMYD3
DNMT3b
this year
PAP
ROR2
LKB1
A1
IKKe
Validated Hits
Leads/probes
9
4
MBT – HTS & synthesis
Eppin – HTS
RGS – HTS
SMYD3/SETD8 – HTS
LKB1 – focused
GRK2/3 – focused
PAP – synthesis
DNMT3b - synthesis
Mer kinase
FS D2
HMT
Kinase lead
Center For Integrative Chemical Biology & Drug Discovery
How do faculty currently establish a
collaboration with the CICBDD?
• Drop by & chat
– Reviewed by Center Faculty
• General Criteria:
– Disease relevance of target/pathway
– Potential scientific impact of small molecule tools
– Tractability of assay development and ligand
discovery
– Portfolio balance and fit with center expertise
– Funding status & prospects
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Center For Integrative Chemical Biology & Drug Discovery
Mer Kinase Background
•
Mer kinase – a member of the Tyro3/Axl/Mer
RTK family
–
Expressed in monocytes, functions to clear
apoptotic material
–
Never expressed in normal T or B lymphocytes
–
Capable of sending anti-apoptotic signals
•
Mer kinase expressed in most T and B cell ALL
lines
•
Mer expression in childhood leukemias
•
–
Mer mRNA expressed in 30-40% T cell ALL (Clin
Canc Res 2006 12:2662)
–
New data :Mer protein expressed in 41% B ALL
(16 of 16 E2A-PBX1 ALLs)
–
Mer protein expressed 54% T cell ALLs and 68%
pediatric AML
–
Mer protein expressed in 50% of
recurrent/induction failures
Protein structure with small molecule bound
published by the Structural Genomics
Consortium
–
Huang, X., et al., Structural insights into the
inhibited states of the Mer receptor tyrosine
kinase. J Struct Biol, 2009. 165(2): p. 88-96.
Center For Integrative Chemical Biology & Drug Discovery
Inhibition of Mer Expression Alters
Chemosensitivity and In Vivo Outcome
In vivo leukemia model: injection of 5x105 697 cells
in Nod/SCID mice. Enhanced survival with Mer
shRNA knockdown.
697 B cell (E2A-PBX) chemosensitivity altered by Mer knockdown
Target validation with shRNA, Linger et al., Blood, 2009 114:2678
Center For Integrative Chemical Biology & Drug Discovery
Mer Kinase Status at UNC
•
Goal: Chemosensitization of ALL patients
–
•
•
Project has been underway for 1.5 years – now
funded by the NCI (1st year = $1.614M)
Structure-based hit generation has yielded one
lead series:
–
–
–
–
–
•
•
i.v. initial formulation, oral secondary
low nanomolar Ki’s, robust structure-activity relationships
Promising initial dmpk (UNC569, mouse, 4.4h t1/2, 56% F)
Broad kinase profiling underway
Cellular assays being optimized – compounds appear to
have <1 mM IC50’s
Compounds suitable for in vivo testing are in hand
Additional hit generation is ongoing via focused
screening and further structure-based design
Initial crystals of the Mer kinase domain have
been obtained – optimizing conditions to develop
a system for routine
co-crystal
structures
Confidential
UNC
CBC
Plasma concentration-time data of UNC 569A
(mouse, dose:3mg/kg).
Center For Integrative Chemical Biology & Drug Discovery
CENTER Mission
Center
Initiated
Projects in
Chemical
Biology
Target Proposals
from
UNC Faculty
“responsive
collaborations”
“prospective
science”
small molecule ‘probes’
target validation
drug leads
Center For Integrative Chemical Biology & Drug Discovery
Chemical Biology of Chromatin Regulation
GOAL = Chemical Probes
PKMT Royal PKDM
Frye, S. V.; Heightman, T.; Jin, J.
Targeting Methyl Lysine. Annu.
Rep. Med. Chem. 2010, in press,
DOI: 10.1016/S00657743(10)45020-4.
HAT Bromo HDAC
Center For Integrative Chemical Biology & Drug Discovery
Why chemical probes?
• Temporal resolution
– rapid exposure and elimination of effects are possible
• Mechanistic flexibility
– can potentially target separate functions of a protein, as
opposed to ablating them all
• Ease of delivery
– freely cell permeable, potential for oral activity
• Applicability to drug discovery
– transition from target validation to therapeutic intervention is
more direct
Weiss, W.A., S.S. Taylor, and K.M. Shokat, Recognizing and exploiting differences between
RNAi and small-molecule inhibitors. Nat Chem Biol, 2007. 3(12): p. 739-44.
Center For Integrative Chemical Biology & Drug Discovery
What is a quality chemical probe?
•
Molecular Profiling: Sufficient in vitro potency and selectivity data to
confidently associate its in vitro profile to its cellular or in vivo profile.
•
Mechanism of Action: Activity in a cell-based or cell-free assay
influences a physiologic function of the target in a dose-dependent
manner.
•
Identity of the Active Species: Has sufficient chemical and physical
property data to permit interpretations of results to be attributed to its intact
structure or a well characterized derivative.
•
Proven Utility as a Probe: Cellular activity data available to confidently
address at least one hypothesis about the role of the molecular target in a
cell’s response to its environment.
•
Availability: Is readily available to the academic community with no
restrictions on use.
–
Frye, S. V. The art of the chemical probe. Nat Chem Biol 2010, 6, 159-161.
Center For Integrative Chemical Biology & Drug Discovery
PKMTs as a Target Class
•
> 50 human PKMTs and >
10 PRMTs identified since
the first PKMT discovered
in 2000
•
Growing evidence
suggests PKMTs involved
in various human
diseases including cancer
•
H3K4
Lack of chemical probes:
only 2 selective PKMT
inhibitors reported (BIX01294 and chaetocin)
Wu et al, PLOS One, 2010, 5, e8570
Copeland et al, Nat. Rev. Drug Discovery, 2009, 8, 724
Center For Integrative Chemical Biology & Drug Discovery
21
Protein Lysine Methyltransferase G9a
• First identified as a H3K9 MT in 2002 (Tachibana et al, Genes Dev. 2002, 16,
1779). Shares 80% sequence identity with GLP (AKA EHMT1) (Chang et al,
Nat. Struct. Mol. Biol. 2009, 16, 31). G9a and GLP work cooperatively via
forming a heterodimer.
• Overexpressed in cancer and knockdown inhibits cancer cell growth
(McGarvey et al, Cancer Res. 2006, 66, 3541) (Kondo et al, PLoS ONE 2008, 3, e2037)
• Also dimethylates K373 of p53, which results in the inactivation of p53
(Huang et al, J. Biol. Chem. 2010, 285, 9636)
• Plays an important role in the development of cocaine addition (Maze et al,
Science 2010, 327, 213) and mental retardation (Schaefer et al, Neuron 2009, 64,
678), and in maintenance of HIV-1 latency (Imai et al, J. Biol. Chem. 2010, 285,
16538)
• BIX01294, a G9a inhibitor, efficacious as a replacement for c-Myc and
Sox2 for reprogramming of mouse fetal neural precursor cells into iPS
cells (Shi et al, Cell Stem Cell 2008, 3, 568)
Center For Integrative Chemical Biology & Drug Discovery
Structure-based Design of UNC0224
Array-based
Optimization*
BIX01294
G9a (ThioGlo): IC50 = 180 nM
G9a (AlphaScreen): IC50 = 250 nM
UNC0123
G9a (ThioGlo): IC50 = 330 nM
G9a (AlphaScreen): IC50 = 230 nM
Reduced MW while maintaining potency
Kubicek, et al. 2007, Mol Cell, 473
UNC0224
Synthesized in 10 steps
GLP-BIX01294 complex
Adopted from Chang, et al. 2009,
Nat. Stru. Mol. Bio., (16), 316
G9a (ThioGlo): IC50 = 43 nM
G9a (AlphaScreen): IC50 = 57 nM
G9a (ITC): KD = 23 nM
Liu et al, J. Med. Chem. 2009, 52, 7950
Center For Integrative Chemical Biology & Drug Discovery
First Co-crystal Structure of G9a + small
molecule: G9a-UNC0224 complex
Asp1078
Asp
1078
Es(b)
Asp1074
Asp
1083
Asp1083
Hb(s)
Leu
1086
Es(b)
Asp1088
Hb(s)
Leu1086
Asp
1074
Es(s)
(s)
Tyr
1154
Asp
1088
Tyr1154
• 7-Dimethylaminopropoxy side chain binds in the lysine binding channel,
validating the binding hypothesis, but does not fully fill available space
PDB code: 3K5K
Liu et al, J. Med. Chem. 2009, 52, 7950
Center For Integrative Chemical Biology & Drug Discovery
Most Potent G9a Inhibitors to Date
• Key SAR findings
− Longer side chain (up to 5 atoms) or bigger N-capping group increases potency
− Basicity of the nitrogen necessary for high potency
• Although UNC0224 and UNC0321 are more potent than BIX01294 in
biochemical assays, they are less potent in the cell-based assay
Liu et al, J. Med. Chem. 2010, 53, 5844
Center For Integrative Chemical Biology & Drug Discovery
Compounds Designed
to Improve Cellular Potency
• Exploit newly identified SAR to increase lipophilicity, thus cell
membrane permeability while maintaining high potency
• Prepared > 50 combination compounds. Aiming to achieve balanced
in vitro potency & physical chemical properties
Feng Liu & Xin Chen
Center For Integrative Chemical Biology & Drug Discovery
UNC0638 More Potent Than BIX01294
110
100
90
80
70
60
50
40
30
20
10
0
120
BIX-01294
IC50 500 nM
100
% H3K9m2
% H3K9m2
BIX01294
UNC0638
UNC0638
IC50 81 nM
0
1
80
60
40
2
3
20
4
Log nM
0
H3K9Me2 levels measured in MDA-MD-231
cells after 48h exposure to BIX or UNC0638.
Control
G9a
EHMT1 G9a/
EHMT1
shRNA
UNC0638 at 250 & 500 nM reduced cellular
H3K9Me2 levels close to, but not equivalent
to G9a/GLP knockdown
Dalia Barsyte (SGC), unpublished results
Center For Integrative Chemical Biology & Drug Discovery
27
Quantitative MS Analysis of
Effects of UNC0638 on Histone PTMs
• UNC0638 ↓H3K9me2, –H3K9me3, ↓H3K9me1, ↑H3K9un & ↑H3K14ac
• Similar to G9a and GLP Knockdown
Control shRNA
521.307
100
H3K9me3
shRNA G9a
H3K9me2
95
90
100
530.829
Control shRNA
shRNA G9a+GLP1
528.314
95
533.345
90
85
85
80
80
75
75
70
70
60
Relativ e Abundanc e
Relative Abundance
65
521.808
55
shRNA G9a
50
45
523.822
40
35
shRNA G9a+GLP1
30
524.323
25
522.309
20
526.337
65
60
10
45
40
35
30
25
5
526.839
524.824
522.810
521
522
524
m/z
525
526
527
534.347
10
527.340
523
531.832
529.317
15
5
0
520
533.846
50
20
15
531.331
528.815
55
0
528
528
529
530
531
532
533
534
535
m/z
Control
Control
521.306
100
H3K9me2
95
530.829
UNC 638
95
90
533.345
90
85
85
80
80
75
75
70
70
65
60
BIX
521.807
55
Relative Abundance
Relative Abundance
H3K9me3
BIX
528.314
100
523.821
50
45
40
35
UNC 638
524.323
30
533.846
50
45
40
30
531.832
534.347
15
524.824
10
529.317
20
526.838
15
527.339
522.810
5
531.331
55
25
522.308
20
528.815
60
35
526.337
25
65
10
5
0
520.5
521.0
521.5
522.0
522.5
523.0
523.5
524.0
524.5
m/z
525.0
525.5
526.0
526.5
527.0
527.5
528.0
0
528.0
528.5
529.0
529.5
530.0
530.5
531.0
531.5
m/z
532.0
532.5
533.0
533.5
534.0
534.5
535.0
Ben Garcia (Princeton)
unpublished results
Center For Integrative Chemical Biology & Drug Discovery
28
UNC0638 Less Toxic Than BIX01294
110
100
90
80
70
60
50
40
30
20
10
0
10 0
BIX01294
H3K9m2
MTT
10 1
10 2
10 3
10 4
H3K9m2
MTT
110
100
90
80
70
60
50
40
30
20
10
0
H3K9m2
MTT
% response
% response
UNC0638
UNC638
10 5
10 0
10 1
10 2
10 3
10 4
10 5
nM
nM
Good separation of functional
and toxic effects
Poor separation of functional
and toxic effects
In Vitro G9a
IC50 (nM)
H3K9me2 48h
IC50 (nM)
Cell Tox 48h
EC50 (nM)
Tox/Func
Ratio
BIX-01294
133 ± 15
500 ± 43
2805
5.6
UNC0638
<15
81 ± 9
11190
138
Dalia Barsyte (SGC), unpublished results
Center For Integrative Chemical Biology & Drug Discovery
UNC0638 Molecular Profile
• Ki = 3 nM and IC50 < 15 nM vs G9a
• IC50 = 19 nM vs GLP
• Competitive with peptide substrate and non-competitive
with cofactor SAM
• Co-crystal structure of G9a-UNC0638-SAH confirms MOA
• Rapid and on and off rate – reversible inhibitor
• At least 100-fold selective over a broad range of
epigenetic targets including PKMTs, PRMTs, PKDMs,
HATs, and DNMTs, and a wide range of non-epigenetic
targets including GPCRs, ion channels, transporters, &
kinases
Center For Integrative Chemical Biology & Drug Discovery
UNC - SGC Probe Consortium Released
UNC0638 as a Chemical Probe on June 1st
http://www.thesgc.org/chemical_probes/UNC0638/#overview
• Data released prior to publication. Living document that is updated as
new data is generated
Center For Integrative Chemical Biology & Drug Discovery
Collaborators Who Are Using UNC0638
Mark Minden (OCI)
Test on EVI-1 expressing
leukemia cells
Jayne Danska (Sick Kids, Toronto)
Test on large panel of ALL cells
Jay Bradner (Dana Farber/Broad Inst)
Test on MLL dependent leukemic cells
Effects on epigenetic markers
Rob Bristow (OCI)
Test on prostate cancer
cell lines
Stuart Schreiber (Broad)
Profile against >1000
genetically defined cancer
cell lines
Alex Tarakhovsky (Rockefeller)
IFN production after viral infection
Viral resistance
Changes of epigenetic markers
James Ellis (Sick Kids, Toronto)
iPS cell studies
David Margolis (UNC)
Effects on HIV-1 latency
Angelique Whitehurst (UNC)
Synergy with chemo
therapies
Michael Taylor (Sick Kids Hosp)
Test on DAOY cell line
(medulloblastoma)
Martin Hirst (UBC)
ChIP-seq
Jing Huang (NCI, NIH)
Bill Janzen (UNC)
p53 methylation
Apoptosis of MCF7 cells
Rod Bremner (TWRI)
Assess effect on IFN expression
Center For Integrative Chemical Biology & Drug Discovery
Summary
• Discovered potent, selective, and cellularly active
G9a/GLP chemical probes via structure-based inhibitor
design in combination with SAR exploration
• UNC0638, which has significantly improved tox/func ratio,
available to research community without restrictions on
use for investigating the biology of G9a/GLP and their role
in chromatin remodeling in health and disease.
Center For Integrative Chemical Biology & Drug Discovery
Acknowledgements
• UNC CICBDD
–
–
–
–
–
–
–
–
–
–
–
–
Feng Liu
Xin Chen
Julianne Yost
Tim Wigle
Sam Pattenden
Dmitri Kireev
Jacqueline Norris
Cathy Simpson
Martin Herold
Emily Hull-Ryde
Bill Janzen
Stephen Frye
• Bryan Roth Lab (UNC)
– Tom Mangano
– Jon Evans
• Cheryl Arrowsmith Lab (SGC)
–
–
–
–
–
–
–
–
Dalia Barsyte
Peter Brown
Masoud Vedadi
Matthieu Schapira
Abdellah Allali-Hassani
Gregory Wasney
Aiping Dong
Aled Edwards
• Anton Simeonov Lab (NCGC)
– Amy Quinn
– Ajit Jadhav
• Ben Garcia Lab (Princeton)
– Peter DiMaggio
Funding: NIH RC1GM090732, UNC Carolina Partnership
Center For Integrative Chemical Biology & Drug Discovery
34