UNC Center for Integrative Chemical Biology and Drug Discovery

UNC Center for Integrative 

Chemical Biology and Drug 

Discovery 

September 1, 2009 

http://www.pharmacy.unc.edu/labs/center-for-integrative-chemical-biology-and-drug-discovery

Why Academic Drug Discovery 

• Global 

– 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 

• Biotech 

– 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 

Center For Integrative Chemical Biology & Drug Discovery 

2

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 

When the target’s role in human 

disease is unprecedented, 

this rarely works (

Drug Discovery – what we are trying to do better 

at UNC. 

Target selection – 

the disease connection 

hypothesis 

biology 

Small molecule tool 

to enhance target 

understanding & validation 

UNC can excel 

at small molecule 

driven elucidation of 

biology relevant to 


RESEARCH: 

hypothesis generation, 

revision & exploration at 

the chemistry/biology 

interface 

chemistry 

Efficacy 

in treating 

disease in 

Humans 


4

Center for Integrative Chemical Biology and 


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. 


5

National Cancer Institute 

Comprehensive Chemical Biology Screening Center 

UNC 

Stephen Frye, Principal Investigator 

William Janzen, Assay Development 

Dmitri Kireev, Computational Chemistry 

Norm Sharpless, Mouse Phase 1 

Russ Mumper, Drug Delivery 

NCCU-BRITE 

Li-An Yeh, Director 

Gordon Ibeanu, Assay Development 

John Scott, Screening 

Alfred Williams, Medicinal Chemistry 

HAMNER INSTITUTES 

Darol Dodd, Toxicology & 

Preclinical Studies 

Sheela Sharma, Senior 

Investigator 


6

CICBDD mission 

Target Proposals 

from 

UNC Faculty 

“responsive hit 

to lead 

collaborations” 

Center 

Initiated 

Projects in 

Chemical 

Biology 

“prospective 

science” 

small molecule ‘probes’ 

new target DISCOVERY 

drug leads 


7

Sample Responsive Projects 

• mer kinase inhibitor for 

treatment of acute 

lymphoblastic leukemia in 

children 

(Earp/Johnson/Graham) 

• PAP in neuropathic pain 

(Zylka) 

• ROR2 kinase inhibitors for 

treatment of renal cell 

carcinoma (Rathmell) 

• Oxytocin agonists for social 

attachment (Jarstfer) 

• Kinase Biosensors (Hahn) 

• Breast Cancer Stem Cell 

selective cytotoxics (Perou) 

• IDH1 mutations in 

glioblastoma (Mumper, Xiong) 

• DNMT3b inhibitor for treatment 

of basal-like breast cancer 

(Coleman) 

• GPCR Functional Selectivity 

– atypical antipsychotics 

(Roth) 

• RGS modulators (Siderovski) 


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Prospective Science: chemical biology of chromatin 

regulation 

• Goal: Cell penetrant, small 

molecule, quality chemical 

probes that modulate the 

regulation of chromatin state. 

• Approach: A systematic and 

therapeutically unbiased effort 

focused on the writers, readers 

and erasers of the histone 

code. 

– tractable intervention points – 

enzymes and binding domains 

with attractive pockets 


9

Chemical Biology of Epigenetics 

DNA 

methylation 

DNA 

Lysine 

Histone 

Histone 

modification 

Modification Write Read Erase 

Acetyl (Ac) HAT Bromo HDAC 

Methyl (Me n 

) KMT Royal KDM 


10

MBT Domain Methyl Lysine Recognition 

Antagonists: Target Class Project Plan 

- biology 

- assays 

- chemistry 

- functional significance 

Center For Integrative Chemical Biology & Drug Discovery

Maligant Brain Tumor (MBT) Domain Proteins 

Repeats of ~100 aa 

Bind lower methylation 

states of lysine (KMe1/KMe2) 

Act as chromatin locks 

11 human & 5 Drosophila 

MBTs known 

Unique binding cavity 

offers 

opportunity for selective 

chemical probes 

no known chemical probes 

or HTS-compatible assays 


12

Established Low-Throughput Assays for MBT Protein Binding 

100 µM L3MBTL1 would 

be required for a screeningworthy 

signal! 

8 wells = ~ 1 mg protein! 

Isothermal titration calorimetry (ITC) has also been employed 

ITC also uses 70 – 100 µM protein 

Li, Fischle, Wang, Duncan, Liang, Murakami‐Ishibe, Allis & Patel. Molecular Cell 28. 2007, 677 

Klymenko, Papp, Fischle, Kocher, Schelder, Fritsch, Wild, Wilm & Muller. 2006 Genes Dev. 20, 1110‐1122 


13

AlphaScreen Assay for MBT Antagonists 


14

Buffer Optimization: 

Reduction of Non-Specific Binding 

Factorial Experimental 

Design for the Selection of 

Agents to Reduce Non- 

Specific Binding 

NaCl 

0 or 50 mM 

Tween 2 0 or 0.1 % 

BSA 0 or 0.1 % 

Large additive effect Small positive intx 

negative intx 

+ Tween + BSA 

–BSA 

–BSA 

+ BSA 

–Tween 

NaCl (mM) NaCl (mM) Tween 20 (%) 


15

Optimized Binding Signal 

Binding K d 

in the nanomolar range –compare to 25 µM for the cognate peptide 


16

Competition with Cognate & Non-Cognate 

Peptides 

Measured IC 50 for H3K9Me1 is 22 ± 3 µM M is in agreement with the literature K d 

of 2 


17

Assay Validation 


18

LOPAC vs. L3MBTL1 & CounterScreen 

LOPAC vs. L3MBTL1 

LOPAC vs. AlphaScreen 


19

L3MBTL1 LOPAC Hits by Reductive Analysis 

***2 confirmed nanomolar hits*** 


20

Functional assay: MBT localization 

MBT proteins co-localize with chromatin. 

GFP-SFMBT fusion co-localizes with DAPI 

staining, while GFP alone (GFP-null) is dispersed 

throughout the cell (adapted from Wu et al., 

2007). 


21

FAIRE – genome-wide, hypothesis free, proximal 

to target - derepression 

©2007 by Cold Spring Harbor Laboratory Press 

Giresi P. G. et.al. Genome Res. 2007;17:877-885 


22

What is a quality chemical probe 

• CICBDD definition – a compound with sufficient potency and selectivity 

data to associate its in vitro profile with confidence to its in vivo profile. 

– broad in vitro profiling usually desirable 

• AlphaScreen for 11 human and 5 Drosophila MBTs + ΔTm -vs- other 

EpiG targets at SGC + CEREP 

– mechanism of action versus target is understood 

• X-ray co-crystallization (SGC) + binding assay kinetics – whole 

nucleosomes 

– some hypothesis ‘free’ profiling technique supports designation 

• FAIRE 

– profiling in vivo includes confirmation of mechanism 

• derepression + nuclear localization 

– phenotypic data in at least one system is consistent with anticipated effects 

• TBD 


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Chemistry Background and Structural Biology 

MBT domains recognize methylated 

lysine residues on histone tails and 

their binding interaction leads to 

chromatin compaction and a 

repressed state of the chromatin. 

Binding cavity is lined with aromatic 

residues allowing for a π-cation 

interaction with the charged amine. 

Further interactions between the MBT 

surface and residues neighboring the 

lysine. 


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Chemistry Goal and Strategy 

Goal: 

- Development of potent and selective 

small molecule probes disrupting the 

interaction between MBT domains and 

methylated lysine-residues (KMe1,2) in 

histone tails. 

- Evaluation of the biochemical and 

phenotypic consequences of blocking 

recruitment of MBT domain containing 

proteins & complexes to chromatin 

Strategy: 

-There are NO known non-peptide 

binders to MBT domains. 

- Hypothesis: Disrupt the binding of 

MBT domains with methylated lysine 

mimetics. 


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Virtual Screening Method 

3D Pharmacophore Screening 

• Based on the histone binding mode* 

– 3 AA form L3MBTL1/H4K20me2 interface 

– Pharmacophore includes amine/cation, H-b 

acceptor, H-b donor and exclusion spheres 

• ChemNavigator 

– 5,000,000 Lipinski-filtered compounds 

– Enumerated** tautomers and stereomers*** 

– 3D Multiconformer + Energy Minimization** 

• Outcome 

– 5,000 primary hits 

• Clustered and visually inspected to skip 

abnormal conformers and mappings 

• 100 representative hits selected for structurebased 

analysis 

• Automated docking 

– Constrained cation coordinates 

– With Refinement and Re-Scoring 

• Energy minimization and visual inspection of 

resulting poses 

• 16 representative hits purchased 

Dmitri Kireev - CICBDD 

*Min, J. et al. Nat. Struct. Mol. Biol. 14, 1229–1230 (2007) 

**Using Pipeline Pilot components 

***When not explicitly specified 


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Focused Library Results 


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Synthetic Chemistry Strategy 

Establish a proof of concept 

with the preparation of 

peptidomimetics using the 

natural histone tail sequence 

as a template. 

• Chemistry efforts started with nicotinic acid analogs, 

• Primary focus on the chain length and the amino group. 

• Second round of synthetic effort directed towards aromatic anchor region and 

the linker. 


28

Peptidomimetics 

A subsequent docking experiment of 

UNC00000092A seem to confirme 

the additional interaction outside the 

binding cavity. 


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Methylation State 

Br 

Br 

Br 

Br 

N 

N 

N 

N 

O 

O 

O 

O 

N 

H 

N 

H 

N 

H 

N 

H 

NH 2 

UNC00000091A 

H 

N 


N 


N + 


Not active 

94 ± 9 µM 

95 ± 6 µM 

Not active 

MBTs are selective for the methylation 

state of the small molecule, but seem 

to not differentiate between methyland 

dimethylamine 

Br 

Br 

Br 

N 

N 

N 

O 

O 

O 

N 

H 

N 

H 

N 

H 

N 


N 


N 


47 ± 19 µM 

> 100 µM 

Not active 

Other amino groups are tolerated 

within a certain limit, most likely size 

O 


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Aromatic Anchor 

• Aromatic Anchor • Linker and Chain 

Br 

N 

O 

N 

H 

N 


TBD 

Br 

N 

O 

N 

H 

N 


TBD 

Br 

H 

N 

O 

H 

N 

N 


47 ± 19 µM 

Br 

O 

N 

>100µM 

N 


The aromatic anchor region offers 

the most room for variation 

N 

H 

N 

N 


82 ± 28 µM 


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target-class synergy 

Design and 

synthesize 

compounds 

knowledge 

cycle 

Structure determination 

-X-ray crystallography 

- docking and scoring 

- interaction analysis 

Comprehensive in vitro 

and in vivo profiling in 

biochemical and cellular 

assays 


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Assay Enables a Target-Family Profiling 

Approach 


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What is success for academic drug discovery 

• Creation of new knowledge with direct 

therapeutic impact 

• Complementary focus to biotech and pharma 

– orphan indications 

– novel targets & target classes 

– novel approaches 

• Lowering the barrier for entry of industrial 

science 


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Acknowledgements 

• CICBDD 

– Stephen Frye – Director 

CICBDD 

– Martin Herold – 

Postdoctoral Fellow – 

medicinal chemistry 

– Tim Wigle – Postdoctoral 

Fellow – assay 

development 

– Dmitri Kireev – Director of 

Computational Drug 

Design 

• UNC administration 

– Holden Thorpe 

– Bob Blouin 

– Alex Tropsha 

• SGC 

– Cheryl Arrowsmith 

– Jinrong Min 

– Aled Edwards 

– Peter Brown 

– Masoud Vedadi 

– Natalie Nady 


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CICBDD Team 


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UNC Center for Integrative Chemical Biology and Drug Discovery

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