Pharmacophore Development Aimed At Inhibiting The Spi-1/Dna - C/Ebpβ Interaction

Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Pharmacophore Development Aimed at Inhibiting

the Spi-1/DNAC/EBP Interaction
Emily Cribas
Penn State
July 24, 2014
1/21
Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
The Interleukin Gene

I
The Spi-1 and C/EBP transcription factors interact via a

critical protein-protein interaction at the promoter of the
human IL1 gene
Figure 1 : Human IL1 gene induction1
1
Auron
Lab, Aimed
Unpublished
Pharmacophore
Development
at Inhibiting the Spi-1/DNAC/EBP Interaction
2/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
The Interleukin Gene

I
The Spi-1 and C/EBP transcription factors interact via a

critical protein-protein interaction at the promoter of the
human IL1 gene
Figure 1 : Human IL1 gene induction1

I
Together, they help code for Interleukin (IL-1), a

macrophage cytokine that mediates acute inflammatory
responses, but leads to chronic disease when highly expressed
1
Auron
Lab, Aimed
Unpublished
Pharmacophore
Development
at Inhibiting the Spi-1/DNAC/EBP Interaction
2/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
IL-1-dependent inflammation may be prevented through the

discovery of a small molecule that can bind to a pocket
formed in the DNA major groove near Arginine232 (R232) in
Spi-1, a recognition site for C/EBP interaction
Figure 2 : Spi-1 DBD and C/EBP bZIP interaction2

2
Listman2005.
3/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
The Spi-1 Protein

I
Spi-1 is a member of the

ETS family of transcription
factors that have a DBD
containing a specific
recognition sequence4
It has a wHTH structure,

where helix 3 contains two
residues that bind
specifically to the
recognition sequence of IL-1
gene4
4
5
Figure 3 : Spi-1 DBD/DNA

Binding5
Kodandapani, et al. Nature 1996, 380 456-460.

Listman, et al. (P. Auron), J. Biol. Chem. 2005, 280 ,41421-8
4/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
The Spi-1 Protein

I
Spi-1 is a member of the

ETS family of transcription
factors that have a DBD
containing a specific
recognition sequence4
It has a wHTH structure,

where helix 3 contains two
residues that bind
specifically to the
recognition sequence of IL-1
gene4
4
5
Figure 4 : Spi-1 DBD/DNA

Binding5
Kodandapani, et al. Nature 1996, 380 456-460.

Listman, et al. (P. Auron), J. Biol. Chem. 2005, 280 ,41421-8
5/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Spi-1/DNA Interaction
Specific Spi-1/DNA Interaction

6/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Hypothesis:
The region near R232 of the Spi-1/DNA complex can
accommodate a small molecule through hydrogen bonding
Specific Aims:
1. Characterize the region near R232 of the Spi-1/DNA complex
I
Carry out 3 different MD simulations for: the complex, the

nucleic acid, the protein
2. Develop a pharmacophore model

I
Using most stable state of the complex
3. Virtually screen a library of small molecules
7/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
The Systems
1. The Complex
I
I
I
42,048 atoms
90 ns of simulation
DNA contained harmonic restraints with force constants
2. The Nucleic Acid

I
I
I
39,432 atoms
50 ns of simulation
DNA contained same restraints
3. The Protein
I
I
22,763 atoms
50 ns of simulation
8/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Basic Procedure
I
I
I
I
I
Each complex was prepared and solvated using MMTSB

tools3 and the CHARMM274 force field
NAMD 2.95 was used for minimization and relaxation through
molecular dynamics simulations
VMD 1.9.16 and R 7 were used to monitor the progress of
equilibration and stability of the complex
Pharmacophore queries were created using the final, stable
structure of the complex in MOE8
A library of small molecules was screened using a specified
molecular database as a reference
Feig2004.
Foloppe2000.
5
Phillips2005.
6
Humphrey1996.
7
R2014.
8
MOE2011.
4
9/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
The Simulations
Protein: 40-50 ns
DNA: 40-50ns
Complex: 80-90 ns
10/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
RMSD of Bound Spi-1,

Mean = 0.774
A
RMSD of Unbound Spi-1,

Mean = 1.377
A
11/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
RMSD of Bound DNA,

Mean = 0.948
A
RMSD of Unbound DNA,

Mean = 1.446
A
12/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
RMSF per Residue of Spi-1
13/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
RMSF per Base of DNA
14/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Binding Pocket
R232 Binging Pocket

Close-up
15/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Pharmacophore Queries
Pharmacophore features
enclosed by the binding pocket
Selected pharmacophore
features
Key:
16/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Screening
Table: Library of Small Molecules
#
1
molecule
rmsd(
A)
rscore
0.328
3.690
0.448
3.693
1.091
11.936
rmsd refers to the

RMSD between query
features and
matching ligand
target points
rscore is the sum of

individual feature
scores (the acceptor
or donor strength of
matching atom per
feature)
17/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Molecule Interactions
Molecule 1
Molecule 2
Molecule 3
18/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Conclusions
Analysis of uncomplexed systems validates our Spi-1:DNA

structure and behavior
RMSD and RMSF values, for the most part agree with
predicted behavior
The R232 binding pocket has provided a viable

pharmacophore model
19/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Future Work
Refine screening results
Perform molecular docking
Run MD simulations for each small molecule in complex
Compare binding strengths of screened small molecules to

that of C/EBP
20/21
Penn State
Background
The Project
Results
Conclusions
Future Work
Acknowledgements
Acknowledgments
I
National Science Foundation, Major Research Instrumentation

(MRI)
CHE-1126465
National Institutes of Health R25, National Institute on Drug

Abuse (NIDA)
1 R25 DA032519-01
Duquesne University URP 2014
Madura Research Group
Auron Research Group
Scott Boesch
Emilio Esposito
21/21
Penn State

Pharmacophore Development Aimed At Inhibiting The Spi-1/Dna - C/Ebpβ Interaction

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Background

Pharmacophore Development Aimed at Inhibiting

July 24, 2014

The Interleukin Gene

The Spi-1 and C/EBP transcription factors interact via a

Figure 1 : Human IL1 gene induction1

The Interleukin Gene

The Spi-1 and C/EBP transcription factors interact via a

Figure 1 : Human IL1 gene induction1

Together, they help code for Interleukin (IL-1), a

IL-1-dependent inflammation may be prevented through the

Figure 2 : Spi-1 DBD and C/EBP bZIP interaction2

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

The Spi-1 Protein

Spi-1 is a member of the

It has a wHTH structure,

Figure 3 : Spi-1 DBD/DNA

Kodandapani, et al. Nature 1996, 380 456-460.

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

The Spi-1 Protein

Spi-1 is a member of the

It has a wHTH structure,

Figure 4 : Spi-1 DBD/DNA

Kodandapani, et al. Nature 1996, 380 456-460.

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

Specific Spi-1/DNA Interaction

Carry out 3 different MD simulations for: the complex, the

2. Develop a pharmacophore model

Using most stable state of the complex

3. Virtually screen a library of small molecules

2. The Nucleic Acid

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

Each complex was prepared and solvated using MMTSB

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

RMSD of Bound Spi-1,

RMSD of Unbound Spi-1,

RMSD of Bound DNA,

RMSD of Unbound DNA,

RMSF per Residue of Spi-1

RMSF per Base of DNA

R232 Binging Pocket

rmsd refers to the

rscore is the sum of

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

Analysis of uncomplexed systems validates our Spi-1:DNA

The R232 binding pocket has provided a viable

Refine screening results

Perform molecular docking

Run MD simulations for each small molecule in complex

Compare binding strengths of screened small molecules to

National Science Foundation, Major Research Instrumentation

National Institutes of Health R25, National Institute on Drug

Duquesne University URP 2014

Madura Research Group

Auron Research Group

Pharmacophore Development Aimed at Inhibiting the Spi-1/DNAC/EBP Interaction

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