Pharmacophore Discovery Aimed at Inhibiting the

Spi-1/DNAC/EBP Interaction
Cribas, Emily S.1; Auron, Philip E.2; Madura, Jeffry D.3
1

Pennsylvania State University, University Park, PA 16802 2Department of Biological Sciences

3
Chemistry and Biochemistry, Center for Computational Sciences
Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282

Introduction
I The

Spi-1/PU.1 and the C/EBP transcription factors

cooperate via a critical protein-protein interaction at the
promoter of the human IL1B gene
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
I 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 in the vicinity of
Arginine232(R232) in Spi-1/PU.1, a recognition site for
C/EBP interaction.
Figure 1. Model Electrostatic Spi-1 DBD and C/EBP bZIP Interaction1

Simulation Analysis

Library Screening Continued

Figure 2. RMSD of Spi-1 and DNA: Bound and Unbound

(a) RMSD of Bound Spi-1

(c) RMSD of Unbound Spi-1

Table: Library of Small Molecules*

(b) RMSD of Bound DNA

Hypothesis:
The region near R232 of the Spi-1/DNA complex can
accommodate a small molecule through hydrogen bonding
Specific Aims:

(b) RMSF per Base of DNA

1. Characterize the region near R232 of the Spi-1/DNA

complex
I Perform three separate molecular dynamics (MD)
simulations on: the complex, protein, and nucleic acid
2. Develop a pharmacophore model
3. Virtually screen a library of small molecules

(a) Denotes RMS fluctuations over time for all atoms in each residue, while (b) denotes fluctuations
for all atoms in each base

Modeling and Screening

Figure 4. R232 Binding Pocket

Figure 5. Pharmacophores
Legend:

0.328

3.690

0.448

3.693

1.091 11.936

I *These 3 molecules indicate only a

small portion of the hundreds of small
molecules generated from virtual
screening
I rscore denotes sum of individual
feature rscores, the acceptor or donor
strength of matching atom per feature
I A low RMSD and high rscore are
features of a desirable inhibitory
molecule

Conclusions:
I Analysis of unbound systems validates our Spi-1:DNA structure
I RMSD and RMSF values agree with the predicted behavior
I The R232 binding pocket has provided viable pharmacophores
I The majority of screened small molecules hydrogen bond to water
molecules present in the pocket
Future Work:
I Refine screening results
I Perform molecular docking
I Run MD simulations for each small molecule in complex
I Compare bond strengths of screened molecules and C/EBP

Acknowledgements
Surface map of binding pocket
surrounding R232

I National Science Foundation, Major Research

Instrumentation (MRI) Grant Number: CHE-1126465
I National Institutes of Health R25, National Institute on Drug
Abuse (NIDA) Grant Number: 1 R25 DA032519-01
I Duquesne University URP

Closeup of developed pharmacophore

queries in the pocket

Figure 6. Ligand Interactions of Screened Molecules

(a) Molecule 1

(b) Molecule 2

(c) Molecule 3

Computational Methods
2.92 - MD package used to conduct 90 ns, 50 ns,
and 50 ns of simulation time for the complex, protein, and
nucleic acid, respectively, at 300K with 2 fs time steps
I CHARMM273 force field
I MMTSB tools4 used for structure preparation and solvation
I Visualization and analysis using VMD5 and MOE6
I R statistical package7 used for data analysis and plotting

rmsd rscore

Conclusions and Future Work

Figure 3. RMSF of Spi-1 and DNA: Bound and Unbound

Surface representation of DNA bound Spi-1 DBD with the C/EBP bZIP aligned to
interact through its acidic residue to Spi-1s basic R232 residue

Hypothesis and Specific Aims

molecule

(d) RMSD of Unbound DNA

RMSD values for (a) and (c) are based on backbone atoms of the Spi-1 structure in complex
(PDBID:1PUE). All RMSD values are based on glycosidic atoms of DNA structure in the same
(b):0.948A,
(c):1.377A,
and (d):1.446A

complex. Mean RMSD Values: (a):0.774A,

(a) RMSF per Residue of Spi-1

I NAMD

Both (a),(b),and (c) are ligand interaction maps of 3 different screened molecules from the queries
shown in Figure 5. (a) and (b) involve hydrogen bonding to waters, while (c) also involves bonding to
a nucleic base

I Madura Group
I Auron Group
I Scott Boesch
I Emilio Esposito

References
1. Listman, J. et al. (P. Auron), 2005, J. Biol. Chem., 280 ,41421-8.
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1545-1615 2009.
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Research Institute
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33-38.
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Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2012.
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