University of Illinois at Chicago College of Pharmacy UIC

Background Oligoamines Containing 3-5-3 Linkers

Summary and Conclusions

Hypothesis

• A novel series of oligoamines containing 3-5-3 linkers has been

synthesized, characterized and tested

• Three compounds (BP-107-3, BP-107-5 and BP-107-15) display 95% or

more inhibition at 10 µM in a fluorimetric demethylation assay

• Of these three best compounds, BP-107-15 displayed a superior IC50

value in a MTS cell viability assay and exhibits competitive kinetics

• Inhibition of LSD1 by BP-107-15 significantly induces re-expression of

aberrantly silenced tumor suppressors (SFRP2, HCAD, GATA4 and p16)

• Computational docking using GOLD revealed key h-bonding and

hydrophobic interactions

• Ongoing structure-based drug discovery efforts are aimed at optimizing

the inhibitors to further improve its potency, as well as testing its efficacy in

animal models

Design, Synthesis and Biological Activities of Novel Oligoamines Containing 3-5-3 Linkers as

Epigenetic Modulators

Boobalan Pachaiyappan 1, Shannon Nowotarski 2, Melissa Sokolosky 1, Steven L. Holshouser 1,

Shiv K. Sharma 3, Robert A. Casero, Jr. 2, Patrick M. Woster 1

Contact Information: (1) Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States; (2) Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University,

Baltimore, MD 21231, United States; (3) Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, United States. (Email: pachaiya@musc.edu)

X = O or S; Ar = various mono and biaryl derivatives

BP-107-15

• 96% inhibition at 10 µM

• IC50 = 5.5 µM

• Cellular IC50 = 4 µM

• Competitive inhibition

Figure 6: LSD1 inhibition by BP-107-3, BP-107-5 and BP-107-15 at 10 µM

promotes re-expression of aberrantly silenced genes detected by qRT-PCR

in this Calu6 human lung adenocarcinoma cells

Figure 1: Normal mammalian cells exhibit an exquisite level of control

of chromatin architecture by maintaining a balance between histone

lysine methyl transferase and lysine-specific demethylase 1 (LSD1)

activity. In tumorigenesis, this equilibrium is disturbed due to

overexpression of LSD1, thereby resulting in decreased activating

marks and increased repressive marks resulting in aberrant silencing of

tumor suppressors

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• Synthesize oligoamines of various linker sizes

• Characterization using NMR and MS

Organic Synthesis

• In vitro and cell-based testing

• Enzyme kinetics

• Gene re-expression studies

Biological Testing

• Model inhibitors and elucidate the key determinants for binding to LSD1

Computational

Modeling

In Vitro Biological Testing

Figure 3: General structure of oligoamines containing 3-5-3 linkers

Figure 4: Chemical synthesis of oligoamines containing 3-5-3 linkers

Demethylated Lysine

(condensed chromatin

represses transcription)

Methylated-Lysine

(elongated chromatin

upregulates transcription)

Histone lysine methyl transferase

LSD1

Inhibitors

(?)

H3 H3

Because LSD1 catalyzes the demethylation of key chromatin marks at

histone 3 lysine 4 (H3K4me and H3K4me2), as well as H3K9, LSD1

inhibition using small molecule oligoamines will have a significant effect in

promoting transcription and re-expression of tumor suppressor genes.

Experimental Paradigm

Figure 2: Integrated strategy for LSD1 inhibitor design

References & Acknowledgements

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Figure 5: (Panel A): Eleven compounds were assessed for LSD1

inhibitory activity at 10 µM by combining the recombinant LSD1,

fluorogenic substrate and inhibitor, and measuring relative fluorescence of

demethylated H3K4me1; (Panel B): To determine the IC50 of BP-107-15,

inhibitory activity was measured over a range of concentrations (0.3125 to

2.5 mM); (Panel C): Kinetics of inhibition of recombinant LSD1 by BP-107-

15; (Panel D): Overall inhibitory profile of BP-107-15.

Organic Synthesis

Computational Modeling Analysis

Figure 7: (left) Computer-predicted binding mode of BP-107-15 (capped

sticks) in the LSD1 binding site (ribbon diagram); (right): molecular

interactions that govern the binding of BP-107-15 in the LSD1 pocket

- NIH 1-RO1-CA149095

(PMW)

- A generous grant from

Progen Pharmaceuticals, Ltd.

• J Med Chem (2010), 53, 5197

• Proc. Natl. Acad. Sci. USA (2007),

104, 8023

• J Med Chem (2012), 55, 7378

• Clin. Cancer. Res. (2009), 15, 7217

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* p<0.1

** p<0.05

Gene Re-Expression Studies