High Throughput Screening of Combinatorial Libraries for
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ACTIVITY OF RESYNTHESIZED HITS
High Throughput Screening of Combinatorial Libraries for Inhibitors
of Clostridium difficile Toxins
Ilana L. Stroke1, Laurie J. Sturzenbecker1, Jeffrey J. Letourneau1,
Jorge G. Quintero1, Joan E. Sabalski1, Brett Marinelli1, Igor
Pechik1, David Diller1, Teresa Paulish-Miller2, David W. Hilbert2,
Scott Gygax2, Philip Stein1, and Maria Webb1
1Venenum Biodesign, LLC, Hamilton, NJ, 08691 • 2Femeris, Hamilton,
NJ, 08690
VENENUM Biodesign Hamilton, NJ 08691
Toll Free:877-950-1032 • Fax:609-587-1827
www.venenumbiodesign.com
Clostridium difficile infections are becoming increasingly
prevalent in hospitals, primarily in patients whose normal
intestinal flora have been compromised by antibiotic treatment.
These infections are debilitating and in some cases fatal. In
recent years an epidemic strain of C. difficile, B1/NAP1/O27,
exhibiting elevated production of virulence factors (Toxins A and
B, or TcdA and TcdB, respectively), has emerged. TcdA and TcdB are
glucose transferases that modify host Rho GTPases and disrupt the
actin cytoskeleton in intestinal epithelial cells and a variety of
other cell types, leading to the severe intestinal inflammation and
diarrhea associated with infection by C. difficile. Through
ultra-high throughput screening of encoded combinatorial libraries
(ECLiPS) and subsequent hit-to-lead structure optimization in our
laboratory, we have identified compounds that inhibit toxin B
enzymatic activity and protect mammalian cells from toxin in vitro.
(Supported by the Genesis Biotechnology Group).
Transcreener fluorescence polarization assay (BellBrook Labs) for
UDP-glucose hydrolysis by Toxin B
Toxin binding and activation
Decoded structures: synthon usage
Toxin B biochemical assay
Toxins A and B
High degree of homology in the glucosyltransferase domains of
toxins A and B
• Our high-throughput combinatorial library screen for inhibitors
of UDP-glucose hydrolysis by C. difficile Toxin B
glucosyltransferase domain was successful, albeit with a low
overall hit rate
• A family of related compounds with IC50 values of 0.54-17 µM were
identified as screening hits
• Analogs with improved potency were synthesized
• The chemotype identified has cell-based activity, protecting
cultured mammalian cells against toxin-induced apoptosis, and
exhibits dual activity vs. toxins A and B
• We would like to thank John Malone, Linda O’Brien, and Linh Ma
(Venenum Biodesign) for their contributions to this work.
Toxin binding and activation
ME Ivarsson (2012) Angew. Chem. Int. Ed. 51: 2–24
Rho glucosylation on Thr residue involved in binding divalent
cation (necessary for GTP binding) disrupts actin cytoskeleton
induces apoptosis
Toxins A and B
Toxins A and B
T. Jank (2007) Glycobiology 17(4): 15R–22R.
High degree of homology in the glucosyltransferase domains of
toxins A and B
N. D’Urzo et al. (2012) FEBS J. 279: 3085-3097; D.J. Reinert et al.
(2005) J. Mol. Biol. 351: 973-981.
(53% identity) high low
glucose hydrolysis by Toxin B
tracer tracer
Variable synthons in active library
56 R1 synthons 80 R2 synthons 13 R3 synthons (sublibraries) x
x
total compounds in the active library: 58,240 Sublibrary size:
4,480 compounds Sublibrary 3 was the most active sublibrary
R3 synthon
multiple compounds per
single compound per well
0.29 % at >50% inhibition
cytotoxicity
48-hour treatment with 0.3 ng/ mL Toxin B and test compound
Apoptosis assay as measured using Caspase 3/7 Glo (Promega)
Compound 6: analog with improved potency Average IC50 in cell-based
assay ~ 70 nM
Protection from Toxin A-induced cytotoxicity
Toxin A at 500 ng/ mL Indicates dual activity at toxins A and
B
Activity of resynthesized hits in the Toxin B biochemical
assay
• 5.5 million compounds • small molecules synthesized on
polystyrene beads • binary encoding – inert chemical tags
Protection from Toxin A-induced cytotoxicity
CELL-BASED ACTIVITY Compound 6 protects CHO-K1 cells against
toxin
B-induced cytotoxicity