high throughput screening of combinatorial libraries for
TRANSCRIPT
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
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