Results continued - • Group study rooms and tables were added.
Miniaturized assays for RhoA inactivation through glucosylation … · 2017-09-11 · MATERIALS AND...
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MATERIALS AND METHODS BACKGROUND RESULTS ABSTRACT GOALS RESULTS (CONTINUED) RESULTS (CONTINUED) CONCLUSION Miniaturized assays for RhoA inactivation through glucosylation by Clostridium difficile toxin Ilana L. Stroke 1 , Linh Ma 1 , Jeffrey J. Letourneau 1 , Andrew G. Cole 1 , Yanfang Li 1 , Brett A. Marinelli1, John Malone1, Teresa Paulish-Miller2, Igor Pechik1, Jorge G. Quintero 1 , Joan E. Sabalski 1 , Diana R. Savoly 1 , Aiying Wang 1 , David W. Hilbert 2 , Scott Gygax 2 , Douglas K. Miller 1 , Philip Stein 1 , and Maria Webb 1 1 Venenum Biodesign, LLC, Hamilton, NJ, 08691 • 2 Femeris, Hamilton, NJ, 08690 VENENUM Biodesign Hamilton, NJ 08691 Toll Free:877-950-1032 • Fax:609-587-1827 www.venenumbiodesign.com Clostridium difficile, a Gram-positive, spore-forming bacterial species, is an opportunistic pathogen that proliferates when patients’ natural intestinal flora are compromised by treatment with antibiotics, causing severe diarrhea, intestinal inflammation, and in some cases, toxic megacolon and sepsis. These infections are highly contagious, particularly in hospital settings; in the United States, C. difficile infections account for over 80% of healthcare-associated infections and 14,000 deaths per year. C. difficile infections are typically treated with broad-spectrum antibiotics such as metronidazole or vancomycin. The recurrence rate for disease caused by C. difficile is high--- approximately 20% --- for patients receiving treatment with antibiotics, because of collateral effects on beneficial intestinal bacteria. For this reason, our laboratory has pursued the discovery of a non- antibiotic therapy for C. difficile-associated disease that would target the intracellular activity of the toxins in mammalian host cells, while allowing the natural intestinal microbiome to recover and eventually out-compete the C. difficile population. The C. difficile virulence factors (Toxins A and B, or TcdA and TcdB, respectively), are glucose transferases that use UDP-glucose as a substrate to glucosylate host Rho GTPases at a single threonine residue, interfering with the interaction between Rho family proteins and their effectors, and as a result, disrupting the actin cytoskeleton in intestinal epithelial cells and a variety of other cell types. Through ultra-high throughput screening of encoded combinatorial libraries (ECLiPS) and subsequent lead optimization in our laboratory, we have identified compounds that inhibit toxin B enzymatic activity, protect mammalian cells from both toxins A and B, and are efficacious in vivo in rodent models. We show here that representative small molecule inhibitors are able to protect RhoA from Toxin B and maintain RhoA’s ability to be activated to its GTP-bound form by guanine nucleotide exchange factors (GEF’s) in homogeneous, low-volume biochemical assays. Additionally, these small molecules, by shielding RhoA from modification by toxin, preserve RhoA’s interaction with GTPase-activating proteins (GAP’s) in vitro. (Supported by the Genesis Biotechnology Group). • Purified RhoA, hDbS (GEF), p50RhoGAP (Cytoskeleton) • GTP (BellBrook Labs) • mant-GTP = 2' - /3' - O - (N' - methylanthraniloyl)guanosine - 5'- O – triphosphate (AnaSpec) • TcdB glucosyltransferase domain (R&D Systems) • PicoProbe™ phosphate detection reagents (Biovision) • Exchange reaction method adapted from Cytoskeleton protocol Challenges in establishing the Rho GTPase assays • Sequential glucosylation and GTPase reactions produce both UDP and GDP • Commercial antibody-based kits detecting GDP (vs. GTP) are not specific for GDP (vs. UDP, ADP) • Phosphate assays detect GTPase activity but require phosphate- free buffers and high purity GTP • Low sensitivity of most phosphate assays • Four low-volume addition steps, with buffer adjustments • Design “mix and read”, low assay volume, room temperature biochemical assays suitable for HTS - Non-radioactive - No wash or separation steps • Sequential reactions in the same well - First step: pretreat Rho protein with C. difficile toxin glucosyltransferase domain - [Toxin quench] - Guanine exchange or GTPase reaction - Detection ( an additional step for GTPase assay) • We have demonstrated the feasibility of performing low-volume, non-radioactive, mix-and-read assays for C. difficile toxin modification of RhoA • These assays can be used to identify and evaluate small- molecule toxin inhibitors from T. Jank and K. Aktories (2008) Trends in Microbiology 16:222 Receptor binding, endocytosis, and autoprocessing of C. difficile toxins deliver the glucosyltransferase domain to the cytoplasm, where it modifies Rho GTPases. adapted from A. Davies et al., Biochem. J. (2011) 436, 517 Rho GTPase glucosylation by C. difficile toxin Glucosylation blocks the interaction of Rho GTPases with regulator proteins: GAP = GTPase Activating Protein GEF = Guanine nucleotide Exchange Factor GDI = GDP Dissociation Inhibitor RhoA mant GTP GEF RhoA mant GTP High fluorescence Low fluorescence RhoA glucosylaOon RHOA INTERACTION WITH GUANINE EXCHANGE FACTOR (FLUORESCENCE-BASED GEF ASSAY) 1536-WELL FORMAT ASSAY FOR GAP-ENHANCED RhoA GTPase ACTIVITY RhoA / mant-GTP binding promoted by GEF: inhibition by Toxin B (TcdB) and protection by TcdB inhibitor (384-well format) 1536-well format RhoA/GAP GTPase activity assay for Toxin B (TcdB) inhibitors using TcdB glucosyltransferase domain RhoA alone, or RhoGAP alone, show little or no GTPase activity, but in the absence of pretreatment with TcdB, RhoA and GAP synergize to hydrolyze GTP. RhoA GTPase assay in 1536-well format using RhoA + RhoGAP pretreated with TcdB and UDP-glucose: protection by TcdB inhibitors Same rank order of potency for TcdB inhibitors in UDP-glucose hydrolysis assay and Rho + GAP assay Compounds 1-4 are analogs of hits identified by high-throughput screening at Venenum Biodesign. 1536-well format RhoA/ GEF (mant-GTP binding) assay 2 µL TcdB 1.5 µL RhoA + UDPglucose 1 hour at room temperature 2.5 µL mantGTP 2 µL hDbs (GEF) DetecOon Ome course (360nm/ 440nm fluorescence) Glucosyltransferase reacOon Exchange reacOon (mantGTP binding) 1 µL RhoA + UDPglucose 1 hour at room temperature Add 1 µL GTP + EDTA 45 minutes at room temperature 4 µL PicoProbe™ phosphate detecOon reagent Detect fluorescence (530 nm/ 590 nm) 1.5 hours at room temperature 2 µL RhoGAP +/ TcdB Glucosyltransferase reacOon Toxin quench and GTPase reacOon Dry test compound in wells Phosphate assay UDP-glucose and GTP were present in all assays. . Compound 1 is a TcdB inhibitor (Venenum Biodesign).
Transcript of Miniaturized assays for RhoA inactivation through glucosylation … · 2017-09-11 · MATERIALS AND...
MATERIALS AND METHODS
BACKGROUND
RESULTS
ABSTRACT GOALS RESULTS (CONTINUED) RESULTS (CONTINUED)
CONCLUSION
Miniaturized assays for RhoA inactivation through glucosylation by Clostridium difficile toxin
Ilana L. Stroke1, Linh Ma1, Jeffrey J. Letourneau1, Andrew G. Cole1, Yanfang Li1, Brett A. Marinelli1, John Malone1, Teresa Paulish-Miller2, Igor Pechik1, Jorge G. Quintero1, Joan E. Sabalski1, Diana R. Savoly1, Aiying Wang1, David W. Hilbert2, Scott Gygax2, Douglas K. Miller1, Philip Stein1, and Maria Webb1
1Venenum Biodesign, LLC, Hamilton, NJ, 08691 • 2Femeris, Hamilton, NJ, 08690
VENENUM BiodesignHamilton, NJ 08691
Toll Free:877-950-1032 • Fax:609-587-1827www.venenumbiodesign.com
Clostridium difficile, a Gram-positive, spore-forming bacterial species, is an opportunistic pathogen that proliferates when patients’ natural intestinal flora are compromised by treatment with antibiotics, causing severe diarrhea, intestinal inflammation, and in some cases, toxic megacolon and sepsis. These infections are highly contagious, particularly in hospital settings; in the United States, C. difficile infections account for over 80% of healthcare-associated infections and 14,000 deaths per year. C. difficile infections are typically treated with broad-spectrum antibiotics such as metronidazole or vancomycin. The recurrence rate for disease caused by C. difficile is high--- approximately 20% --- for patients receiving treatment with antibiotics, because of collateral effects on beneficial intestinal bacteria. For this reason, our laboratory has pursued the discovery of a non-antibiotic therapy for C. difficile-associated disease that would target the intracellular activity of the toxins in mammalian host cells, while allowing the natural intestinal microbiome to recover and eventually out-compete the C. difficile population. The C. difficile virulence factors (Toxins A and B, or TcdA and TcdB, respectively), are glucose transferases that use UDP-glucose as a substrate to glucosylate host Rho GTPases at a single threonine residue, interfering with the interaction between Rho family proteins and their effectors, and as a result, disrupting the actin cytoskeleton in intestinal epithelial cells and a variety of other cell types.
Through ultra-high throughput screening of encoded combinatorial libraries (ECLiPS) and subsequent lead optimization in our laboratory, we have identified compounds that inhibit toxin B enzymatic activity, protect mammalian cells from both toxins A and B, and are efficacious in vivo in rodent models. We show here that representative small molecule inhibitors are able to protect RhoA from Toxin B and maintain RhoA’s ability to be activated to its GTP-bound form by guanine nucleotide exchange factors (GEF’s) in homogeneous, low-volume biochemical assays. Additionally, these small molecules, by shielding RhoA from modification by toxin, preserve RhoA’s interaction with GTPase-activating proteins (GAP’s) in vitro. (Supported by the Genesis Biotechnology Group).
• Purified RhoA, hDbS (GEF), p50RhoGAP (Cytoskeleton)• GTP (BellBrook Labs) • mant-GTP = 2' - /3' - O - (N' - methylanthraniloyl)guanosine -
5'- O – triphosphate (AnaSpec)• TcdB glucosyltransferase domain (R&D Systems)• PicoProbe™ phosphate detection reagents (Biovision)• Exchange reaction method adapted from Cytoskeleton protocol
Challenges in establishing the Rho GTPase assays• Sequential glucosylation and GTPase reactions produce both UDP
and GDP• Commercial antibody-based kits detecting GDP (vs. GTP) are not
specific for GDP (vs. UDP, ADP)• Phosphate assays detect GTPase activity but require phosphate-
free buffers and high purity GTP• Low sensitivity of most phosphate assays• Four low-volume addition steps, with buffer adjustments
• Design “mix and read”, low assay volume, room temperature biochemical assays suitable for HTS - Non-radioactive - No wash or separation steps
• Sequential reactions in the same well - First step: pretreat Rho protein with C. difficile toxin glucosyltransferase domain
- [Toxin quench] - Guanine exchange or GTPase reaction - Detection ( an additional step for GTPase assay)
• We have demonstrated the feasibility of performing low-volume, non-radioactive, mix-and-read assays for C. difficile toxin modification of RhoA
• These assays can be used to identify and evaluate small-molecule toxin inhibitors
BACKGROUND
from T. Jank and K. Aktories (2008) Trends in Microbiology 16:222
Receptor binding, endocytosis, and autoprocessing of C. difficile toxins deliver the glucosyltransferase domain to the cytoplasm, where it modifies Rho GTPases.
adapted from A. Davies et al., Biochem. J. (2011) 436, 517
Rho GTPase glucosylation by C. difficile toxin
Glucosylation blocks the interaction of Rho GTPases with regulator proteins: GAP = GTPase Activating Protein GEF = Guanine nucleotide Exchange Factor GDI = GDP Dissociation Inhibitor
RhoA interaction with Guanine Exchange Factor
(fluorescence-based GEF ASSAY)
RhoA
mant-‐ GTP
GEF RhoA
mant-‐ GTP
High fluorescence Low fluorescence
RhoA glucosylaOon
RhoA / mant-‐GTP binding promoted by GEF: inhibiOon by Toxin B (TcdB) and protecOon by TcdB inhibitor (384-‐well format)
(picture available as JPEG)
Compound 1 is a TcdB inhibitor (Venenum Biodesign).
RHOA INTERACTION WITH GUANINE EXCHANGE FACTOR (FLUORESCENCE-BASED GEF ASSAY) 1536-WELL FORMAT ASSAY FOR GAP-ENHANCED
RhoA GTPase ACTIVITY
RhoA / mant-GTP binding promoted by GEF: inhibition by Toxin B (TcdB) and protection by TcdB inhibitor
(384-well format)
1536-well format RhoA/GAP GTPase activity assay for Toxin B (TcdB) inhibitors using TcdB glucosyltransferase domain
RhoA alone, or RhoGAP alone, show little or no GTPase activity, but in the absence of pretreatment with TcdB, RhoA
and GAP synergize to hydrolyze GTP.
RhoA GTPase assay in 1536-well format using RhoA + RhoGAP pretreated with TcdB and UDP-glucose: protection
by TcdB inhibitors
Same rank order of potency for TcdB inhibitors in UDP-glucose hydrolysis assay and Rho + GAP assay
Compounds 1-4 are analogs of hits identified by high-throughput screening at Venenum Biodesign.
1536-well format RhoA/ GEF (mant-GTP binding) assay1536-well format RhoA/ GEF (mant-GTP binding) assay
2 µL TcdB
1.5 µL RhoA + UDP-‐glucose
1 hour at room temperature
2.5 µL mant-‐GTP
2 µL hDbs (GEF)
DetecOon Ome course (360nm/ 440nm fluorescence)
Glucosyltransferase reacOon
Exchange reacOon (mant-‐GTP binding)
(picture available as JPEG)
1536-well format RhoA/GAP GTPase activity assay for Toxin B (TcdB) inhibitors using TcdB glucosyltransferase domain
1 µL RhoA + UDP-‐glucose
1 hour at room temperature
Add 1 µL GTP + EDTA
45 minutes at room temperature
4 µL PicoProbe™ phosphate detecOon reagent
Detect fluorescence (530 nm/ 590 nm)
1.5 hours at room temperature
2 µL RhoGAP +/-‐ TcdB Glucosyltransferase
reacOon
Toxin quench and GTPase reacOon
Dry test compound in wells
Phosphate assay
UDP-glucose and GTP were present in all assays.
(picture available as JPEG)
RhoA alone, or RhoGAP alone, show little or no GTPase activity, but in the absence of pretreatment with TcdB, RhoA and GAP synergize to hydrolyze GTP. .
RhoA GTPase assay in 1536-well format using RhoA + RhoGAP pretreated with TcdB and UDP-glucose:
protection by TcdB inhibitors
Compounds 1-4 are analogs of hits identified by high-throughput screening at Venenum Biodesign.
(picture available as JPEG; 11-18-14 at 111 minutes)
RhoA GTPase assay in 1536-well format using RhoA + RhoGAP pretreated with TcdB and UDP-glucose:
protection by TcdB inhibitors
Compounds 1-4 are analogs of hits identified by high-throughput screening at Venenum Biodesign.
(picture available as JPEG; 11-18-14 at 111 minutes)
Same rank order of potency for TcdB inhibitors in UDP-‐glucose hydrolysis assay and Rho + GAP assay
(omit from poster: Source: 11/18/14)
Compound 1 is a TcdB inhibitor (Venenum Biodesign).
