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Lucerna, Inc., 3960 Broadway, Suite 330E, New York NY 10032
Cyclic di-guanosine monophosphate (c-di-GMP)
Hengge, Nat. Rev. Microbiol., 2009
Methods: Drawbacks:
c-di-GMP analysis: current methods
Labor intensive and cumbersome Need of expensive equipment Require extensive optimization Poor detection range High background/low specificity Not easily adaptable for HTS
HPLC-MS FRET Fluorescence Circular dichroism Allosteric ribozymes Intercalator dyes
Sensitivity and selectivity of c-di-GMP sensor
c-di-GMP assay: storage stability
c-di-GMP sensor is stable for 6 months at -20oC and -80oC
c-di-GMP sensor stability at-20oC c-di-GMP sensor stability at -80oC
DFHBI + GMP + c-di-GMP
RFU
0
50
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150
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250
1 Week6 Months
DFHBI + GMP + c-di-GMPR
FU0
50
100
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1 Week 6 months
Distinct signal response to differing levels of c-di-GMP in bacterial cells expressing wild type, catalytically dead and constitutively active WspR mutants Assay signal is stable over time, allowing a large window for measurement
c-di-GMP assay performance in bacterial lysates
Estimation in bacterial lysates using c-di-GMP assay
c-di-GMP standard curve using HPLC peak areas
Time (min)-5 0 5 10 15 20 25 30 35
mA
U (2
53nm
)
-50
0
50
100
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200
% A
ceto
nitri
le
-20
0
20
40
60
80
100Wild type WspRCatalytically dead WspRConstitutively active WspR
Gradient
c-di-GMP estimation by c-di-GMP sensor vs HPLC
Comparison of c-di-GMP levels in bacterial lysate
Chromatogram of bacterial extracts
c-di-GMP analysis in bacteria cultures
c-di-GMP assay in bacterial cultures
[c-di-GMP, pg/!l]
0 200 400 600 800
RFU
50
100
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300
350Control+ Nitrofurazone+ DMF
[c-di-GMP, pg/!l]0 50 100 150 200
RFU
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300Control+ Nitrofurazone+ DMF
c-di-GMP standard curve c-di-GMP estimates in WT WspR cells
Bacterial dilution1 10 100 1000 10000
[c-d
i-GM
P, p
g/!
l]
-50
0
50
100
150
200Control+ Nitrofurazone+ DMF
Summary
Grow bacteria culture O/N
Inoculate bacteria in wells and incubate
Add IPTG to induce WspR expression/
add antibiotic/vehicle
Add assay buffer with fluorophore and c-di-GMP sensor to wells
Read plate for fluorescence (Ex:469/Em:501nm) c
-di-G
MP
sens
or
assa
y w
orkf
low
30 min – 24h
Introduction
A bacterial second messenger Key roles in bacterial motility, virulence, biofilm formation, cell cycle, antibiotic resistance, and tolerance c-di-GMP levels are regulated by diguanlyate cyclases (DGC) and phosphodiesterases (PDE) Biofilms are responsible for nosocomial, medical device linked infections and chronic infections in diseases such as cystic fibrosis, endocarditis, and chronic prostatitis c-di-GMP pathways are a potential targets for drug discovery
Biofilms play an important role in promoting bacterial tolerance and antibiotic resistance, two major threats in global public health. Despite NIH’s estimate that 80% of all infectious diseases are caused by microorganisms in the biofilm state, there are no effective ways to prevent biofilm formation. Cyclic di-guanosine monophosphate (c-di-GMP) is a key second messenger involved in biofilm formation and enzymes that are involved in production and degradation of c-di-GMP are an attractive target for drug discovery. Current methods to monitor c-di-GMP, such as HPLC and MS, are slow and cumbersome, and requires organic extraction. Thus, there is a need for robust mix-and-use methods to accelerate the discovery of drugs that can inhibit biofilm formation and prevent bacterial tolerance.
Estimated c-di-GMP concentrations in bacteria expressing wild type, catalytically dead, and constitutively active WspR mutants
WspR, a diguanylate cyclase (DGC) from pseudomonas aeruginosa, uses two GTP molecules to produce c-di-GMP and pyrophosphate A bacterial cell model (E. coli) expressing wild type, catalytically dead, and constitutively active WspRs were used in the analysis c-di-GMP levels in bacteria was measured using the c-di-GMP assay c-di-GMP levels in bacteria were independently evaluated by HPLC and compared with the c-di-GMP assay estimates
Highly selective to c-di-GMP Assay meets HTS criteria with Z factor >0.9 and S/B >5, even in the presence of 500-1000 fold excess counter ligands (GMP, pGpG and c-AMP-GMP)
Sensitive even at lower physiological concentrations Excellent dynamic range (50nM-1000nM) High selectivity for c-di-GMP vs. GMP, pGpG (c-di-GMP hydrolysis products)
c-di-GMP levels estimated by LucernaTM c-di-GMP assay and HPLC are comparable
Bacteria expressing wild type WspR were plated at varying cell densities and treated with the antibiotic nitrofurazone, its vehicle dimethyl formamide (DMF), or left untreated c-di-GMP concentrations in bacteria were determined by the c-di-GMP assay in a homogenous format that requires no lysis or pelleting or wash steps
c-di-GMP standards (0-1000nM) were assayed in LB media with antibiotic nitrofurazone (antibiotic), DMF (vehicle), or control (untreated) Presence of nitrofurazone, DMF, or LB media alone did not affect the performance of the c-di-GMP sensor assay c-di-GMP concentrations in bacteria were estimated based on the standard curve Changes in c-di-GMP levels by varying cell densities or by treatment with antibiotics are detected by the c-di-GMP sensor assay
Easy-to-use homogeneous assay format Quick signal response and prolonged signal stability Broad dynamic range (50nM - 1000nM) High selectivity towards c-di-GMP vs. counter ligands Compatible with biochemical and cell-based applications Assay is stable for at least 6 months when stored at -20 oC or lower Meets NCATS’ HTS assay guidance criteria and suitable for drug discovery HPLC standard curve of c-di-GMP shows good fit with a r2 >0.97
Quick sensor response allows for fast c-di-GMP detection Stable fluorescent signal allows a large measurement window
c-di-GMP analysis in bacterial lysates
Selectivity of c-di-GMP sensor
c-di-G
MP GMP
pGpG
c-AMP
-GMP
RFU
0
100
200
300
400
500
600
1000X
500X 50
0X
Inoculate bacteria culture O/N
Inoculate fresh 25ml culture
Add 1mM IPTG at OD600 = 0.5 and induce
cells for 2-3 h
Harvest cells, normalize OD, and make aliquots and
freeze
Lyse cells with lysis buffer
c-di-GMP assay
Organic extraction
HPLC measurement
Bacterial lysate (!l)
0 2 4 6 8 10 12 14
[c-d
i-GM
P, p
g]
-40
0
40
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Wild type WspRCat. dead WspRConst. active WspR
[c-di-GMP, pg/!l]
0 200 400 600 800
RFU
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[c-di-GMP, pg/!l]
0 50 100 150 200
RFU
-1000
100200300400500600
c-di-GMP estimates c-di-GMP standard curve
c-di-GMP assay principle
Fluorogenic c-di-GMP assay based on Lucerna’s SpinachTM technology Binding of c-di-GMP to the riboswitch module of the c-di-GMP sensor induces the folding of the Spinach aptamer and its binding to DFHBI DFHBI-bound c-di-GMP sensor emits green fluorescence detectable in the GFP/FITC channel and reflects the amount of c-di-GMP present
Time (min)0 200 400 600 800 1000 1200
RFU
0
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c-di-GMPGMP
Assay response and signal stability
Time (min)0 200 400 600 800 1000 1200
RFU
0
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700
Wild type WspRCatalytically dead WspRConstitutively active WspR
Time (min)0 200 400 600 800 1000 1200
Fold
cha
nge
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Z fa
ctor
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Wt WspR vs catalytically dead WspRconst. active WspR vs cat. dead WspR
[c-di-GMP, !M]0 5 10 15 20 25
mAU
(253
nm)
0
1000
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3000
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5000
Time (min)-5 0 5 10 15 20 25 30 35
% A
ceto
nitri
le
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mAU
(253
nm)
-100
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100
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20105210.50.10.05 0
c-di-GMP(!M) Gradient
Chromatogram of c-di-GMP standards c-di-GMP standard curve
Cyclic-di-GMP assay kit: A first-in-class HTS assay for bacterial tolerance drug discovery Balajee Somalinga and Karen Wu
µ µ
µ
µ
µ
µ
µ
µ
[Ligand, nM]0 200 400 600 800 1000 1200
RFU
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350c-di-GMPGMPcGMPpGpG
[Ligand, nM]
0 50 100 150 200 250 300
RFU
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250 c-di-GMPGMPcGMPpGpG
[Ligand, nM]0 200 400 600 800 1000 1200
Fold
cha
nge
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Z fa
ctor
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c-di-GMP vs GMPc-di-GMP vs cGMPc-di-GMP vs pGpG
c-di-GMP
c-di-GMP riboswitch
Spinach aptamer
DFHBI
Kellenberger et al. JACS, 2013
WspR HPLC c-di-GMP sensorWild type 14.1 11.0
Const. active 32.2 27.6
c-di-GMP (pg/µl of lysate)