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

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 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

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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

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c-di-GMP standard curve c-di-GMP estimates in WT WspR cells

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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

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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

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pGpG

c-AMP

-GMP

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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)

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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

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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

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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)