Skip to main content
NCBI home page
ACS Pharmacology & Translational Science logoLink to ACS Pharmacology & Translational Science
. 2023 Nov 21;6(12):1851–1858. doi: 10.1021/acsptsci.3c00176

A Homogeneous Bioluminescent System to Monitor Cyclic Guanosine Monophosphate

Nathan H Murray †, Matthew A Larsen ‡, Kevin Hsiao †, Dareen Mikheil †, Gediminas Vidugiris †, Hui Wang ‡, Said A Goueli †,*
PMCID: PMC10714432  PMID: 38093844

Abstract

graphic file with name pt3c00176_0007.jpg

Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is mediated through highly regulated phosphodiesterase activities. cGMP metabolism has been targeted for the treatment of several diseases, including erectile dysfunction, hypertension, and heart failure. As more drugs are being sought, it will be critical to develop assays that accurately determine cGMP levels. Here, we present cGMP Lumit, a sensitive and specific bioluminescent assay to detect cGMP. We demonstrate the utility of the detection system in enzyme assays, cell-based assays, and high-throughput screening formats. It is anticipated that this assay will be of significant value to aid in further understanding the role of cGMP in physiology and support further drug discovery efforts toward the treatment of human disease.

Keywords: cGMP, cyclic nucleotides, phosphodiesterase, guanylate cyclase, bioluminescence

Since its discovery as a natural product in 1963,1 cyclic guanosine 3′,5′-monophosphate (cGMP) has emerged as a critical secondary messenger in cell signaling and physiology.2 As a second messenger, cellular production of cGMP amplifies signals from multiple sources including nitric oxide (NO) activation of soluble guanylate cyclase (sGC)3,4 and activation of the transmembrane form of guanylate cyclase by natriuretic peptides.5 Production of cGMP leads to downstream effects including activation of protein kinase G,6 regulation of phosphodiesterase (PDE) activity,7 and regulation of cyclic nucleotide-gated channels.8 Through these effects, cGMP helps mediate various physiological responses such as phototransduction, smooth muscle relaxation, and cell growth and differentiation.2 The loss of cGMP signaling through its breakdown is primarily determined through highly regulated PDE activities.9

Given its various roles in human health, cGMP signaling is an important target for therapeutic intervention. The most notable commercial success by targeting cGMP metabolism is the development of PDE5 inhibitors such as sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) to treat erectile dysfunction.10 cGMP signaling has also been targeted in other disease states including pulmonary hypertension,10,11 retinal disease,12 diabetes,13 heart failure,11 kidney dysfunction,14,15 and neurological disorders.16,17 Due to the importance of cGMP in human health and the need for further drug development, it will be necessary to establish robust and reliable assays to accurately determine cGMP levels.

Several systems are already in place to directly detect cGMP both in biochemical assays and in cells. Sensors based on cGMP-binding domains combined with fluorescence resonance energy transfer paired fluorophores18−21 or engineered firefly luciferase (Promega GloSensor) have enabled real-time quantification of cellular cGMP but require overexpression or cell engineering that would interfere with native cell function. Many commercially available biochemical and lytic detection systems utilize immunoassays. The most widely used is the enzyme-linked immunosorbent assay, which is lengthy, requires numerous wash steps, and is limited to low-throughput formats. Fluorescence-based immunoassays such as homogeneous time-resolved fluorescence have also been utilized; however, fluorescent signals are prone to interference, signal quenching, and light sensitivity and require specialized instrumentation. Bioluminescent systems overcome several limitations of fluorescence readouts,22,23 and the Promega PDE-Glo system has been used to study PDE activity against cGMP; however, the assay has high sensitivity for cAMP and therefore cannot be applied to cell-based systems. In this study, we utilize the NanoLuc Binary Technology (NanoBiT) system,24 a split luciferase made up of the Large BiT (LgBiT) subunit and the Small BiT (SmBiT) peptide. We combine a highly specific antibody with a cGMP-SmBiT tracer molecule and secondary antibody conjugated to LgBiT to detect cGMP biochemically and in cell lysate via bioluminescence. We demonstrate the utility of the detection system in enzymatic assays, cell-based assays, and high-throughput screening. The system is sensitive, specific, homogeneous, and amenable to miniaturization for various screening formats and requires only a simple luminometer for detection. This assay will serve as a useful tool for studying cGMP signaling and enabling further screening efforts for therapeutic development.

Experimental Section

cGMP-SmBiT Tracer Synthesis

Detailed methods can be found in the Supporting Information for synthesizing cGMP-PEG3-SmBiT and cGMP-C7-SmBiT. Experiments in this study utilized the cGMP-C7-SmBiT tracer.

Specificity Testing and Z′ Factor Determination

20 μL amount of indicated metabolite [cGMP Promega V6411; cyclic adenosine monophosphate (cAMP) Promega V6421; guanosine monophosphate (GMP) Sigma G-8377; cyclic guanosine monophosphate-adenosine monophosphate (2′3′-cGAMP) Cayman Chemical 19887] diluted in 1× Immunoassay Buffer C (IAB-C) (Promega VB115B) was added to a 96-well plate (Corning 3912). 20 μL portion of Antibody Mix [2 nM Tracer; 3:500 diluted Lumit Anti-Rabbit Ab-LgBit (Promega W1042); 2× IAB-C; Anti-cGMP Antibody (final 2 ng/well) (Invitrogen MA5–44557)] was then added to each well. Plates were mixed on an orbital shaker for 5 min at 400 rpm and then incubated for 1 h at room temperature. 10 μL of Nano-Glo Luciferase Assay Substrate (Promega N113C) diluted 12.5-fold in 1× IAB-C were then added to each well. Plates were mixed on an orbital shaker for 3 min at 400 rpm, and then luminescence was measured on a GloMax Discover Microplate Reader. Reported [metabolite] concentrations are in the initial 20 μL prior to the addition of Antibody Mix. Z′ factors were determined, as previously described.25 EC50, EC90, and EC10 values were determined by fitting to a sigmoidal nonlinear regression in GraphPad Prism. For significance claims, p-values were calculated using an unpaired, two-tailed Student’s t-test, and p < 0.05 was considered significant. For Z′ factor studies, normality was determined using a D’Agostino and Pearson test with p = 0.05 as the cutoff.

LOPAC Library Screening

The LOPAC1280 library (Sigma-Aldrich catalog no. LO4100–1EA) is a collection of 1280 pharmacologically active compounds from 56 pharmacological classes with well-characterized activities. 5 μL of 1× IAB-C (Promega VB115B) with 0.1% DMSO, 10 μM cGMP (Promega V6411) in 1× IAB-C with 0.1% DMSO, or 10 μM library of pharmacologically active compound (LOPAC) in 1× IAB-C with 0.1% DMSO was added to 384-well low volume plates (Corning 4512). 5 μL of HTS Antibody Mix [2 nM Tracer; 3:500 diluted Lumit Anti-Rabbit Ab-LgBit (Promega W1042); 2× IAB-C; Anti-cGMP Antibody (final 0.2 ng/well) (Invitrogen MA5–44557)] was then added to each well. Plates were then incubated for at least 1 h at room temperature. 5 μL of Nano-Glo Luciferase Assay Substrate (Promega N113C) diluted 20.8-fold in 1× IAB-C was then added to each well. Plates were briefly mixed, and then luminescence was measured on a TECAN SPARK 20 M te-cool microplate reader. The Te-Cool temperature control module enables setting the temperature inside the measurement chamber of a reader to be equal to the room temperature, e.g., 22 °C, which minimizes luminescence signal variations during the plate read time. Liquid dispensing was performed using a ThermoFisher Scientific Multidrop Combi nL Reagent Dispenser. Z′ factors and significant interfering compounds were determined for individual plates. Data shown are normalized to 1× IAB-C with 0.1% DMSO (100% normalized activity) and 10 μM cGMP in 1× IAB-C with 0.1% DMSO (0% normalized activity) measured on the same plate.

PDE5A1 and PDE6C Activity Assays

PDE5A1 (BPS Biosciences 60050) and PDE6C (BPS Biosciences 60062) were serially diluted in PDE reaction buffer (40 mM Tris pH 7.5; 10 mM MgCl2; 0.1 mg/mL BSA), and 15 μL was added to a 96-well plate (Corning 3912). 15 μL 100 nM cGMP diluted in PDE reaction buffer was added to each well to initiate the reaction. PDE reactions were mixed on an orbital shaker for 5 min at 400 rpm then incubated for an additional 25 min at 25 °C. Reactions were terminated by the addition of 20 μL of 1.25 mM IBMX (Sigma I7018) in PDE reaction buffer, and plates were mixed on an orbital shaker for 5 min at 400 rpm. 50 μL Antibody Mix was then added to each well. Plates were then mixed for 5 min at 400 rpm and then incubated for 1 h at room temperature. 25 μL of Nano-Glo Luciferase Assay Substrate (Promega N113C) diluted 12.5-fold in 1× IAB-C was then added to each well. Plates were mixed on an orbital shaker for 3 min at 400 rpm, and then luminescence was measured on a GloMax Discover Microplate Reader. 30 μL of cGMP standards in PDE reaction buffer was treated with IBMX and measured using cGMP Lumit on the same plate as experimental samples. Relative light units (RLUs) were converted to cGMP concentrations by fitting cGMP standards to a four parameter logistic curve and interpolating [cGMP] experimental samples using GraphPad Prism.

PDE5A1 Inhibition Study

PDE5A1 (BPS Biosciences 60050) was diluted to 0.11 ng/μL in PDE reaction buffer, and 10 μL was added to a 96-well plate (Corning 3912). Sildenafil citrate (Sigma SML3033), tadalafil (Sigma SML1877), zaprinast (Sigma Z0878), and IBMX (Sigma I7018) were serially diluted in PDE reaction buffer with equivalent DMSO, and 10 μL was added to wells. Reactions were mixed on an orbital shaker for 10 min at 400 rpm. 10 μL of 37.5 nM cGMP in PDE reaction buffer was added to initiate the reactions, which were mixed on an orbital shaker for 5 min at 400 rpm and then incubated for an additional 25 min at 25 °C. Reactions were terminated, and cGMP was detected as described above for PDE activity assays. Reported [inhibitor] are concentrations in the 30 μL enzyme reaction prior to halting with excess IBMX. Percent inhibition was determined by normalizing to a no inhibitor control (0%) and a no enzyme control (100%). To determine IC50 values, the data were fit to a nonlinear regression (log[inhibitor] vs response, variable slope, and four parameters) in GraphPad Prism.

RFL-6 Cell-Based Assays

RFL-6 cells (ATCC CCL-192) were maintained in F-12K Medium (ATCC 30–2004) with 20% FBS (Avantor). Cells were isolated via trypsinization and counted by using a BioRad TC20 automated cell counter. Cells were plated in 96-well plates (Corning 3917) at either 50,000 cells/well [S-nitroso-N-acetyl-d,l-penicillamine (SNAP) treatment] or 75,000 cells/well [atrial natriuretic peptide (ANP) treatment] in 100 μL of media and allowed to adhere overnight. The next day, ANP (Sigma A1663) and SNAP (Cayman Chemical 82250) were serially diluted in HBSS (Gibco 141175–095) containing 500 μM IBMX (Sigma I5879) and 100 μM Ro 20–1724 (Sigma B8279). Media was removed, and cells were treated with serial dilutions of SNAP or ANP for 15 min at 37 °C. Reactions were terminated, and cells were lysed by the addition of 10 μL of 4% TCA in water. Plates were mixed on an orbital shaker for 5 min at 350 rpm. Reactions were then neutralized by the addition of 10 μL of 0.5 M Tris (pH 9) followed by mixing on an orbital shaker for 5 min at 350 rpm. 50 μL of Antibody Mix was then added to each well. Plates were then mixed for 7 min at 350 rpm and then incubated for 1 h at room temperature. 25 μL of Nano-Glo Luciferase Assay Substrate (Promega N113C) diluted 12.5-fold in 1× IAB-C was then added to each well. Plates were mixed on an orbital shaker for 3 min at 400 rpm, and then luminescence was measured on a GloMax Discover Microplate Reader. Reported [SNAP] and [ANP] are concentrations in the 30 μL treatment prior to TCA addition. 30 μL of cGMP standards diluted in HBSS with IBMX and Ro 20–1724 was dispensed on cells equivalent to experimental conditions, treated with TCA and Tris, and measured using cGMP Lumit in parallel with experimental samples. RLUs were converted to cGMP concentrations by fitting cGMP standards to a four parameter logistic curve and interpolating [cGMP] experimental samples using GraphPad Prism. EC50 values were determined by fitting to a sigmoidal nonlinear regression in GraphPad Prism.

Software and Statistics

Statistics were performed using Microsoft Excel and GraphPad Prism 9.1.0. Figures are generated using Microsoft Excel, GraphPad Prism 9.1.0., Microsoft Powerpoint, and Adobe Illustrator. In all cases, n refers to independent samples within an experiment, and SD refers to standard deviation.

Results and Discussion

Assay Principle and Format

cGMP Lumit is a competitive immunodetection system for monitoring cGMP following an in vitro enzyme reaction or cell treatment. A schematic representation of the Lumit cGMP assay is shown in Figure 1. Like other Lumit technologies,26 this system takes advantage of the NanoLuc Binary Technology (NanoBiT),24 a structural complementation system composed of the Large BiT (LgBiT) luciferase subunit and the Small BiT (SmBiT) peptide. When in close proximity, these two subunits interact, forming an active NanoLuc luciferase enzyme. In the presence of the substrate, the luciferase produces light proportional to the level of NanoBiT complementation. Here, we utilize a cGMP-SmBiT tracer molecule that mimics endogenous cGMP. We add a specific anti-cGMP primary antibody and a secondary antibody conjugated to the LgBiT subunit. In the absence of cGMP, the SmBiT tracer will complement the LgBiT subunit and produce light upon the addition of the substrate. The presence of cGMP competes with the cGMP-SmBiT tracer for antibody binding, resulting in a loss of NanoBiT complementation and corresponding bioluminescence.

Figure 1.

Figure 1

Open in a new tab

Scheme for the detection of cGMP using the cGMP Lumit assay.

Assay Development

To develop the assay, it is essential to find effective antibody–tracer pairs. The top combination was a cGMP-SmBiT tracer with a seven-carbon linker between the cGMP and SmBiT peptide paired with a cGMP antibody from Invitrogen (MA5–44557). This pairing consistently showed a greater than 25-fold loss of the signal in response to 10 μM cGMP compared with no cGMP controls. We demonstrated a dose-dependent response to a cGMP standard titration with an EC50 of 6.1 nM cGMP (Figure 2A). The experimental concentration of the assay ranged from 0.56 to 65 nM cGMP using the EC10 and EC90 values as cutoffs. This assay has comparable sensitivity with established highly sensitive assays such as the radioimmunoassay,27 the radioimmunoassay with acetylation,28 and the recently developed homogeneous quenching resonance energy-transfer assay.29 To determine the specificity of the assay, we also performed titrations with the potential competing metabolites GMP, cAMP, and 2′3′-cGAMP (Figure 2A). None of the other metabolites led to more than 15% loss of luminescence at concentrations as high as 10 μM. To determine if these competing metabolites would interfere with the ability to measure cGMP, we included the potential competitors in the assay at a concentration of 10 μM. In the presence of competitors, we were able to produce nearly identical cGMP standard curves with EC50 values within 1.1 nM of buffer controls, although 11 individual values did differ from controls with statistical significance (Figure 2B). Thus, these results demonstrate that the assay is sensitive, specific, and should not be largely compromised by competitor metabolites present in cells.

Figure 2.

Figure 2

Open in a new tab

cGMP detection is sensitive and specific. (A) Titrations of cGMP and potential competitor metabolites in a cGMP Lumit assay (n = 4 ± SD). (B) Titrations of cGMP in the presence of potential competitor metabolites at 10 μM or buffer alone in a cGMP Lumit assay (n = 4 ± SD). For significance claims, p-values were calculated using an unpaired, two-tailed Student’s t-test, and p < 0.05 was considered significant.

To measure the quality of the detection system, we determined Z′ factors for varying concentrations of cGMP, a standard metric in assay development for high-throughput screening.25 Assays are considered excellent and suitable for high-throughput screening with Z′ factors of between 0.5 and 1.0. In a 96-well format, we compared the luminescent signal from buffer alone and indicated concentrations of cGMP (Figure 3). The detection system was robust for concentrations ranging from 5 to 50 nM cGMP with all Z′ factor factors above 0.9. All buffer alone data sets and all but one cGMP concentration passed the D’Agostino and Pearson test for normality with p > 0.05 (25 nM cGMP p = 0.034). From these data, we concluded that our detection system has both the sensitivity and reproducibility required for high-throughput screening.

Figure 3.

Figure 3

Open in a new tab

Z′ factors indicate that cGMP Lumit is robust and reproducible. cGMP Lumit was performed on buffer only controls (n = 48) and indicated concentrations of cGMP (n = 48) to determine fold changes and Z′ factors. Normality testing was performed using the D’Agostino and Pearson test.

LOPAC Screening for Chemical Interference

To ensure that our detection system has utility in high-throughput screens, we formatted the assay to a 15 μL final volume in 384-well low volume plates. Changes from the 96-well format included decreasing the amount of antibody to 0.2 ng/well (see HTS Antibody Mix in the Experimental Section) and adjusting the NanoGlo Luciferase Assay Substrate stock concentration to yield the same final concentration in the assay. Once we determined that the assay was reproducible with buffer and 10 μM cGMP controls, we tested the assay for interfering compounds that could lead to false hits in a high-throughput chemical screen. Toward this goal, we performed our cGMP Lumit assay with samples containing buffer alone, 10 μM cGMP, or 10 μM compound from the LOPAC set (Figure 4). The LOPAC set is a small molecule library of 1280 compounds that covers major drug targets, including G protein-coupled receptors, kinases, and neurotransmitters. The assay was performed on each compound in quadruplicate, and normalization controls with buffer alone or cGMP were present on each plate. From this screen, we sought to determine the reproducibility of our system in a large-scale experiment as well as any nonspecific compounds that inhibit or activate the luminescent signal.

Figure 4.

Figure 4

Open in a new tab

cGMP Lumit LOPAC screen shows limited chemical interference (n = 4 per compound).

Previous studies have shown that both the NanoLuc luciferase and the NanoBiT system have minimal false hit rates in high-throughput formats.30,31 In line with these previous data, a low level of chemical interference was observed in our LOPAC screening. Using a cutoff rate of 3 standard deviations above or below the buffer only control, we saw no activating compounds and only nine compounds inhibiting luminescence, leading to a hit rate of 0.7%. Of these inhibitory compounds, only three led to a greater than 10% loss of normalized luminescence, and the most severe inhibitor only led to a 24% of the loss of signal observed with 10 μM cGMP. In this data set, we observed an average signal to background ratio of 69.1 and an average Z′ factor of 0.93 across all plates, indicating an adequate assay window with excellent reproducibility. These results confirm that the assay is robust and minimally susceptible to chemical interference that could lead to false hits.

Measurement of PDE Activity and Inhibition

PDE activity is critical to cGMP biology as these enzymes are the primary route for cGMP catabolism. PDEs are a large class of enzymes that range in their substrate specificities. The cGMP relevant enzymes include PDE5, −6, and −9, which are specific for cGMP, and PDE1, −2, −3, −10, and −11, which have activity against both cGMP and cAMP.9 PDEs have been successfully targeted in drug discovery, and their implication in wide-ranging therapeutic indications makes this enzyme family attractive for further inhibitor studies.7 To demonstrate the utility of cGMP Lumit in this context, we performed enzyme titrations and used our system to measure the PDE activity. PDE5A1 and PDE6C served as representative enzymes for this protein family that have known selective activity against cGMP. 30 μL reactions proceeded for 30 min at 25 °C before termination with 20 μL excess IBMX, a nonselective PDE inhibitor. We then performed the cGMP detection assay as before by adding an equal volume of Antibody Mix, incubating for 1 h, adding the NanoLuc substrate, and reading luminescence. RLUs were converted to [cGMP] using standard curves run in parallel. Both PDE5A1 and PDE6C showed measurable activity against cGMP and required a very low amount of enzyme for activity detection (Figure 5A). Specific activities for PDE5A1 and PDE6C were 78 and 11 nmol/min/mg, respectively, calculated using the data closest to the middle of the assay window (0.26 ng/well PDE5A1; 2.0 ng/well PDE6C).

Figure 5.

Figure 5

Open in a new tab

PDE activity and inhibition studies using cGMP Lumit. (b) (A) PDE reactions containing 50 nM cGMP and varied enzyme concentrations (n = 3 ± SD). (B) PDE5A1 reactions containing 1.1 ng/well enzyme and 12.5 nM cGMP with varied concentrations of indicated inhibitors (n = 3 ± SD).

A major target for cGMP PDE inhibition studies thus far has been PDE5, with several inhibitors resulting in FDA-approved drugs for the treatment of erectile dysfunction.10 To further demonstrate the capability of our detection system, we tested established inhibitors of PDE5 in our cGMP Lumit assay. Our study included sildenafil citrate (Viagra) and tadalafil (Cialis), both of which are FDA-approved PDE5 inhibitors. We also included the nonselective PDE inhibitor IBMX and the partially selective inhibitor zaprinast. All four compounds inhibited PDE5A1 activity against cGMP (Figure 5B). In line with previous studies,7 sildenafil citrate and tadalafil were the most potent, followed by zaprinast, then IBMX, with IC50 values of 1.2, 1.7, 190, and 3800 nM, respectively. The in vitro PDE studies here demonstrate that cGMP Lumit can be used to recapitulate established PDE activities and that the assay should serve as a useful tool for further therapeutic development.

Measurement of cGMP Production in RFL-6 Cells

In addition to the biochemical screening of modulators of PDEs, measurement of cGMP in a cellular context will be critical for establishing new cGMP-relevant therapeutics. One important target is guanylate cyclase, the protein that produces cGMP. sGC agonists are protective against both hypertension and chronic heart failure, and several drugs have been approved for clinical use. To test the ability of cGMP Lumit to measure cGMP in cells, we stimulated cGMP production in RFL-6 cells with either ANP to activate the membrane-bound guanylate cyclase or the NO donor SNAP to activate sGC. PDE inhibitors were included in the stimulation buffer to prevent the breakdown of cGMP. Following treatment, cells were lysed, and reactions were terminated by the addition of 4% TCA. The reaction was then neutralized with 0.5 M Tris (pH 9) before proceeding with cGMP Lumit detection. Both SNAP (Figure 6A) and ANP (Figure 6B) treatments resulted in detectable production of cGMP by the cGMP Lumit system with EC50 values of 290 nM and 2.3 nM, respectively. Full titrations are represented in RLUs, and points within the dynamic region of the assay (EC10 to EC90 of standard curves) were also converted to pmol of cGMP per cell using standard curves run in the equivalent cell lysate. Although the TCA and Tris treatment increased the background of the cGMP Lumit assay, at least a five-fold loss of luminescence was observed which provides a sufficient assay window for cGMP detection. Overall, the cGMP Lumit assay successfully measured cGMP production in a cellular context, providing an additional cell-based tool for the development of guanylate cyclase modulators and other cGMP-related therapies.

Figure 6.

Figure 6

Open in a new tab

Stimulated cGMP production in RFL-6 cells was measured by cGMP Lumit. (A) Activation of sGC by varying concentrations of SNAP measured by cGMP Lumit (n = 2 ± SD). (B) Activation of membrane-bound guanylate cyclase by varying concentrations of ANP measured by cGMP Lumit (n = 2 ± SD).

Conclusions

cGMP is an important second messenger in health and disease, and establishing systems for its detection will be critical in drug development surrounding cGMP metabolism. Many established methodologies for cGMP detection require cell engineering and extensive equipment or lack the requisite sensitivity and specificity. Here, we present cGMP Lumit, a simple, versatile, and robust assay capable of specific cGMP detection in the nanomolar range. We have demonstrated the utility of the detection system in enzyme assays, cell-based assays, and high-throughput formats. Our novel assay promises to aid in the continued therapeutic pursuit of targeting cGMP-related pathways and pathologies.

Acknowledgments

The authors acknowledge members of the Promega Assay Design Group for helpful input and discussion throughout this project.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00176.

  • Experimental details for synthesizing cGMP-PEG3-SmBiT and cGMP-C7-SmBiT; general experimental details for chemical synthesis; synthesis of SmBiT-PEG3-NH2; synthesis of SmBiT-C7-NH2; synthesis of cGMP-NHS; 1H NMR spectrum; 13C NMR spectrum; synthesis of cGMP-PEG3-SmBiT; and synthesis of cGMP-C7-SmBiT (PDF)

Author Contributions

N.H.M. and S.A.G. wrote the manuscript and conceived the overall project. M.A.L. and H.W. performed tracer synthesis and contributed to experimental design. G.V. performed and analyzed the LOPAC screen. N.H.M. performed experiments and analysis with experimental input from K.H., D.M., and S.A.G. All authors reviewed the manuscript.

The authors declare the following competing financial interest(s): This work was supported by Promega Corporation. N.H.M., M.A.L., K.H., D.M., G.V., H.W., and S.A.G. are employed by Promega Corporation. The authors declare no other competing financial interest.

Supplementary Material

pt3c00176_si_001.pdf (225.5KB, pdf)

References

  1. Ashman D. F.; Lipton R.; Melicow M. M.; Price T. D. Isolation of adenosine 3′, 5′-monophosphate and guanosine 3′, 5′-monophosphate from rat urine. Biochem. Biophys. Res. Commun. 1963, 11, 330–334. 10.1016/0006-291x(63)90566-7. [DOI] [PubMed] [Google Scholar]
  2. Beavo J. A.; Brunton L. L. Cyclic nucleotide research - still expanding after half a century. Nat. Rev. Mol. Cell Biol. 2002, 3 (9), 710–717. 10.1038/nrm911. [DOI] [PubMed] [Google Scholar]
  3. Horst B. G.; Marletta M. A. Physiological activation and deactivation of soluble guanylate cyclase. Nitric Oxide 2018, 77, 65–74. 10.1016/j.niox.2018.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dent M. R.; DeMartino A. W.; Tejero J.; Gladwin M. T. Endogenous Hemoprotein-Dependent Signaling Pathways of Nitric Oxide and Nitrite. Inorg. Chem. 2021, 60 (21), 15918–15940. 10.1021/acs.inorgchem.1c01048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chinkers M.; Garbers D. L.; Chang M. S.; Lowe D. G.; Chin H. M.; Goeddel D. V.; Schulz S. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 1989, 338 (6210), 78–83. 10.1038/338078a0. [DOI] [PubMed] [Google Scholar]
  6. Hofmann F. The biology of cyclic GMP-dependent protein kinases. J. Biol. Chem. 2005, 280 (1), 1–4. 10.1074/jbc.R400035200. [DOI] [PubMed] [Google Scholar]
  7. Maurice D. H.; Ke H.; Ahmad F.; Wang Y.; Chung J.; Manganiello V. C. Advances in targeting cyclic nucleotide phosphodiesterases. Nat. Rev. Drug Discov. 2014, 13 (4), 290–314. 10.1038/nrd4228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kaupp U. B.; Seifert R. Cyclic nucleotide-gated ion channels. Physiol. Rev. 2002, 82 (3), 769–824. 10.1152/physrev.00008.2002. [DOI] [PubMed] [Google Scholar]
  9. Francis S. H.; Blount M. A.; Corbin J. D. Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol. Rev. 2011, 91 (2), 651–690. 10.1152/physrev.00030.2010. [DOI] [PubMed] [Google Scholar]
  10. Ghofrani H. A.; Osterloh I. H.; Grimminger F. Sildenafil: from angina to erectile dysfunction to pulmonary hypertension and beyond. Nat. Rev. Drug Discov. 2006, 5 (8), 689–702. 10.1038/nrd2030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Stasch J. P.; Pacher P.; Evgenov O. V. Soluble guanylate cyclase as an emerging therapeutic target in cardiopulmonary disease. Circulation 2011, 123 (20), 2263–2273. 10.1161/CIRCULATIONAHA.110.981738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Luu J. C.; Saadane A.; Leinonen H.; Choi E. H.; Gao F.; Lewandowski D.; Halabi M.; Sander C. L.; Wu A.; Wang J. M.; Singh R.; Gao S.; Lessieur E. M.; Dong Z.; Palczewska G.; Mullins R. F.; Peachey N. S.; Kiser P. D.; Tabaka M.; Kern T. S.; Palczewski K. Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition. Proc. Natl. Acad. Sci. U.S.A. 2023, 120 (19), e2221045120 10.1073/pnas.2221045120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shao Y. X.; Huang M.; Cui W.; Feng L. J.; Wu Y.; Cai Y.; Li Z.; Zhu X.; Liu P.; Wan Y.; Ke H.; Luo H. B. Discovery of a phosphodiesterase 9A inhibitor as a potential hypoglycemic agent. J. Med. Chem. 2014, 57 (24), 10304–10313. 10.1021/jm500836h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Reinhart G. A.; Harrison P. C.; Lincoln K.; Chen H.; Sun P.; Hill J.; Qian H. S.; McHugh M. C.; Clifford H.; Ng K. J.; Wang H.; Fowler D.; Gueneva-Boucheva K.; Brenneman J. B.; Bosanac T.; Wong D.; Fryer R. M.; Sarko C.; Boustany-Kari C. M.; Pullen S. S. The Novel, Clinical-Stage Soluble Guanylate Cyclase Activator BI 685509 Protects from Disease Progression in Models of Renal Injury and Disease. J. Pharmacol. Exp. Ther. 2023, 384 (3), 382–392. 10.1124/jpet.122.001423. [DOI] [PubMed] [Google Scholar]
  15. Balzer M. S.; Pavkovic M.; Frederick J.; Abedini A.; Freyberger A.; Vienenkotter J.; Mathar I.; Siudak K.; Eitner F.; Sandner P.; Grundmann M.; Susztak K. Treatment effects of soluble guanylate cyclase modulation on diabetic kidney disease at single-cell resolution. Cell Rep. Med. 2023, 4 (4), 100992. 10.1016/j.xcrm.2023.100992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Layton M. E.; Kern J. C.; Hartingh T. J.; Shipe W. D.; Raheem I.; Kandebo M.; Hayes R. P.; Huszar S.; Eddins D.; Ma B.; Fuerst J.; Wollenberg G. K.; Li J.; Fritzen J.; McGaughey G. B.; Uslaner J. M.; Smith S. M.; Coleman P. J.; Cox C. D. Discovery of MK-8189, a Highly Potent and Selective PDE10A Inhibitor for the Treatment of Schizophrenia. J. Med. Chem. 2023, 66 (2), 1157–1171. 10.1021/acs.jmedchem.2c01521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wu Y.; Li Z.; Huang Y. Y.; Wu D.; Luo H. B. Novel Phosphodiesterase Inhibitors for Cognitive Improvement in Alzheimer’s Disease. J. Med. Chem. 2018, 61 (13), 5467–5483. 10.1021/acs.jmedchem.7b01370. [DOI] [PubMed] [Google Scholar]
  18. Honda A.; Adams S. R.; Sawyer C. L.; Lev-Ram V.; Tsien R. Y.; Dostmann W. R. Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (5), 2437–2442. 10.1073/pnas.051631298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Russwurm M.; Mullershausen F.; Friebe A.; Jager R.; Russwurm C.; Koesling D. Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochem. J. 2007, 407 (1), 69–77. 10.1042/BJ20070348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nikolaev V. O.; Gambaryan S.; Lohse M. J. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat. Methods 2006, 3 (1), 23–25. 10.1038/nmeth816. [DOI] [PubMed] [Google Scholar]
  21. Calamera G.; Li D.; Ulsund A. H.; Kim J. J.; Neely O. C.; Moltzau L. R.; Bjornerem M.; Paterson D.; Kim C.; Levy F. O.; Andressen K. W. FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons. Commun. Biol. 2019, 2, 394. 10.1038/s42003-019-0641-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. England C. G.; Ehlerding E. B.; Cai W. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjugate Chem. 2016, 27 (5), 1175–1187. 10.1021/acs.bioconjchem.6b00112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fan F.; Wood K. V. Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 2007, 5 (1), 127–136. 10.1089/adt.2006.053. [DOI] [PubMed] [Google Scholar]
  24. Dixon A. S.; Schwinn M. K.; Hall M. P.; Zimmerman K.; Otto P.; Lubben T. H.; Butler B. L.; Binkowski B. F.; Machleidt T.; Kirkland T. A.; Wood M. G.; Eggers C. T.; Encell L. P.; Wood K. V. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 2016, 11 (2), 400–408. 10.1021/acschembio.5b00753. [DOI] [PubMed] [Google Scholar]
  25. Zhang J. H.; Chung T. D.; Oldenburg K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screen 1999, 4 (2), 67–73. 10.1177/108705719900400206. [DOI] [PubMed] [Google Scholar]
  26. Hwang B. B.; Engel L.; Goueli S. A.; Zegzouti H. A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation. Commun. Biol. 2020, 3, 8. 10.1038/s42003-019-0723-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Steiner A. L.; Parker C. W.; Kipnis D. M. Radioimmunoassay for Cyclic Nucleotides. J. Biol. Chem. 1972, 247 (4), 1106–1113. 10.1016/s0021-9258(19)45621-4. [DOI] [PubMed] [Google Scholar]
  28. Harper J. F.; Brooker G. Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2’0 acetylation by acetic anhydride in aqueous solution. J. Cyclic Nucleotide Res. 1975, 1 (4), 207–218. [PubMed] [Google Scholar]
  29. Kopra K.; Sharina I.; Martin E.; Harma H. Homogeneous single-label cGMP detection platform for the functional study of nitric oxide-sensitive (soluble) guanylyl cyclases and cGMP-specific phosphodiesterases. Sci. Rep. 2020, 10 (1), 17469. 10.1038/s41598-020-74611-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ho P. I.; Yue K.; Pandey P.; Breault L.; Harbinski F.; McBride A. J.; Webb B.; Narahari J.; Karassina N.; Wood K. V.; Hill A.; Auld D. S. Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays. ACS Chem. Biol. 2013, 8 (5), 1009–1017. 10.1021/cb3007264. [DOI] [PubMed] [Google Scholar]
  31. Cartwright T. N.; Meyer S. K.; Higgins J. M. G. Robustness of NanoBiT luciferase complementation technology in the presence of widely used kinase inhibitors. SLAS Discovery 2022, 27 (8), 471–475. 10.1016/j.slasd.2022.09.004. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pt3c00176_si_001.pdf (225.5KB, pdf)

Articles from ACS Pharmacology & Translational Science are provided here courtesy of American Chemical Society

RESOURCES