Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes

Abstract

Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.

Fig. 1.

The Transcreener AMP/GMP assay principle. The detection reagents are an AMP/GMP antibody and an AMP/GMP-Alexa^® Fluor633 tracer. The tracer is displaced from antibody by enzymatically generated AMP or GMP, causing a decrease in polarization.

Fig. 2.

Standard optimization of rabbit polyclonal antibody (pAb1) concentration for 1 to 1,000 μM ATP. EC₈₅ values are as follows (μg/mL pAb1): 1 μM ATP (♦), 9.7; 10 μM ATP (•), 16.0; 100 μM ATP (▴), 25.3; and 1,000 μM ATP (▪), 86.6. Values are similar when using cAMP or cGMP for substrate.

Fig. 3.

Refined optimization of pAb1 concentration for initial velocity phosphodiesterase (PDE) reactions. (A) Antibody-tracer equilibrium binding curve in the presence of 1 μM cAMP (▴) and 0.9 μM cAMP/0.1 μM AMP (▪). (B) Difference plot of the 2 curves in A (▵mP = mP_1μM cAMP − mP_{0.9 μM cAMP/0.1 μM AMP}). The EC₈₅ value was 8.7 μg/mL for the 1 μM cAMP curve A. The maximal ▵mP value in the difference curve in (B) occurred at an antibody concentration of 3.7 μg/mL.

Fig. 4.

Competition binding curves for adenine and guanine nucleotides with AMP/GMP antibodies. (A) Polyclonal antibody pAb1. (B) Monoclonal antibody mAb1. (C) Monoclonal antibody mAb2. Values represent the average of duplicate reactions.

Fig. 5.

Standard curves. Demonstrated here are mock reaction progress curves representing: 0%, 1.0%, 2.5%, 5.0%, 7.5%, 10.0%, 12.5%, 15.0%, 25%, 50%, 75%, and 100% conversion of substrate nucleotide. AMP/cAMP standard curve (▪); GMP/cGMP standard curve (▴); AMP/ATP standard curve (•).

Fig. 6.

Stability of AMP detection reagents on the liquid handling deck and assay signal in the plate. (A) 1× Detection mixture was formulated and held in ambient light and temperature for 1 (•), 2 (▪), 5 (▴), 8 (♦), and 24 (x) h after which an equal volume was added to standard curve aliquots and fl uorescence polarization (FP) measured. (B) In-plate signal stability was tested by measuring the signal over time of a standard curve (same as in A) and detection reagents plated and held, sealed, at ambient temperature for 1 (•), 2(▪), 6 (▴), 8 (♦), and 24 (x) h.

Fig. 7.

Cyclic nucleotide phosphodiesterase (PDE) assays. (A) Time course of cAMP-dependent reactions using 1.6 ng/mL 4A1A (▪), 3.3 ng/mL 3A (▴), and 24.0 ng/mL 9A2 (•). (B) Time course of cGMP-dependent reactions using the same enzyme concentrations as A.

Fig. 8.

Select inhibitor curves from Table 2 data set. Rolipram inhibitor curves using cAMP as substrate: isoform 4A1A (•) and 3a (▴).

Fig. 9.

ATP-utilizing enzyme assays. S-Acetyl coenzyme A synthetase and ubiquitin-activating enzyme (UBE1) were assayed. (A) UBE1 titration: 1.2 (+), 2.3 (▵), 4.7 (□), 9 (○), 19 (♦), 38 (•), 75 (▴), and 150 (▪) nM. (B) S-Acetyl coenzyme A synthetase titration: 0.02 (+), 0.05 (▵), 0.1 (□), 0.2 (○), 0.4 (♦), 0.7 (•), 1.5 (▴), and 2.9 (▪) mU/mL.

Fig. 10.

E2-conjugating enzyme (UbcH2) assays. UbcH2 enzyme was titrated across 1 nM, 5 nM, and 40 nM UBE1. Plotted is the signal generated (1 h at 30°C) with the optimal UbcH2 concentration for respective UBE1 concentrations (); the signal due to corresponding UBE1 quantities without UbcH2 (); and the signal with this same concentration UbcH2 absent UBE1 (□).