Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase

Abstract

We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (K(m) 183 +/- 14 nM) and yet is metabolized fairly quickly (k(cat) 0.690 +/- 0.010 s(-1)), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorometric noncoupled assay that is 2 orders of magnitude more sensitive than the spectrophotometric benzylamine assay. The discovery of ANETA involved elaboration of the lead compound 6-methoxy-2-naphthalenemethaneamine by structure-based design, which recognized the ancillary cation binding site of BPAO as the most significant structural features controlling binding affinity. Structure-based design further ensured a high level of selectivity: ANETA is a good substrate of BPAO but is not a substrate of either porcine kidney diamine oxidase (pkDAO) or rat liver monoamine oxidase (MAO-B). ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond directly to the enzyme activity in real time.

Figure 1

Time-dependent (4 min interval) UV-vis spectra recorded upon mixing 0.1 mM of 1 (left) or 2 (right) with 0.1 µM of BPAO in 0.1 M of pH 7.2 phosphate buffer at 30 °C. Insets: time dependent plots of absorbance growth at wavelengths indicated.

Figure 2

Time-dependent (1 min interval) fluorescence spectra recorded upon mixing 0.1 mM of 1 (left) or 2 (right) with 5 nM of BPAO in 0.1 M of pH 7.2 phosphate buffer at 30 °C. The λ_ex were 350 nm for 1 and 420 nm for 2. Insets: time-dependent plots of emission growth at wavelengths indicated.

Figure 3

Docking of 1 into the active site of BPAO. The arrow indicates the mouth opening from where substrate approaches into the active site. The hydrogen bonding between the substrate amino group and Asp385 is noted as green dashed lines.

Figure 4

The best scored productive docking mode of compound 7 with native BPAO. Note that the substrate OH is interacting with Asp445.

Figure 5

Left panel: time-dependent (30 s interval) UV-vis spectra recorded upon mixing 20 µM of 12 with 0.1 µM of BPAO in 0.1 M pH 7.2 phosphate buffer at 30 °C. Right panel: time-dependent (1 min interval) fluorescence spectra (λ_ex 350 nm) recorded upon mixing 12 (10 µM) with BPAO (1.0 nM) in 0.1 M pH 7.2 phosphate buffer at 30°C.

Figure 6

Left panel: HPLC diagram of a mixture of authentic 12 and 18 (both 200 µM), and time dependent HPLC diagrams of a metabolic mixture of 12 (200 µM) with BPAO (0.1 µM) in 0.1 M of pH 7.2 phosphate buffer at 25 °C. Right panel: the corresponding DAD spectra of peaks at 12.3 min (18).

Figure 7

Plots of initial oxidation velocity (V₀) vs. substrate concentration of 1 in 33 mM of pH 7.2 phosphate buffer in the presence of BPAO (5.0 nM) (left), and of 12 in 0.1 M of pH 7.2 phosphate buffer in the presence of BPAO (2.0 nM) both at 30 °C (right) as measured by fluorescence spectrophotometry.

Figure 8

The best scored productive docking mode of 12 with BPAO. Note that the substrate quaternary ammonium group is close to Asp445 to allow for an additional electrostatic interaction.

Figure 9

Linear relationship between enzyme concentration and initial oxidation velocity (V₀) of 12 (10 µM) in 0.1 M of pH 7.2 phosphate buffer at 30 °C. Left: [BPAO] 0–1.00 nM. Right: [BPAO] 1.00–10.0nM.

Figure 10

Time dependent activity loss of BPAO (50 nM) incubated with various concentrations of 1-amino-2,3-butadiene (29) in 0.1 M of pH 7.2 phosphate buffer at 2 °C as monitored by the new fluorometric assay (left), and the corresponding Kitz and Wilson plot (right).

Scheme 1

Scheme 2