Substrate-Selective Supramolecular Tandem Assays: Monitoring Enzyme Inhibition of Arginase and Diamine Oxidase by Fluorescent Dye Displacement from Calixarene and Cucurbituril Macrocycles

Abstract

A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).

Determining Protease Substrate Selectivity and Inhibition by Label-Free Supramolecular Tandem Enzyme Assays

“…The immediate response is due to the fast rates for the formation and dissociation of the supramolecular assemblies, which, as previously discussed, constitutes an advantage of using macrocycles instead of antibodies. 24 As can be further seen from the actual titration plots (Figure 2), even working at relatively low substrate concentrations of 5-20 M should produce a readily detectable change in fluorescence response upon conversion of a substrate to a product. This working concentration range is exactly desirable in protease assays, including those employed in high-throughput screening for pharmaceutical investigations.…”

“…Cucurbit [7]uril (CB7, Figure 1) is a water-soluble macrocycle that has been investigated extensively in biological applications including drug delivery, [29][30][31][32][33] interactions with enzymes, 34,35 plasma membrane protein fishing, 36 and label-free enzyme assays. [22][23][24][25] The repeating glycoluril units produce a barrel-shaped container that has a hydrophobic cavity and negatively charged portals. 37 The latter are capable, not only for CB7 but also for its homologues, of binding inorganic cations as well as the cationic sites of organic guests, mostly ammonium groups; nonpolar groups are preferentially immersed in the inner cavity.…”

“…55 Upon the addition (or enzymatic formation) of a strongly binding analyte to the preformed CB7•AO complex, the fluorescence intensity drops again, leading to a "switch-off" fluorescence response. Important to note, the binding constant of CB7 with AO (2.9 × 10 5 M -1 ) 24 (Table 1). Peptides 1-6, the candidates to potentially act as substrates for thermolysin, have invariably a low binding affinity to CB7, accounted by the presence of only hydrophobic interaction between the amino acid Phe and the host cavity.…”

“…[22][23][24][25] Until now, the technique was limited to substrates and products which, owing to their low molecular weight, could essentially be fully immersed in the macrocyclic host cavity, such that the entire analyte, e.g. arginine or cadaverine, served as recognition motif.…”

In vitro inhibition of lysine decarboxylase activity by organophosphate esters

“…The dose-response curve was fitted with a sigmoidal model (Origin Lab 8.0, Northampton, MA, USA) and analyzed with the Hill equation to obtain IC 50 value [33]. The IC 50 can be readily converted into the inhibition constants K i by considering the enzyme concentration [34]:…”