Complexation of the Mycotoxin Cyclopiazonic Acid with Lanthanides Yields Luminescent Products

Abstract

Cycopiazonic acid (CPA) is a neurotoxin that acts through inhibition of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). CPA blocks the calcium access channel of the enzyme. The inhibition may involve the binding of CPA with a divalent cation such as Mg²⁺. The potential for CPA to act as a chelator also has implications for methods to detect this toxin. Certain of the lanthanide metals undergo a dramatic increase in luminescence upon coordination with small molecules that can transfer excitation energy to the metal. This report is the first to describe the coordination of CPA with lanthanide metals, resulting in a substantial enhancement of their luminescence. The luminescence expressed was dependent upon the type of lanthanide, its concentration, and the environment (solvent, water content, pH). Based upon the phenomenon, a competitive assay was also developed wherein terbium (Tb³⁺) and a series of metal cations competed for binding with CPA. With increasing cation concentration, the luminescence of the CPA/Tb³⁺ complex was inhibited. The chlorides of ten metals were tested. Inhibition was best with Cu²⁺, followed by Co²⁺, Al³⁺, Fe³⁺, Mn²⁺, Au³⁺, Mg²⁺, and Ca²⁺. Two cations in oxidation state one (Na⁺, K⁺) did not inhibit the interaction significantly. The interaction of CPA with lanthanides provides a novel recognition assay for this toxin. It also provides a novel way to probe the binding of CPA to metals, giving insights into CPA’s mechanism of action.

Keywords: calcium-ATPase; cyclopiazonic acid; lanthanides; luminescence; mechanism of action; mycotoxin.

Figure 1

Structure of α-cyclopiazonic acid (CPA). Note the indole moiety capable of absorbing light at circa 280 nm, and the presence of the tetramic acid moiety that can coordinate with metal cations.

Figure 2

Formation of CPA-lanthanide complex and luminescence of the lanthanide. Excitation corresponds to an absorption band within CPA. Emission is from the lanthanide (Ln³⁺).

Figure 3

Effect of CPA on the luminescence of Eu³⁺. (A) Emission spectrum with excitation at 290 nm; (B) Excitation spectrum with emission at 615 nm. All spectra were collected in MeOH/H₂O (9 + 1).

Figure 4

Effect of CPA on the luminescence of Tb³⁺. (A) Emission scan with excitation at 290 nm; (B) Excitation scan with emission at 545 nm. All scans collected in MeOH/H₂O (9 + 1).

Figure 5

Dependence of luminescence upon the TbCl₃ concentration. CPA was present at 1.5 uM (504 ng/mL). Excitation at 290 nm, emission at 545 nm. Points represent the average of triplicate plates with 8 wells per concentration (n = 24) ± 1 standard deviation.

Figure 6

Effect of solvent type upon the luminescence of CPA/Tb³⁺. Solvents shown were all mixtures of 9 + 1 (v/v) with H₂O. TbCl₃ was present at 0.25 uM. Excitation at 290 nm, emission at 545 nm. Points represent the average of triplicate plates with 8 wells per concentration (n = 24) ± 1 standard deviation. One µM CPA is equivalent to 336 ng/mL.

Figure 7

Effect of water on the luminescence of CPA/Tb³⁺. TbCl₃ was present at 0.25 µM. Excitation at 290 nm, emission at 545 nm. Points represent the average of triplicate plates with 8 wells per concentration (n = 24) ± 1 standard deviation.

Figure 8

Competitive inhibition of the association of CPA and Tb³⁺ with metal cations. In sufficient excess the metal cations (shown as “M⁺”) can replace the Tb³⁺. The resulting decrease in emission results from the dissociation of the CPA from the Tb³⁺, which reduces absorption of excitation light by the Tb³⁺. Furthermore, coordination of Tb³⁺ with water results in quenching.

Figure 9

Competition between various metals and Tb³⁺ for CPA. (a) Metals with an oxidation state of one; (b) Metals with an oxidation state of two; (c) Metals with an oxidation state of three. Data expressed as the percentage of luminescence (F) relative to that seen in the absence of added metal salt (Fo).