Cantharidin inhibits competitively heme‐Fe(III) binding to the FA1 site of human serum albumin

Abstract

Cantharidin, a monoterpene isolated from the insect blister beetle, has long been used as a medicinal agent in the traditional Chinese medicine. Cantharidin inhibits a subgroup of serine/threonine phosphatases, thus inducing cell growth inhibition and cytotoxicity. Cantharidin has anticancer activity in vitro, since it is able of inducing p53-dependent apoptosis and double-strand breakage of DNA in cancer cells. Although the toxicity of cantharidin to the gastrointestinal and urinary tracts prevents its medical use, it is a promising lead compound for chemical modification to develop new anticancer therapeutics. In fact, cantharidin does not cause myelosuppression and displays anticancer activity against cells with a multidrug resistance phenotype. Here, the competitive inhibitory effect of cantharidin on heme-Fe(III) binding to the fatty acid site 1 (FA1) of human serum albumin (HSA) is reported. Docking and molecular dynamics simulations support functional data indicating the preferential binding of cantharidin to the FA1 site of HSA. Present results may be relevant in vivo as HSA could transport cantharidin, which in turn could affect heme-Fe(III) scavenging by HSA.

Neonicotinoid trapping by the FA1 site of human serum albumin

“…To exclude any bias (i.e., to carry out “blind” docking simulations), the search space encompassed the whole protein. Flexibility was allowed only for the residues building up the walls of the FA sites (FA1–FA9) . Residues for which flexibility was allowed are reported in Table 3SM.…”

“…HSA binding to heme‐Fe(III), in the absence and presence of neonicotinoids, was followed spectrophotometrically between 360 and 450 nm, at pH 7.3 (2.0 × 10 −2 M phosphate buffer) and 20.0°C . Heme‐Fe(III) binding to HSA, in the absence and presence of neonicotinoids, was investigated at a fixed low heme‐Fe(III) concentration by increasing the amount of HSA.…”

“…Values of the dissociation equilibrium constants for HSA binding to heme‐Fe(III), in the absence and presence of neonicotinoids (i.e., K h 0 and K h app respectively; see Scheme 1), were obtained spectrophotometrically from the dependence of the relative absorbance intensity change (i.e., Δ A /Δ A max ) of the HSA:heme‐Fe(III) complex on the concentration of free HSA (i.e., [HSA]), according to Equation (1): where Δ A is the absorbance intensity change occurring at each concentration of HSA, Δ A max is the maximum absorbance intensity change occurring under saturating conditions, and n is the Hill coefficient. The concentration of free HSA was determined subtracting the HSA:heme‐Fe(III) concentration from that of total HSA.…”

Fipronil recognition by the FA1 site of human serum albumin

“…Heme‐Fe(III) binding to HSA, in the absence and presence of fipronil, was followed spectrophotometrically between 350 and 460 nm, at pH 7.3 (2.0 × 10 −2 M phosphate buffer) and 20.0°C. The heme‐Fe(III) concentration was 9.2 × 10 −7 M, the fipronil concentration ranged between 2.0 × 10 −6 M and 2.0 × 10 −5 M, and the HSA concentration ranged between 4.3 × 10 −7 M and 1.8 × 10 −6 M. Heme‐Fe(III) binding to HSA, in the absence and presence of fipronil, was investigated at a fixed low heme‐Fe(III) concentration by increasing the amount of HSA …”

“…Dansyl‐sarcosine and dansyl‐arginine binding to HSA, in the absence and presence of fipronil, was followed spectrofluorimetrically at pH 7.3 (2.0 × 10 −2 M phosphate buffer) and 20.0°C. The fluorophore of dansyl‐sarcosine and dansyl‐arginine was excited at 370 nm, and the fluorescence emission intensities were measured at the maximum wavelengths (ie, 460 nm for dansyl‐arginine and at 475 nm for dansyl‐sarcosine); the excitation and emission slits were 5 nm . The HSA concentration was 2.1 × 10 −6 M, and the dansyl‐sarcosine and dansyl‐arginine concentration ranged between 3.0 × 10 −6 M and 3.5 × 10 −5 M and between 1.0 × 10 −5 M and 2.0 × 10 −4 M, respectively; the fipronil concentration ranged between 2.0 × 10 −6 M and 4.0 × 10 −5 M.…”