Effect of Fluorine Atoms and Piperazine Rings on Biotoxicity of Norfloxacin Analogues: Combined Experimental and Theoretical Study

Abstract

To clarify the effect of the fluorine atom and piperazine ring on norfloxacin (NOR), NOR degradation products (NOR-DPs, P1-P8) were generated via UV combined with hydrogen peroxide (UV/H₂O₂) technology. NOR degradation did not significantly affect cytotoxicity of NOR against BV2, A549, HepG2, and Vero E6 cells. Compared with that of NOR, mutagenicity and median lethal concentration of P1-P8 in fathead minnow were increased, and bioaccumulation factor and oral median lethal dose of P1-P8 in rats were decreased. Molecular docking was used to evaluate the inhibitory effect of DNA gyrase A (gyrA) on NOR-DPs to determine the molecular-level mechanism and establish the structure-activity relationship. Results indicated that the most common amino acid residues were Ile13, Ser27, Val28, Gly31, Asp36, Arg46, Arg47, Asp157, and Gly340; hydrogen bonds and hydrophobic interactions played key roles in the inhibitory effect. Binding area (BA) decreased from 350.80 Ų (NOR) to 346.21 Ų (P1), and the absolute value of binding energy (|BE|) changed from 2.53 kcal/mol (NOR) to 2.54 kcal/mol (P1), indicating that the fluorine atom mainly affects BA. The piperazine ring clearly influenced BA and |BE|. "Yang ChuanXi Rules" were used to explain effects of molecular weight (MW), BA, |BE|, and sum of η₁ + η₂ (η₁: normalization of BA, η₂: normalization of |BE|) and predict biotoxicity of NOR-DPs based on half-maximum inhibitory concentration (IC₅₀), half-minimal inhibitory concentration (MIC₅₀), and half-minimal bactericidal concentration (MBC₅₀) values.

Figure 1

ESP of (a) NOR, (b) P1, (c) P2, (d) P3, (e) P4, (f) P5, (g) P6, (h) P7, and (i) P8.

Figure 2

Theories of (a) developmental toxicity, (b) mutagenicity, (c) bioaccumulation factor, (d) fathead minnow LC₅₀ after 96 h (mg/L), (e) T. pyriformis IGC₅₀ after 48 h (mg/L), (f) D. magna LC₅₀ after 48 h (mg/L), and (g) oral rat LD₅₀ (mg/kg) for NOR and NOR-DPs.

Figure 3

Key functional-effector groups and structural-effector pharmacophores for (a) NOR, (b) P1, (c) P2, (d) P3, (e) P4, (f) P5, (g) P6, (h) P7, and (i) P8, which combined with gyrA.

Figure 4

Domain structure of gyrA in E. coli and amino acids in the active pocket of gyrA that participate in NOR/NOR-DPS and gyrA combination (a); arrows indicate domain boundaries, and numbers indicate amino acid residues. The schematic diagram of the gyrA structure was adapted from Hashimi (2019). Amino acids and residues with high frequency (≥6) participate in NOR/NOR-DPS and gyrA combination; amino acids with high frequency are labeled in red (b). X-ray diffraction structure and amino acids in the active pocket with high frequency of gyrA (c).

Figure 5

Values of hydrogen bonds (a), binding area (b), absolute value of binding energy (c), molecular weight, binding area, and absolute value of binding energy (d), and binding area and absolute value of binding energy (e) for NOR/NOR-DPs and gyrA.