Pyridine substituted BODIPYs: synthesis, characterization and cholinesterease, α-glucosidase inhibitory, DNA hydrolytic cleavage effects

In this study, the synthesis of new monostyryl (BDPY-2) and distyryl BODIPY dyes (BDPY-4, BDPY-5) containing pyridine groups has been reported for the first time. The acetylcholinesterase from Electrophorus electricus (AChE), butyrylcholinesterase from equine serum (BuChE), α-glucosidase from Saccharomyces cerevisiae and DNA hydrolytic cleavage actions of BDPY-2, BDPY-4, BDPY-5 were investigated using various techniques. The results indicated that the compounds had varying inhibition properties against AChE, BuChE, and α-glucosidase. BDPY-4 was the most potent compound on AChE with IC50 of 54.78 ± 4.51 µM, and Lineweaver–Burk plots indicated that the compound is bound to a site other than the active site as a noncompetitive inhibitor. The compound-protein binding experiment showed that BDPY-4 changed the microenvironment around AChE. On the other hand, the compounds showed lower α-glucosidase inhibition than the positive control. The DNA hydrolytic cleavage effects were not observed on supercoiled plasmid DNA in the presence of the compounds as compared to negative controls. These findings suggested that BDPY-4 might be a promising compound to treat Alzheimer’s diseases.


Experimental
The materials, equipment, AChE, BuChE, α-glucosidase inhibitory, and DNA hydrolytic cleavage actions are given as supplementary information.

AChE/BuChE inhibitory properties of the compounds
The inhibition actions of the compounds (BDPY-2, BDPY-4, and BDPY-5) on AChE and BuChE were investigated according to our previously reported methods [30]. The results were expressed as IC 50 and selective index (SI=BuChE/ AChE) values. As shown in Table 1 The mechanism for AChE inhibition was graphically determined by applying the Lineweaver-Burk and Dixon plots analysis of the most potent compound (BDPY-4). Acetylthiocholine iodide was used as a substrate for AChE inhibition. Lineweaver-Burk plot showed that K m (an index of the affinity of the enzyme for its substrate) was in similar values, but V max (maximal velocity of the reaction) decreased on increasing concentrations of the compound on AChE. While the K m value was 11.14 mM, the V max values changed from 277.78 µM/min to 126.58 µM/min ( Figure 3, Table 2). The results indicated that BDPY-4 was a noncompetitive inhibitor and bound to a site other than the active site. On the other hand, BDPY-4 presented K i (inhibition constant) values of 57.20 ± 0.20 µM, according to the Dixon plot ( Figure 4, Table 3).
To determine the structural change of AChE (10 µM) induced by BDPY-4, we measured the UV-Vis spectroscopy by adding the compound (5, 10, 15, and 20 µM) into AChE solution (10 µM). AChE has an absorption peak at 282 nm due to aromatic amino acids. As shown in Figure 5, the absorbance of the enzyme increased with various BDPY-4 concentrations (hyperchromism). In addition, the absorption peak shifted from 282 nm to 285 nm (redshift). The UV-Vis spectrum implies that BDPY-4 showed binding with enzyme and changed the microenvironment of some amino acid residues of AChE.

α-Glucosidase inhibitory properties of the compounds
The inhibitory properties of the compounds on α-glucosidase were investigated according to our previously reported methods [31]. The results were expressed as IC 50 values. As shown in Table 4, IC 50 values of the compounds ranged from 94.99 ± 4.77 to 218.62 ± 8.71 µM. BDPY-4 was the highest α-glucosidase inhibitory effects among the tested compounds, but the compound showed lower inhibitory than acarbose used as a positive control (IC 50 =32.22 ± 0.40 µM).

DNA hydrolytic cleavage properties of the compounds
The DNA hydrolytic cleavage actions of the compounds on supercoiled pBR322 plasmid DNA were determined according to our previously reported methods, and the intensity bands were observed under UV illuminator [32]. To investigate the ability of the compounds to damage the phosphodiester bonds of supercoiled plasmid DNA, we designed hydrolytic   cleavage studies. The supercoiled plasmid DNA has three forms in agarose gel: form I (supercoiled form), form II (nicked form cleavage of one strand), form III (linear form cleavage of two strands). The results are presented in Figure 6. It is known that supercoiled pBR322 plasmid DNA (Thermo Fischer Scientific, SD0041) is in the supercoiled form at a rate of more than 90%. Since the plasmid DNA has impurity, the density of Form II is increased on negative controls. In this study, the presence of the compounds did not have DNA hydrolytic cleavage effects at 25 and 50 µM as compared to negative controls (Figures 6(a),(b), lanes 1) under our experimental conditions. The results claimed that these compounds may low toxicity potential in the dark as a preliminary study.

Conclusion
In conclusion, we have synthesized new monostyryl (BDPY-2) and distyryl BODIPY dyes (BDPY-4, BDPY-5) and investigated their acetylcholinesterase from Electrophorus electricus (AChE), butyrylcholinesterase from equine serum (BuChE), α-glucosidase from Saccharomyces cerevisiae, and DNA hydrolytic cleavage actions. The compounds showed varying inhibition actions against AChE, BuChE, and α-glucosidase. BDPY-4, which was a noncompetitive inhibitor, was the most potent compound on AChE with an IC 50 of 54.78 ± 4.51 µM. The UV-vis spectroscopy studies claimed that it interacted with enzyme change the microenvironment around AChE. In addition, the compounds had low α-glucosidase inhibitory effects when compared to acarbose. The DNA hydrolytic cleavage was not showed on supercoiled plasmid DNA in the presence of the compounds at 25 and 50 µM as compared to negative controls under our experimental conditions. These findings suggested that these compounds have low toxicity potential in the dark. Further studies are needed regarding anticholinesterase and toxicity effects of BDPY-4 on development of formulations, cell cultures, and in vivo studies for application in internal diseases.

α-Glucosidase inhibitory assay
α-Glucosidase inhibition assay was performed as previously reported with some modifications [5]. Acarbose was used as a positive control, and DMSO (final concentration 1) as blank. The compounds (50 µL) in phosphate buffer pH 6.9, αglucosidase (100 µL, 0.5 U/mL) were added and allowed to react for 20 min in a microplate. After incubation, 4-pNPG (50 µL, 5 mM) was added and incubated for 20 min at room temperature. The absorbance was measured at 405 nm using a 96-well microplate reader. α-glucosidase inhibition percentage of the compounds was calculated using the formula 1.

Supercoiled pBR322 plasmid DNA cleavage experiments
Supercoiled pBR322 plasmid DNA nuclease effects of the compounds were investigated using agarose gel electrophoresis. DMSO (final concentration 1%) was used as a negative control. Supercoiled pBR322 plasmid DNA was treated with increasing concentrations of the compounds (50 and 100 µM) in the buffer containing 50 mM Tris-HCl pH 7.0. All samples were incubated at 37 ºC for 30 min and 60 min. Afterwards, loading buffer (bromophenol blue, xylene cyanol, glycerol, ethylenediaminetetraacetic acid, sodium dodecyl sulfate) was added, and the samples were loaded on agarose gel (0.8%) with ethidium bromide staining in TAE buffer (Tris-acetic acid-EDTA).