Aggregation of small molecules inhibit DNase I Shunsuke Habe 1 , Natsumi Shimizu 1 , Tomoko Moriwaki 1 , Takashi Aoi 2 , Mariko Ikeda 3 , Kenta Morita 1 and Tatsuo Maruyama 1 1 Kobe U. Eng., 2 Kobe U. Med., 3 Fujita Health U. Streptococcal toxic shock syndrome is a serious disease with a fatality rate of more than 30%. Currently, treatment is limited to resection of the infected area and administration of antimicrobial agents. Neutrophils kill pathogens by releasing neutrophil extracellular traps (NETs) composed of their own DNA. Streptococcus pyogenes, however, destroy NETs by secreting DNA-degrading enzymes (DNase). In our previous study, a small molecule designated as Mannan 007 (Mn007) 1 was shown to be an inhibitor of DNase I when it aggregated in an aqueous solution. Mn007 is a potential new therapeutic agent for streptococcal infections by inhibiting the secreted DNase of streptococci. However, the key structure in Mn007 for inhibition of DNase I is not specified. Therefore, the structure of Mn007 was divided into parts, and each part was enabled to aggregate in a solution attaching a carbon chain to it. The inhibitory activity of the synthesized molecule was examined to clarify the critical structure of Mn007 to inhibit DNase I. Furthermore, we attempted to identify the inhibitory sites by arranging them on liposomes. Compound 1 (Comp.1) was synthesized by introducing a carbon chain into the right substructure of Mn007. The synthesis of Comp.1 was confirmed by MALDI-TOF/MS and 1 H NMR. DNase I activity was assayed with the degradation of double-stranded DNA modified with fluorescent molecules. DNase I, Comp.1 dissolved in DMSO, MgSO 4 , and the substrate DNA were mixed in a Tris HCl Buffer (pH 7.5). The fluorescence of the solution was measured using a fluorometer according to the time (Ex/Em = 495/515). The DNase I inhibitory activity was then evaluated by calculating the change of fluorescence per second. Comp.1 inhibited DNase I at higher concentrations than 5.6 µM. Based on our previous studies, the concentration at which Mn007 showed inhibitory activity was higher than 45 µM. Comparing the concentrations, Comp.1 inhibited DNase I at lower concentrations than Mn007 did. It might be the reason that the carbon chain improved the aggregation ability of Comp.1 and effectively arranged the inhibitory sites of Comp.1 on the surface of the aggregates. The critical aggregation concentration (CAC) of Comp.1 was investigated by light scattering intensity measurements. Because the scattered light intensity increased above 9.7 µM, the CAC of Comp.1 was determined to be 9.7 µM. Comp.1 formed aggregates at low concentrations and that the aggregates inhibited DNase I. Thus, it is possible that the substructure used for Comp.1 is the DNase I inhibitory site of Mn007. To investigate the supramolecular surface of Comp.1 that inhibits DNase I, we tried to arrange Comp.1 on liposome surface. Liposomes were conformed of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’- rac -glycerol) sodium salt (POPG) in a 9:1 molar ratio. The diameter of the liposomes was measured as 100 nm approx by a dynamic light scattering measurement. In the future, we will arrange Comp.1 on the liposome membranes and evaluate its DNase I inhibitory activity. References 1. Lv, F. et al , Bioorg. Med. Chem. 2015, 23 , 7661
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