Screening The Anticancer Drugs Against C2H2 Domain Of Human DPF2 Using Isothermal Titration Calorimetry And Fluorescence Quenching

DPF2 belongs to d4-protein family, which is characterized of evolutionary conservation and structural similarity. All proteins of this family exhibit a distinctive domain in the N-terminal region, a single Cys2His2 (C2H2) zinc finger domain in the middle section of the protein and a C-terminal tandem plant homeodomain (PHD). DPF2 is an important epigenetic modification and recongnition protein. It has been identified as a transcription factor, which is necessary for the participate in developmentally programmed cell death. In previous study, the DPF2 was mapped to chromosomes 1 Iql3, a region coupled with different kinds of cancers. The human DPF2 can be implicated in gastric cancer, leukemia or other cancers, and its C2H2 domain is related to sequence-specific DNA binding, so the potent inhibitors for C2H2 domain of DPF2 attract our attentions.In this study, we selected C2H2 domain of DPF2 as the research object. Moreover, eight widely used anticancer drugs were chose, including cisplatin, methotrexate, 5-fluorouracil, mitomycin, hydroxyurea, bortezomib, decitabine and interferon-a. Isothermal titration calorimetry (ITC) and fluorescence quenching (FQ) were performed to investigate the interaction of these drugs with C2H2 domain of DPF2.The results of isothermal titration calorimetry showed that the C2H2 domain of DPF2 preferentially recognizes cisplatin (with binding constant Ka is 1.76x10 L/mol) followed by methotrexate,5-fluorouracil, hydroxyurea and decitabine in one binding site manner, whereas no obvious interaction was found for mitomycin, bortezomib and interferon-a with the C2H2 domain of DPF2 by the software "NanoAnalyze" analyzed. The binding site n is closed to one. It indicates that there is one binding site on C2H2 domain of DPF2.At the same time, fluorescence quenching method confirmed the result. The findings showed that there is a decreased regularity and stability of intrinsic fluorescence intensities of C2H2 domain of DPF2 due to the addition of six kinds of drugs, including cisplatin,5-fluorouracil, hydroxyurea, methotrexate and decitabine. Moreover, cisplatin is the top priority of C2H2 domain of DPF2 (Ka is 1.58*106 L/mol), and the binding site n is approximately equal to one, whereas mitomycin, bortezomib and interferon-a couldn’t quench the intrinsic fluorescence of C2H2 domain of DPF2.This research is aimed at the interaction of small molecule drug with protein, using two methods-isothermal titration calorimetry and fluorescence quenching. To make the results much more accurate and more persuasive, the same reaction system was studied by the two methods meanwhile. The study can screening the anticancer drugs against C2H2 domain of human DPF2 using isothermal titration calorimetry and fluorescence quenching, it may be bring convenient and reliable treatment by the DPF2 mediated tumor, and also pave a way for the investigation of inhibitors against other C2H2 domain of d4 protein family.