| Bcl-2family including anti-apoptotic as well as pro-apoptotic members regulate cell apoptosis. Researches show that the reasons of non-death tumors is the over-expression of anti-apoptotic proteins, especially Mcl-1and Bcl-2protein, which make tumors avoid being killed. So those kinds of proteins are important targets for cancer therapy. Anti-apoptotic proteins interact with pro-apoptotic proteins by the BH3groove. And small molecule inhibitors which targets Bcl-2aniti-apoptotic proteins can release pro-apoptotic proteins so that the tumors can be eliminated successfully. Mcl-1and Bcl-2are traditional anti-apoptotic proteins. Because of the peculiarities of Mcl-1differ from other anti-apoptotic proteins, Bcl-2inhibitors cannot bind Mcl-1. So it is necessary to further study the interaction mode of small molecules with anti-apoptotic proteins, so that we can direct the design of new inhibitors binding both of Mcl-1and Bcl-2proteins. We explored the binding site and binding mode of the small molecule C1(2,3-dihydroxyanthracene-9,10-dione)with Mcl-1protein by Docking and’H-15N HSQC NMR spectroscopy. Based on the results, we optimized those small molecule and designed a set of new Mcl-1inhibitors to expand binding sites within BH3groove and gained higher binding affinity.Firstly, we constructed a plasmid for the Mcl-1protein expression. Through optimized conditions for protein expression, the quantity of expressed protein Mcl-1was up to8.3mg/L without labeling, and7.9mg/L of labeled15N. And through optimization of molecular sieve chromatography and elution conditions, the purity of the purified protein increased more than95%. Circular Dichroism spectrum result showed that the spectral characteristics of protein secondary structure is same as the structure when consisted with8a helix. Protein NMR one-dimensional and two-dimensional mapping results showed that the marked protein we purified is good-well folded and stable. It suggests that the expression of protein spatial structure is normal and the protein we gained can meet the requirements of the subsequent experiment.Secondly, Having been performed the1H-15N HSQC NMR experiment for the binding mode study of the small molecule S1(compound name), we found the key amino acid on protein Mcl-1which interact with C1. This result proved that this small molecule C1binding with Mcl-1is based on the BH3groove on the surface of Mcl-1. And the chemical shift of residue R263is the largest in the report of NMR. For the further study of interaction mode of those molecules, we docked them on computer software Autodock. It showed that the small molecule C1formed a hydrogen bond with R263, and the anthraquinone backbone of C1crossed on the p3pocket. However, the benzene ring of C1did not well occupy with the p3pocked. So, we designed C2molecule to figure the problem out. Fluorescence polarization experiment performed that C2binds with Bcl-2and Mcl-1and their binding constant Ki values are132n and107nM which is three or four times of C1. Moreover,1H-15N HSQC NMR showed that the bromine atom of C2successfully interacts with F228residues in the p3pocket of protein Mcl-1. Further molecular docking intuitively proved C2bromine atoms well occupied Mcl-1p3pockets. Then, on the basis of C2, we designed C3, C4, C5, C6in order to occupy the p2pocket of Mcl-1protein by longer substituent replace the bromine atoms of C2. Fluorescence polarization experiment showed that the affinity of those four small molecules binding with Mcl-1the Bcl-2protein had significantly raised than C2. Especially C6molecule, its Ki value are24nm and13nm when binding with the Bcl-2and Mcl-1respectively. Compared with C1, the affinity of C2has been greatly improved about13times and38times binding with Mcl-1and Bcl-2, respectively. HSQC2D-NMR showed that in addition to the chemical shift of amino acids in the C2, residues M250and F270of Mcl-1protein is added for better affinity of interactions with C6molecule by occupying p2pocket HSQC help us gained a high affinity Bcl-2/Mcl-1dual inhibitor C6. |
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