| The Bcl-2protein family includes anti-apoptotic as well as pro-apoptotic members that show homology in up to four conserved regions, termed the Bcl-2homology (BH) domains BH1, BH2, BH3, and BH4. The anti-apoptotic members, such as Bcl-2and Mcl-1contain three or four BH domains. Multi-domain pro-apoptotic proteins such as Bax and Bak contain three BH domains. BH3-only proteins contain only the short BH3domain. The unifying BH3domain mediates their interactions, through which regulates apoptosis. Studies have revealed that Bcl-2and Mcl-1proteins allow cancer cells to escape apoptosis. Therefore, they become very attractive targets for the development of novel anticancer drugs. Small molecule inhibitors are designed to occupy the hydrophobic BH3groove of the anti-apoptotic proteins, thereby disrupt Bcl-2family protein interactions and induce apoptosis in cancer cells. Though several Bcl-2inhibitors have been succesffuly designed and entered clinical trial, very little Mcl-1inhibitors were reported. Because there are differences between the BH3groove of Mcl-1and the homolog of Bcl-2protein, most Bcl-2inhibitors can not simultaneously bind in Mcl-1protein. Therefore, a structural study of the interactions of small molecule inhibitors with Mcl-1protein can guide molecular design of Mcl-1inhibitors and structure-activity relationship (SAR) analysis.The study explored the binding mode and binding site of small molecule inhibitors toward Mcl-1protein by HSQC NMR spectroscopy. Based on this, novel Mcl-1inhibitors were designed to expand binding sites within BH3groove and gained improved binding affinity.First, we constructed a human Mcl-1protein expression plasmid. By means of isotope label method, we obtained I5N labeld Mcl-1protein. Then we got high quantity of95%and7.2mg per litter bacteria medium high levels of15N labeled Mcl-1protein through optimizing protein expression and purification. The circular dichroism spectra show that15N labeled Mcl-1have a stable secondary structure mainly consisting of a-hexlix. The HSQC spectra of 15N labeled Mcl-1alone showed well-dispersed peaks, indicative of a folded and stable protein. These proteins are eligible for the HSQC NMR experimentes.Next, we performed1H-15N HSQC NMR to study the binding mode of3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1), which was identified as a dual inhibitor of Bcl-2and Mcl-1in our previous study. Upon S1addition, NMR spectra generated many chemical shifts. The combined chemical shift perturbation (CCSP) signals revealed that more than60%of the residues perturbed above0.03ppm were located in the BH3binding groove of the protein. Among them, the residues located in p2pockets (V249, M250, V253, R263) experience average chemical shift changes of at least0.05ppm. In particular, the chemical shift of R263residue change is most obvious following the addition of S1. Docking results supported the binding mode:carbonyl substitution of S1binds near R263of Mcl-1through hydrogen bonds, whereas the3-position substituent extends into the p2pocket and the9-position cyano group points to but does not access the p4of Mcl-1proteins. With the aim of accessing the p4pocket to achieve enhanced dual-inhibition effects, we designed S1derivatives that replace the cyano group with longer and larger groups. We synthesized small molecules of3-(4-bromophenylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-(3-phenyl)propylamine (H2) and (E, E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g). NMR studies demonstrated that H2and4g can occupy the BH3-binding groove of Mcl-1protein. In addition to same residues in p2pocket expreincing significant CCSP, a cluster of residues in p4pockets including V216, G219, V220emerged after adding H2and4g. respectively. It demonstrated that these two compounds achived occupying p2and p4pocket simultaneously. |
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