Screening Of A Small-Molecule PBcl-2 Inhibitor And Its Molecular Pharmacological Mechanism

The Bcl-2 family proteins are the main control factors of endogenous apoptosis pathway, and contain members of both pro-and anti-apoptotic. Bcl-2, Bcl-XL and Mcl-1 are three important inhibitors of apoptosis. They have been identified as antitumor targets, and be studied as the small moleculars which can simulate the function of the BH3 domain. Small molecules that can occupy the BH3 groove may neutralize Bcl-2 anti-apoptotic proteins, liberate Bax/Bak, or activate them through the BH3-only proteins, and thus induce tumor cell apoptosis. Such artificial BH3 mimetics have entered the stage of clinical trials with high efficiency and high specificity as anticancer drugs.Recently, phosphorylated Bcl-2 proteins (pBcl-2) were identified as another important therapeutic target. Many types of cancer cells express high levels of pBcl-2, and the upregulation of Bcl-2 phosphorylation is associated with a high resistance to BH3 mimetics. For example, Bcl-2 phosphorylation at serine 70 acts antagonistically against the Bcl-2 inhibitors ABT-737 and Obatoclax in leukemia cells. However, To date, no small molecules have been identified, and this lack of structural information of pBcl-2 BH3 limits the design of pBcl-2 inhibitors.Based on the molecular pharmacology mechanism and structural biology research about the BH3 mimetics, this study gives the following hypothesis:The Bcl-2 phosphorylated modification allosteric regulation BH3 groove structure causes resistance by exhibiting a higher binding affinity toward BH3 only proteins or by sterically blocking the binding of BH3 mimetics. Therefore, based on cell phenotype, we designed a screening method to obtain pBcl-2 inhibitor which can be compatible with pBcl-2 BH3 groove from SI derivatives. Furthermore, Its molecular pharmacological mechanism had also been studied so that we can explicit the function of Bcl-2 phosphorylated modification on its regulating mechanism of antiapoptotic function.For the initial screen, we used HL-60 cells transfected with a phosphor-mimetic mutant EEE-Bcl-2 and nonphospho-mimetic mutant AAA-Bcl-2 (the phosphorylated sites in Bcl-2, threonine 69, serine 70, and serine 87, converted to glutamate residues or alanine) to obtain compounds from 23 structurally different S1 derivatives that could target pBcl-2. Then they were subjected to a further analysis for their specificity-of-action using Bax-1-Bak-1- mouse embryonic fibroblast (MEF) cytotoxicity assays. Ultimately, we selected S1-16 as a candidate pBcl-2 inhibitor which could antagonize pBcl-2 and relied on the Bcl-2 pathway.Next, we detected the binding affinities to EEE-Bcl-2 of S1, S1-16 and ABT-737, as well as the ability to compete with BH3-containing proteins to occupy BH3 groove by ITC and ELIAS. S1-16 could occupy BH3 groove of EEE-Bcl-2, whereas S1 and ABT-737 could not. Besides that, we validated BH3 grooves as the protein interaction site accounted for the competitive Sl-16 binding activity.Then we performed co-immunoprecipitation (co-IP) experiments in order to evaluate the influence of S1, S1-16 and ABT-737 on the heterodimers of anti-apoptotic proteins and pro-apoptotic proteins and activation of Bak and Bax proteins, cytochrome c release, PARP cleavage. These data suggested that activation of mitochondria mediated apoptosis by S1-16 was induced by antagonizing pBcl-2 to release their pro-apoptotic partners Bax or Bak. Lastly, only Sl-16 was found to induce mechanism-based apoptosis in high pBcl-2-expressing cancer cell lines and cells transfected EEE-Bcl-2 as a single agent.S1-16 is the first report of a pBcl-2 inhibitor. The allosteric effect on BH3 groove by Bcl-2 phosphorylation in a loop region was illustrated for the first time. And we also gave the structural difference between Bcl-2 BH3 groove and pBcl-2 BH3 groove. These results will provide the molecular basis for further design and optimization of small molecular pBcl-2 inhibitors. Furthermore, As a more broad-spectrum BH3 mimetic, S1-16 will not only provide a new strategy for cancer therapy, but also be helpful for further investigations of Bcl-2 phosphorylation.