Design, Synthesis And Antitumor Activity Study Of Small Molecule BH3α-helix Mimetics

Protein-protein interactions (PPI) play a central role in all biological processes, and misregulation of these interactions is often implicated in disease states. Most multiprotein complexes feature a-helical segments at their interfaces. In these a-helices, a small subset of the residues (hotspots) contributes most of the free energy of binding. Proteomimetics that reproduce the arrangement of key side chains in an a-helix could disrupt protein-protein interfaces and become potential leading drugs.The Bcl-2family proteins are central regulators of apoptosis. The six known Bcl-2-like antiapoptotic proteins are divided into two classes, represented by Bcl-2and Mcl-1. The PPI between antiapoptotic and proapoptotic Bcl-2members are mediated by the a-helix of the BH3-only proteins. Bim is a non-selective BH3-only protein that can neutralize both arms of the anti-apoptotic Bcl-2family, Bcl-2and Mcl-1protein. BH3helix in Bim is a regular a-helix and has the characteristic of a-helix. Therefore, small molecules that reproduce the spatial distribution of hotspots in the Bim BH3a-helix have been investigated as representative a-helix mimetics and potential antitumor drugs.So far, proteomimetics could only impart functionality from one face of the a-helix. However, the hotspots of Bim, L62,165, D67, and F69locate on two faces of the a-helix. Hydrophilic D67has been established by our group as the "very hotspot". Small-molecule BH3mimetics including clinical candidates fail to simultaneously mimic D67and other hotspots on multiple Bim faces. The lack of hydrophilic group mimicking may result in not only suboptimal affinity to target proteins but also non-selectivity, which would significantly limit the utility of the mimetic due to potential promiscuity.In this study, a novel rigid-plan small molecule scaffold A5was constructed to mimic the structural and recognized binding features of both D67and165on two faces of the Bim BH3a-helix. This scaffold is able to maintain its two-face binding mode and serves as a platform for further molecular evolution, leading to novel Mcl-1/Bcl-2dual inhibitors with optimized affinities and pharmaceutical properties. Compound A10, for example, was derived based on A5as a specific Bim mimetic and dual inhibitor with nanomolar affinity. It was found to bind to the Mcl-1and Bcl-2proteins with Ki values of0.013and0.024μM, respectively in our FP-based binding assay.1H-15N HSQC provide compelling evidence that A10is the first compound that can reproduce a similar arrangement with the side chains of L62,165, and D67on two faces of the Bim peptide. A series of biological tests in vitro illustrated that A10is a functional and structural Bim mimetic. A10completely killed the multiple cancer cell lines through Bax/Bak, while no significant cytotoxicity was found in the normal HEK293cell line.Based on A5, we designed a scaffold Bl with high structural rigidity that can easily install up to four functional groups to mimic the main chain located on two faces and two turns of an a-helix. In the lowest energy conformation of B1, four substituents project from the template with similar angular relationships and distances compared to the L62,165, F69, and D67a-carbons in the a-helix. Compound C4that is derived from A5binds to the Mcl-1and Bclr2proteins with Ki values of0.079and0.056μM, respectively.2D1H-15N HSQC NMR results demonstrated that C4is the first compound that accurately mimics the L62,165, F69, and D67residues on two face of Bim peptide. The hotspot mutation could also be mimicked by small compounds for the first time through individual or multiple deletions of side chains on the scaffold B1. Small molecules that mimic hotspot mutations could probe the detailed local structural environment that determines the fine-tuned binding specificity to multiple targets of pro-survival proteins. The series of compounds E, F, G were designed with different combination of functional groups. The affinities of these smaller molecules toward Mcl-1and Bcl-2indicates that D67mimicry plays a more important role binding to Mcl-1than binding to Bcl-2, while the affinity for Bcl-2is dependent on the mimicry of hydrophobic groups.Based on structural information of S1, we applied Fragment-based drug discovery to get a series of novel small-molecule Bcl-2and Mcl-1dual inhibitors. The most potent compound16binds to Mcl-1and Bcl-2proteins with Ki values of298nM and1.1μM, respectively. Additionally, this study provided new starting hits for fragment-based Mcl-1/Bcl-2dual inhibitors discovery.