Xiangqian Li

An anthraquinone scaffold for putative, two-face Bim BH3 α-helix mimic.

Bim BH3 peptide features an α-helix with hotspot residues on multiple faces. Compound 5 (6-bromo-2,3-dihydroxyanthracene-9,10-dione), which adopts a rigid-plan amphipathic conformation, was designed and evaluated as a scaffold to mimic two faces of Bim α-helix. It reproduced the functionalities of both D67 and I65 on two opposing helical sides. Moreover, it maintained the two-faced binding mode during further evolution. A putative BH3 α-helix mimic and nanomolar Bcl-2/Mcl-1 dual inhibitor, 6, was obtained based on the structure of 5.

3-Thiomorpholin-8-oxo-8H-acenaphtho [1,2-b] pyrrole-9-carbonitrile (S1) derivatives as pan-Bcl-2-inhibitors of Bcl-2, Bcl-xL and Mcl-1.

Based on the binding mode of our previously discovered dual inhibitor of Bcl-2 and Mcl-1, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3, S1), a library of 9-substituted 3 derivatives was synthesized to further probe the p4 pocket of the two targets. By NMR, structure-activity relationship study, and site-directed mutation, compound 6d (3-(4-aminophenylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-3-phenyl)propylamine) was identified to span p2-p4 pockets of Mcl-1, Bcl-2 and Bcl-x(L), and then exhibited 9- to 35-fold better affinity to the three targets than 3 (IC(50)=10, 20 and 18 nM, respectively), which led to greater activity in induction of apoptosis in multiple cancer cell lines. Different contribution of p4 pocket to binding Bcl-2 and Mcl-1 was also investigated by plotting the potency and the HAC of the derivatives.

Novel soluble myeloid cell leukemia sequence 1 (Mcl-1) inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g) developed using a fragment-based approach

Based on a known nanomolar Bcl-2 homology domain 3 (BH3) mimetic 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b] pyrrole-9-carbonitrile (S1, MW: 331), we applied a fragment-based approach to obtain BH3 mimetics with improved affinity and improved solubility in a water-ethanol (9:1) cosolvent. After the deconstruction of 1 (S1), we obtained fragment cyanoacetamide (4), which was determined to be a ligand efficiency (LE) hot part. After a rational optimization through fragment evolution beginning with fragment 4, a smaller Mcl-1 inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g, MW: 288) with a 6-fold increase in affinity compared to 1 was obtained, as predicted by our optimization curve and identified by Mcl-1 protein nuclear magnetic resonance (NMR).

Fragment-based design, synthesis, and biological evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxynicotinamide derivatives as novel Mcl-1 inhibitors.

We have previously reported a nanomolar inhibitor of antiapoptotic Mcl-1 protein, 3-thiomorpholin-8-oxo-8H-acenaphtho [1,2-b] pyrrole-9-carbonitrile (S1). S1 plays its function by binding to the BH3 groove of Mcl-1. Basing on this spacial structural characteristic, we developed a novel class of Mcl-1 inhibitor using fragment-based drug discovery approach. By dissecting S1, we identified the compound 4 with a 2-hydroxypyridine core as the starting fragment. In the following molecular growth, we used the ligand efficiency evaluation and fit quality score to assess the fragments. A novel potent compound, N-benzyl-5-(4-isopropylthiophenol)-2-hydroxyl nicotinamide (12c), which binds Mcl-1 with an IC(50) value of 54 nM was obtained. Compound 12c demonstrated a better aqueous solubility than S1.

Two‐Face, Two‐Turn α‐Helix Mimetics Based on a Cross‐Acridine Scaffold: Analogues of the Bim BH3 Domain

The design of a cross‐acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two‐face, two‐turn α‐helix is described. Docking studies and 2D 1H,15N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl‐1 and Bcl‐2 proteins (Ki 0.079 and 0.056 μM, respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.

Design and application of a rigid quinazolone scaffold based on two-face Bim α-helix mimicking

Based on our previous discovery of an anthraquinone scaffold mimicking two faces of Bim α-helix, we derived a quinazolone scaffold through structure simplification and optimization. It was inferred that a rigid bicyclic ring was necessary and efficient to maintain the two-faced binding mode. A novel dual inhibitor 6c [6,7,8-trihydroxy-3-(2-hydroxy-5-methylbenzyl)-2-phenylquinazolin-4(3H)-one] was obtained based on this scaffold. 6c exhibited dual binding activity with K(i) values of 0.123 μM for Mcl-1 and 0.179 μM for Bcl-2.

Deactivation of Mcl-1 by Dual-Function Small-Molecule Inhibitors Targeting the Bcl-2 Homology 3 Domain and Facilitating Mcl-1 Ubiquitination

By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. NoxaBH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, BimBH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.

Discovery of a small-molecule pBcl-2 inhibitor that overcomes pBcl-2-mediated resistance to apoptosis.

Although the role of Bcl‐2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT‐737 and S1. We identified an S1 analogue, S1‐16, as a small‐molecule inhibitor of pBcl‐2. S1‐16 efficiently kills EEE‐Bcl‐2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)‐expressing HL‐60 cells and high endogenously expressing pBcl‐2 cells, by disrupting EEE‐Bcl‐2 or native pBcl‐2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1‐16 binds to the BH3 binding groove of EEE‐Bcl‐2 (Kd=0.38 μM by ITC; IC50=0.16 μM by ELISA), as well as nonphosphorylated Bcl‐2 (npBcl‐2; Kd=0.38 μM; IC50=0.12 μM). However, ABT‐737 and S1 had much weaker affinities to EEE‐Bcl‐2 (IC50=1.43 and >10 μM, respectively), compared with npBcl‐2 (IC50=0.011 and 0.74 μM, respectively). The allosteric effect on BH3 binding groove by Bcl‐2 phosphorylation in the loop region was illustrated for the first time.

Pan-BH3 Mimetic S1 Exhibits Broad-Spectrum Antitumour Effects by Cooperation between Bax and Bak

Small molecule S1 is a pan‐BH3 mimetic that can bind antiapoptotic Bcl‐2, Bcl‐xL and Mcl‐1 proteins. Herein, different Bcl‐2 member expression cancer cell lines (NCI‐H345, MCF‐7, SMMC‐7721 and Hela) and cells deficient in Bax and/or Bak by shRNA were used to unravel the cascade of events by which S1 promotes apoptosis compared with Bcl‐2/Bcl‐xL inhibitor ABT‐737. We identified that S1 exhibited broader antitumour spectrum than ABT‐737 through disruption of more Bcl‐2 interactions including Mcl‐1/Bak interaction. Moreover, the individual and combined roles of Bax and Bak in S1‐induced apoptosis were revealed. Our results showed that S1 induced a Bak‐mediated apoptosis. Bak played a predominant role in either S1 or ABT‐737‐induced apoptosis through the cooperation with Bax on the formation of large oligomers on mitochondrial membrane.

Design, synthesis and structure–activity relationship studies of morpholino-1H-phenalene derivatives that antagonize Mcl-1/Bcl-2.

We report herein characteristic studies of Mcl-1 and Bcl-2 dual inhibitors. It was found that a protruding carbonyl group forming hydrogen bond with R263 plays a predominant role compared with the hydrophobic group that occupies the p2 pocket. A series of dual inhibitors representing different parts of the morpholino-1H-phenalene were designed, synthesized and evaluated.