Directly targeting BAX for drug discovery: Therapeutic opportunities and challenges.

For over two decades, the development of B-cell lymphoma-2 (Bcl-2) family therapeutics has primarily focused on anti-apoptotic proteins, resulting in the first-in-class drugs called BH3 mimetics, especially for Bcl-2 inhibitor Venetoclax. The pro-apoptotic protein Bcl-2-associated X protein (BAX) plays a crucial role as the executioner protein of the mitochondrial regulated cell death, contributing to organismal development, tissue homeostasis, and immunity. The dysregulation of BAX is closely associated with the onset and progression of diseases characterized by pathologic cell survival or death, such as cancer, neurodegeneration, and heart failure. In addition to conducting thorough investigations into the physiological modulation of BAX, research on the regulatory mechanisms of small molecules identified through biochemical screening approaches has prompted the identification of functional and potentially druggable binding sites on BAX, as well as diverse all-molecule BAX modulators. This review presents recent advancements in elucidating the physiological and pharmacological modulation of BAX and in identifying potentially druggable binding sites on BAX. Furthermore, it highlights the structural and mechanistic insights into small-molecule modulators targeting diverse binding surfaces or conformations of BAX, offering a promising avenue for developing next-generation apoptosis modulators to treat a wide range of diseases associated with dysregulated cell death by directly targeting BAX.

Optimization of BAX trigger site activator BTSA1 with improved antitumor potency and in vitro ADMET properties.

Direct activation of the pro-apoptotic protein BAX represents a potential therapeutic strategy to trigger apoptosis in cancer. Herein, structural optimization of the reported BAX trigger site activator BTSA1 turned out into a series of pyrazolone derivatives, where compound 6d exhibited significantly enhanced antiproliferative effects and apoptosis induction ability compared to BTSA1. Mechanism of action studies revealed that compound 6d could initiate the BAX activation cascade, promoting BAX insertion into mitochondrial membranes and activating MOMP, ultimately leading to the release of cytochrome c and apoptosis. Furthermore, 6d showed significantly improved in vitro stability and CYPs profile compared to BTSA1. This work may lay a foundation to develop potent BAX trigger site activators for the treatment of BAX-expressing malignancies.

Development of a series of novel Mcl-1 inhibitors bearing an indole carboxylic acid moiety.

The B cell lymphoma protein 2 (Bcl-2) family proteins regulate cell apoptosis by participating in the endogenous apoptosis pathway. As an important anti-apoptotic protein, Myeloid cell leukemia 1 (Mcl-1) is overexpressed in a variety of tumor cells, and targeting this protein has been a promising strategy for cancer therapy. Herein, based on the 1H-indole-5-carboxylic acid structure previously discovered, we have developed a series of novel compounds with increased affinities and selectivity toward Mcl-1 through structure-based drug design. Among those compounds, 26 exerted relatively better affinity and selectivity for Mcl-1 with moderate inhibition in HL-60 cells. Mechanism studies showed that compound 26 could induce cancer cells apoptosis in an Mcl-1-dependent manner. It also exhibited good microsomal and plasma stability with acceptable pharmacokinetics profiles. Furthermore, treatment with target compound in a 4T1 xenograft mouse model significantly suppressed the tumor growth. Overall, the small molecule described herein represents a promising Mcl-1 inhibitor for further study.

Discovery and structure-activity relationship studies of novel Bcl-2/Mcl-1 dual inhibitors with indole scaffold.

The Bcl-2 anti-apoptotic proteins were widely overexpressed in diverse tumor cells, especially for Bcl-2 and Mcl-1, which regarding as important targets of apoptosis induction and resistance of chemotherapy. We identified novel Bcl-2/Mcl-1 dual inhibitors with indole scaffold by the optimization of hit 1. Structure modification against several moieties including hydrophobic fragment, side chain and benzoic acid fragment was conducted and the structure-activity relationship was analyzed. The representative compound 12f exhibited sub-micromolar binding affinities to Bcl-2/Mcl-1 without binding affinity to Bcl-XL. Mechanism of action studies suggested that compound 12f dose-dependently triggered apoptosis in HL-60 cells. Compound 12f represents a novel Bcl-2/Mcl-1 dual inhibitor which deserving further study.

Trends in targeting Bcl-2 anti-apoptotic proteins for cancer treatment.

Apoptosis is the major mode of programmed cell death, which conduces to maintain tissue homeostasis, clearance of abnormal cells and development of organisms. Over the past two decades, great progress and significant clinical benefits in cancer treatment have been made by targeting Bcl-2 anti-apoptotic proteins. However, with the deep research of clinic, the therapeutic value of single target inhibitors is restricted due to the limited monotherapy activity, potential and complex drug resistance as well as monotherapy safety. This review focuses on recent advance in discovery of novel apoptosis inducers targeting Bcl-2 anti-apoptotic proteins aiming to overcome existing therapeutic limitations, and introduce the strategies and advanced technologies in the drug design and optimization.

Design, synthesis and biological evaluation of dual Bcl-2/Mcl-1 inhibitors bearing 2-(1H-indol-4-yl)benzoic acid scaffold.

The anti-apoptotic protein inhibitors of the B cell lymphoma 2 (Bcl-2) family have been developed as new anticancer therapies. Numerous studies illustrated the great potential in the development of dual Bcl-2/myeloid cell leukemia 1 (Mcl-1) inhibitors. Herein, we reported a series of Bcl-2/Mcl-1 inhibitors that optimized from a hit compound 1 via structure-based rational design. The biological evaluation suggested that most compounds exhibited potent binding affinities at submicromolar to both Bcl-2 and Mcl-1 without any Bcl-xL binding affinities, especially compound 9o, with a Ki value of 0.07 µM to Mcl-1 and 0.66 µM to Bcl-2, that has great potential for developing dual inhibitors targeting Bcl-2 and Mcl-1.

Design, synthesis and biological evaluation of novel hydroxamic acid based histone deacetylase 6 selective inhibitors bearing phenylpyrazol scaffold as surface recognition motif.

In recent years, inhibition of HDAC6 became a promising therapeutic strategy for the treatment of cancer and HDAC6 inhibitors were considered to be potent anti-cancer agents. In this work, celecoxib showed moderate degree of HDAC6 inhibition activity and selectivity in preliminary enzyme inhibition activity assay. A series of hydroxamic acid derivatives bearing phenylpyrazol moiety were designed and synthesized as HDAC6 inhibitors. Most compounds showed potent HDAC6 inhibition activity. 11i was the most selective compound against HDAC6 with IC50 values of 0.020µM and selective factor of 101.1. Structure-activity relationship analysis indicated that locating the linker group at 1' of pyrazol gave the most selectivity. The most compounds 11i (GI50=3.63µM) exhibited 6-fold more potent than vorinostat in HepG2 cells. Considering of the high selectivity against HDAC6 and anti-proliferation activity, such compounds have potential to be developed as anti-cancer agents.

Synthesis, anticancer evaluation and pharmacokinetic study of novel 10-O-phenyl ethers of dihydroartemisinin.

Twenty hybrid compounds, tethering dihydroartemisinin (DHA) with diaryl-pyrazoline/diaryl-pyrazole through ether linkage, were synthesized based on hybridization strategy and assessed for their anticancer activity. The representative compound 6f exhibited significantly elevated antiproliferative activity compared with DHA against a panel of cancer cell lines. Unexpected sensitivity of 6f in Adriamycin-resistant MCF-7 cells (MCF-7/Adr) inspired subsequent research on anticancer activity of these DHA derivatives against breast cancer cell lines. All the novel compounds exhibited potent activity in three breast cancer cells including MDA-MB-231, MCF-7 and MCF-7/Adr. Most of them exerted almost 10-fold higher potency in MCF-7/Adr than in MCF-7 and MDA-MB-231 cells. Compound 5f, the most potent compound against MCF-7/Adr (GI50 = 18 nM), was used for mechanism research which revealed that compound 5f arrested MCF-7 and MCF-7/Adr cells in G0/G1 phase with decreased levels of cyclin D1 and increased levels of p27. Preliminary pharmacokinetic properties of 5f and 6f were investigated in rats after a single intravenous administration. The high sensitivity of MCF-7/Adr indicates that these compounds have potential to be developed as therapeutic agents to treat drug-resistant cancer.

Conjugates of 18ß-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid as Pin1 inhibitors displaying anti-prostate cancer ability.

Twenty-six conjugates of 18ß-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid were designed and synthesized as Pin1 inhibitors. Most of these semi-synthetic compounds showed improved Pin1 inhibitory activity and anti-proliferative effects against prostate cancer cells as compared to 3-(1H-benzo[d]imidazol-2-yl)propanoic acid and GA. Compounds 10a and 12i were the most potent to inhibit growth of prostate cancer PC-3 with GI50 values of 7.80µM and 3.52µM, respectively. The enzyme inhibition ratio of nine compounds at 10µM was over 90%. Structure-activity relationships indicated that both appropriate structure at ring C of GA and suitable length of linker between GA skeleton and benzimidazole moiety had significant impact on improving activity. Western blot assay revealed that 10a decreased the level of cell cycle regulating protein cyclin D1. Thus, these compounds might represent a novel anti-proliferative agent working through Pin1 inhibition.

Discovery and structure-activity relationship studies of N-substituted indole derivatives as novel Mcl-1 inhibitors.

Myeloid cell leukemia-1 (Mcl-1) is an important antiapoptotic protein functioning through protein-protein interactions. We discovered LSL-A6 (2-((2-carbamoyl-1-(3-(4-methoxyphenoxy)propyl)-1H-indol-6-yl)oxy)acetic acid) with a novel N-substituted indole scaffold to interfere Mcl-1 binding as a novel Mcl-1 inhibitor. Molecular modeling indicated that this compound binds with Mcl-1 by interaction with P2 and R263 hot-spots. Structure modification focused on several moieties including indole core, hydrophobic tail and acidic chain were conducted and structure-activity relationship was analyzed. The most potent compound 24d which exhibited Ki value of 110nM for interfering Mcl-1 binding was obtained after hit-to-lead modification.

Novel thiol-based histone deacetylase inhibitors bearing 3-phenyl-1H-pyrazole-5-carboxamide scaffold as surface recognition motif: Design, synthesis and SAR study.

A series of novel thiol-based histone deacetylase (HDAC) inhibitors bearing 3-phenyl-1H-pyrazole-5-carboxamide scaffold as surface recognition motif was designed, synthesized, and evaluated for their HDAC inhibition activity. Among them, 15j (IC50=0.08µM) was identified as a better inhibitor than Vorinostat (IC50=0.25µM) against total HDACs. In addition, Structure-activity relationships (SAR) analyses indicated that (i) compounds with different substituents on pyrazole N-1 position exhibited superior activities than those on pyrazole N-2 position, (ii) variation of functional groups on N-1'-alkyl chain terminus followed the trends of carboxyl group>hydroxyl group≫alkyl group, and (iii) methylation on pyrazole C-4 position diminished the HDAC inhibition activity. The SAR will guide us to further refine compounds bearing 3-phenyl-1H-pyrazole-5-carboxamide scaffold to achieve better HDAC inhibitors.

Synthesis and biological evaluation of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines as inhibitors of vascular endothelial growth factor receptor-2.

A novel series of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines were synthesized. The abilities of these compounds to inhibit the VEGFR-2 kinase activity and the proliferation of human microvascular endothelial cells (MVECs) were determined. 6-Methoxy-4-substituted-1,2,3-benzotriazines and 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines have the abilities of inhibiting the VEGFR-2 kinase activity, but only the 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines exhibit good growth inhibitory effects on MVECs. Compound 6-chloro-4-(3-trifluoromethylanilino)-pyrido[3,2-d][1,2,3]triazin (11d) is less half active than PTK787 to inhibit the VEGFR-2 kinase activity, but is more active than PTK787 to inhibit the growth of MVECs. The potential binding modes of 6d, 11d, and CTZ12 in complex with their putative intracellular target, VEGFR-2, were predicted using Surflex-Dock.