Anti-Leukemic Activity of Mitochondrially-Directed Asymmetrical 2-2'-Binaphthoquinones.
Authors: Ricklis Rebecca , Vuica-Ross Milena , Brodsky Robert , Sausville Ed , Dang Chi Van , Karp Judith E , Emadi Ashkan 
Published: October 2019

Abstract
Abstract 4799

Introduction
Binaphthoquinones are unique molecules consisting of two linked naphthoquinone units. Previously, we regiospecifically synthesized a series of asymmetrical 2,2'-binaphthoquinones, which possess HIV integrase inhibitory activity. Potent activity against non-HIV-infected CEM-T4 acute lymphoblastic leukemia cells prompted us to investigate other cytotoxic mechanisms of these compounds. A genome-wide yeast screen uncovered that mitochondrial-related genes are required for sensitivity and resistance to these agents. Furthermore, by generating reactive oxygen species (ROS), biquinones halted yeast growth which was rescued by addition of N-acetylcysteine. Therefore, as part of our efforts to identify new compounds with anti-leukemic activity, we hypothesized that 2,2'-binaphthoquinones would be able to kill leukemic cells by interference with mitochondrial function. The majority of studies on antineoplastic effects of quinones have focused on benzoquinones, anthraquinones or monomeric naphthoquinones. However, nothing is published on the anti-leukemic action of asymmetrical 2,2'-binaphthoquinones, in which two different mononaphthoquinones are attached at the quinone double bond. Biquinones possess four carbonyl groups that have the potential to generate a greater number of ROS per one mole of quinone, and thereby cause more effective oxidative stress, than their monoquinone counterparts. In addition, the potential differences in cytotoxicities between monomeric and dimeric naphthoquinones result from many parameters, including the number of units, planarity, and the specific nature and pattern of aromatic functional groups.

Materials and Methods
Seventeen of our biquinones were dissolved in DMSO and incubated with four leukemia cell lines grown under standard conditions. All cultures were maintained with a constant DMSO concentration. A standard MTT cell proliferation assay was used to identify IC50 values. MV411 cells were incubated for 42-49 hours (2 nights) with two of the most potent compounds, biquinone #7 (BiQ7) and biquinone #10 (BiQ10), and they were assayed and analyzed by flow cytometry for loss of mitochondrial membrane potential (via rhodamine 123 staining), as well as a variety of apoptosis markers such as exposed phosphatidylserine, activated caspases, and sub-2n DNA increases. Simultaneously, preliminary toxicology experiments have been performed on mice.

Results
The IC50 values for both compounds were less than 5 micromolar (μM) against all four cell lines. For subsequent experiments, the cells were treated with either 5 μM BiQ7 or 5 μM BiQ10. A loss of mitochondrial membrane electrochemical potential, as expressed and evidenced by substantial decrease in fluorescence intensity of Rhodamine 123, was observed in 98-100% of the treated cells compared to control (5%). Loss of plasma membrane phosphatidylserine asymmetry was observed via Annexin V- PE (with 7AAD) staining in 94-95% of the treated cells and in only 18% of the control cells. Furthermore, the treated cells showed increases in staining for activated caspases 3 & 7 (BiQs 96-98%; control 10%). Finally, the cell cycle analyses via propidium iodide showed increases in sub-2n DNA in the treated cells compared to the control (control 12%; BiQ7 34%; and BiQ10 54%). Importantly, the preliminary toxicology results in mice suggest selectivity for neoplastic cells since no cytopenia or obvious end organ toxicity was observed with injection of 600 μM binaphthoquinones intraperitoneally daily for three weeks.

Conclusion
Our study shows that this new class of compounds possesses promising in vitro anti-leukemic effects by targeting mitochondrial membrane permeabilization, which occurs early in the apoptotic program and is located downstream of most identified chemotherapy resistance mechanisms in hematologic malignancies. In vivo experiments in xenograft model and ex vivo experiments on primary human cells are planned.

Disclosures:
No relevant conflicts of interest to declare.