STRUCTURAL CHARACTERIZATION OF THE HUMAN C-MYC QUADRUPLEX AND MECHANISTIC EVALUATION OF ANTITUMOR AGENTS: ANTHRACENYL ISOXAZOLE AMIDES AND QUINOLINEDIONES

Cancer is a disease characterized by the abnormal proliferation of cells in the body. For many forms of cancer, there remain only limited and often ineffective treatment options available. Treatment of cancer is problematic for several reasons that include the difficulty in establishing molecular targets, finding interventions that cause selective toxicity to cancer cells, and the uniform capability of cancer cells to evade apoptosis. To address this, new strategies must be employed that take advantage of novel mechanisms of action to develop better therapies. The goal of this research is to aid in this effort through the study of emerging targets and antitumor agents. In these studies, we characterize the structure of an important target in the field of anticancer drug design, the quadruplex formed in the human c-MYC promoter region. The oncogene c-MYC is dysregulated or overexpressed in approximately 70% of human cancers and contributes to many survival pathways used by cancer cells to evade apoptosis. Stabilization of the c-MYC promoter quadruplex has been shown to reduce c-MYC expression and cause apoptosis in tumor cells. We also examine the mechanisms of action of two novel classes of antitumor agents, the anthracenyl isoxazole amides (AIMs) and a group of 5,8-quinolinedione analogs. We demonstrate interactions of the AIMs with quadruplex-forming sequences found in human telomeres, the c-MYC promoter, and mitochondrial DNA. Additionally, we provide evidence that the AIMs can inhibit the electron transport chain of mitochondria, specifically Complex II. Further, we show that treatment with the AIMs causes damage to mitochondrial DNA and loss of the mitochondrial membrane potential, leading to the intrinsic pathway of apoptosis in human glioblastoma cells. We also show a novel set of 5,8-quinolinedione analogs have potent antitumor activity in human breast cancer cells not related to their suitability as substrates for the NQO1-reductase that is often overexpressed in cancer. Together, this work has provided new insights to the field of anticancer drug discovery through characterization of an important target, the c-MYC promoter quadruplex, and through analysis of two novel classes of antitumor compounds, the AIMs and the 5,8-quinolinediones

Anthracenyl isoxazole amides (AIMs) stabilize quadruplex DNA structures in telomeric and c-MYC promotor sequences

Approximately 23,000 people are affected by malignant brain and CNS tumors in the United States each year and those afflicted have a median survival rate of only 12–15 months due to the limited treatment options available. The anthracenyl isoxazole amides (AIMs) are a novel class of compounds that have been shown to possess significant anti tumor activity in the NCI 60 cell line panel and to inhibit growth of SNB-19 glioblastoma cells at low micromolar and nanomolar concentrations. The goal of our current research is to characterize the mechanism underlying the anti tumor activity of the AIMs. We hypothesize the mechanism of growth inhibition to involve binding and stabilization of a DNA tertiary structure known as a guanine quadruplex. Various regulatory regions of DNA, such the c-MYC oncogene promoter sequence and repeating sequences formed at the end of telomeres, adopt the quadruplex conformation. Stabilization of quadruplex structures by small-molecule binding ligands has been reported to modulate the expression of genes and inhibit telomerase activity. Down-regulation of certain oncogenes or the inhibition of telomerase can cause tumor cells to undergo apoptosis or become unable to efficiently replicate. To establish whether interactions between the AIMs and quadruplex-forming sequences act to stabilize the quadruplex tertiary structure, circular dichroism spectroscopy (CD) was employed. CD is a method that utilizes the differential absorbance of left and right circularly polarized light to examine the chiral structure of molecules. CD thermal melting studies were conducted to determine whether the AIMs would increase the melting temperature of quadruplex forming sequences as an indication of increased stability. Our results demonstrated the AIMs, at two equivalents, increase the melting temperature (Tm) of both the c-MYC promoter and telomeric sequences by approximately 2–3 °C with strong statistical significance and reproducibility. Utilizing CD allows the use of low micromolar concentrations of DNA and this method will be used in the future to rapidly develop additional structure-activity relationships between novel AIMs and quadruplex forming sequences. Our laboratory has also shown chemical shifts in the imino region upon treatment with the AIMs for both the c-MYC promotor and telomeric sequence by NMR, providing additional evidence of the AIMs interaction with quadruplex structures. Interestingly, fluorescence microscopy of SNB-19 cells treated with AIMs show their localization is primarily in the mitochondria, and mitochondrial DNA contains several other important quadruplex forming sequences. Mitochondrial-dependent apoptosis has been suggested for other quadruplex binding ligands and therefore our future work will examine the potential stabilization of mitochondrial quadruplexes by these and other novel AIMs

Targeting mitochondrial DNA for development of novel antitumor drugs

Mitochondria are responsible for the vast majority of energy metabolism in healthy cells and this is often considered their primary function. However, their role routinely changes in tumor cells where mitochondrial metabolism is no longer the major pathway used to produce energy; this shift in metabolism has been named the “Warburg effect”. This metabolic reprogramming has been suggested to also suppress another function of mitochondria in cells where they act as the gatekeeper to the intrinsic pathway of apoptosis, also known as programmed cell death. In order for tumor cells to survive, they must evade apoptosis and this is often referred to as a “hallmark of cancer”. With this in mind, targeting mitochondria for treatment of cancer is an emerging strategy suggested by several high impact reviews as a route to develop more selective therapies. Mitochondria possess their own separate genome, and mutations in mitochondrial DNA (mtDNA) are associated with various diseases including cancer. One strategy for inducing apoptosis in tumor cells involves disrupting transcription and/or replication of mtDNA. In the past decade, much research has been focused on a tertiary structure formed by DNA known as a guanine-quadruplex. Quadruplexes found in nuclear DNA have been demonstrated previously to effect expression of oncogenes and to inhibit telomerase activity, both of which contribute to the pathology of cancer. More recently however, quadruplexes have also been shown to form in mtDNA and are suspected to play a major role in mitochondrial genome homeostasis. A region of mtDNA known as conserved sequence block II (CSB II) has been reported to form a hybrid quadruplex (HQ) and to act as a switch that halts transcription, allowing formation of primers and initiation of mtDNA replication. Deletions in mtDNA have also been shown to occur in close proximity to quadruplexes and these structures are believed to act as “breakpoints”, or regions of increased DNA instability. These reports in the literature have informed the development of a novel class of antitumor compounds, the anthracenyl isoxazole amides (AIMs), which we have reported previously to inhibit the growth of SNB-19 glioblastoma cells at low micromolar to high nanomolar concentrations (10’BiPhenOxy AIM IC50 = 0.67 µM). The AIMs were originally designed to target quadruplexes found in nuclear DNA, however our laboratory has demonstrated that the majority of the AIMs do not enter the nucleus and rather concentrate in the mitochondria of SNB-19 cells. This has lead us to begin examining potential quadruplex targets found in mtDNA. Using a previously published quantitative PCR assay, we have shown the AIMs cause an increase in mtDNA damage following treatment at multiple time-points (3, 6, 12, & 24 hours) preceding the induction of apoptosis. Our laboratory is also working to crystallize the CSB II HQ in the presence and absence of the AIMs. This work will allow us to develop a structure-activity relationship to aid in further synthetic refinement of the AIMs to selectively target the CSB II HQ as a method to prevent mtDNA replication and induce apoptosis in tumor cells

Crystal structure of the major quadruplex formed in the promoter region of the human c-MYC oncogene.

The c-MYC oncogene mediates multiple tumor cell survival pathways and is dysregulated or overexpressed in the majority of human cancers. The NHE III1 region of the c-MYC promoter forms a DNA quadruplex. Stabilization of this structure with small molecules has been shown to reduce expression of c-MYC, and targeting the c-MYC quadruplex has become an emerging strategy for development of antitumor compounds. Previous solution NMR studies of the c-MYC quadruplex have assigned the major conformer and topology of this important target, however, regions outside the G-quartet core were not as well-defined. Here, we report a high-resolution crystal structure (2.35 Å) of the major quadruplex formed in the NHE III1 region of the c-MYC promoter. The crystal structure is in general agreement with the solution NMR structure, however, key differences are observed in the position of nucleotides outside the G-quartet core. The crystal structure provides an alternative model that, along with comparisons to other reported quadruplex crystal structures, will be important to the rational design of selective compounds. This work will aid in development of ligands to target the c-MYC promoter quadruplex with the goal of creating novel anticancer therapies

10-N-heterocylic aryl-isoxazole-amides (AIMs) have robust anti-tumor activity against breast and brain cancer cell lines and useful fluorescence properties

© 2020 Elsevier Ltd A novel series of anthracenyl-isoxazole amide (AIM) antitumor agents containing N-heterocycles in the 10 position (N-het) were synthesized using palladium cross-coupling. The unique steric environment of the N-het-AIMs required individual optimization in each case. Lanthanide mediated double activation was used to couple the dimethylamino pyrrole moiety, required for antitumor action. Robust antitumor activity was observed against breast and brain cancer cell lines. The compounds were docked with the c-myc oncogene promoter sequence, which adopts a G4 quadruplex DNA conformation, and represents the working hypothesis for biological action. The N-het-AIMs have useful fluorescence properties, allowing for observation of their distribution within tumor cells

AIMing towards improved antitumor efficacy Dedicated to the memory of Professor Albert I. Meyers

Using the structure-activity relationship emerging from previous Letter, and guided by pharmacokinetic properties, new AIMs have been prepared with both improved efficacy against human glioblastoma cells and cell permeability as determined by fluorescent confocal microscopy. We present our first unambiguous evidence for telomeric G4-forming oligonucleotide anisotropy by NMR resulting from direct interaction with AIMs, which is consistent with both our G4 melting studies by CD, and our working hypothesis. Finally, we show that AIMs induce apoptosis in SNB-19 cells

C.: The SMT-LIB Standard: Version 2.0

Permission is granted to anyone to make or distribute verbatim copies of this document, in any medium, provided that the copyright notice and permission notice are preserved, and that the distributor grants the recipient permission for further redistribution as permitted by this notice. Modified versions may not be made. Preface The SMT-LIB initiative is an international effort, supported by several research groups worldwide, with the two-fold goal of producing an extensive on-line library of benchmarks and promoting the adoption of common languages and interfaces for SMT solvers. This document specifies Version 2.0 of the SMT-LIB Standard. This is a major upgrade of the previous version, Version 1.2, which, in addition to simplifying and extending the languages of that version, includes a new command language for interfacing with SMT solvers. Acknowledgments Version 2.0 of the SMT-LIB standard was developed with the input of the whole SMT community and three international work groups consisting of developers and users of SMT tools: the SMT-API work group, led by A. Stump, the SMT-LOGIC work group, led by C. Tinelli, the SMT-MODELS work group, led by C. Barrett. Particular thanks are due to the following work group members, who contributed numerou