Elsevier

Biochimie

Volume 93, Issue 6, June 2011, Pages 1055-1064
Biochimie

Research paper
Structure-based optimization of FDA-approved drug methylene blue as a c-myc G-quadruplex DNA stabilizer

https://doi.org/10.1016/j.biochi.2011.02.013Get rights and content

Abstract

G-quadruplexes are non-canonical DNA secondary structures putatively present in the promoter regions of oncogenes in the human genome. The targeting of promoter G-quadruplex structures to repress oncogene transcription represents a potential anticancer strategy. Here, we have used high-throughput virtual screening to identify FDA-approved drug methylene blue (MB) as a promising scaffold for binding the c-myc oncogene G-quadruplex DNA. Based on molecular docking analysis of MB to the c-myc G-quadruplex, we designed and screened 50 MB derivatives containing side chains that could interact with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring compounds were synthesized and the interactions with the c-myc G-quadruplex were investigated using the FID assay. The results showed that the methylene blue derivatives 6ac were able to bind to the c-myc G-quadruplex with greater binding affinity compared to the known G-quadruplex binding ligand, crystal violet. The activity of the most potent compound identified from the FID assay, 6b, as an inhibitor for polymerase-drive DNA extension was examined using a PCR-stop assay and compared against that of the parent compound methylene blue. The results of the PCR-stop assay showed that the addition of the side chain improved the activity of the derivatives as an inhibitor compared to the parent compound. The MB derivative 6b was shown to be highly selective towards c-myc G-quadruplex over double-stranded DNA and other biologically relevant G-quadruplexes using UV–visible spectroscopy and mass spectrometry, respectively. The MB derivative 6b could induce or stabilize c-myc G-quadruplex formation in both cell-free and cellular biological models, and displayed higher cytoxicity against human hepatocarcinoma cells compared to the parent compound, MB.

Graphical abstract

Highlights

► The FDA-approved drug, methylene blue (MB), was identified by high-throughput screening as a promising scaffold for binding the c-myc oncogene G-quadruplex DNA. ► Addition of side chains improved the inhibiting activity of the derivatives compared to the parent compound. ► The most potent MB derivative investigated could induce or stabilize c-myc G-quadruplex formation in both cell-free and cellular biological models, and display higher cytotoxicity against human hepatocarcinoma cells compared to parent compound, MB.

Introduction

G-quadruplexes are DNA secondary structures formed from planar arrangements of four guanines stabilized by Hoogsteen hydrogen bonding and monovalent cations [1]. G-quadruplexes have received much attention recently due to their putative existence in telomeres and in the promoter regions of oncogenes such as c-myc [2](a), [2](a)(b). These non-canonical DNA secondary structures have emerged as a potentially new avenue for therapeutic intervention of cancers [3] (a), [3] (a)(b), [3] (a)(c), [3] (a)(d), [3] (a)(e), [3] (a)(f), [3] (a)(g). Small molecules that can stabilize the G-quadruplex secondary structure could act as chemotherapeutic agents by inhibiting telomerase activity or by blocking oncogene transcription [3] (a), [3] (a)(b), [3] (a)(c), [3] (a)(d), [3] (a)(e), [3] (a)(f), [3] (a)(g). Quarfloxin (CX-3543), a first-in-class G-quadruplex-binding chemotherapeutic drug that has entered Phase II clinical trials for chronic lymphocytic leukaemia, is believed to exert its potent antitumour effects through disruption of the nucleolin/rDNA G-quadruplex complex in the nucleolus, arresting ribosomal RNA biogenesis [4].

The c-myc gene encodes a transcription factor that is understood to regulate 15% of all gene expression, including those involved in cell growth and apoptosis, and the overexpression of c-myc has been implicated in the tumourigenesis of malignant cancers [5] (a), [5] (a)(b). The nuclease hypersensitivity element III1 (NHE III1) is a guanine-rich 27 base-pair sequence located upstream of the c-myc P1 promoter, and controls 80–90% of c-myc transcription [6] (a), [6] (a)(b). Several small molecule ligands have been reported to stabilize the c-myc NHE III1 G-quadruplex and inhibit c-myc oncogene transcription, including cationic porphyrins [7] (a), [7] (a)(b), [7] (a)(c), quindoline derivatives [8] and platinum complexes [9]. We have recently identified a natural product-based c-myc G-quadruplex binder using high-throughput virtual screening [10a].

Pharmaceutical discovery and development is a highly difficult and expensive process. Repurposing is an attractive strategy whereby existing drugs are redeveloped for new uses [11] (a)(b), [11] (a)(c), [11] (a). With known pharmacokinetic and toxicological profiles, such drugs can enter Phase II clinical trials rapidly, allowing a 40% reduction in the overall cost due to the bypassing of preliminary assessments [12]. Furthermore, existing drugs tend to have more favourable absorption, delivery, metabolism and excretion (ADME) profiles. Consequently, marketed drugs can be considered to represent privileged scaffolds for the development of new therapeutics.

Structure-based design has emerged as a powerful tool in drug design and discovery, complementing existing combinatorial and high-throughput techniques [13] (a)(b), [13] (a)(c), [13] (a). By identifying potent small molecule binders in silico, the number of compounds to be tested in vitro can be vastly reduced. Abagyan and co-workers have identified nonsteroidal antiandrogens from a library of marketed oral drugs using high-throughput virtual screening [14]. Encouraged by these ideas, we set out to apply high-throughput virtual screening methods to identify interesting molecular scaffolds from existing drugs that could be developed as effective G-quadruplex binders. From the virtual screening results, we identified methylene blue (MB) as a promising candidate for further structure-based lead optimization (Fig. 1). MB contains a positively charged, aromatic scaffold suitable for G-quadruplex end-stacking. Functional groups can be attached to the parent MB scaffold to interact with the G-quadruplex grooves, increasing the binding affinity of the MB derivatives to the G-quadruplex. We report herein our structure-based lead optimization of FDA-approved drug MB as a c-myc G-quadruplex binding ligand. Using a unique c-myc NHE III1 G-quadruplex model developed by our group, we designed and screened 50 MB derivatives containing various side chains for c-myc G-quadruplex binding ability. As a proof-of-concept of our approach, we synthesized the highest-scoring MB derivatives 6ac containing 4-bromophenyl moieties linked by short alkyl chains (Fig. 1). These analogues displayed improved c-myc G-quadruplex binding ability and selectivity profiles compared to the parent compound (MB), as demonstrated through spectroscopic experiments and in vitro biological assays.

Section snippets

Materials

Calf thymus DNA (ct DNA) was purchased from Sigma Chemical Co. Ltd. and purified according to literature methodology [15a]. The DNA per base-pair concentration was determined by UV–Vis absorption spectroscopy using the following molar extinction coefficient at the indicated wavelength: calf thymus DNA, ɛ260 = 13200 cm−1 M−1 (base-pair) [15b]. DNA oligomers were obtained from Tech Dragon Limited (Hong Kong). The sequence for oligomer Pu27 is [5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′]. The

Virtual screening of FDA-approved drug database

Despite the publication of a solution-based structure of myc22 by Ambrus et al. [24], we have chosen to construct a model of the c-myc NHE III1 G-quadruplex loop isomer using the X-ray crystal structure of the intramolecular human telomeric G-quadruplex DNA (PDB code: 1KF1) [15] (a)(b), [15] (a). NMR structures of biomolecules are typically solved as a series of energy minimized conformations [24], and as a result, for a given NMR structure there are multiple conformations which needs to be

Conclusion

In conclusion, we have employed the unique intramolecular G-quadruplex c-myc NHE III1 loop isomer model developed by our group to perform high-throughput virtual screening on an FDA-approved drug database of over 3000 compounds. Methylene blue emerged as an attractive scaffold for further structural modifications. As a proof-of-concept, we used a structure-based lead optimization approach to generate MB derivatives that displayed superior binding affinity and selectivity for the c-myc

Acknowledgment

This work is supported by the Hong Kong Baptist University (FRG2/09-10/070 and FRG2/10-11/008), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU) and City University of Hong Kong (Project No. 9667032).

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