Research paperStructure-based optimization of FDA-approved drug methylene blue as a c-myc G-quadruplex DNA stabilizer
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 6a–c 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).
References (33)
- et al.
Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis
Cancer Res.
(2009) - et al.
Stabilization of G-Quadruplex DNA and down-regulation of oncogene c-myc by quindoline derivatives
J. Med. Chem.
(2007) - et al.
The price of innovation: new estimates of drug development costs
J. Health Econ.
(2003) - et al.
Methylene blue. A possible treatment for manic depressive psychosis
J. Affect. Disord.
(1983)Two cases of poisoning by contamination of nitrous oxide with higher oxides of nitrogen during anaesthesia
Br. J. Anaesth.
(1967)et al.Methylene blue
Am. J. Ther.
(2003)et al.The phenothiazinium chromophore and the evolution of antimalarial drugs
Trop. Med. Int. Health
(2005)et al.Methylene blue in photodynamic therapy: from basic mechanisms to clinical applications
Photodiagnosis Photodyn. Ther.
(2005) G-quadruplex DNA structures - variations on a theme
Biol. Chem.
(2001)- et al.
G-quadruplex DNA: a target for drug design
Nat. Med
(1998)et al.G-quadruplexes in promoters throughout the human genome
Nucleic Acids Res.
(2007) - et al.
G-quadruplexes as targets for drug design
Pharmacol. Ther.
(2000)G-quadruplex DNA as a target for drug design
Curr. Pharm. Des.
(2000)et al.G-quadruplexes: targets in anticancer drug design
Chem. Med. Chem.
(2008)et al.A hitchhiker’s guide to G-quadruplex ligands
Org. Biomol. Chem.
(2008)et al.G-quadruplex nucleic acids as therapeutic targets
Curr. Opin. Chem. Biol.
(2009)et al.Targeting telomeres and telomerase
Biochimie
(2008)Human telomeric G-quadruplex: the current status of telomeric G-quadruplexes as therapeutic targets in human cancer
FEBS J.
(2010) - et al.
The c-Myc target gene network
Semin. Cancer Biol.
(2006)et al.Global mapping of c-Myc binding sites and target gene networks in human B cells
Proc. Natl. Acad. Sci. U S A
(2006) - et al.
A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor
Mol. Cell. Biol.
(1989)et al.Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene
Proc. Natl. Acad. Sci. U S A
(1989) - et al.
Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription
Proc. Natl. Acad. Sci. U S A
(2002)et al.The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo
Mol. Cancer Ther.
(2002)et al.Design and synthesis of an expanded porphyrin that has selectivity for the c-MYC G-quadruplex structure
J. Am. Chem. Soc.
(2005)
Stabilization of G-Quadruplex DNA with platinum(ii) schiff base complexes: luminescent probe and down-regulation of c-myc oncogene expression
Chemistry
Identification of natural product fonsecin B as a stabilizing ligand of c-myc G-quadruplex DNA by high-throughput virtual screening
Chem. Commun.
Drug repurposing: identify, develop and commercialize new uses for existing or abandoned drugs. Part II
Idrugs
New uses for old drugs
Nature
Self-organizing maps in drug discovery: compound library design, scaffold-hopping, repurposing
Curr. Med. Chem.
Structure-based strategies for drug design and discovery
Science
Integration of virtual and high-throughput screening
Nat. Rev. Drug Discov.
Virtual screening of chemical libraries
Nature
Structure-based discovery of natural product-Like TNF-? inhibitors
Angewandte Chemie
A natural product-like inhibitor of NEDD8-activating enzyme
Chem. Commun
Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs
Proc. Natl. Acad. Sci. U S A
Molecular Cloning, a Laboratory Manual
A neighbor-interaction analysis of the hypochromism and spectra of DNA
J. Mol. Biol.
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- 1
These authors contributed equally to the work.
- 2
D.-L. Ma and C.-H. Leung contributed equally to this work.