Small-molecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC promoter

Abstract

It has been widely accepted that DNA can adopt other biologically relevant structures beside the Watson-Crick double helix. One recent important example is the guanine-quadruplex (Gquadruplex) structure formed by guanine tracts found in the MYC (or c-myc) promoter region, which regulates the transcription of the MYC oncogene. Stabilization of this G-quadruplex by ligands, such as the cationic porphyrin TMPyP4, decreases the transcriptional level of MYC. Here, we report the first structure of a DNA fragment containing five guanine tracts from this region. An unusual G-quadruplex fold, which was derived from NMR restraints using unambiguous modelindependent resonance assignment approaches, involves a core of three stacked guanine tetrads formed by four parallel guanine tracts with all anti guanines and a snapback 3′-end syn guanine. We have determined the structure of the complex formed between this G-quadruplex and TMPγP4. This structural information, combined with details of small-molecule interaction, provides a platform for the design of anticancer drugs targeting multi-guanine-tract sequences that are found in the MYC and other oncogenic promoters, as well as in telomeres.Human c-MYC is a transcription factor that is central to regulation of cell growth, proliferation, differentiation and apoptosis [1][2][3][4] . The MYC (also known as c-myc) gene that encodes this protein is tightly regulated in normal cells and its aberrant overexpression is associated with the progression of many cancers 5 . An important element in the MYC promoter region, termed nuclease-hypersensitivity element III 1 (NHE III 1 ), controls up to 90% of total MYC transcription [6][7][8][9] . Previous studies have suggested that this element, composed of a pyrimidine-rich and a purine-rich strand, may form other structures beyond the canonical B-DNA Watson-Crick duplex 6,8,[10][11][12] . In particular, the 27-nucleotide (nt) purine-rich strand (Pu27) of this element, which contains six guanine tracts (Table 1), forms multiple G-quadruplex structures [12][13][14][15] built from the stacking of G·G·G·G tetrads 16 . Furthermore, the G-quadruplex structure(s) involving four central guanine tracts of Pu27 is biologically relevant, as its destabilization (by guanine-to-adenine mutations) and its Correspondence should be addressed to A.T.P. (phantuan@mskcc.org) or D.J.P. (pateld@mskcc.org). Accession codes. Protein Data Bank codes: The coordinates for the structures of Pu24I and the Pu24I-TMPyP4 complex have been deposited under accession codes 2A5P and 2A5R, respectively. Note: Supplementary information is available on the Nature Chemical Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Here we present the structure of a 24-nt five-guanine-tract sequence from the guanine-rich strand of the MYC NHE III 1 in K + solution. The structure represents a unique intramolecular parallel-stranded foldback G-quadruplex, in which a guanine from the 3′ tail is plugged back into the core...

Structure of an Unprecedented G-Quadruplex Scaffold in the Human c-kit Promoter

“…22 The present study has shown that this is not the case. The structure contains two single-residue double-chainreversal loops spanning three G-tetrad layers, a robust type of loop reported previously 15,28 that could play an important role in the folds of many G-quadruplexes 29 in K + solution. This quadruplex is remarkable in requiring the active participation of 18 out of the 22 nucleotides in the structural organization.…”

“…We prepared samples (Table S1, Supporting Information) in which only one guanine was 2% 15 N-labeled at a time, using deliberately diluted 15 N-labeled phosphoramidite. 24 Guanine imino protons were unambiguously assigned by the site-specific low-enrichment approach 24 ( Figure 1b).…”

“…Guanine H8 proton assignments were completed (or confirmed) by multi-bond correlations to 15 N nuclei in sitespecific 2% 15 N-labeled samples. 24 The spectral assignments for other resonances were completed by through-bond (COSY and TOCSY) and through-space (NOE-SY) correlations between protons.…”

“…15 The latter are parallel-stranded quadruplexes, with several strand-reversal loops and base-pair platforms. 15 They have structural complexity beyond that of quadruplexes formed from telomeric DNA repeats, [16][17][18][19][20][21] suggesting the possibility of identifying ligands that are selective for the c-myc quadruplexes.…”

Targeting G-Quadruplex DNA with Small Molecules

“…[97] Extensive substitution of the meso positions with groups other than pyridinium failed to generate compounds with improved activities, but the resulting structure-activity relationships demonstrated that base stacking and charge-charge interactions are important for porphyrin-DNA binding. [97] Subsequent X-ray crystallography and NMR studies have shown that TMPyP4 can bind to G-quadruplex DNA at many different positions, including the terminal G-tetrads, [98] and the loops, grooves, and phosphodiester backbone. [99] TMPyP4 has exhibited some promising anticancer activities in vivo, [62,100] but it has very poor DNA specificity, [86] causes anaphase bridges in sea urchin embryos, [87] and is highly toxic in vivo.…”