Megan Carver

Structure of the Hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K+ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K+ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.

Polymorphism of human telomeric quadruplex structures

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). Compounds that can stabilize the intramolecular DNA G-quadruplexes formed in the human telomeric sequence have been shown to inhibit the activity of telomerase and telomere maintenance, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. Knowledge of intramolecular human telomeric G-quadruplex structure(s) formed under physiological conditions is important for structure-based rational drug design and thus has been the subject of intense investigation. This review will give an overview of recent progress on the intramolecular human telomeric G-quadruplex structures formed in K+ solution. It will also give insight into the structure polymorphism of human telomeric sequences and its implications for drug targeting.

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K+ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K+ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins. LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.

The Major G-Quadruplex Formed in the Human BCL-2 Proximal Promoter Adopts a Parallel Structure with a 13-nt Loop in K+ Solution

The human BCL-2 gene contains a 39-bp GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of BCL-2 gene expression. Inhibition of BCL-2 expression can decrease cellular proliferation and enhance the efficacy of chemotherapy. Here we report the major G-quadruplex formed in the Pu39 G-rich strand in this BCL-2 promoter region. The 1245G4 quadruplex adopts a parallel structure with one 13-nt and two 1-nt chain-reversal loops. The 1245G4 quadruplex involves four nonsuccessive G-runs, I, II, IV, V, unlike the previously reported bcl2 MidG4 quadruplex formed on the central four G-runs. The parallel 1245G4 quadruplex with the 13-nt loop, unexpectedly, appears to be more stable than the mixed parallel/antiparallel MidG4. Parallel-stranded structures with two 1-nt loops and one variable-length middle loop are found to be prevalent in the promoter G-quadruplexes; the variable middle loop is suggested to determine the specific overall structure and potential ligand recognition site. A limit of 7 nt in loop length is used in all quadruplex-predicting software. Thus, the formation and high stability of the 1245G4 quadruplex with a 13-nt loop is significant. The presence of two distinct interchangeable G-quadruplexes in the overlapping region of the BCL-2 promoter is intriguing, suggesting a novel mechanism for gene transcriptional regulation and ligand modulation.

A New G-Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription

The abnormal overexpression of the BCL2 gene is associated with many human tumors. We found a new 28-mer G-quadruplex-forming sequence, P1G4, immediately upstream of the human BCL2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. G-quadruplex can readily form in the P1G4 sequence under physiological salt condition as shown by DMS footprinting. P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions in the BCL2 P1 promoter. In the extended BCL2 P1 promoter region containing both Pu39 and P1G4, P1G4 appears to play a more dominant role in repressing the transcriptional activity. Using NMR spectroscopy, the P1G4 G-quadruplex appears to be a novel dynamic equilibrium of two parallel structures, one regular with two 1-nt loops and a 12-nt middle loop and another broken-strand with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long loop. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures. The presence of P1G4 and Pu39 in adjacent regions of the BCL2 P1 promoter suggests a mechanism for precise regulation of BCL2 gene transcription. The unique P1G4 G-quadruplex may provide a specific target for small molecules to modulate BCL2 gene transcription.

Abstract 3089: A novel G-quadruplex formed in the PDGFR-β promoter that is selectively targeted by a small molecule to repress transcription

Abnormal expression of PDGFR-β protein contributes to several malignancies. PDGFR-β has become increasingly attractive therapeutic target in the treatments of certain cancers. A G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter has been found to form multiple G-quadruplexes from the overlapping sequences. This G-quadruplex-forming NHE has been shown to regulate approximately 60% of basal promoter activity. Targeting transcriptional control of PDGFR-β provides an attractive target for developing inhibitors for the PDGFR-β signaling pathway, in addition to molecular targeting of the PDGFR-β protein or its cognate ligand. We have previously determined the most stable G-quadruplex formed in the PDGFR-β NHE. Interestingly, the 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively targeted by an ellipticine analog GSA1129, which has been shown to repress PDGFR-β activity in cancer cell lines, and GSA1129 appears to shift the dynamic equilibrium in the full-length sequence to favor this structure. Therefore, characterization of the 3′-end G-quadruplex structure is important for understanding its function and for rational design of small molecules targeting this element. The 3′-end PDGFR-β G-quadruplex appears to adopt an unusual parallel G-quadruplex structure containing an imperfect GGGA tetrad at the 3′-end, as shown by DMS footprinting and CD spectroscopy. We further investigated the stability and structure of the 3′-end G-quadruplex and its interactions with GSA1129 by mutational analysis combined with NMR spectroscopy. The 3′-end G-quadruplex can be stabilized by an A-to-G mutation at position 18. However, the truncated wild-type and mutated 18-mer (Pu18) sequences appear to form predominantly dimer structure as shown by NMR. Using a 19-mer sequence Pu19 with the A-to-G mutation (Pu19A18G), a stable monomeric G-quadruplex can be formed in potassium solution, adopting a parallel-stranded structure as shown by NMR. In the wild-type 3’-end NHE sequences, the formation of a monomeric G-quadruplex in potassium can only be observed in a 20-mer Pu20 sequence with additional 3’ G20; the assignment of Pu20 G-quadruplex using site-specific 15N-G-labeled DNA sequences indicated a novel parallel-stranded G-quadruplex structure using G20 instead of A18 in the 3’-end-tetrad. The Pu20 G-quadruplex appears to have high thermo-stability as shown by CD studies; however, its specific formation can only be achieved at low potassium salt. GSA1129 can bind Pu20 and increase the 3′-end G-quadruplex stability by nearly 20 degrees. This study highlighted the dynamic nature of the 3′-end PDGFR-β G-quadruplex and the importance of identifying the proper sequence for the biologically relevant G-quadruplex structure formation. Significantly, the dynamic nature of the 3′-end G-quadruplex may make it an attractive target for drug regulation. Citation Format: Buket Onel, Prashansa Agrawal, Megan Carver, Robert Brown, Laurence Hurley, Danzhou Yang. A novel G-quadruplex formed in the PDGFR-β promoter that is selectively targeted by a small molecule to repress transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3089.

Abstract 2130: The novel G-quadruplex with hairpin loop formed immediately upstream of the human BCL-2 P1 promoter represses BCL-2 transcription

The abnormal overexpression of the BCL-2 protein is linked to a large number of cancers. Elevated levels of BCL-2 are found to promote resistance to chemotherapy and radiation. Therefore, BCL-2 is considered to be an attractive target for cancer therapeutics. We have previously identified a 39-base-pair GC-rich sequence located in the major P1 promoter region of the human BCL-2 gene, whose G-rich strand (Pu39) can form two interchangeable G-quadruplex (G4) structures, a hybrid-type G-quadruplex and a parallel G-quadruplex with a 13-nt middle loop. However, in our functional study of the BCL-2 P1 promoter activity using a luciferase reporter system containing this region in tumor cells, we found that the construct with a complete G-quadruplex-knock-out Pu39 mutant is still affected by G4-interactive compounds. In this study we report a new 29-mer G-quadruplex-forming sequence, P1G4, just downstream of Pu39 and immediately upstream of the human BCL-2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. Using NMR spectroscopy, the P1G4 G-quadruplex is shown to be a novel dynamic equilibrium of two parallel structures, one regular G4 with two 1-nt loops and a 12-nt middle loop and another broken-strand G4 with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) structure in the long loop. Using dimethyl sulfate (DMS) footprinting assays, we show that G-quadruplexes can readily form in the P1G4 sequence under physiological salt conditions, and that P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions. To further understand the respective function of Pu39 and P1G4, we created luciferase-constructs driven by the BCL-2 promoter sequence including both Pu39 and P1G4, and examined their functions by knocking out either or both of them. We found P1G4 independently and predominantly represses BCL-2 transcription. Unlike the 1245 parallel G-quadruplex formed in Pu39, P1G4 structure is easily compromised by modifications in the hairpin loop, indicating the loop is crucial for the maintenance of this dynamic structure. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures, and may provide a unique target for small molecules to modulate BCL-2 gene transcription. The presence of P1G4 and Pu39 G-quadruplexes in adjacent regions suggests a mechanism for precise regulation of BCL-2 gene transcription. Citation Format: Buket Onel, Guanhui Wu, Megan Carver, Daria Timonina, Marti Larriva, Danzhou Yang. The novel G-quadruplex with hairpin loop formed immediately upstream of the human BCL-2 P1 promoter represses BCL-2 transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2130.

Abstract 4514: NMR study of XR5944 interaction with diverse estrogen response elements (EREs)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL ER has emerged as one of the most effective targets for breast cancer therapy since the discovery of the estrogen receptor (ER) as the modulator of estrogen action. However, the current ER-targeting therapies have a main limitation related to resistance. Our previous studies have shown that XR5944, a DNA bis-intercalator is capable of inhibiting the ER activity by its ability to specifically block the binding of ER to its consensus EREs. An DNA intercalator that targets ERE may be a useful therapeutic agent that can overcome the resistance to existing endocrine therapies. To understand the mechanism of the inhibition of ER-ERE interactions by XR5944, we have tested the specificity of XR5944 binding to consensus EREs (AGGTCAnnnTGACCT) with different tri-nucleotide spacer sequences as well as natural EREs using 1H NMR titration studies. Our results suggested that the ERE spacers play a significant role in determining the binding characteristics of the complex. XR5944 binds the ERE sequences in a slow-exchange binding mode on the NMR time-scale. The binding stoichiometry of XR5944 with ERE sequences was shown to be 2:1. Of those tested, the CGG/GCC and AGG/CCT spacers showed the best binding, while the CGT/ACG and TTT/AAA spacers showed the worst binding with XR5944. Results of luciferase reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of reporters containing ERE-CGG than those containing ERE-TTT, consistent with our NMR results. Natural ERE sequences, including those located in the promoter of ER target genes TFF1, GREB1, Cathepsin D, Lactoferrin, and TGF-α, also appear to bind to XR5944 with different specificities as evident by differences in the 1H NMR spectra of their drug-DNA complexes. Thus, the affinity and specificity of the XR5944-ERE complex is influenced both by the nucleotide sequence of the half-sties and the tri-nucleotide spacer. Of the natural EREs tested, XR5944 appears to bind with the highest specificity to the ERE sequence of TFF1; hence we carried out further NMR studies of the binding of XR5944 to the TFF1 ERE sequence. Using site-specific labeling at each base, we were able to assign both the imino and H8 protons of the free TFF1 DNA and its drug complex. These assignments provide important information on the XR5944 binding sites with TFF1 ERE. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4514. doi:10.1158/1538-7445.AM2011-4514

Abstract LB-200: Solution structure of c-Myc G-quadruplex complex with a quindoline compound

Overexpression of the c-Myc oncogene is associated with a broad spectrum of human cancers. Targeting transcriptional control of c-Myc represents an attractive means for cancer molecular therapeutics. A highly conserved nuclear hypersensitivity element III1 (NHE III1) of the c-Myc promoter controls 80–90% of the c-Myc transcription. It can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-Myc transcriptional silencing. Small molecules that stabilize the c-Myc G-quadruplex have been shown to suppress c-Myc transcription and are anti-tumorigenic. We have determined the NMR structure of a 2:1 complex of the c-Myc quadruplex with a quindoline compound. This is the first molecular structure of a drug complex with a natural oncogene promoter quadruplex. The solution structure of this 2:1 quindoline-G-quadruplex complex shows unexpected features, including the drug-induced reorientation of the flanking sequences to form a new binding pocket. While both 3′ and 5′ complexes show overall similar features, there are identifiable differences that emphasize the importance of both stacking and electronic interactions. We identified the importance of the two flanking bases as well as the shape of the ligand in determining drug-binding specificity. The structural information will be crucial for structure-based rational drug design of molecules that specifically target the c-Myc silencer element. It also provides important implications for rational design of drugs that bind to unimolecular parallel G-quadruplexes commonly found in promoter elements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2011-LB-200

Abstract 4616: XR5944 interacts with diverse EREs differently, which may influence ER inhibition by XR5944

Since the discovery of the estrogen receptor (ER) as the modulator of estrogen action, ER has evolved to be one of the most effective targets for breast cancer therapy. However, the current ER-targeting therapies have a main limitation related to resistance. Our previous studies have shown that XR5944, a DNA bis-intercalator with potent anticancer activity, is capable of inhibiting the ER activity by its ability to specifically block the binding of ER to its consensus DNA sequence ERE compared to other transcription factor-target DNA interactions. This novel mechanism of ER inhibition by a DNA bis-intercalator may be useful in overcoming resistance to current antiestrogens. The consensus ERE is an inverted repeat comprised of two ERE half-sites separated by three bases: AGGTCAnnnTGACCT where nnn is known as the tri-nucleotide spacer. ER can also bind to non-consensus EREs, i.e. natural EREs that differ from the consensus ERE by one or more base pairs but nevertheless serve as potent transactivation sites for estrogen target genes Therefore, to further understand the mechanism and specificity of the inhibition of ER by XR5944, we tested the specificity of XR5944 to block interactions with consensus and non-consensus ERE sequences by 1D 1H NMR titration studies. Our NMR studies of XR5944-DNA interactions suggested that the consensus ERE spacer plays a significant role in determining the binding characteristics of the complex. Of those tested, the CGG/GCC and AGG/CCT spacers showed the best binding, while the CGT/ACG and TTT/AAA spacers showed the worst binding with XR5944. The binding stoichiometry of XR5944 with ERE sequences appears to be 2:1, which explains why the spacer region can affect the drug-DNA binding. To validate our NMR results, we have also conducted functional studies by using reporter constructs containing EREs with tri-nucleotide spacers CGG and TTT. Results of luciferase reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of reporters containing ERE-CGG than those containing ERE-TTT, consistent with our NMR results. Together with our NMR studies, these reporter gene experiments indicate that the tri-nucleotide spacer plays a significant role in the binding characteristics and functional consequence of XR5944 interactions with the consensus ERE. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4616.