Windsor, J. B., Thomas, C., Hurley, L., Roux, S. J., & Lloyd, A. M. (2002). Automated colorimetric screen for apyrase inhibitors. BioTechniques, 33(5), 1024-1030.
PMID: 12449379;Abstract:
Apyrases are enzymes that efficiently hydrolyze ATP and ADP and may operate both inside and outside the cell. Although apyrases are important to a variety of cellular mechanisms and uses in industry, there are no available apyrase-specific inhibitors. Colorimetric assays based on the Fiske-Subbarow method for measuring inorganic phosphate are able to detect the release of inorganic phosphate, from ATP and other nucleotides. We found that this type of assay could be automated and used to screen, for apyrase-inhibiting compounds by assaying for a reduction in released phosphate in the presence of potential inhibitors. The automation of this assay allowed for the successful screening of a commercially available compound library. Several low molecular weight compounds were identified that, when used at micromolar concentrations, effectively inhibited apyrase activity.
Bose, D. S., Thompson, A. S., Ching, J., Hartley, J. A., Berardini, M. D., Jenkins, T. C., Neidle, S., Hurley, L. H., & Thurston, D. E. (1992). Rational design of a highly efficient irreversible DNA interstrand cross-linking agent based on the pyrrolobenzodiazepine ring system. Journal of the American Chemical Society, 114(12), 4939-4941.
Fletcher, T. M., Sun, D., Salazar, M., & Hurley, L. H. (1998). Effect of DNA secondary structure on human telomerase activity. Biochemistry, 37(16), 5536-5541.
PMID: 9548937;Abstract:
Telomeres are specialized DNA-protein complexes located at the chromosome ends. The guanine-rich telomeric sequences have the ability to form G-quadruplex structures under physiological ionic conditions in vitro. Human telomeres are maintained through addition of TTAGGG repeats by the enzyme telomerase. To determine a correlation between DNA secondary structure and human telomerase, telomerase activity in the presence of various metal cations was monitored. Telomerase synthesized a larger proportion of products corresponding to four, five, eight, and nine full repeats of TTAGGG in 100 mM K+ and to a lesser extent in 100 mM Na+ when a d(TTAGGG)3 input primer was used. A more even product distribution was observed when the reaction mixture contained no added Na+ or K+. Increasing concentrations of Cs+ resulted in a loss of processivity but not in the distinct manner observed in K+. When the input primer contained 7-deaza-dG, the product distribution resembled that of reactions without K+ even in the presence of 100 mM K+. Native polyacrylamide gel electrophoresis indicated that d(TTAGGG)4, d(TTAGGG)5, d(TTAGGG)8, and d(TTAGGG)9 formed compact structures in the presence of K+. The oligonucleotide d(TTAGGG)4 had a UV spectrum characteristic of that of the G-quadruplex only in the presence of K+ and Na+. A reasonable explanation for these results is that four, five, eight, and nine repeats of TTAGGG form DNA secondary structures which promote dissociation of the primer from telomerase. This suggests that telomerase activity in cells can be modulated by the secondary structure of the DNA template. These findings are of probable relevance to the concept of telomerase as a therapeutic target for drug design.
Sun, D., & Hurley, L. H. (2009). The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: Implications for drug targeting and control of gene expression. Journal of Medicinal Chemistry, 52(9), 2863-2874.
PMID: 19385599;PMCID: PMC2757002;Abstract:
The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc. © 2009 American Chemical Society.
Brooks, T. A., Kendrick, S., & Hurley, L. (2010). Making sense of G-quadruplex and i-motif functions in oncogene promoters. FEBS Journal, 277(17), 3459-3469.
PMID: 20670278;PMCID: PMC2971675;Abstract:
The presence and biological importance of DNA secondary structures in eukaryotic promoters are becoming increasingly recognized among chemists and biologists as bioinformatics in vitro and in vivo evidence for these structures in the c-Myc, c-Kit, KRAS, PDGF-A, hTERT, Rb, RET and Hif-1 promoters accumulates. Nevertheless, the evidence remains largely circumstantial. This minireview differs from previous ones in that here we examine the diversity of G-quadruplex and i-motif structures in promoter elements and attempt to categorize the different types of arrangements in which they are found. For the c-Myc G-quadruplex and Bcl-2 i-motif, we summarize recent biological and structural studies. © 2010 FEBS.
