Pharmacology & Therapeutics 85 (2000) 141–158 0163-7258/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0163-7258(99)00068-6 Associate editor: D. Shugar G-quadruplexes as targets for drug design Laurence H. Hurley a, *, Richard T. Wheelhouse b , Daekyu Sun c , Sean M. Kerwin d , Miguel Salazar d , Oleg Yu. Fedoroff a , Frank X. Han a , Haiyong Han a , Elzbieta Izbicka c , Daniel D. Von Hoff c a Drug Dynamics Institute, The University of Texas, Austin, TX 78712, USA b School of Pharmacy, The University of Bradford, Bradford, West Yorkshire BD7 1DP, UK c Institute for Drug Development, 14960 Omicron Drive, San Antonio, TX 78245, USA d Division of Medicinal Chemistry, The University of Texas, Austin, TX 78712, USA Abstract G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (an- thraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex–ligand complex and its effect on the dy- namics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the exper- iments that relate the biological effects to their interactions with G-quadruplexes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: G-quadruplex; Porphyrin; Telomere; Telomerase; Anthraquinone; Perylene Abbreviations: AZT, 3'-azido-3'-deoxythymidine; Mut-TRr2, 5'-TACAGATAGTTAGACTTAACGTTA-3'; NOESY, nuclear Overhauser effect correla- tion spectroscopy; ODN, control-scrambled phosphorothioate oligonucleotide dTGTGAG; PAGE, polyacrylamide gel electrophoresis; PIPER, N,N'-bis [2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide; TAG6, antisense phosphorothioate oligonucleotide to telomeric RNA dTTAGGG; TBP, telomere-binding protein; TE buffer, 0.01 M Tris-EDTA buffer; TMPyP2, positional isomeric cationic porphyrin 5,10,15,20-tetra-(N-methyl-2-pyridyl)por- phine; TMPyP4, cationic porphyrin compound 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine; TRr2, 5'-TACAGATAG(TTAGGG) 2 . Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 2. G-quadruplex as a target for inhibition of telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 2.1. Inhibition of telomerase by 2,6-diamidoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . 143 2.2. NMR titration of 2,6-diamidoanthraquinone (Compound 1) with parallel four-stranded G-quadruplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 2.3. Time-course inhibition of telomerase by 2,6-diamidoanthraquinone (Compound 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 3. Interaction of a perylene (N,N'-bis[2-(1-piperidino)ethyl]-3,4,9,10- perylenetetracarboxylic diimide) with G-quadruplex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 3.1. NMR-based structure of a perylene–G-quadruplex complex . . . . . . . . . . . . . . . . . . 146 3.2. Accelerated assembly of G-quadruplex by N,N'-bis[2-(1-piperidino)ethyl]- 3,4,9,10-perylenetetracarboxylic diimide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 3.2.1. Potassium ions and N,N'-bis[2-(1-piperidino)ethyl]-3,4,9,10- perylenetetracarboxylic diimide each promote the formation of different 5'-TACAGATAG(TTAGGG) 2 G-quadruplexes . . . . . . . . . . . . . . 148 3.2.2. The second-order dimerization reaction of 5'- TACAGATAG(TTAGGG) 2 becomes a first-order reaction in the * Corresponding author. Tel.: 512-471-4842; fax: 512-471-2746. E-mail address: dg-dna@mail.utexas.edu (L.H. Hurley)