Current Medicinal Chemistry - Anti-Cancer Agents, Vol. 5, No. 4, 2005
Contents
The
Chemistry and Biology of Small Molecules Acting on Nucleic Acids
Guest
Editors: P.B. Arimondo, T. Garestier, J.-L. Mergny
Dedicated
to the Memory of Professor Claude Helene
Editorial
Pp.315-317
Therese Garestier, Paola B. Arimondo, Jean-Louis Mergny
Triplex-Forming
Oligonucleotides as Potential Tools for Modulation of Gene Expression
Pp.319-326
Faye A. Rogers, Janice
A. Lloyd and Peter M. Glazer
Recognition of Triple
Helical Nucleic Acids by Aminoglycosides Pp.327-338
H. Xi and D.P. Arya
Competition Dialysis:
An Assay to Measure the Structural Selectivity of Drug-Nucleic Acid
Interactions Pp.339-352
Jonathan B. Chaires
Camptothecin: Roles of
the D and E Rings in Binding to the Topoisomerase I-DNA Covalent Binary Complex
Pp.353-362
Sidney M. Hecht
Etoposide,
Topoisomerase II and Cancer Pp.363-372
E.L. Baldwin and N.
Osheroff
Programmable DNA
Binding Oligomers for Control of Transcription Pp.373-387
Peter B. Dervan, Raymond
M. Doss and Michael A. Marques
Dications That Target
the DNA Minor Groove: Compound Design and Preparation, DNA Interactions,
Cellular Distribution and Biological Activity Pp.389-408
W. David Wilson, Binh Nguyen, Farial A. Tanious, Amanda Mathis, James Edwin Hall, Chad E. Stephens and David W. Boykin
Target Practice:
Aiming at Satellite Repeats with DNA Minor Groove Binders Pp.409-420
Guillaume Susbielle, Roxane Blattes, Vanessa Brevet, Caroline Monod and Emmanuel Kas
Interfacial Inhibitors
of Protein-Nucleic Acid Interactions Pp.421-429
Yves Pommier and Christophe Marchand
Abstracts
[Back
to top] Editorial
Therese Garestier,
Paola B. Arimondo, Jean-Louis Mergny
This issue is dedicated to Prof. Claude Helene, who was a pioneer in the field of Nucleic Acids recognition by natural and artificial small molecules. He passed on Feb 11, 2003, three days after the “International Symposium on Nucleic Acids: From Bases to Genomics” organized by his coworkers for his 65th birthday. For many of us he inspired our work and showed the way to a vigorous and rigorous research.
The research activity of Claude Hélène was entirely dedicated to the recognition of nucleic acids in all its aspects, his aim being to find the key to the « artificial modulation » of gene expression by nucleic acids ligands. He started, in a program launched in 1967 at the Centre de Biophysique Moléculaire in Orléans, by studying the interactions between aromatic amino acids and nucleosides stacked in ice. These mimic oligonucleotides, the chemical synthesis of which was not readily available at that time [1]. Today, studies of unusual DNA structures are the main research interest of the Laboratoire de Biophysique of the Muséum National d’Histoire Naturelle in Paris, of which he was head since 1976. All along this path, many results opened fruitful perspectives and brought original concepts, and sometime new methodologies.
The first studies indicated that aromatic amino acids are able to intercalate in single-stranded nucleic acids and that their lateral chains could form specific H-bonds with nucleic acids bases [2]. These predictions were confirmed by crystallographic studies of specific nucleic acids-proteins complexes [3].
When Claude Hélène became head of the Laboratoire de Biophysique in Paris, he oriented all his research efforts to the specific recognition of nucleic acids. DNA depurination, a phenomenon that occurs very frequently in nature, allowed the insertion of specific aromatic amino acids in the abasic site. The tripeptide Lys-Trp-Lys was taken as a model and shown to be able to recognize selectively an apurinic site and to cleave the nucleic acid chain at this position [4]. The same tripeptide was able to photosensitize the cleavage of thymine dimers induced by UV radiation [5]. This tripeptide was therefore considered as the smallest enzyme known.
In 1980 a new challenge was launched with the design of molecules recognizing specifically short sequences of messenger RNAs, in order to inhibit protein synthesis. Interestingly, few years earlier it had been shown that a modified oligonucleotide was able to inhibit specifically the protein synthesis of the Rous sarcoma virus in cells [6, 7]. It was the birth of oligonucleotides directed against mRNAs, called antisense oligonucleotides. However, the synthesis of oligonucleotides was still not an easy task for chemists as automated DNA synthesis was not yet available. The original idea of Claude Hélène was to stabilize the formed complex by covalent linkage of an intercalating agent at the 3’ end of the oligonucleotide. The chosen agent was 2-methoxy-6-chloro-9-aminoacridine mainly because of its fluorescent properties. Spectroscopic studies, 2D NMR and circular dichroism demonstrated that the acridine was intercalated between the bases of the short double helix locally formed by the oligonucleotide [8]. A selective inhibition of protein synthesis was observed in vitro and in Xenopus oocytes. This inhibition was not due to a physical arrest of the polymerase but rather to the induction of the RNase H cleavage of the hybrid DNA/RNA complex [9]. The application of this approach to cellular systems showed the selective inhibition of three different viruses: type A Influenza [10], SV40 [11] and HIV viruses [12]. The acridine-oligonucleotide conjugates were able to selectively kill trypanosomes, bearing at the 5’ end of their mRNAs a common 39-nt target sequence [13]. To induce an irreversible damage on the target sequence, agents such as orthophenanthroline and psoralen replaced acridine in the conjugates [14]. In 1987, an important discovery opened a new field to the community of nucleic acids researchers. Simultaneously to Claude Hélène’s group, Peter Dervan’s laboratory reached the same conclusion: a short pyrimidine oligonucleotide could bind in the major groove of DNA specifically to a oligopurine sequence forming a local triple helix [15, 16]. In both cases, the triplex-forming oligonucleotide (TFO) was linked at its 5’ end to a photo or chemically active agent, causing a sequence-specific double-stranded break, which also allowed to evidence triplex formation. These TFO were demonstrated to bind to a single specific target on the DNA [17], and to thus inhibit transcription in vitro, in cell nuclei, and in cells (reviewed in [18]). The stability of the triplex is fundamental for its biological activity, many chemical modifications of the bases, sugars or backbone of the TFOs were explored in order to increase it. In the same aim of triplex stabilization, intercalating agents with crescent shape and positively charges were demonstrated to intercalate and stabilize triple helices, and, later, new ones were then rationally designed to intercalate in triple-helical structures [19, 20]. In this issue Xi and Arya describe how the rational design of triplex-specific ligands has evolved and it is now possible to achieve a triplex-selective groove recognition with aminoglycosides.
Triple helices can be formed on single-stranded oligopurine target sequences by a composite oligonucleotide forming a “clamp”. Attachment of a psoralen derivative at the 5’ end of the TFO allowed to photoinduce a covalent linkage to the target sequence. The psoralen moiety becomes covalently linked to all three portions of the triplex making the clamp irreversible [21]. The attachment of psoralen to the 5’ end of the TFO creates, upon photoactivation, irreversible damages on the targeted nucleic acids sequence [22]. This property and the conformational changes induced by the triple-helical structure alone are extensively reviewed by Rogers et al. showing the use of triplexes as tools to modulate gene expression.
Another strategy to irreversibly modify the DNA target consists in recruiting a cellular enzyme, which cleaves transiently DNA, such as topoisomerases I and II. To do so topoisomerase poisons, such as camptothecin or etoposide, were covalently linked to the TFO that, upon binding to its target, captures the enzyme specifically at the triplex site [23]. The understanding of the chemical structure of camptothecin that contributes to the inhibition of topoisomerase I is the topic of Sidney Hecht’s review in this issue. Baldwin and Osheroff resume the drug discovery of etoposide and discuss its mechanism of action on topoisomerase II and new implications to how design a better anticancer agent.
After three why not four! Quadruplexes structures became rapidly a new interest for Claude Hélène’s laboratory. At the end of most chromosomes are telomeres, constituted of a single-stranded overhang rich in guanines that can fold to form quadruplex structures. Interestingly, the complementary strand, C-rich, can form the i-motif, which is also a 4-stranded structure [24]. The dysfunctions of some human diseases, such as cancer and neurodegenerative diseases, seem related to unusual DNA structures. This led to the search of ligands that could bind specifically to these structures in order to fight these diseases [25].Therefore, new methods are developed to study the interaction between drugs and several DNA structures. In this context, Jonathan Chaires adapted the competition dialysis method to compare the selectivity of drugs (123 compounds up to now) for different nucleic acids structures, including single-strands, duplexes, triplexes and quadruplexes.
Not only the major groove of DNA can host sequence-specific ligands, but also the minor groove, as showed by Peter Dervan’s group upon use of small molecules composed of N-methylpyrrole and N-methylimidazole units. On the recognition of the minor groove are based also new antiparasite therapies that use heterocyclic diamidines binding to AT-rich sequences predominating in these genomes. Wilson et al. show how, once the target and the mechanism of action of these diamidines identified, rational design allows chemical synthesis of new improved drugs, with increased groove-binding and specificity.
A beautiful demonstration of the use of minor groove binders is presented in this issue by Susbielle et al. Polyamides and diamidines are used both as probes and as tools to modulate gene expression in a complex organism such as Drosophila melanogaster.
In the lapse of a scientific carrier, the understanding of the interactions between nucleic acids and their ligands, a field in which Claude Hélène was always one of the central players, went from the elementary units (nucleosides and amino acids), to complexed nucleic acids structures and small peptides. The study of the interactions between these structures and their ligands are used to regulate the biological functions in cells and now in animals. Hopefully, the next step will be the extensive use in diagnostic and therapeutics. In this context, Pommier and Marchand propose an exciting new concept to describe the targeting of protein-nucleic acids interactions by drugs.
References
[1] Montenay-Garestier, T.; Helene, C. Nature 1968, 217(131), 844-5.
[2] Helene, C.; Lancelot, G. Prog. Biophys. Mol. Biol. 1982, 39(1), 1-68.
[3] Werner, M. H.; Gronenborn, A. M.; Clore, G. M. Science 1996, 271(5250), 778-84.
[4] Behmoaras, T.; Toulme, J. J.; Helene, C. Proc. Natl. Acad. Sci. USA 1981, 78(2), 926-30.
[5] Behmoaras, T.; Toulme, J. J.; Hélène, C. Nature 1981, 292(5826), 858-9.
[6] Zamecnik, P. C.; Stephenson, M. L. Proc. Natl. Acad. Sci. USA 1978, 75(1), 280-4.
[7] Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci. USA 1978, 75(1), 285-8.
[8] Asseline, U.; Delarue, M.; Lancelot, G.; Toulme, F.; Thuong, N. T.; Montenay-Garestier, T.; Helene, C. Proc. Natl. Acad. Sci. USA 1984, 81(11), 3297-301.
[9] Cazenave, C.; Chevrier, M.; Nguyen, T. T.; Helene, C. Nucleic Acids Res. 1987, 15(24), 10507-21.
[10] Zerial, A.; Thuong, N. T.; Helene, C. Nucleic Acids Res. 1987, 15(23), 9909-19.
[11] Birg, F.; Praseuth, D.; Zerial, A.; Thuong, N. T.; Asseline, U.; Le Doan, T.; Helene, C. Nucleic Acids Res. 1990, 18(10), 2901-8.
[12] Bordier, B.; Helene, C.; Barr, P. J.; Litvak, S.; Sarih-Cottin, L. Nucleic Acids Res. 1992, 20(22), 5999-6006.
[13] Verspieren, P.; Cornelissen, A. W.; Thuong, N. T.; Helene, C.; Toulme, J. J. Gene 1987, 61(3), 307-15.
[14] Francois, J. C.; Saison-Behmoaras, T.; Barbier, C.; Chassignol, M.; Thuong, N. T.; Helene, C. Proc. Natl. Acad. Sci. USA 1989, 86(24), 9702-6.
[15] Le Doan, T.; Perrouault, L.; Praseuth, D.; Habhoub, N.; Decout, J. L.; Thuong, N. T.; Lhomme, J.; Hélène, C. Nucleic Acids Res. 1987, 15(19), 7749-60.
[16] Moser, H. E.; Dervan, P. B. Science 1987, 238(4827), 645-50.
[17] Perrouault, L.; Asseline, U.; Rivalle, C.; Thuong, N. T.; Bisagni, E.; Giovannangeli, C.; Le Doan, T.; Hélène, C. Nature 1990, 344(6264), 358-60.
[18] Giovannangeli, C.; Helene, C. Curr. Opin. Mol. Ther. 2000, 2(3), 288-96.
[19] Mergny, J. L.; Duval-Valentin, G.; Nguyen, C. H.; Perrouault, L.; Faucon, B.; Rougee, M.; Montenay-Garestier, T.; Bisagni, E.; Helene, C. Science 1992, 256(5064), 1681-4.
[20] Escude, C.; Nguyen, C. H.; Kukreti, S.; Janin, Y.; Sun, J. S.; Bisagni, E.; Garestier, T.; Hélène, C. Proc. Natl. Acad. Sci. USA 1998, 95(7), 3591-6.
[21] Giovannangeli, C.; Thuong, N. T.; Hélène, C. Proc. Natl. Acad. Sci. USA 1993, 90(21), 10013-7.
[22] Takasugi, M.; Guendouz, A.; Chassignol, M.; Decout, J. L.; Lhomme, J.; Thuong, N. T.; Hélène, C. Proc. Natl. Acad. Sci. USA 1991, 88(13), 5602-6.
[23] Arimondo, P. B.; Bailly, C.; Boutorine, A.; Sun, J. S.; Garestier, T.; Helene, C. C. R. Acad. Sci. III 1999, 322(9), 785-90.
[24] Leroy, J. L.; Gueron, M.; Mergny, J. L.; Helene, C. Nucleic Acids Res. 1994, 22(9), 1600-6.
[25] Mergny, J. L.; Helene, C. Nat. Med. 1998, 4(12), 1366-7.
[Back
to top] Triplex-Forming Oligonucleotides as Potential Tools
for Modulation of Gene Expression
Faye A. Rogers, Janice
A. Lloyd and Peter M. Glazer
Triplex–forming oligonucleotides (TFOs) bind in the major groove of duplex DNA at polypurine/ polypyrimidine stretches in a sequence-specific manner. The binding specificity of TFOs makes them potential candidates for use in directed genome modification. A number of studies have shown that TFOs can introduce permanent changes in a target sequence by stimulating a cell’s inherent repair pathways. TFOs have also been demonstrated to inhibit gene expression providing a possible role for these compounds in cancer therapy. This review summarizes the dual roles of TFOs for use in delivering DNA reactive compounds to a specific site in the genome or for introducing permanent changes in the target sequence through the introduction of an altered helical structure. In addition to compiling the ways in which TFOs have been successfully utilized, this review will explore conflicting reports of TFO bioactivity focusing on the variables which affect the efficacy in vitro of TFO mediated genomic modification which in turn may represent the obstacles encountered using TFOs to modulate gene expression in vivo.
[Back
to top] Recognition of Triple Helical Nucleic Acids by
Aminoglycosides
H. Xi and D.P. Arya
Aminoglycosides, traditional RNA binders, were found to be a new class of triple helical nucleic acid-stabilizing ligands. Neomycin, of all the aminoglycosides, has shown the most significant effects in stabilizing DNA, RNA, and hybrid triple helices. When compared with minor groove binders or intercalators, neomycin excels at triple helical stabilization in most cases. Molecular modeling studies suggest that neomycin reaches into the larger Watson-Hoogsteen groove. The charge and shape complementarity are the key factors in neomycin-triplex recognition. By conjugating neomycin with intercalators such as BQQ (a potent triple helix intercalating agent designed by Hélène), we have progressed in developing more potent triple helix stabilizing ligands. The design of such dual or even triple recognition ligands opens a new paradigm for recognition of triple helix nucleic acids. The article herein presents studies of neomycin as the first molecule that can selectively stabilize nucleic acid triplex structures. These studies are supported by our recent discovery that neomycin prefers to bind to A-like conformations, of which triple helix structures are known to display some characteristics. These findings will contribute to the development of a new series of triplex-specific ligands, and may contribute to either antisense or antigene therapies.
[Back
to top] Competition Dialysis: An Assay to Measure the
Structural Selectivity of Drug-Nucleic Acid Interactions
Jonathan B. Chaires
Competition dialysis is a powerful new tool for the discovery of ligands that bind to nucleic acids with structural- or sequence-selectivity. The method is based on firm thermodynamic principles and is simple to implement. In the competition dialysis experiment, an array of nucleic acid structures and sequences is dialyzed against a common test ligand solution. After equilibration, the amount of ligand bound to each structure or sequence is determined spectrophotometrically. Since all structures and sequences are in equilibrium with the same free ligand concentration, the amount bound is directly proportional to the ligand binding affinity. Competition dialysis thus provides a direct and quantitative measure of selectivity, and unambiguously identifies which of the structures or sequences within the sample array that are preferred by a particular ligand. Following the introduction of the method, competition dialysis has been used worldwide to probe a variety of ligand-nucleic acid interactions. This contribution will focus on new analytical approaches for extracting information from the database that resulted from the first-generation competition dialysis assay, in which binding data was gathered for the interaction of 126 compounds with 13 different structures and sequences. Such global analyses allow identification of compounds with unique types of binding selectivity.
[Back
to top] Camptothecin: Roles of the D and E Rings in
Binding to the Topoisomerase I-DNA Covalent Binary Complex
Sidney
M. Hecht
The alkaloid camptothecin is the prototypical DNA topoisomerase I poison. This core structure has formed the basis for two marketed antitumor agents and numerous clinical candidates, and has been the focus of many synthetic and medicinal chemistry studies. Recent reports have furthered our understanding of the roles played by the D and E rings of camptothecin in stabilization of the enzyme-DNA-camptothecin ternary complex. Important parameters for further study and optimization include the facility of E-ring lactone hydrolysis and the prospects for replacing the E ring with more stable structures, the role of the 14-CH group in binary complex binding, and the effect of ternary complex dynamics on the expression of cytotoxicity by the camptothecins.
[Back
to top] Etoposide, Topoisomerase II and Cancer
E.L. Baldwin and N. Osheroff
Etoposide is an important chemotherapeutic agent that is used to treat a wide spectrum of human cancers. It has been in clinical use for more than two decades and remains one of the most highly prescribed anticancer drugs in the world. The primary cytotoxic target for etoposide is topoisomerase II. This ubiquitous enzyme regulates DNA under- and overwinding, and removes knots and tangles from the genome by generating transient double-stranded breaks in the double helix. Etoposide kills cells by stabilizing a covalent enzyme-cleaved DNA complex (known as the cleavage complex) that is a transient intermediate in the catalytic cycle of topoisomerase II. The accumulation of cleavage complexes in treated cells leads to the generation of permanent DNA strand breaks, which trigger recombination/repair pathways, mutagenesis, and chromosomal translocations. If these breaks overwhelm the cell, they can initiate death pathways. Thus, etoposide converts topoisomerase II from an essential enzyme to a potent cellular toxin that fragments the genome. Although the topoisomerase II-DNA cleavage complex is an important target for cancer chemotherapy, there also is evidence that topoisomerase II-mediated DNA strand breaks induced by etoposide and other agents can trigger chromosomal translocations that lead to specific types of leukemia. Given the central role of topoisomerase II in both the cure and initiation of human cancers, it is imperative to further understand the mechanism by which the enzyme cleaves and rejoins the double helix and the process by which etoposide and other anticancer drugs alter topoisomerase II function.
[Back
to top] Programmable DNA Binding Oligomers for Control
of Transcription
Peter B. Dervan, Raymond M. Doss and Michael A. Marques
Mapping and sequencing the genetic blueprint in human, mice, yeast and other model organisms has created challenges and opportunities for chemistry, biology and human medicine. An understanding of the function of each of the ~ 25, 000 genes in humans, and the biological circuitry that controls these genes will be driven in part by new technologies from the world of chemistry. Many cellular events that lead to cancer and the progression of human disease represent aberrant gene expression. Small molecules that can be programmed to mimic transcription factors and bind a large repertoire of DNA sequences in the human genome would be useful tools in biology and potentially in human medicine. Polyamides are synthetic oligomers programmed to read the DNA double helix. They are cell permeable, bind chromatin and have been shown to downregulate endogenous genes in cell culture.
[Back
to top] Dications That Target the DNA Minor Groove:
Compound Design and Preparation, DNA Interactions, Cellular Distribution and
Biological Activity
W. David Wilson, Binh Nguyen, Farial A. Tanious, Amanda Mathis, James Edwin Hall, Chad E. Stephens and David W. Boykin
Fluorescence microscopy of trypanosomes from drug treated mice shows that biologically active heterocyclic diamidines that target the DNA minor groove bind rapidly and specifically to parasite kinetoplast DNA (k-DNA). The observation that the kinetoplast is destroyed, generally within 24 hours, after drug treatment is very important for understanding the biological mechanism, and suggests that the diamidines may be inhibiting some critical opening/closing step of circular k-DNA. Given the uncertainties in the biological mechanism, we have taken an empirical approach to generating a variety of synthetic compounds and DNA minor groove interactions for development of improved and new biological activities. Furamidine, DB75, is a diphenyl-diamidine that has the curvature to match the DNA minor groove as expected in the classical groove interaction model. Surprisingly, a linear diamidine with a nitrogen rich linker has significantly stronger binding than furamidine due to favorable linker and water-mediated DNA interactions. The water interaction is very dependant on compound structure since other linear compounds do not have similar interactions. Change of one phenyl of furamidine to a benzimidazole does not significantly enhance DNA binding but additional conversion of the furan to a thiophene (DB818) yields a compound with ten times stronger binding. Structural analysis shows that DB818 has a very favorable curvature for optimizing minor groove interactions. It is clear that there are many ways for compounds to bind to k-DNA and exert specific effects on kinetoplast replication and/or transcription that are required to obtain an active compound.
[Back
to top] Target Practice: Aiming at Satellite Repeats with DNA Minor Groove
Binders
Guillaume Susbielle, Roxane Blattes, Vanessa Brevet, Caroline Monod and Emmanuel Kas
Much progress has been made in recent years in developing small molecules that target the minor groove of DNA. Striking advances have led to the design of synthetic molecules that recognize specific DNA sequences with affinities comparable to those of eukaryotic transcription factors. This makes it feasible to modulate or inhibit DNA/protein interactions in vivo, a major step towards the development of general strategies of anti-gene therapy. Examples from anti-parasitic drugs also suggest that synthetic molecules can affect a variety of cellular functions crucial to cell viability by more generally targeting vast portions of genomes based on their biased base composition. This provides a rationale for developing approaches based on selective interactions with broad genomic targets such as satellite repeats that are associated with structural or architectural components of chromatin essential for cellular proliferation. Using examples drawn from the Drosophila melanogaster model system, we review here the use of synthetic polyamides or diamidines that bind the DNA minor groove and can be used as highly selective agents capable of interfering with specific protein/DNA interactions that occur in A+T-rich repeated sequences that constitute a significant portion of eukaryotic genomes. The satellite localization of cellular proteins that bind the minor groove of DNA via domains such as the AT hook motif is highly sensitive to these molecules. A major consequence of the competition between these proteins and their synthetic mimics is an alteration of the nuclear localization and function of proteins such as topoisomerase II, a major target of anti-cancer drugs.
[Back
to top] Interfacial Inhibitors of Protein-Nucleic Acid Interactions
Yves Pommier and Christophe Marchand
This essay develops the paradigm of “Interfacial Inhibitors” (Pommier and Cherfils, TiPS, 2005, 28: 136) for inhibitory drugs beside orthosteric (competitive or non-competitive) and allosteric inhibitors. Interfacial inhibitors bind with high selectivity to a binding site involving two or more macromolecules within macromolecular complexes undergoing conformational changes. Interfacial binding traps (generally reversibly) a transition state of the complex, resulting in kinetic inactivation. The exemplary case of interfacial inhibitor of protein-DNA interface is camptothecin and its clinical derivatives. We will also provide examples generalizing the interfacial inhibitor concept to inhibitors of topoisomerase II (anthracyclines, ellipticines, epipodophyllotoxins), gyrase (quinolones, ciprofloxacin, norfloxacin), RNA polymerases (a-amanitin and actinomycin D), and ribosomes (antibiotics such as streptomycin, hygromycin B, tetracycline, kirromycin, fusidic acid, thiostrepton, and possibly cycloheximide). We discuss the implications of the interfacial inhibitor concept for drug discovery.