Nucleic Acid Ligands Based on Carbohydrates

MEDIZINISCHE CHEMIE . MEDICINAL CHEMISTRY 248 C'HIMIA 50 (I 99() Nr. () (Jlllli) edizinisc edicinal Chemistry Chimia 50 (1996) 248-256 Watson-Crick pairing rules. Translation is © Neue Schweizerische Chemische Gesellschaft arrested by this complexation and the RNA ISSN 0009-4293 strand is then degraded by RNase H, an enzyme which recognizes DNA-RNA het- eroduplexes, leading to catalytic turnover Nucleic Acid Ligands Based on of the antisense agent. In a different approach, single-stranded oligonucleotides Carbohydrates are designed such as to form a local triple- helical structure with the third strand asso- ciated in the major groove of double- Jiirg Hunziker* stranded DNA. In this case, the sequence specificity is derived from selective base Abstract. Sequence-specific nucleic acid ligands are important tools in chemistry and triplet formation on the Hoogsteen face of molecular biology and are thought to possess a considerable pharmaceutical potential. purine bases (Fig. 2). Since all such oligo- An overview of the structurally and mechanistically diverse approaches in the field with nucleotide analogs are linear polymers an emphasis on carbohydrates will be presented. their affinity and specificity can be altered Carbohydrates are just beginning to emerge as a novel class of nucleic acid binding at will by changing chain length and base compounds. The detailed study of the factors affecting the site-selectivity of some sequence. recently discovered antitumor antibiotics, e.g. calicheamicin, has shed a new light on Several clinical trials are currently un- the role thatoligosaccharides may play in nucleic acid recognition. Understanding these der way and although there have been factors may aid in the rational design of novel nucleic acid ligands and their therapeutic some promising results, difficulties have application. been encountered in getting antisense oligonucleotides to work the way they were intended to [le][2]. Selective delivery to 1. Introduction dance of the protein sequence and individ- the target tissue and potentially toxic side ual nucleotides. In addition, their bio- effects such as decreased blood clotting Nucleic acids are attractive targets for availability is low due to the relative ease and inreased blood pressure are the main the development of novel therapeutics. with which they are degraded and the slow obstacles. An improved understanding of The sequence specific, high affinity com- transport across cell membranes. And they the fate of antisense oligonucleotides in plexation of single-stranded messenger- are difficult and rather expensive to pro- vivo in conjunction with the development RNA or the corresponding double-strand- duce. Oligonucleotides or rather syntheti- of novel, more specific analogs will be ed genomic DNA can, in principle, pre- cally derived analogs, on the other hand, necessary to successfully generate thera- vent the production of illness related gene are obvious candidates for this task [I]. In peutics based on this concept. products at the stage of translation or tran- the antisense strategy, single-stranded oli- scription, respectively (Fig. 1). The inher- gonucleotides are designed to pair to an 1.2. Small DNA-Binding Molecules ent advantage of this approach lies in the RNA target (sense strand) by forming a In view of these difficulties, numerous low abundance of a certain gene or the local duplex structure according to the academic and industrial research groups resulting messenger-RNA within an af- fected cell. This theoretically requires a much lower dosage of a potential thera- RNA~.rget: inhibition of translation peutic agent. However, it also necessitates very high affinities and selectivities. Nu- cleic acid ligands based on this concept would act as direct antagonists to the cel- lular proteins that control translation and transcription. 1.1. Antisense Agents Proteins themselves are poor candi- =====~>s DNA-target: inhibition of transcription antisense- or dates for these purposes. There is no gen- antigene agent eral code for the recognition of nucleic ~ acids by proteins, a one to one correspon- *\ *Correspondence: Dr. J. Hunziker Institut fUr Organische Chemie Universitat Bern Fig. 1. Schematic representation of a generalized antigene-concept. By complex formation with Freieslrasse 3 messenger-RNA or the corresponding DNA anti gene agents can shut down production of illness- CH-30 12 Bern related proteins at the stage of translation or transcription, respectively. MEDIZINISCHE CHEMIE . MEDICINAL CHEMISTRY 249 C'HIMIA 50 (lqq6l N,.. 6 (Jllnil are joined in an effort to evaluate alterna- tives to oligonucleotides, compounds of major groove major groove different structural types, with respect to their anti gene properties. Studies of the binding modes of such molecules might lead to new strategies for the sequence- A T G c specific binding of nucleic acids. The vast pool of natural products provides many small molecules that bind to nucleic acids, most of them of fungal or microbial origin. Unlike oligonucleotides, minor groove minor groove the majority of these small molecules interact with double-stranded nucleic acids Fig. 2. Substitution pattern of H-bonding donor and acceptor groups for adenine-thymine and via contacts within the minor groove. Two guanine-cytosine base pairs in the major and minor groove examples of such compounds are netropsin and distamycin (Fig. 3). These crescent- shaped polyamides composed of N-methylpyrrole amino acids bind to stretches of double-stranded DNA consisting of adenine/thymine base pairs. The sequence selectivity derives from H-bonding between the amide protons and the nitrogen and carbonyl oxygen of adenine and thymine on the floor of the minor groove. It was shown in NMR studies by Wemmer and coworkers that in the complex two distamycin molecules are aligned side by side in an anti parallel manner [3a]. Based on these findings, Dervan and coworkers Netropsin Distamycin Im-Py-Py"y-Py-Py-Py successfully altered the sequence specificity of these molecules to include guanine/ Fig. 3. Structures of the minor groove binding drugs netropsin and distamycin. By substituting N- cytosine base pairs by substitutingN-meth- methylimidazole for N-methylpyrrole carboxamides at selected positions of and covalently linking ylimidazole for N-methylpyrrole carbox- two individual molecules to a dimer Dervan and coworkers developed distamycin analogs, e.g .. Im- Py-Py-y-Py-Py-Py, with altered sequence specificity and higher affinity [3). amides at selected positions [3]. The affinity could be improved to subnanomolar levels by covalently linking the two indi- charged DNA and thus helps aligning the chromophore part then leads to double- vidual halfs of these dimers [3e] (Fig. 3). other parts of the molecu Ie. Alternati vel y, strand cleavage. In 1985, Nishida and co- certain DNA-ligands form dimeric (or workers reported the structure of the neo- higher order) complexes upon binding of carcinostatin chromophore (Fig. 4) which 2. DNA Ligands a metal and only these can then interact is responsible for the observed biological with their target. However, these mecha- activity [6b]. This chromophore consisted Quite a few natural products have the nisms alone cannot account for the se- of a highly unusual bicyclo[7.3.0]dode- capability not just to form complexes with quence specificity displayed by the re- cadienediyne scaffold. Just two years lat- DNA but to chemically modify it. Many spective DNA-binding drugs. These as er, the discovery of two compounds with compounds with antitumor activity be- well as compounds without an aromatic potent cytostatic activity and a similar long into this category. Figs. 4 and 5 show moiety or an apparent metal binding site core structure, calicheamicin YI' and espe- some examples of such naturally occur- often contain additional carbohydrate side ramicin A I (Fig. 4), has been reported ring compounds. Their mechanism of com- chains which have to be responsible for simultaneously by researchers at Lederle plexation with nucleic acids can be distin- site selectivity. Carbohydrates might there- Laboratories [7] and Bristol-Myers [8]. guished into four main categories, interca- fore be an interesting class of potential Their unusual structure containing a bicy- lation, binding via metal complexes, and DNA ligands [5]. In recent years, several clic enediyne and their impressive anti- binding mediated by a protein component research groups have tried to develop syn- cancer activity intrigued the chemical and or carbohydrate side chains. Intercalation. thetic DNA ligands based on the saccha- pharmaceutical community. Immediate- consists of aligning an extended aromatic ride units of known DNA binders. ly, a race aimed at the total synthesis of chromophore in between two base pairs of Certainly the most extensively studied calicheamicin set on, culminating in two the double helix (Fig. 6). Driving force of group of DNA binding small molecules in reported synthesis by the groups of Nico- this kind of interaction is n-stacking that recent years are the enediyne antibiotics, laou [9] and Danishefsky [10]. In 1989, a extends over both strands and leads to an among them calicheamicin, esperamicin, fourth structure ofthis type, dynemicin A, overall stabilization of the double helix. and neocarcinostatin. Neocarcinostatin, a has been reported, again by researchers at Many naturally occurring DNA ligands I:] complex of a protein and a chromo- Bristol-Myers [II], followed by the dis- contain metal binding subunits. By com- phore compound has first been isolated covery of the kedarcidin [12] and C-I 027 plexing a metal, a net positive charge 1965 [6a]. The protein is thought to medi- [13] chromophores (Fig. 4) adding to the results which provides an attractive elec- ate the sequence-specific recognition of arsenal of enediyne antibiotics. Of these trostatic force towards the negatively double-stranded DNA, activation of the latter compounds, the synthesis of dyne- MEDIZINISCHE CHEMIE . MEDICINAL CHEMISTRY 250 C1HMIA 50 (144h) I\r. h (luni) micin A has been reported by Myers and coworkers [14]. The mode of action of these enediyne antibiotics follows a similar pathway as shown in Scheme J for calicheamycin.

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