Figure 5.6. Selected 5'-oligonucleotide conjugates.

additional synthetic steps and modifications to the oligonucleotide synthesis protocol or monomer synthesis. In addition, there may be a loss in binding affinity to the target RNA because of these changes with geometrically altered linkages. Thiono triester (adamantyl, cholesteryl, and others) modified oligonucleotides have been reported to improve nuclease stability, cellular association, and binding affinity (328).

The demonstration that modifications may induce nuclease stability sufficient to enhance activity in cells in tissue culture and in animals has proved to be much more complicated because of the presence of 5'-exonucleases and -endonucleases. In our laboratory, 3'-modifi-cations and internal point modifications have not provided sufficient nuclease stability to demonstrate pharmacological activity in cells (183). In fact, even a 5-nucleotide-long phos-phodiester gap in the middle of a phosphoro-thioate oligonucleotide resulted in sufficient loss of nuclease resistance to cause complete loss of pharmacological activity (164).In mice, neither a 5'-cholesterol nor 5'-Cl8 amine conjugate altered the metabolic rate of a phospho-rothioate oligodeoxynucleotide in liver, kid ney, or plasma (166). Furthermore, blocking the 3'- and 5'-termini of a phosphodiester oligonucleotide did not markedly enhance the nuclease stability of the parent compound in mice (90). However, 3'-modification of a phos-phorothioate oligonucleotide was reported to enhance its stability in mice relative to that of the parent phosphorothioate (329).Moreover, a phosphorothioate oligonucleotide with a 3'-hairpin loop was reported to be more stable than its parent in rats (328). Thus, 3'-modifi-cations may enhance the stability of the relatively stable phosphorothioates sufficiently to be of value.

7.3.2 Enhanced Cellular Uptake. Although oligonucleotides have been shown to be taken up by a number of cell lines in tissue culture, with perhaps the most compelling data relating to phosphorothioate oligonucleotides, a clear objective has been to improve cellular uptake of oligonucleotides (72, 264). Inasmuch as the mechanisms of cellular uptake of oligonucleotides are still very poorly understood, the medicinal chemistry approaches have been largely empirical and based on many unproven assumptions.

Because phosphodiester and phosphoro-thioate oligonucleotides are hydrophilic, the conjugation of lipophilic substituents to enhance membrane permeability has been a subject cf considerable interest. Unfortunately, studies in this area have not been systematic and, at present, there is precious little information about the changes in physicochemical properties of oligonucleotides actually effected by specific lipid conjugates. Phospholipids, cholesterol and cholesterol derivatives, cholic acid, and simple alkyl chains have been conjugated to oligonucleotides at various sites in the oligonucleotide. The effects of these modifications on cellular uptake have been assessed using fluorescent, or radiolabeled, oligonucle-otides or by measuring pharmacological activities. From the perspective of medicinal chemistry, very few systematic studies have been performed. The activities of short alkyl chains, adamantine, daunomycin, fluorescein, cholesterol, and porphyrin-conjugated oligonucleo-tides were compared in one study (330). A cholesterol modification was reported to be more effective than the other substituents at enhancing uptake. It also seems likely that the effects of various conjugates on cellular uptake may be affected by the cell type and target studied. For example, we have studied cholic acid conjugates of phosphorothioate deoxyoli-gonucleotides or phosphorothioate 2'-0-methyl oligonucleotides and observed enhanced activity against HIV and no effect on the activity of ICAM-1-directed oligonucleotides (331).

Additionally, polycationic substitutions and various groups designed to bind to cellular carrier systems have been synthesized. Although many compounds have been synthesized, the data reported to date are insufficient to draw firm conclusions about the value of such approaches or structure-activity relationships (SARs) (332).

7.3.3 RNA Cleaving Croups. Oligonucle-otide conjugates were recently reported to act as artificial ribonucleases, albeit in low efficiencies (333). Conjugation of chemically reactive groups such as alkylating agents, photo-induced azides, porphyrins, and psoralens have been used extensively to effect a crosslinking of oligonucleotide and the target

RNA. In principle, this treatment may lead to translation arrest. In addition. lanthanides and complexes thereof have been reported to cleave RNA by way of a hydrolytic pathway. Recently, a novel europium complex was co-valently linked to an oligonucleotide and shown to cleave 88% of the complementary RNA at physiological pH (334).

7.3.4 RNase H Activity. Oligonucleotide conjugates may play a distinct role in improving RNase H activity because conjugation of small molecules, especially at the oligonucleotide termini, may not affect the factors required for enzyme activity. Acridine-conju-gated oligonucleotides targeted to j3-globin mRNA were shown to be better inhibitors of synthesis than the unmodified oligo-nucleotides (335) in a microinjected Xenopus oocytes assay as a result of RNase H activity. Cholesterol-conjugated oligonucleotides targeted to an mRNA fragment of Ha-ras onco-gene were able to promote a greater extent of target RNA hydrolysis, nearly three- to fivefold, by RNase H compared to the nonconju-gated parent (336). The 3'-conjugate performed better than the 5'-conjugate. More recently, polycyclic aromatic chromophores such as phenazine (PZN) and dipyridophena-zine (DPPZ) have been evaluated in E. coli RNase H cleavage of the target strand when conjugated to oligonucleotides (337,338). The 3'-conjugated antisense oligomers promoted faster hydrolysis of the target RNA than the unmodified compound and a stabilizing interaction between the ligands and the enzyme has been proposed.

7.3.5 In Vivo Effects. To date, relatively few studies have been reported in vivo. The properties of 5'-cholesterol and 5'-C18 amine conjugates of a 20-mer phosphorothioate oli-godeoxynucleotide have been determined in mice. Both compounds increased the fraction of an i.v. bolus dose found in the liver. The cholesterol conjugate resulted in more than 80% dose accumulation in the liver. Neither conjugate enhanced stability in plasma, liver, or kidney (166). Interestingly, the only significant change in the toxicity profile was a slight increase in effects on serum transamineses and histopathological changes, indicative of

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