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Phosphorothioate oligonucleotides bind to proteins. The interactions with proteins can be divided into nonspecific, sequence-specific, and structure-specific binding events, each of which may have different characteristics and effects. Nonspecific binding to a wide variety

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Figure 5.3. TNF screen. Results from a screen to identify the optimal antisense inhibitor to tumor necrosis factor a receptor 1 2'-Methoxyethyl chimeric antisense inhibitors were synthesized to 80 sites in the target RNA and their effects on RNA were evaluated after incubation with cells. The results are expressed as percentage control RNA determined by RT-PCR analyses. The effects of the antisense inhibitors are displayed schematically from to right to left on the figure. The antisense inhibitors that result in the maximum (smallest bars) are then evaluated with careful dose-response curves and

6 Characteristics of Phosphorothioate Oligonucleotides 131

of proteins has been demonstrated. Exem- plary of this type of binding is the interaction of phosphorothioate oligonucleotides with se- rum albumin. The affinity of such interactions is low. The value for albumin is approxi- mately 200 in a range similar to that of aspirin or penicillin (166, 167). Furthermore, in this study, no competition between phos- phorothioate oligonucleotides and several drugs that bind to bovine serum albumin was observed. In this study, binding and competi- tion were determined in an assay in which electrospray mass spectrometry was used. In contrast, in a study in which an equilibrium dissociation constant was derived from an as- say using albumin loaded on a CH-Sephadex column, the value ranged from 1 to 5

M

for bovine serum albumin and 2 to 3

M for human serum albumin. Moreover, warfarin and indomethacin were reported to compete for binding to serum albumin

However, in experiments in our laboratory, we were unable to reproduce the results (for re- view, see Ref. 169). Clearly, much more work is required before definitive conclusions can be drawn.

Phosphorothioate oligonucleotides can in- teract with nucleic acid binding proteins such as transcription factors and single-strand cleic acid binding proteins. However, very little is known about these binding events. Ad- ditionally, it has been reported that phos- phorothioates bind to an membrane protein that was suggested to be involved in cellular uptake processes (87). However, again, little is known about the affinities, se- quences, or structure specificities of these pu- tative interactions. More recently, interac- tions with 30- and surface proteins in

mouse fibroblasts were reported

Phosphorothioates interact with nucleases and DNA polymerases. These compounds are slowly metabolized by both and

nucleases and inhibit these enzymes (160, 171). The inhibition of these enzymes appears

to

be competitive and this may account for some early data suggesting that phosphoro- thioates are almost infinitely stable to

In these studies, the

enzyme ratio was very high and thus the enzyme was inhibited. Phosphorothioates also bind to RNase H when in an RNA-DNA duplex

and the duplex serves as a substrate for RNase H (172). At higher concentrations, presum- ably by binding as a single strand to RNase H, phosphorothioates inhibit the enzyme (149, 160). Again, the oligonucleotides appear to be competitive antagonists for the DNA-RNA substrate.

Phosphorothioates have been shown to be inhibitors of DNA polymerase and with respect to the DNA template, and noncompetitive inhibitors of DNA poly- and (172). Despite this inhibition, several studies have suggested that

rothioates might serve as primers for poly- and be extended (140, 173, 174). In our laboratories, we have shown extensions of only nucleotides. At present, a full expla- nation as to why no longer extensions are ob- served is not available.

Phosphorothioate oligonucleotides have been reported to be competitive inhibitors for

transcriptase and inhibit

sociated RNase H activity (175, 176). They have been reported to bind to the cell surface protein CD4 and to protein kinase C

(177). Various viral polymerases have also been shown to be inhibited by phosphorothio- ates (140). Additionally, we have shown po- tent, non-sequence-specific inhibition of RNA splicing by phosphorothioates

Like other oligonucleotides, phosphoro- thioates can adopt a variety of secondary structures. As a general rule,

tary oligonucleotides are avoided, if possible, to avoid duplex formation between oligonucle- otides. However, other structures that are less well understood can also form. For ex- ample, oligonucleotides containing runs of guanosines can form tetrameric structures called G-quartets, and these appear to interact with a number of proteins with relatively greater than that of unstructured oli- gonucleotides

In conclusion, phosphorothioate oligonu- cleotides may interact with a wide range of proteins through several types of mecha- nisms. These interactions may influence the pharmacokinetic, pharmacologic, and toxico- logic properties of these molecules. They may also complicate studies on the mechanism of action of these drugs, and may obscure an an- tisense activity. For example, phosphorothio-

Oligonucleotide Therapeutics

ate oligonucleotides were reported to enhance lipopolysaccharide-stimulated synthesis or tu- mor necrosis factor This would obvi- ously obscure antisense effects on this target. 6.3 Pharmacokinetic Properties

To study the pharmacokinetics of phosphoro- thioate oligonucleotides, a variety of labeling techniques have been used. In some cases,

32

or 5

P

end-labeled or fluorescently labeled oligonucleotides have been used in either in vitro or in vivo studies. These are probably less satisfactory than internally labeled compounds because terminal phosphates are rapidly re- moved by phosphatases and fluorescently la- beled oligonucleotides have

properties that differ from those of the unmodi- fied oligonucleotides. Consequently, either uni- formly S-labeled or base-labeled

rothioates are preferable for

studies. In our laboratories, a tritium exchange method that labels a slowly exchanging proton at the position in purines was developed and proved to be quite useful (180). Very recently, a method that added radioactive methyl groups through S-adenosyl methionine was also suc- cessfully used (181). Finally, advances in extrac- tion, separation, and detection methods have re- sulted in methods that provide excellent analyses without radiolabeling (182).

6.3.1 Stability. The principal met- abolic pathway for oligonucleotides is cleav- age by and exonucleases.

thioate oligonucleotides, although quite stable to various nucleases, are competitive inhibi- tors of nucleases (67, 93, 172, 183, 184). Con- sequently, the stability of phosphorothioate oligonucleotides to nucleases is probably a bit less than initially thought, given the high con- centrations (that inhibited nucleases) of oligo- nucleotides that were employed in the early studies. Similarly, phosphorothioate oligonu- cleotides are degraded slowly by cells in tissue culture with a half-life of 12-24 h and are slowly metabolized in animals (183,185,186). The pattern of metabolites suggests primarily exonuclease activity, with perhaps modest contributions by endonucleases. However, a number of lines of evidence suggest that, in many cells and tissues, endonucleases play an

important role in the metabolism of oligonu- cleotides. For example, 3'- and 5'-modified oli- gonucleotides with phosphodiester backbones have been shown to be relatively rapidly de- graded in cells and after administration to an- imals (90, 187). Thus, strategies in which oli- gonucleotides are modified at only the 3'- and 5'-terminus as a means of enhancing stability have proved to be unsuccessful.

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