Characterization of binding sites, extent of binding, and drug interactions of oligonucleotides with albumin.
Phosphorothioate oligonucleotides (S-ODNs) have the ability to modulate gene expression selectively and thus have potential therapeutic capabilities. This potential led us to investigate the protein binding characteristics of selected S-ODNs. We evaluated S-ODN interactions with bovine serum albumin (BSA) and human serum albumin (HSA) in vitro. The equilibrium dissociation constants Km for the binding of a 20 mer S-ODN with BSA and HSA range between 1.1-5.2 x 10(-5) and 2.4-3.1 x 10(-4) M, respectively. The Km for an unrelated 15 mer S-ODN binding with HSA ranges between 3.7 and 4.8 x 10(-5) M. Studies with a fluorescently labeled 27 mer S-ODN suggest cooperative binding (Hill slope = 1.67) and/or the presence of secondary binding sites on the S-ODN. HSA or BSA linked to Sepharose was incubated with a 15, 20, or 24 mer S-ODN followed by the addition of selected drugs known to be highly protein bound (nifedipine, warfarin, midazolam, probenecid, indomethacin, and mitoxantrone). Up to 30% of S-ODN was displaced by warfarin in competition binding assays. Conversely, HSA-bound warfarin was incubated with a variety of oligonucleotides, including RNA and genomic dsDNA. Maximum displacement of warfarin-bound HSA was observed following incubation with 5'-cholesterol-conjugated 20 mer S-ODN. In summary, S-ODNs are likely to interact and displace other therapeutic agents that bind to albumin, particularly those binding at site I.
Srinivasan
S K
SK
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198-6260, USA.
Tewary
H K
HK
Iversen
P L
PL
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*; D014859:Warfarin / Q000378:metabolism
D009841:Oligonucleotides; D012709:Serum Albumin; D013873:Thionucleotides; D012710:Serum Albumin, Bovine; D014859:Warfarin