title: Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.
abstract: RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs.
authors.affiliation: Department of Core Antisense Research, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA Lliang@ionisph.com.
authors.forename: Xue-Hai
authors.initials: XH
authors.lastname: Liang
authors.affiliation: Department of Core Antisense Research, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Shen
authors.affiliation: Department of Core Antisense Research, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Medicinal Chemistry, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Garth A
authors.initials: GA
authors.lastname: Kinberger
authors.affiliation: Department of Medicinal Chemistry, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.affiliation: Department of Core Antisense Research, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Joshua G
authors.initials: JG
authors.lastname: Nichols
authors.affiliation: Department of Core Antisense Research, IONIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D002460:Cell Line; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009841:Oligonucleotides / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains
chemical_list: D018841:HSP90 Heat-Shock Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; C477371:locked nucleic acid