Trivalent siRNA-Conjugates with Guanosine as ASGPR-Binder Show Potent Knock-Down In Vivo

To increase the chemical space around the well-known
GalNAc-ligand
as ASGPR-binder, a high-throughput screening campaign was performed,
testing approximately 550,000 compounds. After evaluation of the potential
screening hits, only one compound, which showed high similarity with
guanosine nucleosides, was chosen for further profiling. Crystal structure
analysis revealed the coordination of the Ca2+-ion within
the ASGPR-binding site by the cis-diol motif of the
ribose unit as well as an additional π–π-interaction
of the purine heterocycle to tryptophan-243. Based on these findings,
guanosine was attached via the 5′-OH group to a recently described
morpholino-based nucleotide using two different linker units. The
resulting morpholino-guanosine building blocks were conjugated to
the 5′-end of a literature-known transthyretin targeting small
interfering RNA (siRNA), leading to trivalent siRNA-guanosine conjugates,
which were tested for their TTR knockdown and exhibited similar potencies
as the analogous GalNAc-conjugates in vitro and in vivo.