Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs

Abstract

One of the major obstacles to the pharmaceutical success of oligonucleotide therapeutics (ONTs) is efficient delivery from the point of injection to the intracellular setting where functional gene silencing occurs. In particular, a significant fraction of internalized ONTs are nonproductively sequestered in endo-lysosomal compartments. Here, we describe a two-step, robust assay for high-throughput de novo detection of small bioactive molecules that enhance cellular uptake, endosomal escape, and efficacy of ONTs. Using this assay, we screened the LOPAC (Sigma–Aldrich) Library of Pharmacologically Active Compounds and discovered that Guanabenz acetate (Wytensin™), an FDA-approved drug formerly used as an antihypertensive agent, is capable of markedly increasing the cellular internalization and target mRNA silencing of hydrophobically modified siRNAs (hsiRNAs), yielding a ∼100-fold decrease in hsiRNA IC50 (from 132 nM to 2.4 nM). This is one of the first descriptions of a high-throughput small-molecule screen to identify novel chemistries that specifically enhance siRNA intracellular efficacy, and can be applied toward expansion of the chemical diversity of ONTs.

A Novel Family of Small Molecules that Enhance the Intracellular Delivery and Pharmacological Effectiveness of Antisense and Splice Switching Oligonucleotides

“…However, further analysis of structure activity relationships may lead to the development of new compounds with greater efficacy and reduced toxicity. It is interesting to note that other groups have begun to describe oligonucleotide enhancing small molecules (28, 29) . The structures of those molecules are very different from ones described here or previously by us (20) , suggesting that there may be a variety of mechanisms by which small molecules can augment effects of oligonucleotides.…”

Imaging small molecule-induced endosomal escape of siRNA

“…Other studies have investigated the use of small molecules for enhancing knockdown mediated by chol-siRNA and LNP-siRNA [19][20][21][22] . In these studies, the identified small molecules acted through either facilitating uptake or by effects on intracellular processes-potentially including endosomal escape.…”

“…While release-enhancing strategies using endosmolytic polymers or peptides have been promising 17,18 , such approaches are challenging from a formulation, toxicity, and bioavailability perspective. Efforts have also been made to identify small-molecule drugs capable of enhancing oligonucleotide delivery [19][20][21][22] . However, direct visualization or analysis of enhanced endosomal escape of non-formulated oligonucleotides has not been possible.…”

6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells

“…16,33 In fact, the small-molecule GSK-3 inhibitor CHIR99021 also increased SSO activity in HeLa-EGFP-654 cells ( Figure 7A). Via the use of small molecules identified in similar high-throughput screens, 17,58,59 it has previously been observed that the silencing ability of ASOs and hydrophobically modified siRNAs can be augmented through effective release from the late endosome. However, at the present time it remains an open question as to precisely how 6BIO augments ASO activity.…”