Mechanisms of ASO toxicities and the structure activity relationships that improve therapeutic index
Last year, we published a landmark paper that described a general mechanism that explains how toxic ASOs produce toxicities and provided a straightforward medicinal chemistry solution to enhance safety with little effect on potency (Shen et.al., 2019). We have now published several papers that extend our observations and broaden the understanding of the SAR of binding to key proteins and the modifications that reduce toxicity and enhance the therapeutic index.
In this paper published in Nucleic Acid Research (NAR), we presented a more detailed understanding of the sites in the paraspeckle protein P54nrb involved in the formation of the ASO-induced toxic complex. While ASO-protein interactions were found to be highly influenced by the chemistry of the PS-ASO binding environment, no direct correlation between affinity and toxicity was observed. Rather, the formation of the toxic complex is key. Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system (Nano-BiT) which allows observation of ASO/protein interactions in real-time in live cells, we determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. We presented additional data on the nature of the chemical interactions between PS ASOs and P54nrb and RNase H1 and evaluated the kinetics of ASO binding in cells.
In this NAR paper, we extend the understanding of the SAR that results in improved therapeutic index. We showed that replacing PS at positions 2 and 3 in the gap with neutral linkages such as alkyl phosphonates can reduce toxicities. Obviously, we continue to expand the chemical space studied in this regard and report our progress in future papers and patents.
Several years ago, we thoroughly evaluated the value of chirally pure PS ASOs (Wan et al., 2014). We learned that it is a zero-sum game because the isomer that enhances affinity for RNA results in less stable. As we studied RNase H1 in detail, we also showed that the hybrid-binding domain and the catalytic domain behave differently with regard to chiral PS modifications and this adds to the ‘zero-sum game’.
Nevertheless, in response to recent claims, we reevaluated chiral PS modifications again and found that they do not enhance potency or safety, but once again showed that a 2’methoxy at position 2 of the gap enhanced therapeutic index.
Future work
In future papers, we will provide a much more detailed understanding of the chemical nature of PS ASO -protein interactions and expand the repertoire of chemical solutions to enhance the therapeutic index.