Date Published: April 9, 2019
Publisher: Public Library of Science
Author(s): Jason Lin, Sakthisri Krishnamurthy, Hiroyuki Yoda, Yoshinao Shinozaki, Takayoshi Watanabe, Nobuko Koshikawa, Atsushi Takatori, Paul Horton, Hiroki Nagase, Chakrabhavi Dhananjaya Mohan.
In the search for new pharmaceutical leads, especially with DNA-binding molecules or genome editing methods, the issue of side and off-target effects have always been thorny in nature. A particular case is the investigation into the off-target effects of N-methylpyrrole-N-methylimidazole polyamides, a naturally inspired class of DNA binders with strong affinity to the minor-groove and sequence specificity, but at < 20 bases, their relatively short motifs also insinuate the possibility of non-unique genomic binding. Binding at non-intended loci potentially lead to the rise of off-target effects, issues that very few approaches are able to address to-date. We here report an analytical method to infer off-target binding, via expression profiling, based on probing the relative impact to various biochemical pathways; we also proposed an accompanying side effect prediction engine for the systematic screening of candidate polyamides. This method marks the first attempt in PI polyamide research to identify elements in biochemical pathways that are sensitive to the treatment of a candidate polyamide as an approach to infer possible off-target effects. Expression changes were then considered to assess possible outward phenotypic changes, manifested as side effects, should the same PI polyamide candidate be administered clinically. We validated some of these effects with a series of animal experiments, and found agreeable corroboration in certain side effects, such as changes in aspartate transaminase levels in ICR and nude mice post-administration.
N-methylpyrrole-N-methylimidazole (PI) polyamides have been frequently discussed as viable alternatives for “undruggable” oncological targets otherwise inaccessible at the protein level, such as KRAS [1–3]. Their ability to interact with the DNA minor-groove binders with unrivaled sequence recognition translates to their distinctive ability to disrupt transcription, and proper functionalization with histone modifiers or alkylating agents transform these molecules to programmable epigenetic switches and anticancer agents in vivo [4–10]. A troubling issue for PI polyamides, however, is their relatively short motifs, leading to multiple genomic binding sites; lengthening a PI polyamide beyond its typical working range of 8–10 bases will reduce the number of genomic binding sites at the penalty of reducing its chemical affinity to the DNA minor groove due to increased structural rigidity [11, 12]; intriguingly, most PI polyamide, despite the presence of multiple binding sites, led to little toxicity [13–15]. It would therefore imply that nonunique binding typically did not lead to ill biological effects, further complicating our understanding on the subject matter. Nevertheless, to improve the clinical prospects of PI polyamides, the evaluation of these so-called “off-target effects” would be unavoidable in a similar manner as genome editing methods such as CRISPR/Cas. Yet, even before we could approach the problem, we were met with a difficult question: for PI polyamides, how would one define the effect of off-target binding for PI polyamides?
Most developmental pipelines encounter a similar problem: it is not always certain whether the investment in preclinical studies will attribute to the eventual success for a marketable product. PI polyamides, while highly versatile and by their mechanistic nature tunable, also face this. By evaluating candidate polyamides with expression microarrays and this systematic method can greatly improve the throughput and meaningfully shorten the initial lead selection and optimization period. We understand how changing the structure of PI polyamides may affect its binding affinity, and how these molecules’ relative hydrophobicity can have on its tumor retention ; yet we still know very little about how these molecules will influence the outward phenotypes, especially when binding motifs are concerned. The lack of progress in understanding the off-target effect of PI polyamides can limit the growth of PI polyamide research and the translation to bedside applications. With our first report on the subject matter, we hope to facilitate the push of PI polyamides to bedside applications and accelerate the field of precision medicine.