Identification of Binding Targets of a Pyrrole-Imidazole Polyamide KR12 in the LS180 Colorectal Cancer Genome

Abstract

Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as seco-CBI. These molecules also provide an alternative for oncogenes deemed “undruggable” at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide’s possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.

Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer

“…16 In 2015, mutant KRAS suppression by seco-CBI conjugate resulted in tumor death in an oncogenic mutation-specific manner. [17][18][19] These findings corroborate the significance of seco-CBI conjugate as a promising drug candidate.…”

“…21 The Bind-n-Seq 22 substrate DNA comprises 102 base pairs containing two adapters, a barcode, and 21 base-pair random sequences. 18,19,21,[23][24][25] The workflow starts from primer extension to prepare dsDNA containing random sequences, then a binding/alkylation reaction by the PI polyamide, affinity purification by exploiting the interaction between biotinylated PI polyamide and streptavidin magnetic beads, followed by emulsion PCR and sequencing. These properties are seen in vitro, but experiments targeting genomic DNA in vivo, Chem-Seq 26 , enabled biological discussions at gene levels.…”

The Road Not Taken with Pyrrole-Imidazole Polyamides: Off-Target Effects and Genomic Binding

“…The differences in the level of background noise between different sequencing methods also cannot be ignored; DNase-seq is highly prone to noise and generates reads that are not strand-specific to have characteristic shifts that are often used to as a marker for positive peaks. Preliminary assessment of sequencing data suggested some similarities between Chem-seq and DNase-seq [65], but there have been no systematic studies to verify this to date.…”

CRED: a rapid peak caller for Chem-seq data

“…In our research of pyrrole-imidazole polyamides, a class of DNA minor-groove binders, we have also noticed that MACS (Zhang et al, 2008), a popular model-based peak caller, suffered from similar issues in the analysis of affinity-enriched DNA fragments sequenced by Ion Torrent systems (a method we hereafter will refer as "Chem-seq"). We previously proposed a coverage-based approach (Lin et al, 2016), in which we employed Perl-based diffReps (Shen et al, 2013) as the initial candidate selection component, followed by bootstrapped Kolmogorov-Smirnov comparisons to characterize Chem-seq peaks.…”

“…PI polyamides' relatively short recognition motif and molecular weight, however, can result in generally smaller binding surfaces in polyamide-DNA interactions compared to interactions of DNA with other biomolecules, for instance proteins and transcription factors. A direct consequence of this difference in interaction leads to a mixture of broad and narrow peaks in sequencing experiments conducted with PI polyamides (Chem-seq) that can be atypical of other NGS experiments (Lin et al, 2016). To properly analyze Chem-seq data necessitates the creation of a Chemseq specific computational tool that can analyze general regions of enriched precipitation of polyamide-DNA ligands (a process known as peak calling) from sequencing reads in the genome.…”