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Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (Kelly cells)

ABSTRACT: Side and off-target effects remain a difficult issue in the search of pharmaceutical leads, especially with DNA-binding molecules or genome editing methods where the issue becomes particularly thorny. A particular case is the investigations into the off-target effects of N-methylpyrrole-N-methylimidazole polyamides, a naturally inspired class of DNA binders that discriminately access the minor groove with strong affinity and sequence specificity but the relatively short motif of < 20 bases often imply possible non-unique genomic binding. Multiple binding sites may translate to binding at non-intended loci, potentially leading to off-target effects, issues that very few approaches are able to address to-date. We here report an analytical method of inferring off-target binding from expression profiling based on the relative impact to various biochemical pathways, as well as an accompanying side effect prediction engine to allow candidate polyamides to be systematically screened. This method marks the first attempt in PI polyamide research to identify elements in various biochemical pathways sensitive to the treatment of a candidate polyamide to infer possible off-target effects. Expression changes were then considered for their possible effect on outward phenotypic changes, manifested as side effects, should the same PI polyamide candidate be administered clinically. We also performed a series of animal experiments to validate some of these effects, and found some corroboration in certain side effect manifestations, such as changes in the level of aspartate transaminase in ICR and nude mice after injection of some of the candidate polyamides in this study.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124457 | GEO | 2019/04/09

REPOSITORIES: GEO

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Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (SK-N-AS cells)

Project description:Side and off-target effects remain a difficult issue in the search of pharmaceutical leads, especially with DNA-binding molecules or genome editing methods where the issue becomes particularly thorny. A particular case is the investigations into the off-target effects of N-methylpyrrole-N-methylimidazole polyamides, a naturally inspired class of DNA binders that discriminately access the minor groove with strong affinity and sequence specificity but the relatively short motif of < 20 bases often imply possible non-unique genomic binding. Multiple binding sites may translate to binding at non-intended loci, potentially leading to off-target effects, issues that very few approaches are able to address to-date. We here report an analytical method of inferring off-target binding from expression profiling based on the relative impact to various biochemical pathways, as well as an accompanying side effect prediction engine to allow candidate polyamides to be systematically screened. This method marks the first attempt in PI polyamide research to identify elements in various biochemical pathways sensitive to the treatment of a candidate polyamide to infer possible off-target effects. Expression changes were then considered for their possible effect on outward phenotypic changes, manifested as side effects, should the same PI polyamide candidate be administered clinically. We also performed a series of animal experiments to validate some of these effects, and found some corroboration in certain side effect manifestations, such as changes in the level of aspartate transaminase in ICR and nude mice after injection of some of the candidate polyamides in this study.

2019-04-09 | GSE124461 | GEO

Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (SiHA cells)

2019-04-09 | GSE124460 | GEO

Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (ME180 cells)

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Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (MC-IXC cells)

2019-04-09 | GSE124458 | GEO

Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (CHP-134 cells)

2019-04-09 | GSE124456 | GEO

Estimating Genome-wide Off-Target Effects for Pyrrole-Imidazole Polyamide Binding by a Pathway-Based Expression Profiling Approach (CaSki cells)

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Alkylating pyrrole-imidazole polyamide KR12 binding in colorectal cancer genomes (SW480)

Project description: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 endo-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.

2016-11-01 | GSE86598 | GEO

Alkylating pyrrole-imidazole polyamide KR12 binding in colorectal cancer genomes (LS180)

2016-11-01 | GSE86597 | GEO

COSMIC-seq of bioactive hairpin N-methylpyrrole/N-methylimidazole polyamides

Project description:Regulating desired loci in the genome with sequence-specific DNA-binding molecules is a major goal for the development of precision medicine. Pyrrole–imidazole (Py–Im) polyamides are synthetic molecules that can be rationally de-signed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of binding energetics. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their utility in vivo. In this report we directly identified polyamide–DNA interaction sites across the entire genome, by covalent crosslinking and capturing these events in the nuclei of human LNCaP cells. This method, termed COSMIC-seq, confirms the ability of hairpin-polyamides, with similar architectures but differing at a single ring position, to retain in vitro specificities and display distinct genome-wide binding profiles. These results underpin the development of Py-Im polyamides as DNA-targeting molecules and the potential for utility as synthetic transcription factors (Syn-TFs) capable of functioning in concert with the cellular regulatory circuitry.

2020-12-02 | GSE149367 | GEO

Transcription profiling of human U251 cells treated with hypoxia inducible factor HIF-1I siRNA or a polyamide targeted against the hypoxia response element (HRE) after induction of hypoxia

Project description:Transcription mediated by hypoxia inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in the activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compared the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element (HRE), siRNA targeted to HIF-1Î±, and echinomycin, a DNA binding natural product with a similar but less specific sequence preference to the polyamide. The polyamide affects a subset of hypoxia-induced genes that are consistent with the binding site preferences of the polyamide. For comparison, siRNA targeted to HIF-1α and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data shows how polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression. Experiment Overall Design: Hypoxia-mimetic DFO (deferoxamine)-stimulated U251 cells that were treated with polyamide 1, HIF-1α siRNA, and echinomycin were compared to control cells that were also DFO-stimulated. Cells not stimulated with DFO were also compared to the DFO-stimulated controls. Three biological replicates were included for each treatment/condition.

2008-06-15 | E-GEOD-7835 | biostudies-arrayexpress

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