Anandhakumar Chandran

Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N-methylpyrrole-N-methylimidazole polyamide seco-1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one conjugate (conjugate 4) designed to target the KRAS codon 12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon 12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer-minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one-base-pair mismatch sequence. Conjugate 4 was modified for next-generation sequencing. Bind-n-Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild-type sequence.

A Synthetic Transcriptional Activator of Genes Associated with the Retina in Human Dermal Fibroblasts

Small molecules capable of modulating epigenetic signatures can activate the transcription of tissue‐restricted genes in a totally unrelated cell type and have potential use in epigenetic therapy. To provide an example for an initial approach, we report here on one synthetic small‐molecule compound—termed “SAHA–PIP X”—from our library of conjugates. This compound triggered histone acetylation accompanied by the transcription of retinal‐tissue‐related genes in human dermal fibroblasts (HDFs).

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing.

Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing

Tet (ten‐eleven translocation) family proteins have the ability to oxidize 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC), 5‐formylcytosine (fC), and 5‐carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic‐level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high‐throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×104‐member mC‐containing random DNA‐sequence library was constructed. The library was subjected to Tet‐reactive pulldown followed by high‐throughput sequencing. Analysis of the obtained sequence data identified the Tet1‐reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein.

Genome-Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by High-Throughput Sequencing.

One of the major goals in DNA‐based personalized medicine is the development of sequence‐specific small molecules to target the genome. SAHA‐PIPs belong to such class of small molecule. In the context of the complex eukaryotic genome, the differential biological effects of SAHA‐PIPs are unclear. This question can be addressed by identifying the binding regions across the genome; however, it is a challenge to enrich small‐molecule‐bound DNA without chemical crosslinking. Here, we developed a method that employs high‐throughput sequencing to map the binding area of small molecules throughout the chromatinized human genome. Analysis of the sequenced data confirmed the presence of specific binding sites for SAHA‐PIPs from the enriched sequence reads. Mapping the binding sites and enriched regions on the human genome clarifies the reason for the distinct biological effects of SAHA‐PIP. This approach will be useful for identifying the function of other small molecules on a large scale.

Sequence-specific DNA binding by long hairpin pyrrole–imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit

With the aim of improving aqueous solubility, we designed and synthesized five N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides capable of recognizing 9-bp sequences. Their DNA-binding affinities and sequence specificities were evaluated by SPR and Bind-n-Seq analyses. The design of polyamide 1 was based on a conventional model, with three consecutive Py or Im rings separated by a β-alanine to match the curvature and twist of long DNA helices. Polyamides 2 and 3 contained an 8-amino-3,6-dioxaoctanoic acid (AO) unit, which has previously only been used as a linker within linear Py-Im polyamides or between Py-Im hairpin motifs for tandem hairpin. It is demonstrated herein that AO also functions as a linker element that can extend to 2-bp in hairpin motifs. Notably, although the AO-containing unit can fail to bind the expected sequence, polyamide 4, which has two AO units facing each other in a hairpin form, successfully showed the expected motif and a KD value of 16nM was recorded. Polyamide 5, containing a β-alanine-β-alanine unit instead of the AO of polyamide 2, was synthesized for comparison. The aqueous solubilities and nuclear localization of three of the polyamides were also examined. The results suggest the possibility of applying the AO unit in the core of Py-Im polyamide compounds.

Comparative Analysis of DNA-Binding Selectivity of Hairpin and Cyclic Pyrrole-Imidazole Polyamides Based on Next-Generation Sequencing

Many long pyrrole‐imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind‐n‐Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind‐n‐Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind‐n‐Seq motif identification with a reference sequence (Bind‐n‐Seq‐MR). High‐throughput sequence analysis of the PIP‐enriched DNA data by Bind‐n‐Seq‐MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method.

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

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.

Overview of Next-Generation Sequencing Technologies and Its Application in Chemical Biology

Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this chapter we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening.

Genome-Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by Utilizing the Power of High-Throughput Sequencing

One of the major goals in DNA-based personalized medicine is the development of sequence-specific small molecules to target the genome by means of synthetic biology; SAHA-PIPs belong to such class of small molecules. In a complex eukaryotic genome, the differential biological effects of SAHA-PIPs remain unclear. These questions can be addressed by directly identifying the binding regions of small molecules across the genome; however, it is a challenge to enrich specifically the small-molecule-bound DNA without chemical cross-linking. Here, we developed a method using high-throughput sequencing to map the binding area of non-cross-linked small molecules throughout the chromatinized human genome. Analysis of the sequenced data confirmed the presence of specific binding sites for SAHA-PIPs among the enriched sequence reads. Mapping the binding sites and enriched regions on the human genome clarifies the origin of the distinctive biological effects of SAHA-PIP. This approach will be useful for identifying the functionality of other small molecules on a large scale.