RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.

Development of multifunctional pyrrole-imidazole polyamides that increase hepatocyte growth factor and suppress transforming growth factor-ß1.

PURPOSE: The DNA recognition peptide compounds pyrrole-imidazole (PI) polyamides bind to the minor groove and can block the binding of transcription factors to target sequences. To develop more PI polyamides as potential treatments for fibrotic diseases, including chronic renal failure, we developed multifunctional PI polyamides that increase hepatocyte growth factor (HGF) and decrease transforming growth factor (TGF)-ß1. METHODS: We designed seven PI polyamides (HGF-1 to HGF-7) that bind to the chicken ovalbumin upstream promoter transcription factor-1 (COUP-TF1) binding site of the HGF promoter sequence. We selected PI polyamides that increase HGF and suppress TGF-ß1 in human dermal fibroblasts (HDFs). FINDINGS: Gel shift assays showed that HGF-2 and HGF-4 bound the appropriate dsDNAs. HGF-2 and HGF-4 significantly inhibited the TGF-ß1 mRNA expression in HDFs stimulated by phorbol 12-myristate 13-acetate. HGF-2 and HGF-4 significantly inhibited the TGF-ß1 protein expression in HDFs with siRNA targeting HGF, indicating that HGF-2 and HGF-4 directly inhibited the expression of TGF-ß1. CONCLUSION: The designed and synthetic HGF PI polyamides targeting the HGF promoter, which increased the expression of HGF and suppressed the expression of TGF-ß, will be a potential practical medicine for fibrotic diseases, including progressive renal diseases.

Suppression of non-small-cell lung cancer A549 tumor growth by an mtDNA mutation-targeting pyrrole-imidazole polyamide-triphenylphosphonium and a senolytic drug.

Certain somatic mutations in mtDNA were associated with tumor progression and frequently found in a homoplasmic state. We recently reported that pyrrole-imidazole polyamide conjugated with the mitochondria-delivering moiety triphenylphosphonium (PIP-TPP) targeting an mtDNA mutation efficiently induced apoptosis in cancer cells with the mutation but not normal cells. Here, we synthesized the novel PIP-TPP, CCC-021-TPP, targeting ND6 14582A > G homoplasmic missense mutation that is suggested to enhance metastasis of non-small-cell lung cancer A549 cells. CCC-021-TPP did not induce apoptosis but caused cellular senescence in the cells, accompanied by a significant induction of antiapoptotic BCL-XL. Simultaneous treatment of A549 cells with CCC-021-TPP and the BCL-XL selective inhibitor A-1155463 resulted in apoptosis induction. Importantly, the combination induced apoptosis and suppressed tumor growth in an A549 xenografted model. These results highlight the potential of anticancer therapy with PIP-TPPs targeting mtDNA mutations to induce cell death even in apoptosis-resistant cancer cells when combined with senolytics.

Targeted epigenetic modulation using a DNA-based histone deacetylase inhibitor enhances cardiomyogenesis in mouse embryonic stem cells.

The epigenome has an essential role in orchestrating transcriptional activation and modulating key developmental processes. Previously, we developed a library of pyrrole-imidazole polyamides (PIPs) conjugated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, for the purpose of sequence-specific modification of epigenetics. Based on the gene expression profile of SAHA-PIPs and screening studies using the α-myosin heavy chain promoter-driven reporter and SAHA-PIP library, we identified that SAHA-PIP G activates cardiac-related genes. Studies in mouse ES cells showed that SAHA-PIP G could enhance the generation of spontaneous beating cells, which is consistent with upregulation of several cardiac-related genes. Moreover, ChIP-seq results confirmed that the upregulation of cardiac-related genes is highly correlated with epigenetic activation, relevant to the sequence-specific binding of SAHA-PIP G. This proof-of-concept study demonstrating the applicability of SAHA-PIP not only improves our understanding of epigenetic alterations involved in cardiomyogenesis but also provides a novel chemical-based strategy for stem cell differentiation.

A PI polyamide-TPP conjugate targeting a mtDNA mutation induces cell death of cancer cells with the mutation.

Mitochondrial DNA (mtDNA) mutations occur frequently in cancer cells, and some of them are often homoplasmic. Targeting such mtDNA mutations could be a new method for killing cancer cells with minimal impact on normal cells. Pyrrole-imidazole polyamides (PIPs) are cell-permeable minor groove binders that show sequence-specific binding to double-stranded DNA and inhibit the transcription of target genes. PIP conjugated with the lipophilic triphenylphosphonium (TPP) cation can be delivered to mitochondria without uptake into the nucleus. Here, we investigated the feasibility of the use of PIP-TPP to target a mtDNA mutation in order to kill cancer cells that harbor the mutation. We synthesized hairpin-type PIP-TPP targeting the A3243G mutation and examined its effects on the survival of HeLa cybrid cells with or without the mutation (HeLamtA3243G cells or HeLamtHeLa cells, respectively). A surface plasmon resonance assay demonstrated that PIP-TPP showed approximately 60-fold higher binding affinity for the mutant G-containing synthetic double-stranded DNA than for the wild-type A-containing DNA. When added to cells, it localized in mitochondria and induced mitochondrial reactive oxygen species production, extensive mitophagy, and apoptosis in HeLamtA3243G cells, while only slightly exerting these effects in HeLamtHeLa cells. These results suggest that PIP-TPPs targeting mtDNA mutations could be potential chemotherapeutic drugs to treat cancers without severe adverse effects.

Targeting the mutant PIK3CA gene by DNA-alkylating pyrrole-imidazole polyamide in cervical cancer.

PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

Mitochondria: Endosymbiont bacteria DNA sequence as a target against cancer.

As the energy factory for the cell, the mitochondrion, through its role of adenosine triphosphate production by oxidative phosphorylation, can be regarded as the guardian of well regulated cellular metabolism; the integrity of mitochondrial functions, however, is particularly vulnerable in cancer due to the lack of superstructures such as histone and lamina folds to protect the mitochondrial genome from unintended exposure, which consequently elevates risks of mutation. In cancer, mechanisms responsible for enforcing quality control surveillance for identifying and eliminating defective mitochondria are often poorly regulated, and certain uneliminated mitochondrial DNA (mtDNA) mutations and polymorphisms can be advantageous for the proliferation, progression, and metastasis of tumor cells. Such pathogenic mtDNA aberrations are likely to increase and occasionally be homoplasmic in cancer cells and, intriguingly, in normal cells in the proximity of tumor microenvironments as well. Distinct characteristics of these abnormalities in mtDNA may provide a new path for cancer therapy. Here we discuss a promising novel therapeutic strategy, using the sequence-specific properties of pyrrole-imidazole polyamide-triphenylphosphonium conjugates, against cancer for clearing abnormal mtDNA by reactivating mitochondrial quality control surveillance.

A novel pyrrole-imidazole polyamide targets Aurora kinase A and suppresses tumor growth in vivo.

Aurora kinase A (Aurora A) plays a critical role in regulating cell mitotic progression and has been considered as a promising drug target for cancer therapy. To develop a novel molecule targeting Aurora A with high selectivity and efficacy, we designed and synthesized a pyrrole-imidazole polyamide (PIP) Hoechst conjugate, PIP-Ht, targeting to a cell-cycle regulated DNA sequence locating at the promoter of human Aurora A gene (AURKA). PIP-Ht potently suppressed AURKA promoter activities, mRNA expression and protein level, induced tumor cell cycle delay and inhibited tumor cell proliferation in vitro. Furthermore, subcutaneous injection of PIP-Ht into mice bearing human cancer xenografts induced significant tumor growth suppression and cell apoptosis. Collectively, PIP-Ht exhibits the potential as an effective therapeutic candidate for the tumor treatment.

A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.

Somatic mutations in mitochondrial DNA may provide a new avenue for cancer therapy due to their associations to a number of cancers and a tendency of homoplasmicity. In consideration of mitochondrial features and its relatively small genome size, a nucleotide-based targeting approach is a considerably more promising option. To explore the efficacy of short linear N-methylpyrrole-N-methylimidazole polyamide (PI polyamide), we synthesized a five-ring short PI polyamide that provided sequence-specific homing for the A3243G mitochondrial mutation upon conjugation with triphenylphosphonium cation (TPP). This PI polyamide-TPP was able to induce cytotoxicity in HeLamtA3243G cybrid cells, while preserving preferential binding for oligonucleotides containing the A3243G motif from melting temperature assays. The PI polyamide-TPP also localized in the mitochondria in HeLamtA3243G cells and induced mitochondrial reactive oxygen species production, mitophagy and apoptosis in a mutation-specific fashion compared to the wild-type HeLamtHeLa cybrids; normal human dermal fibroblasts were also relatively unaffected to suggest discriminating selectivity for the mutant mitochondria, offering a novel outlook for cancer therapy via mitochondrial homing of short linear PIP-TPPs.

Chemical Approaches to the Development of Artificial Transcription Factors Based on Pyrrole-Imidazole Polyamides.

To maintain the functions of living organisms, cells have developed complex gene regulatory networks. Transcription factors have a central role in spatiotemporal control of gene expression and this has motivated us to develop artificial transcription factors that mimic their function. We found that three functions could be mimicked by applying our chemical approaches: i) efficient delivery into organelles that contain target DNA, ii) specific DNA binding to the target genomic region, and iii) regulation of gene expression by interaction with other transcription coregulators. We chose pyrrole-imidazole polyamides (PIPs), sequence-selective DNA binding molecules, as DNA binding domains, and have achieved each of the required functions by introducing other functional moieties. The developed artificial transcription factors have potential as chemical tools that can be used to artificially modulate gene expression to enable cell fate control and to correct abnormal gene regulation for therapeutic purposes.