Chb-M', an Inhibitor of the RUNX Family Binding to DNA, Induces Apoptosis in p53-Mutated Non-Small Cell Lung Cancer and Inhibits Tumor Growth and Repopulation In Vivo.

Runt-related transcription factor (RUNX) proteins are considered to play various roles in cancer. Here, we evaluated the anticancer activity of Chb-M', a compound that specifically and covalently binds to the consensus sequence for RUNX family proteins, in p53-mutated non-small cell lung cancer cells. Chb-M' killed the cancer cells by inducing apoptosis. The compound showed an anticancer effect comparable to that of the clinically used drugs alectinib and ceritinib in vivo. Notably, Chb-M' extended the cancer-free survival of mice after ending treatment more effectively than did the other two drugs. The results presented here suggest that Chb-M' is an attractive candidate as an anticancer drug applicable to the treatment of non-small cell lung cancer and various other types of cancers.

Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A-A-Mismatch-Containing DNA.

Triplet repeat diseases are caused by the abnormal elongation of repeated sequences comprising three bases. In particular, the elongation of CAG/CTG repeat sequences is thought to result in conditions such as Huntington's disease and myotonic dystrophy type 1. Although the causes of these diseases are known, fundamental treatments have not been established, and specific drugs are expected to be developed. Pyrrole imidazole polyamide (PIP) is a class of molecules that binds to the minor groove of the DNA duplex in a sequence-specific manner; because of this property, it shows promise in drug discovery applications. Earlier, it was reported that PIP designed to bind CAG/CTG repeat sequences suppresses the genes that cause triplet repeat diseases. In this study, we performed an X-ray crystal structure analysis of a complex of double-stranded DNA containing A-A mismatched base pairs and a cyclic-PIP that binds specifically to CAG/CTG sequences. Furthermore, the validity and characteristics of this structure were analyzed using in silico molecular modeling, ab initio energy calculations, gel electrophoresis, and surface plasmon resonance. With our direct observation using atomic force microscopy and DNA origami, we revealed that the PIP caused structural changes in the DNA strands carrying the expanded CAG/CTG repeat. Overall, our study provides new insight into PIP from a structural perspective.

Novel Auger-Electron-Emitting 191Pt-Labeled Pyrrole-Imidazole Polyamide Targeting MYCN Increases Cytotoxicity and Cytosolic dsDNA Granules in MYCN-Amplified Neuroblastoma.

Auger electrons can cause nanoscale physiochemical damage to specific DNA sites that play a key role in cancer cell survival. Radio-Pt is a promising Auger-electron source for damaging DNA efficiently because of its ability to bind to DNA. Considering that the cancer genome is maintained under abnormal gene amplification and expression, here, we developed a novel 191Pt-labeled agent based on pyrrole-imidazole polyamide (PIP), targeting the oncogene MYCN amplified in human neuroblastoma, and investigated its targeting ability and damaging effects. A conjugate of MYCN-targeting PIP and Cys-(Arg)3-coumarin was labeled with 191Pt via Cys (191Pt-MYCN-PIP) with a radiochemical purity of >99%. The binding potential of 191Pt-MYCN-PIP was evaluated via the gel electrophoretic mobility shift assay, suggesting that the radioagent bound to the DNA including the target sequence of the MYCN gene. In vitro assays using human neuroblastoma cells showed that 191Pt-MYCN-PIP bound to DNA efficiently and caused DNA damage, decreasing MYCN gene expression and MYCN signals in in situ hybridization analysis, as well as cell viability, especially in MYCN-amplified Kelly cells. 191Pt-MYCN-PIP also induced a substantial increase in cytosolic dsDNA granules and generated proinflammatory cytokines, IFN-α/ß, in Kelly cells. Tumor uptake of intravenously injected 191Pt-MYCN-PIP was low and its delivery to tumors should be improved for therapeutic application. The present results provided a potential strategy, targeting the key oncogenes for cancer survival for Auger electron therapy.

A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models.

Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat-derived RNAs in a noncoding region and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases. Here, we show that a CWG repeat DNA-targeting compound, cyclic pyrrole-imidazole polyamide (CWG-cPIP), suppressed the pathogenesis of coding and noncoding CWG repeat diseases. CWG-cPIP bound to the hairpin form of mismatched CWG DNA, interfering with transcription elongation by RNA polymerase through a preferential activity toward repeat-expanded DNA. We found that CWG-cPIP selectively inhibited pathogenic mRNA transcripts from expanded CWG repeats, reducing CUG RNA foci and polyglutamine accumulation in cells from patients with myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Treatment with CWG-cPIP ameliorated behavioral deficits in adeno-associated virus-mediated CWG repeat-expressing mice and in a genetic mouse model of HD, without cytotoxicity or off-target effects. Together, we present a candidate compound that targets expanded CWG repeat DNA independently of its genomic location and reduces both pathogenic RNA and protein levels. CWG-cPIP may be used for the treatment of CWG repeat diseases and improvement of clinical outcomes.

Anticancer Activities of DNA-Alkylating Pyrrole-Imidazole Polyamide Analogs Targeting RUNX Transcription Factors against p53-Mutated Pancreatic Cancer PANC-1 Cells.

The runt-related transcription factor (RUNX) family is known to play important roles in the progression of cancer. Conjugate 1, which covalently binds to the RUNX-binding sequences, was reported to inhibit the binding of RUNX proteins to their target sites and suppress cancer growth. Here, we evaluated the anticancer effects of 1 and its analogs 2-4 against p53-mutated PANC-1 pancreatic cancer cells. We found that they possessed different DNA-alkylating properties in vitro. And conjugates 1-3 were shown to have anticancer effects by inducing apoptosis in PANC-1 cells. Furthermore, conjugates 2 and 3 suppressed cancer growth in PANC-1 xenograft mice, with activity equivalent to a 50-fold dose of gemcitabine. Especially, 3 showed the highest alkylation efficiency, specificity, and better anticancer effects against pancreatic cancer than 1 in vivo without significant body weight loss. Our results revealed the potential of our compounds as new candidates for cancer therapy.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

RNA G-quadruplex organizes stress granule assembly through DNAPTP6 in neurons.

Consecutive guanine RNA sequences can adopt quadruple-stranded structures, termed RNA G-quadruplexes (rG4s). Although rG4-forming sequences are abundant in transcriptomes, the physiological roles of rG4s in the central nervous system remain poorly understood. In the present study, proteomics analysis of the mouse forebrain identified DNAPTP6 as an RNA binding protein with high affinity and selectivity for rG4s. We found that DNAPTP6 coordinates the assembly of stress granules (SGs), cellular phase-separated compartments, in an rG4-dependent manner. In neurons, the knockdown of DNAPTP6 diminishes the SG formation under oxidative stress, leading to synaptic dysfunction and neuronal cell death. rG4s recruit their mRNAs into SGs through DNAPTP6, promoting RNA self-assembly and DNAPTP6 phase separation. Together, we propose that the rG4-dependent phase separation of DNAPTP6 plays a critical role in neuronal function through SG assembly.

Substitution to hydrophobic linker and formation of host-guest complex enhanced the effect of synthetic transcription factor made of pyrrole-imidazole polyamide.

GAA repeat expansion in the first intron of the frataxin (FXN) gene represses the transcription of FXN, and that induces Friedreich's ataxia (FRDA). Pyrrole-imidazole polyamides (PIPs) are the class of oligopeptide that targets double-stranded DNA with sequence selectivity. Previously, bromodomain inhibitors such as JQ1 conjugated with PIPs were reported to selectively increase transcription. Here, we report the synthesis of a compound that increases the transcription of FXN in cells derived from an FRDA patient. The compound was effective in lower (one tenth) concentration than the compound that previously reported. High concentration of the compound is toxic, but toxicity was reduced with a host-guest complex.

Twelve Colors of Streptavidin-Fluorescent Proteins (SA-FPs): A Versatile Tool to Visualize Genetic Information in Single-Molecule DNA.

Streptavidin-fluorescent proteins (SA-FPs) are a versatile tool to visualize a broad range of biochemical applications on a fluorescence microscope. Although the avidin-biotin interaction is widely used, the use of SA-FPs has not been applied to single-molecule DNA visualization. Here, we constructed 12 bright SA-FPs for DNA staining or labeling reagents. To date, 810 FPs are available, many of which are brighter than organic dyes. In this study, 12 bright FPs were selected to construct SA-FP plasmids covering green to red colors. Their brightness ranges from 40 to 165 mM-1 cm-1. Moreover, SA-FP is brighter than FP itself because streptavidin forms a tetramer complex; thus, four FPs are in a single complex. In addition, FPs often form a dimer or a tetramer, resulting in multiple FPs in a single spot on a microscopic image. This feature is advantageous because multiple fluorescent ß-barrels on a single biotin tag provide enough brightness to be easily visualized by epifluorescence microscopy. Using SA-FPs, we visualized DNA backbones, nickase-based optical mapping, and AT-frequency profiling. Finally, we demonstrated the combination of nickase-based optical mapping using SA-FP and AT-frequency profiling.

N-terminal Cationic Modification of Linear Pyrrole-Imidazole Polyamide Improves Its Binding to DNA.

Pyrrole-imidazole polyamides (PIPs) bind to double-stranded DNA (dsDNA) with varied sequence selectivity. We synthesized linear PIPs that can bind to narrow minor grooves of polypurine/polypyrimidine sequences and target long recognition sequences but have lower molecular weights than commonly used hairpin PIPs. We modified the N-terminus of linear PIPs using several groups, including ß-alanine extension and acetyl capping. Melting curve analysis of dsDNA demonstrated that cationic modifications improved the binding affinity of the PIPs to the targeted dsDNA. In addition, circular dichroism assays revealed the characteristic spectra depending on the binding stoichiometry of the N-cationic linear PIP and dsDNA (1 : 1, monomeric; 2 : 1, dimeric). Surface plasmon resonance assays confirmed the high binding affinities of linear PIPs. These findings may aid in the design of effective linear PIPs.

Targeted elimination of mutated mitochondrial DNA by a multi-functional conjugate capable of sequence-specific adenine alkylation.

Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential to cure mitochondrial diseases, no chemical-based drugs in clinical trials are capable of selective modulation of mtDNA mutations. Here, we construct a class of compounds encompassing pyrrole-imidazole polyamides (PIPs), mitochondria-penetrating peptide, and chlorambucil, an adenine-specific DNA-alkylating reagent. The sequence-selective DNA binding of PIPs allows chlorambucil to alkylate mutant adenine more efficiently than other sites in mtDNA. In vitro DNA alkylation assay shows that our compound 8950A-Chb(Cl/OH) targeting a nonpathogenic point mutation in HeLa S3 cells (m.8950G>A) can specifically alkylate the mutant adenine. Furthermore, the compound reduces the mtDNA possessing the target mutation in cultured HeLa S3 cells. The programmability of PIPs to target different sequences could allow this class of compounds to be developed as designer drugs targeting pathogenic mutations associated with mitochondrial diseases in future studies.

Strong and Specific Recognition of CAG/CTG Repeat DNA (5'-dWGCWGCW-3') by a Cyclic Pyrrole-Imidazole Polyamide.

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (Tm ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.

Telomere-specific chromatin capture using a pyrrole-imidazole polyamide probe for the identification of proteins and non-coding RNAs.

BACKGROUND: Knowing chromatin components at a DNA regulatory element at any given time is essential for understanding how the element works during cellular proliferation, differentiation and development. A region-specific chromatin purification is an invaluable approach to dissecting the comprehensive chromatin composition at a particular region. Several methods (e.g., PICh, enChIP, CAPTURE and CLASP) have been developed for isolating and analyzing chromatin components. However, all of them have some shortcomings in identifying non-coding RNA associated with DNA regulatory elements. RESULTS: We have developed a new approach for affinity purification of specific chromatin segments employing an N-methyl pyrrole (P)-N-methylimidazole (I) (PI) polyamide probe, which binds to a specific sequence in double-stranded DNA via Watson-Crick base pairing as a minor groove binder. This new technique is called proteomics and RNA-omics of isolated chromatin segments (PI-PRICh). Using PI-PRICh to isolate mouse and human telomeric components, we found enrichments of shelterin proteins, the well-known telomerase RNA component (TERC) and telomeric repeat-containing RNA (TERRA). When PI-PRICh was performed for alternative lengthening of telomere (ALT) cells with highly recombinogenic telomeres, in addition to the conventional telomeric chromatin, we obtained chromatin regions containing telomeric repeat insertions scattered in the genome and their associated RNAs. CONCLUSION: PI-PRICh reproducibly identified both the protein and RNA components of telomeric chromatin when targeting telomere repeats. PI polyamide is a promising alternative to simultaneously isolate associated proteins and RNAs of sequence-specific chromatin regions under native conditions, allowing better understanding of chromatin organization and functions within the cell.

Application of DNA-Alkylating Pyrrole-Imidazole Polyamides for Cancer Treatment.

Pyrrole-imidazole (PI) polyamides, which target specific DNA sequences, have been studied as a class of DNA minor-groove-binding molecules. To investigate the potential of compounds for cancer treatment, PI polyamides were conjugated with DNA-alkylating agents, such as seco-CBI and chlorambucil. DNA-alkylating PI polyamides have attracted attention because of their sequence-specific alkylating activities, which contribute to reducing the severe side effects of current DNA-damaging drugs. Many of these types of conjugates have been developed as new candidates for anticancer drugs. Herein, we review recent progress into research on DNA-alkylating PI polyamides and their sequence-specific action on targets associated with cancer development.

Evaluation of the DNA Alkylation Properties of a Chlorambucil-Conjugated Cyclic Pyrrole-Imidazole Polyamide.

Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP-Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP-Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP-Chb conjugates 1 and 2. Subsequent HPLC analysis revealed that the alkylation site of conjugate 3, which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP-Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2. These results suggest that cPIP-Chbs could be novel DNA alkylating anticancer drugs.

Suppression of malignant rhabdoid tumors through Chb-M'-mediated RUNX1 inhibition.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive pediatric malignancy primarily affecting infants and young children. Intensive multimodal therapies currently given to MRT patients are not sufficiently potent to control this highly malignant tumor. Therefore, additive or alternative therapy for these patients with a poor prognosis is necessary. We herein demonstrated that the inhibition of runt-related transcription factor 1 (RUNX1) by novel alkylating conjugated pyrrole-imidazole (PI) polyamides, which specifically recognize and bind to RUNX-binding DNA sequences, was highly effective in the treatment of rhabdoid tumor cell lines in vitro as well as in an in vivo mouse model. Therefore, suppression of RUNX1 activity may be a novel strategy for MRT therapy.

Enhanced nuclear accumulation of pyrrole-imidazole polyamides by incorporation of the tri-arginine vector.

The tri-arginine moiety enhanced nuclear accumulation of a 12-ring pyrrole-imidazole polyamide (PIP) without compromising sequence-selectivity and achieved efficient repression of SOX2-downstream genes and HER2 transcription in live cells. This simple vector expands the application of long PIPs in live cells by overcoming the compound delivery problems associated with them.

A Near-Infrared Fluorogenic Pyrrole-Imidazole Polyamide Probe for Live-Cell Imaging of Telomeres.

Telomeres are closely associated with cellular senescence and cancer. Although some techniques have been developed to label telomeres in living cells for study of telomere dynamics, few biocompatible near-infrared probes based on synthetic molecules have been reported. In this study, we developed a near-infrared fluorogenic pyrrole-imidazole polyamide probe (SiR-TTet59B) to visualize telomeres by conjugating a silicon-rhodamine (SiR) fluorophore with a tandem tetramer pyrrole-imidazole polyamide targeting 24 bp in the telomeric double-stranded (ds) DNA. SiR-TTet59B was almost nonfluorescent in water but increased its fluorescence dramatically on binding to telomeric dsDNA. Using a peptide-based delivery reagent, we demonstrated the specific and effective visualization of telomeres in living U2OS cells. Moreover, SiR-TTet59B could be used to observe the dynamic movements of telomeres during interphase and mitosis. This simple imaging method using a synthetic near-infrared probe could be a powerful tool for studies of telomeres and for diagnosis.

DNA Alkylation of the RUNX-Binding Sequence by CBI-PI Polyamide Conjugates*.

Many types of molecular targeted drugs that inhibit cancer growth by acting on specific molecules have been developed. The runt-related transcription factor (RUNX) family, which induces cancer development by binding to a specific DNA sequence, has attracted attention as a new target for cancer treatment. We have developed Chb-M', which targets the RUNX-binding sequence. Chb-M' was developed by conjugating pyrrole-imidazole (PI) polyamides and chlorambucil as an anticancer agent. It was recently reported that Chb-M' had a remarkable anticancer effect in vivo. In this study, to explore the possibility of an alternative structure, we designed a new series of CBI-PI polyamides, in which seco-CBI was applied as a DNA-alkylating agent. We examined the characteristics of the CBI-PI polyamides targeting the RUNX-binding sequence and found that these conjugates have great potential for cancer treatment.

Dynamic Stabilization of DNA Assembly by Using Pyrrole-Imidazole Polyamide.

We used N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamide as an exogenous agent to modulate the formation of DNA assemblies at specific double-stranded sequences. The concept was demonstrated on the hybridization chain reaction that forms linear DNA. Through a series of melting curve analyses, we demonstrated that the binding of Py-Im polyamide positively influenced both the HCR initiation and elongation steps. In particular, Py-Im polyamide was found to drastically stabilize the DNA duplex such that its thermal stability approached that of an equivalent hairpin structure. Also, the polyamide served as an anchor between hairpin pairs in the HCR assembly, thus improving the originally weak interstrand stability. We hope that these proof-of-concept results can inspire future use of Py-Im polyamide as a molecular tool to modulate the formation of DNA assemblies.