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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hairpin pyrrole-imidazole (Py-Im) polyamides are promising medium-sized molecules that bind sequence-specifically to the minor groove of B-form DNA. Here, we synthesized a series of hairpin Py-Im polyamides and explored their binding affinities and orientation preferences to methylated DNA with the m CGG target sequence. Thermal denaturation assays revealed that the five hairpin Py-Im polyamides, which were anticipated to recognize m CGG in a forward orientation, bind to nontarget DNA, GG m C, in a reverse orientation. Therefore, we designed five Py-Im polyamides that could recognize m CGG in a reverse orientation. We found that the two Py-Im polyamides containing Im/β pairs preferentially bound to m CGG in a reverse orientation. The reverse binding Py-Im polyamide successfully inhibited TET1 binding on the methylated DNA. Taken together, this study illustrated the importance of designing reverse binding Py-Im polyamides for the target sequence, m CGG, which paved the way for Py-Im polyamides that can be used with otherwise difficult to access DNA with CG sequences., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pyrrole-imidazole polyamides (Py-Im polyamides) are cell-permeable compounds that bind to the minor groove of double-stranded DNA in a sequence-specific manner without causing denaturation of the DNA. These compounds can be used to control gene expression and to stain specific sequences in cells. Here, we review the history, structural variations, and functional investigations of Py-Im polyamides., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

N-Methylpyrrole-N-methylimidazole (PI) polyamides are a class of DNA minor groove binders with DNA sequence-specificity. DNA-alkylating PI polyamide conjugates are attractive candidates as anticancer drugs acting through DNA damage and its subsequent inhibition of cell proliferation. One example is a chlorambucil-PI polyamide conjugate targeting the runt-related transcription factor (RUNX) family. RUNX1 has pro-oncogenic properties in acute myeloid leukemia, and recently the chlorambucil-PI polyamide conjugate was demonstrated to have anticancer effects. Herein, we apply another DNA-alkylating agent, seco-CBI, to target the consensus sequence of the RUNX family. Two types of CBI conjugates were prepared and their binding properties were characterized by Bind-n-Seq analysis using a high-throughput sequencer. The sequencing data were analyzed by two methods, MERMADE and our new MR (motif identification with a reference sequence), and the resultant binding motif logos were as predicted from the pairing rules proposed by Dervan et al. This is the first report to employ the MR method on alkylating PI polyamide conjugates. Moreover, cytotoxicity of conjugates 3 and 4 against a human non-small cell lung cancer, A549, were examined to show promising IC 50 s of 120 nm and 63 nm, respectively. These findings suggest seco-CBI-PI polyamide conjugates are candidates for oncological therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are organic molecules that can recognize predetermined DNA sequences in a sequence-specific manner. Human telomeres contain regions of (TTAGGG)n repetitive nucleotide sequences at each end of chromosomes, and these regions protect the chromosome from deterioration or from fusion with neighboring chromosomes. The telomeres are disposable buffers at the ends of chromosomes that are truncated during cell division. Tandem hairpin Py-Im polyamide TH59, which recognizes human telomere sequences, was reported by Laemmli's group in 2001. Here, we synthesized three types of Py-Im polyamides 1-3 based on TH59 for specific recognition of human telomere repeat sequences. Thermal melting temperature (Tm) measurements and surface plasmon resonance analysis were used to evaluate the abilities of the three types of Py-Im polyamides to discriminate between three kinds of DNA sequences. Significantly, the results showed that polyamides 1 and 2 have better affinities to TTAAGG than to TTAGGG. In contrast, polyamide 3 displayed good specificity to human telomere sequence, TTAGGG, as expected on the basis of Py-Im binding rules., (Copyright © 2015. Published by Elsevier Ltd.)

Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3 and 4 via the introduction of an amino group into a GABA (γ-turn) contained in 3, to target CAG/CTG repeat sequences, which are associated with various hereditary disorders. A high-resolution denaturing polyacrylamide sequencing gel revealed sequence-specific alkylation both strands at the N3 of adenines or guanines in CAG/CTG repeats by conjugates 3 and 4, with 11bp recognition. In vitro transcription assays using conjugate 4 revealed that specific alkylation inhibited the progression of RNA polymerase at the alkylating sites. Chiral substitution of the γ-turn with an amino group resulted in higher binding affinity observed in SPR assays. These assays suggest that conjugates 4 with 11bp recognition has the potential to cause specific DNA damage and transcriptional inhibition at the alkylating sites., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that can recognize predetermined DNA sequences with high sequence specificity. As many eukaryotic promoter regions contain highly GC-rich sequences, it is valuable to synthesize and characterize Py-Im polyamides that recognize GC-rich motifs. In this study, we synthesized four hairpin Py-Im polyamides 1-4, which recognize 5'-GCGC-3' and investigated their binding behavior with surface plasmon resonance assay. Py-Im polyamides 2-4 contain two, one, and one β-alanine units, replacing the Py units of 1, respectively. The binding affinities of 2-4 to the target DNA increased 430, 390, and 610-fold, respectively, over that of 1. The association and dissociation rates of 2 to the target DNA were improved by 11 and 37-fold, respectively, compared with those of 1. Interestingly, the association and dissociation rates of 3 and 4 were higher than those of 2, even though the binding affinities of 2, 3, and 4 to the target DNA were comparable to each other. The binding affinity of 2 to DNA with a 2bp mismatch was reduced by 29-fold, compared with that to the matched DNA. Moreover, the binding affinities of 3 and 4 to the same mismatched DNA were reduced by 270 and 110-fold, respectively, indicating that 3 and 4 have greater specificities than 2 and are suitable as DNA-binding modules for engineered epigenetic regulation., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Development of multifunctional transcriptional activators is of increasing importance as they could trigger complicated gene networks. Recently, we developed a differential gene activating multifunctional small molecule SAHA-PIP (Sδ) by conjugating a histone deacetylase (HDAC) inhibitor, SAHA, to a selective DNA-binding pyrrole-imidazole polyamide (PIP). Epigenetic activity of Sδ was attributed to the active metal-binding (-NHOH) domain of SAHA. We synthesized a derivative of Sδ, called Jδ to evaluate the role of surface recognition domain (-phenyl) of SAHA in Sδ-mediated transcriptional activation. In vitro studies revealed that Jδ displayed potent inhibitory activity against HDAC8. Jδ retained the pluripotency gene-inducing ability of Sδ when used alone and in combination with Sδ; a notable increase in the pluripotency gene expression was observed. Interestingly, Jδ significantly induced the expression of HDAC8-controlled Otx2 and Lhx1. Our results suggest that the epigenetic activity of our multifunctional molecule could be altered to improve its efficiency as a transcriptional activator for intricate gene network(s)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fluorophores that are conjugated with N-methylpyrrole-N-methylimidazole (Py-Im) polyamides postulates versatile applications in biological and physicochemical studies. Here, we show the design and synthesis of new types of pyrene-conjugated hairpin Py-Im polyamides (1-5). We evaluated the steady state fluorescence of the synthesized conjugates (1-5) in the presence and absence of oligodeoxynucleotides 5'-CGTATGGACTCGG-3' (ODN 1) and 5'-CCGAGTCCATACG-3' (ODN 2) and observed a distinct increase in emission at 386nm with conjugates 4 and 5. Notably, conjugate 5 that contains a β-alanine linker had a stronger binding affinity (K(D)=1.73×10(-8)M) than that of conjugate 4 (K(D)=1.74×10(-6)M). Our data suggests that Py-Im polyamides containing pyrene fluorophore with a β-alanine linker at the γ-turn NH(2) position can be developed as the competent fluorescent DNA-binding probes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Epigenetic modifications that govern the gene expression are often overlooked with the design of artificial genetic switches. N-Methylpyrrole-N-methylimidazole (PI) hairpin polyamides are programmable small DNA binding molecules that have been studied in the context of gene regulation. Recently, we synthesized a library of compounds by conjugating PI polyamides with SAHA, a chromatin-modifier. Among these novel compounds, PI polyamide-SAHA conjugate 1 was shown to epigenetically activate pluripotency genes in mouse embryonic fibroblasts. Here, we report the synthesis of the derivatives of conjugate 1 and demonstrate that these epigenetically active molecules could be developed to improve the induction of pluripotency factors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To investigate the effect of elongating base-pair (bp) recognition sequences, we synthesized N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates with eight-bp recognition (3-5). The DNA alkylating activities of conjugates 3-5 were evaluated by high-resolution denaturing polyacrylamide gel electrophoresis with a 208-bp DNA fragment. Conjugates 3-5 showed high alkylating activities at nanomolar concentrations. We then addressed the following issue about PI conjugates. Generally, PI polyamide conjugates hardly dissolve in aqueous solution. To improve the aqueous solubility, by the introduction of hydrophilic groups, we synthesized PI polyamide conjugates that were modified with a seco-CBI moiety (6-11). Conjugates 9-11 that were modified by methoxypolyethylene glycol (PEG) 750 acquired moderate solubility and stability in aqueous solution. In addition, conjugates 10 and 11 had high cytotoxicity against A549 and DU145., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We designed and synthesized human telomere alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1-6). The C-type conjugates 1-3 possessed a chlorambucil moiety at the C terminus, whereas the N-type conjugates 4-6 had one of these moieties at the N terminus. The DNA alkylating activity of these conjugates was evaluated by high-resolution denaturing polyacrylamide gel electrophoresis using a 220bp DNA fragment containing the human telomere repeat sequence 5'-(GGGTTA)(4)-3'/5'-(TAACCC)(4)-3'. C-type conjugates are designed to alkylate the G-rich-strand-containing 5'-GGGTTA-3' and N-type conjugates were designed to alkylate the complementary C-rich strand-containing 5'-TAACCC-3' sequence. The difference between conjugates 1-3 and 4-6 lies in the linker region between the polyamide moiety and chlorambucil. Conjugates 1 and 4 efficiently alkylated the 5'-GGTTAGGGTTA-3' and 5'-CCCTAACCCTAA-3' sequences, respectively, by recognizing 11bp in the presence of distamycin A (Dist), in a heterotrimeric manner: one long alkylating polyamide conjugate (1-6) and two short partners (Dist)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide-chlorambucil conjugates 1-4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py-Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1-4 alkylated DNA at the sequences recognized by the Py-Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide-seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py-Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide-seco-CBI conjugate 5 was similar to that of polyamide-chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1-4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

In order to investigate the influence of molecular size and pyrrole (Py)/imidazole (Im) content on the cell permeability of Py-Im-polyamide-fluorescein conjugates we systematically designed the Py-polyamides and Im-polyamides. Flow cytometric analysis revealed that Py-polyamides, even those with large molecular size, P-15 and P-18, showed good cellular uptake, but Im-polyamides showed very poor uptake. Fluorescence microscopy revealed that conjugate P-6 exhibited nuclear localization, while P-18 showed less nuclear stain but intracellular localization, suggesting that increased molecular size is one of the determinants in reducing nuclear access. Furthermore, results for hairpin polyamide conjugates H-1, H-2, and H-3 containing different Py/Im content indicated that cellular uptake increases as the Im residue is reduced. It appears that Py-Im-polyamide has general properties regardless of whether they have a linear or a hairpin structure., (Copyright 2009 Elsevier Ltd. All rights reserved.)

We designed and synthesized conjugates between pyrrole-imidazole polyamides and seco-CBI that alkylate within the coding regions of the histone H4 genes. DNA alkylating activity on the histone H4 fragment and cellular effects against K562 chronic myelogenous leukemia cells were investigated. One of the conjugates, 5-CBI, showed strong DNA alkylation activity and good sequence specificity on a histone H4 gene fragment. K562 cells treated with 5-CBI down-regulated the histone H4 gene and induced apoptosis efficiently. Global gene expression data revealed that a number of histone H4 genes were down-regulated by 5-CBI treatment. These results suggest that sequence-specific DNA alkylating agents may have the potential of targeting specific genes for cancer chemotherapy., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

We designed and synthesized alkylating conjugates 5-7 and their partner N-methylpyrrole-N-methylimidazole (PI) polyamides 8, 9. The DNA alkylating activities of conjugates 5-7 were evaluated by high-resolution denaturing polyacrylamide gel electrophoresis with a 219 base pair (bp) DNA fragment containing the human telomere repeat sequence. Conjugate 5 efficiently alkylated the sequence, 5'-GGTTAGGGTTA-3', in the presence of partner PI polyamide 8 or distamycin A (Dist). In contrast, the heterodimer system of 5 with 9 showed very weak alkylating activity. Accordingly, this heterotrimeric system of 5 with two short partners is an expedient way to attain improved precision and extension of the recognition of DNA sequences.

Perylene-conjugated pyrrole (Py)-polyamide 2 was designed and synthesized using the Fmoc solid-phase synthesis and a subsequent Sonogashira coupling reaction with 3-bromoperylene. Interestingly, conjugate 2 did not luminesce in water at 313 nm irradiation but was turned on in the presence of target double-stranded (ds) DNA, and showed strong emission with increasing DNA concentration, in particularly, by the binding to the target telomere sequences through heterodimer formation with partner 3. Importantly, the excitation spectrum of 2 clearly indicates that the Py and Imidazole (Im) moieties in the polyamide effectively sensitize the perylene moiety to give rise to fluorescence emission. Energy transfer would occur from the Py moiety to the perylene. Thus, screening of perylene-conjugates will allow us to develop a novel "molecular light switch" with sequence-specificity.

Methods for sequence-specific detection in double-stranded DNA (dsDNA) are becoming increasingly useful and important as diagnostic and imaging tools. Recently, we designed and synthesized pyrrole (Py)-imidazole (Im) polyamides possessing two pyrene moieties, 1, which showed an increased excimer emission in the presence of (CAG)(12)-containing oligodeoxynucleotides (ODN) 1 and 2. In this study, we synthesized bis-pyrenyl Py-Im polyamides with rigid linkers 2, 3, and 4 to improve their fluorescence properties. Among the conjugates, 2 showed a marked increase in excimer emission, which was dependent on the concentration of the target ODN and the number of CAG repeats in the dsDNA. Unlike conjugate 1, which has flexible linkers, the excimer emission intensity of 2 was retained at over 85%, even after 4h. Py-Im polyamides have the potential to be important diagnostic molecules for detecting genetic differences between individuals.

To investigate the effect of incorporation of beta-alanine in alkylating N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide, seco-CBI conjugates 2-8 were synthesized by an Fmoc solid-phase method and subsequent coupling with an alkylating moiety. DNA-alkylating activities of conjugates 2-8 were evaluated by high-resolution denaturing gel electrophoresis with 202-base pair (bp) DNA fragments. Alkylation by conjugates 2 and 3, which have antiparallel pairings of beta-alanine (beta) opposite beta (beta/beta) and Py/beta, occurred mainly at the adenine (A) of the matching sequences, 5'-AGCTCCA-3' (site 1) and 5'-AGCACCA-3' (site 3). However, conjugate 4, with beta/Py, did not show any DNA-alkylating activities. Similarly, conjugate 5, which possessed a Py/Py pair, weakly alkylated the matching sites at micromolar concentrations. Conjugates 6 and 7, which possessed beta/beta and Py/beta pairs, respectively, alkylated at the A of the matching sequences, 5'-ACTACCA-3' (site 2) and 5'-ACAACCA-3' (site 4). In contrast, conjugated 8, with a Py/Py pair, showed lower activity and less alkylated DNA at sites 2 and 4 with mismatched alkylation at site 1 at a higher concentration than that of 6 and 7. These results demonstrate that incorporation of beta-alanine is required for the sequence-specific alkylation by seco-CBI Py-Im conjugates with a seven-base pair sequence.

Five N-methylpyrrole-N-methylimidazole (Py-Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of conjugates 1-5 was examined in the presence and absence of (CAG)(12)-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the conjugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at 475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concentration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon binding in the minor groove of DNA.

Purpose: We have developed infrared fluorescent labeling agents and infrared-ray fluorescence endoscopes to establish a novel diagnostic technique. Since the fluorescence intensity of the initial labeled antibody (ICG-sulfo-OSu-labeled antibody) was not sufficient for practical use, we synthesized indocyanine green acylthiazolidinethione (ICG-ATT), which was expected to label various target molecules having amino groups efficiently., Materials and Methods: To confirm imaging of infrared fluorescence intensity of ICG-ATT- and ICG-sulfo-OSu-labeled anti-MUC1 antibodies, cotton thread was soaked in various concentrations of the antibody solution in 0.1M PBS, and observed under the epi-illumination infrared fluorescence microscope. Localization and the intensity of infrared fluorescence and DAB coloring was compared in paraffin sections of human gastric mucosa., Results: In the study of cotton threads, both labeled antibodies showed relatively clear infrared fluorescence, and significant difference was not observed between the two antibodies. ICG-ATT-labeled anti-MUC1 antibody produced stronger staining than that by ICG-sulfo-OSu-labeled antibody. Localization pattern of infrared fluorescent staining was in good agreement with that by the conventional method with oxidized DAB staining., Conclusion: ICG-ATT is useful as a fluorescent-labeling agent for diagnosis of microcancers by infrared fluorescence endoscopes.

A novel hairpin polyamide-cyclopropapyrroloindole (CPI) conjugate PyImImIm-gamma-PyPyPyLDu86 (conjugate 11), which targets human telomere repeats d(TTAGGG)(n)/d(CCCTAA)(n), was synthesized. High resolution denaturing polyacrylamide gel electrophoresis using 44 bp DNA fragments and HPLC product analysis of a synthetic nonanucleotide demonstrated that conjugate 11 alkylates the target adenine in the telomere repeats, 5'-CCCTAA-3'. Examination of the antitumor activity of conjugate 11 using a panel of 39 cancer cell lines demonstrated that the average concentration of conjugate 11 required for 50% growth inhibition was 5.75 microM, which is superior to pepleomycin and bleomycin and comparable to cisplatin.

Fluorescence intensity of indocyanine green (ICG) derivative (ICG-sulfo-OSu) was too low for its use to detect microlesions. Therefore, we examined the effects of reinforcement agents on ICG-sulfo-OSu labeled antibodies. Solutions of distearoylphosphatic acid sodium salt (DSPA) and octylglucoside (OG) in physiological phosphate buffered saline (PBS) were found to increase the intensity of fluorescence of ICG-sulfo-OSu labeled antibodies, with shift in the fluorescence peak wavelength from 804 to 821 nm.