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.

A selective hybrid fluorescent sensor for fructose detection based on a phenylboronic acid and BODIPY-based hydrophobicity probe.

Fructose is widely used in the food industry. However, it may be involved in diseases by generating harmful advanced glycation end-products. We have designed and synthesized a novel fluorescent probe for fructose detection by combining a phenylboronic acid group with a BODIPY-based hydrophobicity probe. This probe showed a linear fluorescence response to d-fructose concentration in the range of 100-1000 µM, with a detection limit of 32 µM, which is advantageous for the simple and sensitive determination of fructose.

Elimination of Off-Target Effect by Chemical Modification of 5'-End of siRNA.

In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5'-O-methyl-2'-deoxythymidine (X) and 5'-amino-2', 5'-dideoxythymidine (Z) at the 5'-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.

Brønsted acid-induced transannulation of the phytochemical zerumbone.

The sesquiterpene zerumbone was treated with HCl in ethyl acetate under the light-protected condition, and the time-dependent conversions were analyzed by gas chromatography. Nine products were isolated, and their structures were revealed by several NMR measurements such as 1H NMR, 13C{1H} NMR, distortionless enhancement by polarization transfer (DEPT)-135, 1H-1H correlation spectroscopy (COSY), 1H-13C heteronuclear multiple quantum coherence (HMQC), and 1H-13C heteronuclear multiple bond coherence (HMBC). The X-ray crystallography determined the stereochemistries of the three products and the two derivatives. After all, this acidic reaction was found to provide the (2Z,6E,10E)-isomer, the two HCl adducts, the two 7,6-bicyclic compounds, the valence isomers cycloheptatriene and norcaradiene, and the two dihydronaphthalenes. Based on the product analyses of the reactions from the isolated intermediates as well as the mechanistic considerations, these products were arranged into two paths: one of the paths ended in the two dihydronaphthalenes the same as previously reported under the Lewis acid condition; the other ended in the 7,6-bicyclic compound, the epimer of which was known. In addition, density functional theory (DFT) calculations indicated that the (2Z,6E,10E)-isomer was more stable than the (2E,6E,10Z)-isomer as well as that the activation energy for the isomerization at the C2-C3 double bond decreased to half by protonation. The closely examined reaction mechanisms under the simple acidic condition were established upon the intensive characterization of the intermediates and products, and these findings would add to the attractive value of zerumbone and would help understand the unknown biosynthetic pathway around sesquiterpenoids.

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.

Remarkable Potential of Zerumbone to Generate a Library with Six Natural Product-like Skeletons by Natural Material-Related Diversity-Oriented Synthesis.

Diversity-oriented synthesis (DOS) is an effective strategy for the quick creation of diverse and high three-dimensional compounds from simple starting materials. The selection of a starting material is the key to constructing useful, chemically diverse compound libraries for the development of new drugs. Here, we report a novel, general, and facile strategy for the creation of diverse compounds with high structural diversity from readily available natural products, such as zerumbone, as the synthetic starting material. Zerumbone is the major component of the essential oil from wild ginger, Zingiber zerumbet Smith. It is noteworthy that zerumbone has a powerful latent reactivity, partly because of its three double bonds, two conjugated and one isolated, and a double conjugated carbonyl group in an 11-membered ring structure. In fact, zerumbone has been shown to be a successful example of natural material-related DOS (NMRDOS). We will report that zerumbone can be converted in one chemical step from four zerumbone derivatives into rare and markedly different scaffolds by transannulation.

RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells.

The dual function of runt-related transcriptional factor 1 (RUNX1) as an oncogene or oncosuppressor has been extensively studied in various malignancies, yet its role in gastric cancer remains elusive. Up-regulation of the ErbB2/HER2 signaling pathway is frequently-encountered in gastric cancer and contributes to the maintenance of these cancer cells. This signaling cascade is partly mediated by son of sevenless homolog (SOS) family, which function as adaptor proteins in the RTK cascades. Herein we report that RUNX1 regulates the ErbB2/HER2 signaling pathway in gastric cancer cells through transactivating SOS1 expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, RUNX1 interacts with the RUNX1 binding DNA sequence located in SOS1 promoter and positively regulates it. Knockdown of RUNX1 led to the decreased expression of SOS1 as well as dephosphorylation of ErbB2/HER2, subsequently suppressed the proliferation of gastric cancer cells. We also found that our novel RUNX inhibitor (Chb-M') consistently led to the deactivation of the ErbB2/HER2 signaling pathway and was effective against several gastric cancer cell lines. Taken together, our work identified a novel interaction of RUNX1 and the ErbB2/HER2 signaling pathway in gastric cancer, which can potentially be exploited in the management of this malignancy.

RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice.

Although the function of Runt-related (RUNX) transcription factors has been well characterized in leukemogenesis and regarded as an ideal target in antileukemia strategies, the effect of RUNX-inhibition therapy on bone marrow niche cells andr its impact on the engraftment of acute myeloid leukemia (AML) cells have largely been unknown. Here, we provide evidence suggesting the possible involvement of RUNX transcription factors in the transactivation of E-selectin, a member of selectin family of cell adhesion molecules, on the vascular endothelial cells of the mice bone marrow niche. In our experiments, gene switch-mediated silencing of RUNX downregulated E-selectin expression in the vascular niche and negatively controlled the engraftment of AML cells in the bone marrow, extending the overall survival of leukemic mice. Our work identified the novel role of RUNX family genes in the vascular niche and showed that the vascular niche, a home for AML cells, could be strategically targeted with RUNX-silencing antileukemia therapies. Considering the excellent efficacy of RUNX-inhibition therapy on AML cells themselves as we have previously reported, this strategy potentially targets AML cells both directly and indirectly, thus providing a better chance of cure for poor-prognostic AML patients.

Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer.

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 IC50s of 120 nm and 63 nm, respectively. These findings suggest seco-CBI-PI polyamide conjugates are candidates for oncological therapy.

Autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells.

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-ß (CBFB) play pivotal roles in leukemogenesis, and inhibition of RUNX1 has now been widely recognized as a novel strategy for anti-leukemic therapies, it has been elusive how leukemic cells could acquire the serious resistance against RUNX1-inhibition therapies and also whether CBFB could participate in this process. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion, and their mutual interaction causes the physiological resistance against chemotherapy for acute myeloid leukemia (AML) cells. Mechanistically, p53 induced by RUNX1 gene silencing directly binds to CBFB promoter and stimulates its transcription as well as its translation, which in turn acts as a platform for the stabilization of RUNX1, thereby creating a compensative RUNX1-p53-CBFB feedback loop. Indeed, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis, and these CBFB expressions were positively correlated to those of p53. Our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted at any sides of this triangle in strategizing anti-leukemia therapies.

Genetic regulation of the RUNX transcription factor family has antitumor effects.

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent-conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

DNA Interstrand Crosslinks by H-pin Polyamide (S)-seco-CBI Conjugates.

Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin imidazole-pyrrole polyamide 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-CBI) conjugates that produce sequence-specific DNA ICLs were designed and synthesized. Conjugates with H-pin polyamide and seco-CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high-resolution denaturing gel electrophoresis and sequence-specific plasmid cleavage. One conjugate (6), which contained a chiral (S)-seco-CBI, exhibited greater sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)-CBI unit.

A Multi-target Small Molecule for Targeted Transcriptional Activation of Therapeutically Significant Nervous System Genes.

An integrated multi-target small molecule capable of altering dynamic epigenetic and transcription programs associated with the brain and nervous system has versatile applications in the regulation of therapeutic and cell-fate genes. Recently, we have been constructing targeted epigenetic ON switches by integrating sequence-specific DNA binding pyrrole-imidazole polyamides with a potent histone deacetylase inhibitor SAHA. Here, we identified a DNA-based epigenetic ON switch termed SAHA-L as the first-ever multi-target small molecule capable of inducing transcription programs associated with the human neural system and brain synapses networks in BJ human foreskin fibroblasts and 201B7-iPS cells. Ingenuity pathway analysis showed that SAHA-L activates the signaling of synaptic receptors like glutamate and γ-aminobutyric acid, which are key components of autism spectrum disorders. The long-term incubation of SAHA-L in 201B7-iPS cells induced morphology changes and promoted a neural progenitor state. Our finding suggests that the tunable SAHA-L could be advanced as a cell-type-independent multi-target small molecule for therapeutic and/or cell-fate gene modulation.

Direct observation of a deoxyadenosyl radical in an active enzyme environment.

5'-deoxyadenosyl radicals have been proposed as the first common intermediate in the molecular reaction mechanism of the family of radical S-adenosyl-l-methionine (SAM) enzymes. However, this radical species has not yet been directly observed in a catalytically active enzyme environment. In a reduced and SAM-containing C140A mutant of the spore photoproduct lyase from Geobacillus thermodenitrificans, a mutant with altered catalytic activity, we were able to identify an organic radical with pronounced hyperfine structure using electron paramagnetic resonance spectroscopy. Guided by quantum-chemical computations at the density functional theory level of theory, this radical could be tentatively assigned to a deoxyadenosyl radical, which provides first experimental evidence for this intermediate in the reaction mechanism of radical SAM enzymes.

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.

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 ß-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 ß-alanine-ß-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.

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.

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 DNA-Binding Domain Guides Distinct Chromatin-Modifying Small Molecules to Activate an Identical Gene Network.

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands.