Differential gene regulation by a synthetic vitamin D receptor ligand and active vitamin D in human cells.

Vitamin D (VD) exerts a wide variety of biological functions including calcemic activity. VD nutritional status is closely associated with the onset and development of chronic diseases. To develop a VD analog with the desired VD activity but without calcemic activity, we screened synthetic VDR antagonists. We identified 1α,25-dihydroxyvitamin D3-26-23-lactams (DLAM)-2a-d (DLAM-2s) as nuclear vitamin D receptor (VDR) ligands in a competitive VDR binding assay for 1α,25(OH)2 vitamin D3 (1α,25(OH)2D3), and DLAM-2s showed an antagonistic effect on 1α,25(OH)2 D3-induced cell differentiation in HL60 cells. In a luciferase reporter assay in which human VDR was exogenously expressed in cultured COS-1 cells, DLAM-2s acted as transcriptional antagonists. Consistently, DLAM-2s had an antagonistic effect on the 1α,25(OH)2D3-induced expression of a known VD target gene [Cytochrome P450 24A1 (CYP24A1)], and VDR bound DLAM-2s was recruited to an endogenous VD response element in chromatin in human keratinocytes (HaCaT cells) endogenously expressing VDR. In an ATAC-seq assay, the effects of 1α,25(OH)2 D3 and DLAM-2b on chromatin reorganization were undetectable in HaCaT cells, while the effect of an androgen receptor (AR) antagonist (bicalutamide) was confirmed in prostate cancer cells (LNCaP) expressing endogenous AR. However, whole genome analysis using RNA-seq and ATAC (Assay for Transposase Accessible Chromatin)-seq revealed differential gene expression profiles regulated by DLAM-2b versus 1α,25(OH)2D3. The upregulated and downregulated genes only partially overlapped between cells treated with 1α,25(OH)2D3 and those treated with DLAM-2b. Thus, the present findings illustrate a novel VDR ligand with gene regulatory activity differing from that of 1α,25(OH)2D3.

Advances in the Administration of Vitamin D Analogues to Support Bone Health and Treat Chronic Diseases.

Vitamin D (VD) exerts a wide variety of biological actions in addition to its well-known roles in calcium homeostasis. Nutritional VD deficiency induces rachitic abnormalities in growing children and osteomalacia in adults, and it has been proposed to underlie the onset and development of multiple non-communicable chronic diseases. Therefore, the administration of VD or synthetic VD analogues represents a promising therapeutic strategy; indeed, VD and a VD agonist have shown clinical promise in mitigating osteoporosis and symptoms of insufficient calcium intake. However, even though high doses of VD analogues have shown pre-clinical efficacy against several diseases, including cancers, they have not yet had wide-spread clinical success. This difference may be due to limitation of clinical doses in light of the inherent calcemic action of VD. An approach to overcome this problem involves the development of VD analogues with lower calcemic activity, which could be administered in high doses to attenuate the onset and progress of disease. In a similar strategy, selective estrogen receptor modulators have had success as anti-osteoporosis drugs, and they have shown benefit for other estrogen target organs by serving as partial antagonists or agonists of estrogen receptor α. It is thus conceivable to generate synthetic partial antagonists or agonists for the VD receptor (VDR) that would exert beneficial effects on bone and other VD target organs. In this review, we discuss the molecular basis of the development of such synthetic VDR ligands from the viewpoint of roles of VDR in gene regulation.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands.

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

Chemical targeting of G-quadruplexes in telomeres and beyond for molecular cancer therapeutics.

G-quadruplexes (G4s) are higher-order structures formed by guanine-rich sequences of nucleic acids, such as the telomeric 5'-TTAGGG-3'/5'-UUAGGG-3' repeats and those in gene regulatory regions. G4s regulate various biological events, including replication, transcription, and translation. Imbalanced G4 dynamics is associated with diseases, such as cancer and neurodegenerative diseases. Telomestatin is a natural macrocyclic compound derived from Streptomyces anulatus 3533-SV4. It interacts with the guanine quartet via π-π stacking and potently stabilizes G4. Because G4 stabilization at the telomeric repeat inhibits the telomere-synthesizing enzyme telomerase, telomestatin was originally identified as a telomerase inhibitor. Whereas non-toxic doses of telomestatin induce gradual shortening of telomeres and eventual crisis in human cancer cells, higher doses trigger prompt replication stress and DNA damage responses, resulting in acute cell death. Suppression of the transcription and translation of G4-containing genes is also implicated in the anticancer effects of telomestatin. Because telomestatin is rare, labile, and insoluble, synthetic oxazole telomestatin derivatives have been developed and verified for their therapeutic efficacies in preclinical cancer models. Furthermore, a variety of G4-stabilizing compounds have been reported as promising seeds for molecular cancer therapeutics. To improve the design of future clinical studies, it will be important to identify predictive biomarkers of drug efficacy.

Target identification of a macrocyclic hexaoxazole G-quadruplex ligand using post-target-binding visualization.

Macrocyclic hexaoxazoles (6OTDs) are G-quadruplex (G4) ligands, and some derivatives, such as L2H2-6OTD (1a) bearing two aminobutyl side chains, show cytotoxicity towards cancer cells. To identify the cellular target of 1a, we employed a post-target-binding strategy utilizing click reaction (Huisgen cyclization) between the azide-conjugated ligand L2H2-6OTD-Az (1b) and the cell-permeable dye CO-1 bearing a strained alkyne moiety and the BODIPY fluorophore under Cu-free conditions. We confirmed that introduction of the small azide group did not alter the physical or biological properties, including anti-cancer activity, of 1a, and we also demonstrated bias-free localization of CO-1. The post-binding visualization strategy suggested that L2H2-6OTD (1a) colocalized with RNA G4 in living cells.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .

Rif1 promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization activities.

Rif1 is a conserved protein regulating replication timing and binds preferentially to the vicinity of late-firing/dormant origins in fission yeast. The Rif1 binding sites on the fission yeast genome have an intrinsic potential to generate G-quadruplex (G4) structures to which purified Rif1 preferentially binds. We previously proposed that Rif1 generates chromatin architecture that may determine replication timing by facilitating the chromatin loop formation. Here, we conducted detailed biochemical analyses on Rif1 and its G4 binding. Rif1 prefers sequences containing long stretches of guanines and binds preferentially to the multimeric G4 of parallel or hybrid/mix topology. Rif1 forms oligomers and binds simultaneously to multiple G4. We present a model on how Rif1 may facilitate the formation of chromatin architecture through its G4 binding and oligomerization properties.

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole.

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.

Esterification of PQQ Enhances Blood-Brain Barrier Permeability and Inhibitory Activity against Amyloidogenic Protein Fibril Formation.

Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of α-synuclein, amyloid ß1-42 (Aß1-42), and mouse prion protein. To develop a novel drug against neurodegenerative diseases based on PQQ, it is necessary to improve the insufficient BBB permeability of PQQ. Here, we show that an esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), has twice the BBB permeability than PQQ in vitro. Moreover, PQQ-TME exhibited greater inhibitory activity against fibrillation of α-synuclein, Aß1-42, and prion protein. These results indicated that esterification of PQQ could be a useful approach in developing a novel PQQ-based amyloid inhibitor.

Author Correction: FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2.

The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: This study was in part supported by the Swiss National Foundation Grant No.: 31003A-156023 to Alessandro Sartori.

FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2.

Interstrand cross-link (ICL) hypersensitivity is a characteristic trait of Fanconi anemia (FA). Although FANCD2-associated nuclease 1 (FAN1) contributes to ICL repair, FAN1 mutations predispose to karyomegalic interstitial nephritis (KIN) and cancer rather than to FA. Thus, the biological role of FAN1 remains unclear. Because fork stalling in FAN1-deficient cells causes chromosomal instability, we reasoned that the key function of FAN1 might lie in the processing of halted replication forks. Here, we show that FAN1 contains a previously-uncharacterized PCNA interacting peptide (PIP) motif that, together with its ubiquitin-binding zinc finger (UBZ) domain, helps recruit FAN1 to ubiquitylated PCNA accumulated at stalled forks. This prevents replication fork collapse and controls their progression. Furthermore, we show that FAN1 preserves replication fork integrity by a mechanism that is distinct from BRCA2-dependent homologous recombination. Thus, targeting FAN1 activities and its interaction with ubiquitylated PCNA may offer therapeutic opportunities for treatment of BRCA-deficient tumors.

Targeting glioma stem cells in vivo by a G-quadruplex-stabilizing synthetic macrocyclic hexaoxazole.

G-quadruplex (G4) is a higher-order nucleic acid structure that is formed by guanine-rich sequences. G4 stabilization by small-molecule compounds called G4 ligands often causes cytotoxicity, although the potential medicinal impact of this effect has not been fully established. Here we demonstrate that a synthetic G4 ligand, Y2H2-6M(4)-oxazole telomestatin derivative (6OTD), limits the growth of intractable glioblastoma (grade IV glioma) and glioma stem cells (GSCs). Experiments involving a human cancer cell line panel and mouse xenografts revealed that 6OTD exhibits antitumor activity against glioblastoma. 6OTD inhibited the growth of GSCs more potently than it did the growth of differentiated non-stem glioma cells (NSGCs). 6OTD caused DNA damage, G1 cell cycle arrest, and apoptosis in GSCs but not in NSGCs. These DNA damage foci tended to colocalize with telomeres, which contain repetitive G4-forming sequences. Compared with temozolomide, a clinical DNA-alkylating agent against glioma, 6OTD required lower concentrations to exert anti-cancer effects and preferentially affected GSCs and telomeres. 6OTD suppressed the intracranial growth of GSC-derived tumors in a mouse xenograft model. These observations indicate that 6OTD targets GSCs through G4 stabilization and promotion of DNA damage responses. Therefore, G4s are promising therapeutic targets for glioblastoma.

A G-quadruplex structure at the 5' end of the H19 coding region regulates H19 transcription.

The H19 gene, one of the best known imprinted genes, encodes a long non-coding RNA that regulates cell proliferation and differentiation. H19 RNA is widely expressed in embryonic tissues, but its expression is restricted in only a few tissues after birth. However, regulation of H19 gene expression remains poorly understood outside the context of genomic imprinting. Here we identified evolutionarily conserved guanine (G)-rich repeated motifs at the 5' end of the H19 coding region that are consistent with theoretically deduced G-quadruplex sequences. Circular dichroism spectroscopy and electrophoretic mobility shift assays with G-quadruplex-specific ligands revealed that the G-rich motif, located immediately downstream of the transcription start site (TSS), forms a G-quadruplex structure in vitro. By using a series of mutant forms of H19 harboring deletion or G-to-A substitutions, we found that the H19-G-quadruplex regulates H19 gene expression. We further showed that transcription factors Sp1 and E2F1 were associated with the H19-G-quadruplex to either suppress or promote the H19 transcription, respectively. Moreover, H19 expression during differentiation of mouse embryonic stem cells appears to be regulated by a genomic H19 G-quadruplex. These results demonstrate that the G-quadruplex structure immediately downstream of the TSS functions as a novel regulatory element for H19 gene expression.

A single molecule study of a fluorescently labeled telomestatin derivative and G-quadruplex interactions.

The potential use of G-quadruplex (GQ) stabilizing small molecules as anti-cancer drugs has created a flurry of activity on various aspects of these molecules. Telomestatin and oxazole telomestatin derivatives (OTD) are some of the most prominent of such molecules, yet the underlying dynamics of their interactions with GQ and the extent of heterogeneities in these interactions are not known. We performed single molecule measurements to study binding kinetics, rotational freedom, and dwell time distributions of a Cy5-labeled OTD (L1Cy5-7OTD) as it interacted with several different GQ structures. Our measurements show that L1Cy5-7OTD dwells on more stable GQ for longer times and binds to such GQ with higher frequency. The dwell times showed a broad distribution, but were longer than a minute for a significant fraction of molecules (characteristic dwell time τ = 192 ± 15 s and τ = 98 ± 15 s for the more and less stable GQ, respectively). In addition, L1Cy5-7OTD might be able to bind to GQ in at least two different primary orientations and occasionally transition between these orientations. The dwell time in one of these orientations was significantly longer than that in the other one, suggesting different stabilities for different binding orientations.

Regulation of the vitamin D receptor by vitamin D lactam derivatives.

The active metabolite of vitamin D3 , 1α,25-dihydroxyvitamin D3 , acts as a ligand for the vitamin D receptor (VDR) and activates VDR-mediated gene expression. Recently, we characterized 1α,25-dihydroxyvitamin D3 -26,23-lactams (DLAMs), which mimic vitamin D3 metabolites, as noncalcemic VDR ligands that barely activate the receptor. In this study, we present structural insights onto the regulation of VDR function by DLAMs. X-ray crystallographic analysis revealed that DLAMs induced a large conformational change in the loop region between helices H6 and H7 in the VDR ligand-binding domain. Our structural analysis suggests that targeting of the loop region may be a new mode of VDR regulation.

Detection of DNA Methylation of G-Quadruplex and i-Motif-Forming Sequences by Measuring the Initial Elongation Efficiency of Polymerase Chain Reaction.

DNA methylation has been proposed as one of the promising biomarkers for cancer diagnosis. In this study, we developed a DNA methylation detection system utilizing G-quadruplex and i-motif-forming sequences that requires neither sodium bisulfite treatment nor methylated DNA ligands. We hypothesized that G-quadruplex and i-motif structures would be stabilized by DNA methylation and arrest DNA polymerase activity during quantitative polymerase chain reaction (qPCR). The PCR products from VEGF, RET G-quadruplex, and i-motif-forming sequences were used as templates and analyzed by qPCR. Our results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in the G-quadruplex and i-motif-forming sequences. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increased DNA methylation of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G-quadruplex and i-motif-forming sequences on genomic DNA can be detected by qPCR.

Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile.

The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which ßArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2-1.3 Å). In combination with FTIR analyses of NHase following hydration in H2 (18) O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO(-) ) atom of αCys114-SO(-) , followed by nucleophilic attack of the S(SO(-) ) atom by a ßArg56-activated water molecule to release the product amide and regenerate αCys114-SO(-) .

2-(4-Hydroxy-3-methoxyphenyl)-benzothiazole suppresses tumor progression and metastatic potential of breast cancer cells by inducing ubiquitin ligase CHIP.

Breast cancer is the most common malignancy among women and has poor survival and high recurrence rates for aggressive metastatic disease. Notably, triple-negative breast cancer (TNBC) is a highly aggressive cancer and there is no preferred agent for TNBC therapy. In this study, we show that a novel agent, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109), has ability to inhibit breast cancer cell growth and invasiveness in vitro and in vivo. In addition, YL-109 repressed the sphere-forming ability and the expression of stem cell markers in MDA-MB-231 mammosphere cultures. YL-109 increased the expression of carboxyl terminus of Hsp70-interacting protein (CHIP), which suppresses tumorigenic and metastatic potential of breast cancer cells by inhibiting the oncogenic pathway. YL-109 induced CHIP transcription because of the recruitment of the aryl hydrocarbon receptor (AhR) to upstream of CHIP gene in MDA-MB-231 cells. Consistently, the antitumor effects of YL-109 were depressed by CHIP or AhR knockdown in MDA-MB-231 cells. Taken together, our findings indicate that a novel agent YL-109 inhibits cell growth and metastatic potential by inducing CHIP expression through AhR signaling and reduces cancer stem cell properties in MDA-MB-231 cells. It suggests that YL-109 is a potential candidate for breast cancer therapy.

Identification of a Novel Compound That Suppresses Breast Cancer Invasiveness by Inhibiting Transforming Growth Factor-ß Signaling via Estrogen Receptor α.

Breast cancer is the most frequently diagnosed cancer and the leading cause of death by cancer among females worldwide. An overwhelming majority of these deaths is because of metastasis. Estrogen stimulates and promotes growth of breast tumors, whereas transforming growth factor-beta (TGF-ß) signaling promotes invasion and metastasis. We previously reported that estrogen and estrogen receptor alpha (ERα) suppressed breast cancer metastasis by inhibiting TGF-ß signaling, whereas antiestrogens that suppress breast cancer growth, such as the selective ER modulator tamoxifen (TAM) or the pure antiestrogen fulvestrant (ICI 182,780), cannot suppress TGF-ß signaling or breast cancer invasiveness. Therefore, we predicted that a compound that inhibits TGF-ß signaling but does not facilitate ERα signaling would be ideal for suppressing breast cancer invasiveness and growth. In the present study, we identified an ideal candidate compound, N-23. Like estrogen, N-23 strongly decreased expression of TGF-ß/Smad target gene plasminogen activator inhibitor-1 (PAI-1), but it did not increase the expression of ERα target gene pS2. While estrogen decreased the levels of phosphorylated Smad2 and Smad3, N-23 had no effect. In addition, TGF-ß-dependent recruitment of Smad3 to the PAI-1 gene promoter was inhibited in the presence of estrogen or N-23. We also investigated the effects of N-23 on proliferation, migration, and invasion of breast cancer cells. In contrast to estrogen, N-23 inhibited the cellular proliferation of breast cancer cells. Moreover, we showed that N-23 suppressed the migration and invasion of breast cancer cells to the same extent as by estrogen. Taken together, our findings indicate that N-23 may be a candidate compound that is effective in inhibiting breast cancer progression.

Macrocyclic polyoxazoles as G-quadruplex ligands.

This review deals with recent progress in the synthesis and evaluation of our telomestatin-inspired macrocyclic polyoxazoles as G-quadruplex (G4) ligands. The hexaoxazole derivatives (6OTDs) interact with and stabilize G4-forming oligonucleotides, depending upon the character of the side chain functional groups. Cationic functional groups are particularly effective due to their secondary interaction with phosphate in the DNA backbone. On the other hand, heptaoxazole derivatives (7OTDs) showed potent G4-binding and stabilization activity regardless of the functional groups on the side chain. A caged G4 ligand, Y2Nv2-6OTD (7), and a fluorescent G4 ligand, L1BOD-7OTD (13), have been synthesized.