DNA

BMH-21 inhibits Pol I transcription by binding to ribosomal DNA[1].
BMH-21 suppresses RNA polymerase I without regard to the DNA damage response[1].
BMH-21 prefers to bind to DNA sequences that are rich in GCs and intercalates into double strand (ds) DNA[1].
BMH-21 (1 μM; 3 hours) induces nucleolar stress and RPA194 degradation in a way that is independent of DNA damage[1].

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Researchers have recently described a planar heterocyclic small molecule DNA intercalator, BMH-21, that binds ribosomal DNA and inhibits RNA polymerase I (Pol I) transcription. Despite DNA intercalation, BMH-21 does not cause phosphorylation of H2AX, a key biomarker activated in DNA damage stress. Here Researchers assessed whether BMH-21 activity towards expression and localization of Pol I marker proteins depends on DNA damage signaling and repair pathways. Researchers show that BMH-21 effects on the nucleolar stress response were independent of major DNA damage associated PI3-kinase pathways, ATM, ATR and DNA-PKcs. However, testing a series of BMH-21 derivatives with alterations in its N,N-dimethylaminocarboxamide arm showed that several derivatives had acquired the property to activate ATM- and DNA-PKcs -dependent damage sensing and repair pathways while their ability to cause nucleolar stress and affect cell viability was greatly reduced. The data show that BMH-21 is a chemically unique DNA intercalator that has high bioactivity towards Pol I inhibition without activation or dependence of DNA damage stress. The findings also show that interference with DNA and DNA metabolic processes can be exploited therapeutically without causing DNA damage.[1]

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Researchers show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, Researchers find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.[2]

BMH-21 (50 mg/kg; i.p.; daily; for 6 days) inhibits the growth of HCT116 colon cancer tumors in vivo[2].

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BMH-21 also demonstrated potent antitumor activity at 25 mg/kg and 50 mg/kg in a human melanoma xenograft model and reduced Ki67 proliferation index (Figure 1G). Similarly, BMH-21 significantly inhibited HCT116 colon cancer tumor growth (Figure 1H). The tumor control ratios, calculated as change of median tumor weight over the course of treatment in the treatment group as compared to the control group were 21% and 30% for the A375 and HCT116 xenografts, respectively. BMH-21 did not cause changes in the weight of the mice or organ histology indicating that it is well tolerated. [2]

FID Assay [2]
FID assay was conducted as in Tse and Boger, 2005. Deoxyoligonucleotide DNA hairpins containing 8-bp stem region with random DNA sequences or human rDNA sequences were purchased from .... EtBr (6 μM) was added to each well in sodium acetate buffer (35 mM NaOAc, 162.8 mM NaCl, 1.75 mM EDTA, pH 5.0), followed by hairpin DNA (1.5 μM), and 2 μM BMH-21 in sodium acetate buffer containing 2% DMSO. For each experiment the ratio of EtBr to hairpin DNA was adjusted to 1 molar equivalent EtBr per 2 DNA base pairs. The 0% control wells contained neither hairpin DNA nor BMH-21, while 100% control wells contained hairpin DNA but no BMH-21. All assays were conducted in triplicate.

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rRNA Synthesis Assays [2]
Cells were labeled with 1 mM 5-FUrd (Sigma) using hypotonic shift and fixed with ice-cold methanol and acetone. Cells were blocked in 3% BSA and FUrd was detected using anti-5-BrdU antibody. DNA was stained with DAPI. Alternatively cells were labeled with ethynyl-uridine (EU) and processed according to the Click-IT protocol. The analysis of de novo synthesized rRNA was according to Pestov et al., 2008. Cells were labeled with [5, 6-3H]-uridine at 2–3 μCi/ml. Total RNA was isolated using NucleoSpin RNAII Total RNA isolation kit. 3–5 μg of total RNA was separated on a 0.8% agarose-formaldehyde gel and total 18S rRNA was detected for loading control. RNA was transferred to Hybond-N+ filter and crosslinked. The membrane was sprayed using Enhancer and exposed to film.

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In vitro Pol I Transcription Assay [2]
In vitro Pol I transcription assay was conducted as in Mayer et al., 2004. The reaction mixtures contained 500 ng of template DNA containing Pol I promoter at −347 to +385 (pHrP2/EcoRI), HeLa nuclear extract, 10 mM HEPES-KOH, (pH 8.0), 0.1 mM EDTA, 10 mM MgCl2, 300 mM KCl, 10 mM creatine phosphate, 12.5% (v/v) glycerol, 0.66 mM each of ATP, GTP, UTP and CTP. After incubation for 2 hr at 30°C, RNA was extracted, and the rRNA product was amplified using PCR primers for 5′ETS rRNA located at +2 (forward),+82 (reverse), analyzed in 8% polyacrylamide gel and stained using SYBR Gold.

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In vitro Degradation Assay [2]
In vitro degradation of RPA194 was as in Williamson et al., 2009 with slight modifications. Cells were treated with BMH-21 for 3 hr and extracts were prepared in SB-buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT, protease inhibitors), 15 mM creatine phosphate, 2 mM ATP). Reactions were carried out in a mix containing human recombinant E1 (32 ng/ml), UbcH5c (50 ng/ml), UbcH10 (10 ng/ml), ubiquitin (50 ng/ml), Ub-aldehyde and supplemented with 5 mM ATP. Reactions were initiated by addition of in vitro translated pRPA194-HA and incubated for 90–120 min at 30°C. MG132 was used at 85 μM when indicated.

The cells are kept in a humidified environment with 5% CO₂ at 37 °C. The culture medium used for U2OS osteosarcoma cells contains 15% fetal bovine serum in addition to DMEM. In triplicate, cells are plated in 96-well plates at a density of 10,000 cells per well, and the compounds are then incubated for 48 hours. Using the WST-1 cell proliferation reagent, viability is assessed.

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Viability assay[1]
Cells were plated in 96-well plates at a density of 10,000 cells/well and incubated for 48 hours followed by viability measurement using the WST-1 cell proliferation reagent according to manufacturer's protocol.

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Immunofluorescence and image analysis[1]
Cells grown on coverslips were fixed in 3.5% paraformaldehyde, permeabilized with 0.5% NP-40 and blocked in 3% BSA.The following primary antibodies were used: UBF (H-300), NCL (4E2), RPA194 (C-1), phospho-ATM, γH2AX, phospho-KAP1, phospho-DNA-PKcs. Secondary Alexa488 and Alexa594-cojugated anti-mouse and anti-rabbit antibodies were from Invitrogen. DNA was stained with DAPI. Images were captured using Axioplan2 fluorescence microscope (Zeiss) equipped with AxioCam HRc CCD-camera and AxioVision 4.5 software using EC Plan-Neofluar 20x/0.5 and 40x/0.75 objectives (Zeiss). Image analysis was conducted using FrIDA designed for the analysis of RGB color image datasets. Hue saturation and brightness ranges for green and red fluorescence channel and DNA (blue) were defined for each image set. Image intensities were determined as the fraction of positive cells divided total nuclear area as defined by DNA staining. An average of 100 cells was quantified from two fields for each sample.

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Immunoblotting[1]
Cells were lysed in 0.5% NP-40 buffer (25 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 4 mM NaF, 100 μM Na3VO4, 100 KIU/ml aprotinin, 10 μg/ml leupeptin) or RIPA lysis buffer. Proteins were separated on SDS-PAGE, blotted, probed for respective proteins and detected using ECL). The primary antibodies used for detection were NCL, RPA194. HRP-conjugated secondary antibodies and were from DAKO or Santa Cruz Biotechnology, HRP-conjugated streptavidin was from DAKO.

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Chromatin Immunoprecipitation[2]
A375 cells (1 × 107) were fixed with 1% formaldehyde, lysed, and chromatin was extracted essentially as in Denissov et al., 2011. All buffers were supplemented with 1x protease inhibitor cocktail. Chromatin was sheared to 500–1000 bp. Each IP was conducted using 100 μg DNA, 5 μg antibody, and collected using secondary antibody-coupled Dynabeads. DNA was purified with ChIP DNA Clean and Concentrator kit, and eluted to 100 μl. qPCR was conducted using 2 μl of elute in 10 μl reaction, using a final concentration of primers at 200 nM on ABI Prism7900. Primer sequences are listed in Supplemental Experimental Procedures.

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RNA Interference[2]
U2OS cells were transfected with control siRNA duplex or specific siRNAs (10 nM) using Lipofectamine RNAiMAX and incubated for 48 hr. The following siRNAs were used: POLR1A si403, si405 and control siRNA.

6-week old athymic NCr nu/nu mice, with HCT116 colorectal carcinoma xenograft
50 mg/kg
Intraperitoneal injection, daily, for 6 days

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Mouse Xenograft Studies [2]
Pharmacokinetic and toxicity studies are detailed in Supplemental Experimental Procedures. For tumor xenograft experiments, A375 (3×106) or HCT116 (2×106) cells were injected in 0.1 ml PBS to the flanks of 6-week old athymic NCr nu/nu mice and the tumors were allowed to establish until they reached 90–120 mm3. Mice were randomized to respective treatment groups (mock, BMH-21 25 mg/kg and  BMH-21  50 mg/kg). Mock treatments consisted of phosphate-citrate buffer (pH 6.0). Treatments were administered i.p. on a cycle 6 days on, one day off (A375) or daily (HCT116). Tumor weight (TW) was calculated by measuring the tumor diameter with a caliper and using the formula: TW =(𝐿 ×𝑊2)/2 where L is the tumor length and W is the tumor width. Tumor growth ratios were calculated as Δtumor weight (median) of treated group/Δtumor weight (median) control group. [2]

Pharmacokinetic and Toxicity Studies [2]
To assess potential toxicity of the compound in vivo, BMH-21 was administered i.p. once daily to male FVB/N mice for 10 days on a cycle 5 days on, two off. Mouse weight was recorded daily and their well being was monitored. Organs were collected and processed for hematoxylin-eosin staining. Pharmacokinetic (PK) studies were conducted using a single 25 mg/kg i.p. dose. Mice (n = 3) were sacrificed at various times (5, 15, 45 min, 1.5, 4, 8, 12, 18 and 24 hr) and plasma was collected. Plasma samples were analyzed by LC/MS/MS and PK parameters were determined by non-compartmental analysis from the mean data using WinNonlin version 5.3 (Pharsight Corporation, Mountain View, CA). BMH-21 plasma T1/2 was 2.5 hr. No toxicities were observed throughout the study, and weight changes of mice, monitored throughout the study, were ≤ 20%. The PK studies were supported in part by Grant Number UL1 RR 025005 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and NIH Roadmap for Medical Research, and its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.

[1]. DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response. Oncotarget. 2014 Jun 30;5(12):4361-9.

[2]. A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity. Cancer Cell. 2014 Jan 13; 25(1): 77–90.

DNA intercalation mediates the anticancer activities of widely used chemotherapeutic agents by causing DNA damage and by hindering cellular DNA metabolic processes. We show here that BMH-21, a recently discovered heteroaromatic intercalator, does not activate the cellular DNA damage response and acts independently of the damage signaling and repair pathways to activate markers of nucleolar and Pol I transcription stress. In this regard, BMH-21 differs from other known Pol I inhibitors actinomycin D and CX-5461 by causing the degradation of Pol I catalytic subunit protein RPA194, and by lacking the property to activate DDR. We describe here a set of BMH-21 derivatives and show that the majority of them in which the stacking tetracycle was maintained did not launch DDR. These findings indicate that the heterocycle per se lacks DNA damaging property despite intercalation. However, we also find that five of the derivatives analyzed had an over 10-fold increased DDR response in human cancer cells. These derivatives contain changes in the BMH-21 sidearm that lead to alterations in the sidearm charge and length, and had substantially decreased potencies to cause nucleolar stress. These findings support further development of BMH-21 as a novel class of molecules with the exceptional ability to repress a specified transcriptional process without instigating cellular DNA damage.

We have earlier shown that BMH-21-mediated activation of p53 is independent of cellular DNA damage response as measured by phosphorylation of H2AX and KAP1 and activity of ATM. These findings led us to propose that BMH-21 activities are independent of DDR. Here we studied those responses in respect of the profound activity of BMH-21 to repress Pol I transcription. By using chemical inhibitors of ATM and ATR, the major kinases sensing ss and ds DNA breaks, or ATR-defective cells, we find that neither are required for BMH-21-mediated nucleolar stress response. Furthermore, blocking of DNA-PKcs, requisite of NHEJ repair, and which hyperactivates DDR due to accumulation of DNA lesions did not reveal BMH-21-mediation of DDR or attenuated the ability of BMH-21 to target RPA194. These data support and strengthen the notion that inhibition of Pol I transcription by BMH-21 and the associated anticancer activity is independent of DDR.

Molecular modeling of BMH-21 showed that it stacks flatly between GC-bases via π-π intercalation and that its sidearm with the protonated terminal amine assumes a very flat configuration. The tetracycle lies almost parallel with the GC-bases, in contrast to the plane anthraquinone ring of doxorubicin, which is perpendicular to the DNA bases with its side chains protruding to the DNA major and minor grooves. Based on the modeling, BMH-21 does not lead to any significant size exclusion in the major or minor grooves, and is predicted to mostly to cause unwinding of the DNA helix. Given this, DNA damage directed by the derivatives could take place by several not necessarily mutually exclusive mechanisms. These include the protrusion of the side arm into either major or minor grooves, electrophilic addition of DNA bases, free radical interaction with deoxyribose, production of reactive oxygen species, or inhibiting DNA transcription or replication complexes. With this in mind, we have also investigated whether BMH-21 could act as catalytic inhibitor of topoisomerase I or topoisomerase II, without evidence of such activity. Further molecular modeling and dynamic studies will be needed to reveal BMH-21 interaction modalities with DNA.

Chromatin conformation is an important modulator of DDR. Chromatin compaction and heterochromatinization limits the DDR response, and when heterochromatin is damaged, it is repaired slower than the euchromatin. In addition, DNA intercalator doxorubicin has been shown to cause nucleosome eviction at gene promoters leading to changes in promoter activity or by direct eviction of γH2AX leading to attenuated repair. We hence considered the possibility that BMH-21 intercalation could lead to a global change in the chromatin state that desensitizes the DDR. However, BMH-21 pretreatment attenuated neither the DNA damage caused by IR-induced ds breaks nor by the CPT-type DNA lesions.[1]

C21H20N4O2

360.41

360.158

C, 69.59; H, 6.12; N, 15.46; O, 8.83

896705-16-1

3508054

Yellow to orange solid powder

1.3±0.1 g/cm3

1.668

1.57

1

4

4

27

709

0

O=C(C1C2N(C(C3C(N=2)=CC2C(=CC=CC=2)C=3)=O)C=CC=1)NCCN(C)C

BXYDVWIAGDJBEC-UHFFFAOYSA-N

InChI=1S/C21H20N4O2/c1-24(2)11-9-22-20(26)16-8-5-10-25-19(16)23-18-13-15-7-4-3-6-14(15)12-17(18)21(25)27/h3-8,10,12-13H,9,11H2,1-2H3,(H,22,26)

N-[2-(dimethylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)