Carboxyl-terminal domain kinases, such as casein kinase II and cell cycle-dependent kinases (CDK)

In human colon cancer cells, 5,6-Dichlorobenzimidazole riboside (10-80 μg/ml, 72 h) inhibits RNA synthesis, causing p53-dependent death [5]. Through the regulation of Mcl-1 and BclxL, 5,6-dichlorobenzimidazole riboside (10-100 μM, 72 h) promotes apoptosis in human MCF-7 breast cancer cells. and subjected caspase family members to dose- and time-dependent activation [6].

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The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a specific inhibitor for RNA polymerase II transcription in vivo and in vitro [Tamm + Sehgal (1978) Adv. Virus Res. 22, 187-258; Zandomeni & Weinmann (1984) J. Biol. Chem. 259, 14804-14811]. The effect on RNA polymerase II-specific transcription seems to be mediated by its inhibition of nuclear casein kinase II [Zandomeni, Carrera-Zandomeni, Shugar & Weinmann (1986) J. Biol. Chem. 261, 3414-3419]. Inhibition studies indicated that DRB acted as a mixed-type inhibitor with respect to casein and as a competitive inhibitor with respect to the nucleotide phosphate donor substrates. The DRB inhibition constant is 7 microM for the calf thymus casein kinase II, with regard to both ATP and GTP [1].

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Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.[2]

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Most modern chemo- and radiotherapy treatments of human cancers use the DNA damage pathway, which induces a p53 response leading to either G1 arrest or apoptosis. However, such treatments can induce mutations and translocations leading to secondary malignancies or recurrent disease, which often have a poor prognosis because of resistance to therapy. Here we report that 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK7 TFIIH-associated kinase, CKI and CKII kinases, blocking RNA polymerase II in the early elongation stage, triggers p53-dependent apoptosis in human colon adenocarcinoma cells in a transcription independent manner. The fact that DRB kills tumour-derived cells without employment of DNA damage gives rise to the possibility of the development of a new alternative chemotherapeutic treatment of tumours expressing wild type p53, with a decreased risk of therapy-related, secondary malignancies.[5]

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The effective treatment of breast cancer remains a profound clinical challenge, especially due to drug resistance and metastasis which unfortunately arise in many patients. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), as a selective inhibitor of cyclin-dependent kinase 9, was shown to be effective in inducing apoptosis in various hematopoietic malignancies. However, the anticancer efficacy of DRB against breast cancer is still unclear. Herein, we demonstrated that administration of DRB to the breast cancer cell line led to the inhibition of cellular proliferation and induction of the typical signs of apoptotic cells, including the increases in Annexin V-positive cells, DNA fragmentation, and activation of caspase-7, caspase-9, and poly (ADP ribose) polymerase (PARP). Treatment of DRB resulted in a rapid decline in the myeloid cell leukemia 1 (Mcl-1) protein, whereas levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 decreased the DRB-induced PARP cleavage, whereas knockdown of Mcl-1 enhanced the effects of DRB on PARP activation, indicating that loss of Mcl-1 accounts for the DRB-mediated apoptosis in MCF-7 cells, but not in T-47D. Furthermore, we found that co-treatment of MCF-7 cells with an inhibitor of AKT (LY294002) or an inhibitor of the proteasome (MG-132) significantly augmented the DRB-induced apoptosis. These data suggested that DRB in combination with LY294002 or MG-132 may have a greater therapeutic potency against breast cancer cells [6].

Protein Kinase Assay [2]
20-μl reactions contained 50 mM KCl, 20 mM Tris-HCl, pH 8.0, 7 mM MgCl2, 2 mM DTT, 5 mM 2-glycerophosphate, 1 μM microcystin, 3.3 μg/ml EcoRI linearized pAdH3 DNA, 100 μg/ml bovine serum albumin, 7.5 μM ATP, 4 μCi of [g32P]ATP, 1 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 100 ng of VP16 affinity column fraction 5 (except in Fig. 2A) or 0.1 μl of purified TFIIH (BTF2) HAP fraction (Gerard et al., 1991). pAdH3 contains the 2.1-kilobase SmaI-HindIII fragment of adenovirus 2, including the major late promoter. Substrates were at the following concentrations: GST-CTD at 40 μg/ml, TFIIF at 45 μg/ml, and TFIIE at 60 μg/ml. No kinase activity was detected in these recombinant substrates. Samples were preincubated for 30 min at 30°C with 5 μM unlabeled ATP (and inhibitors, where indicated) followed by addition of substrate and [g-32P]ATP. Incubation was for 1 h at 30°C. Under these conditions the kinase reaction was linear for more than 3 h. Reactions were terminated by adding 5 μl of 5 × SDS loading buffer. Fixed, dried gels were quantified by PhosphorImager. The protein kinase assay of immunoprecipitated CAK from Xenopus eggs was described previously (Poon et al., 1994). Kinase reactions were with 50 μg/ml of GST-CTD or GST-Cdk2(K33R), respectively.

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Protein Kinase Substrates [2]
Immunoaffinity-purified calf thymus pol II (Thompson et al., 1990) was a gift of J. Greenblatt. GST-CTD containing all 52 heptad repeats of the mouse CTD was produced from pGCTD (Peterson et al., 1992) or from a derivative, pET21a-GCTD. TFIIE p34 and p56 were produced from vectors supplied by Dr. R. Tjian. The p34 expression vector was modified by insertion of an oligonucleotide encoding a His6 tag into the NdeI site. The p56-His6 p34 complex was isolated by mixing bacterial cell lysates containing the two subunits for 20 min on ice prior to purification on Ni2+ agarose. TFIIF (rap74-rap30) was expressed from vectors provided by Dr. Z. Burton. The rap74 vector was modified by insertion of a His6 tag at the NcoI site and the His6 rap74-rap30 complex isolated as for TFIIE. The kinase-deficient Cdk2 substrate GST-Cdk2(K33R) was described previously (Poon et al., 1994). ATPase Assay ATPase assays were performed under protein kinase reaction conditions. 0.2 μl of TFIIH HAP fraction, 300 ng of VP16-fraction 5, or 5 μg of HeLa nuclear extract were incubated for 2 h at 30°C. Under these conditions the ATPase reaction was linear for more than 3 h. Reactions were terminated by adding 0.8 ml of ice-cold 5% activated charcoal (Sigma) in 7 mM H3P04. The mixture was centrifuged, and released inorganic phosphate was measured by counting aliquots of the supernatant.

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In Vitro Transcription [2]
RNase protection, purification of recombinant GAL4-AH and GAL4-VP16 and the plasmids pSPVA (adenovirus VA1) and pGal5-HIV2 CAT have been described (Yankulov et al., 1994). Transcription reactions (20 μl) contained 250 ng of supercoiled plasmids pGal5-HIV2 CAT and pSPVA, 80 μg of HeLa nuclear extract (Dignam et al., 1983), 100 ng of GAL4-AH or GAL4-VP16, 1 mM MgCl2, 1 mM spermidine, 4% polyethylene glycol 8000, 0.5 mM NTPs, 50 mM KCl, 12 mM Hepes, pH 7.9, 10 μM ZnCl2, 2 mM DTT, 20 mM creatine phosphate, 0.01% Nonidet P-40, 15 units of RNAguard (Pharmacia). Reactions were incubated at 30°C for 1 h, stopped by addition of α-amanitin (5 μg/ml), 1 mM CaCl2, and 2 μl of RQ DNase I (Promega). After 10 min at 30°C, SDS (0.4%) and proteinase K (400 μg/ml) were added and incubated 10 min at 37°C. The reactions were processed for RNase protection using antisense probes for HIV2 and VA (Yankulov et al., 1994).

Western Blot Analysis[5]
Cell Types: LS174T, HT29, SW48
Tested Concentrations: 80 μg/ml
Incubation Duration: 24 h
Experimental Results: diminished incorporation of [5,6-3H] uridine and increased level of p53 protein.

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Cell Viability Assay[6]
Cell Types: MCF-7, T-47D
Tested Concentrations: 10, 50, 75,100 μM
Incubation Duration: 72 h
Experimental Results: Inhibited cell-growth in a dose-dependent manner. Resulted in a higher early apoptotic population (5.7 ± 1.1 vs. 2 ± 0.4%) and late apoptotic population (15.9 ± 2.4 vs. 7.7 ± 0.9%) at a concentration of 75 μM.

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Western Blot Analysis[6]
Cell Types: MCF-7
Tested Concentrations: 75 μM
Incubation Duration: 0.5, 2, 6, 10 h
Experimental Results: decreased Mcl-1 protein levels in a time-dependent manner and increased the level of p53 after 6 h.

[1]. Zandomeni RO. Kinetics of inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole on calf thymus casein kinase II. Biochem J. 1989 Sep 1;262(2):469-73.

[2]. The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase. J Biol Chem. 1995 Oct 13;270(41):23922-5.

[3]. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene. 1999 Jan 28;18(4):1093-102.

[4]. Schang LM. Cyclin-dependent kinases as cellular targets for antiviral drugs. J Antimicrob Chemother. 2002 Dec;50(6):779-92.

[5]. RNA synthesis block by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) triggers p53-dependent apoptosis in human colon carcinoma cells. Oncogene. 1999 Oct 14;18(42):5765-72.

[6]. 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) induces apoptosis in breast cancer cells through inhibiting of Mcl-1 expression. Sci Rep. 2023 Aug 3;13(1):12621.

[7]. Synthesis and biological properties of certain 5, 6-dichlorobenzimidazole ribosides. Journal of the American Chemical Society, 1957, 79(5): 1185-1188.

An RNA polymerase II transcriptional inhibitor. This compound terminates transcription prematurely by selective inhibition of RNA synthesis. It is used in research to study underlying mechanisms of cellular regulation.

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FIIH kinase phosphorylates pol II CTD, TATA-binding protein, and the large subunits of TFIIF and TFIIE in vitro (Ohkuma and Roeder, 1994; Fig. 2B). The relative importance of these phosphorylations for transcriptional elongation is not established. The modification of a known elongation factor, TFIIF, could stimulate processivity by stabilizing its interaction with pol II which is quite labile (Price et al., 1989). A role for the CTD in elongation is suggested by the observation that pol II is hypophosphorylated when it enters the preinitiation complex and when it pauses shortly after initiation, whereas the actively elongating form is hyperphosphorylated (Lu et al., 1991; O'Brien et al., 1994; Payne et al., 1989; Weeks et al., 1993). The timing of CTD phosphorylation therefore appears to coincide with the time when 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)  is effective, during or immediately following initiation (Cisek and Corden, 1989; Kephart et al., 1992). Furthermore, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)  inhibits CTD phosphorylation in vivo (Dubois et al., 1994a, 1994b). These data are consistent with the idea that CTD phosphorylation by TFIIH is the 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) -sensitive modification (Marshall and Price, 1992; Roberts and Bentley, 1992; Bentley, 1995), which stimulates elongation by pol II. [2]

C12H12N2O4CL2

319.14068

318.017

53-85-0

5894

White to off-white solid powder

1.8±0.1 g/cm3

606.2±65.0 °C at 760 mmHg

222-224ºC

320.4±34.3 °C

0.0±1.8 mmHg at 25°C

1.750

2.05

3

5

2

20

364

4

C1=C2C(=CC(=C1Cl)Cl)N(C=N2)[C@H]3[C@@H]([C@@H]([C@H](O3)CO)O)O

XHSQDZXAVJRBMX-DDHJBXDOSA-N

InChI=1S/C12H12Cl2N2O4/c13-5-1-7-8(2-6(5)14)16(4-15-7)12-11(19)10(18)9(3-17)20-12/h1-2,4,9-12,17-19H,3H2/t9-,10-,11-,12-/m1/s1

(2R,3R,4S,5R)-2-(5,6-dichlorobenzimidazol-1-yl)-5-(hydroxymethyl)oxolane-3,4-diol

53-85-0; 5,6-Dichlorobenzimidazole riboside; DRB; Dichlororibofuranosylbenzimidazole; NSC 401575; 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole; MFCD00036785; 5,6-Dichloro-1-Beta-D-Ribofuranosyl-1h-Benzimidazole;

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)

DMSO : ~100 mg/mL (~313.34 mM)
DMF : 100 mg/mL (~313.34 mM)
Ethanol : ~7.69 mg/mL (~24.10 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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