PKC; staurosporine analog

Antiviral activity of protein kinase inhibitors [3]
We have investigated different protein kinase inhibitors (PKIs) with respect to their antiviral effect against HCMV. The structures of the PKIs used are shown in Fig. 1. The antiviral activity of PKI was tested by focus reduction assays using three HCMV strains. The results are listed in Table 1. The indolocarbazole derivatives K252a,K252c and Gö6976 exhibited a clear antiviral effect against the HCMV laboratory strain AD169 with IC50 values in the nanomolar range, which was markedly lower than the IC50 of GCV. In contrast, other inhibitors of serine/threonine kinases, Gö6850 (bisindoylmaleimide I), the isoquinolonesulfonamide H-7 and roscovitine, which is a strong inhibitor of the cyclin dependent kinase cdk2a, showed a much weaker antiviral activity with IC50 values in the micromolar range. Likewise, oxoflavone inhibitors of tyrosine kinases (genisteine, quercetine) provided only weak antiviral activity. Gö6976, K252a and K252c also proved to be effective against an HCMV patient isolate (A6245) and a GCV-resistant strain (HCMV-6), which contains the amino acid exchange H520-Q (Hanson et al., 1995). The IC50 values for these strains showed only minor differences within a two-fold range as compared to the respective values for AD169. In order to test the virus selectivity of the effective indolocarbazoles we also performed inhibition tests with herpes simplex virus. However, no antiviral effect could be observed (Table 1). These data indicate that Gö6976, K252a and K252c are potent and specific inhibitors of HCMV replication. In another series of experiments we studied the antiviral activity of indolocarbazoles by determination of the virus yield reduction under drug. Gö6976 and K252c reduced the virus yield from cell cultures infected at an MOI of one or higher in a dose-dependent manner by at least three orders of magnitude (Fig. 2) but did not completely abolish virus replication. Only K252a provided a complete loss of virus yield at concentrations >500 nM. No virus yield reduction was achieved using the other PKIs. Additionally, we determined the inhibitory effects of PKIs on proliferating HEL cells. For this purpose, HEL cells were seeded at low density (2000 cells/0.28 cm2-well) and allowed to proliferate in presence of PKIs for 5 days. The results are also summarized in Table 2, indicating a more pronounced antiproliferative effect of PKIs as compared to cytotoxicity. With the indolocarbazoles Gö6976 and K252c the CC50 for confluent as well as proliferating cells was 20–200-fold higher than the corresponding doses required for inhibition of HCMV replication indicating a reasonable therapeutic index for these compounds. For K252a, the observed cytotoxicity was also moderate, however, K252a exhibited a pronounced antiproliferative effect at doses >500 nM. This result might also explain the complete abrogation of HCMV replication observed at the high concentration range. Since it has already been shown that the inhibition of cdk2 by roscovitine resulted in a complete inhibition of HCMV replication (Bresnahan et al., 1997), it seems reasonable to assume that the effect of K252a is at least partially induced by interaction with cellular kinases resulting in a cell cycle arrest in treated cells.

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Effect of protein kinase inhibitors on the HCMV-encoded protein kinase pUL97 [3]
Since HCMV encodes for the functional protein kinase pUL97, we have investigated the effect of PKIs on this enzyme. We showed before that the expression of the human cytomegalovirus UL97 protein in recombinant vaccinia viruses (rVV) is a suitable system for studying all known pUL97 functions such as pUL97-dependent autophosphorylation and pUL97-dependent GCV phosphorylation (Michel et al., 1998). Furthermore, the use of rVV allows the analysis of pUL97 functions in the absence of other HCMV gene products. In order to determine the intracellular inhibition of pUL97 functions by PKIs we investigated the effects of different PKIs on pUL97-dependent GCV phosphorylation in cells infected by rVV. The pUL97-dependent GCV phosphorylation was strongly inhibited by all indolocarbazoles tested (Gö6976, K252a, K252c), while bisindoylmaleimide I (Gö6850), an other inhibitor of serine/threonine kinases, showed a much weaker inhibitory effect. No inhibition of GCV phosphorylation could be achieved by other PKIs. The IC50 values calculated for the inhibition of pUL97 dependent GCV phosphorylation by PKIs are summarized in Table 4. The inhibition of pUL97-dependent GCV phosphorylation by PKIs was strongly dose-dependent (Fig. 3(A)). In order to rule out any effect of PKIs on the quantitative expression of pUL97 in this assay, each inhibition experiment was accompanied by western blot analysis for monitoring pUL97 expression. Using the indolocarbazoles Gö6976, K252a and K252c, no influence on pUL97 expression was detected (Fig. 3(B)). Interestingly, increasing concentrations of indolocarbazoles resulted in changes of the electrophoretic mobility of pUL97 and finally in the appearance of a slightly faster migrating pUL97-band. These results are in line with data reported by Michel et al. (1999), who observed similar differences in the pUL97 electrophoretic pattern when expressing UL97 mutants which are autophosphorylation-deficient. Additionally, van Zeijl et al. (1997) have demonstrated that the dephosphorylated form of pUL97 migrates slightly faster in SDS gels than phosphorylated pUL97. In conclusion, we presume that the dose-dependent differences in the electrophoretic pattern of pUL97 may reflect an intracellular inhibition of pUL97 autophosphorylation. In consequence, we investigated the effects of PKIs on pUL97 autophosphorylation in vitro. The inhibition of pUL97 autophosphorylation was determined quantitatively by phosphoimaging, and the effects of PKIs on pUL97 autophosphorylation are also summarized in Table 4. Similarly as for the pUL97-dependent GCV phosphorylation, all indolocarbazoles tested (Gö6976, K252a, K252c) were strong inhibitors of pUL97 autophosphorylation in a dose-dependent manner. However, it should be noted that the inhibition of pUL97 autophosphorylation in vitro appeared much stronger than should be expected from the differences in electrophoretic mobility described above. Hence, we cannot rule out that there exist other factors which influence the intracellular interaction of PKIs and pUL97.

Enzyme Assays. [4]
Kinase inhibitors were tested for inhibition of β-lactamase, α-chymotrypsin, and MDH. Unless otherwise stated, assays were performed in 50 mM potassium phosphate (KPi) buffer, pH 7.0, at 25 °C. Stocks of inhibitors were typically prepared at 10 mM in dimethyl sulfoxide (DMSO). No more than 5% DMSO was present in any assay, and results were controlled for the effect of DMSO. All reactions were monitored on an HP8453 spectrophotometer.
For most β-lactamase assays, inhibitor and 1 nM enzyme were incubated for 5 min and the reaction was initiated with 200 μM nitrocefin. Nitrocefin was prepared as a 20 mM stock in DMSO. For β-lactamase assays without incubation, inhibitor and 200 μM nitrocefin were mixed, and the reaction was initiated with 1 nM enzyme. For assays with a 10-fold increase in β-lactamase, inhibitor and 10 nM enzyme were incubated for 5 min, and the reaction was initiated with 100 μM cephalothin-G-ester.15 Cephalothin-G-ester was prepared as a 10 mM stock in DMSO. Hydrolysis was monitored at 265 nm for cephalothin-G-ester and at 482 nm for nitrocefin.
For chymotrypsin assays, inhibitor and 28 nM enzyme were incubated for 5 min, and the reaction was initiated with 200 μM succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was prepared as a 20 mM stock in DMSO. Reaction progress was monitored at 410 nm. For MDH assays, inhibitor and 2 nM enzyme were incubated for 5 min and the reaction was initiated with 200 μM oxalacetate and 200 μM NADH; progress was monitored at 340 nm. 29 Oxalacetate and NADH were each prepared as 20 mM stocks in 50 mM KPi buffer, and the NADH stock contained 2 mM DTT.
Five compounds previously shown to act as promiscuous enzyme inhibitors15 were tested for inhibition of Abl1 kinase with the Z‘-lyte β kit from PanVera (Madison, WI) and a PC1 fluorimeter from ISS (Table 4). Inhibitors were dissolved to 10 mM in DMSO. A 1.2 nM Abl1 kinase was mixed with 2 μM peptide substrate and then incubated with inhibitor for 5 min. The reaction was initiated by 10 μM ATP. After 1 h, 440 nM chymotrypsin was added to cleave unphosphorylated peptide. The peptide contained a coumarin label (FRET donor) and a fluorescein label (FRET acceptor). FRET between these labels was disrupted by proteolysis, whereas phosphorylated peptide was not cleaved and retained FRET after excitation at 400 nm. 30 The ratio of unphosphorylated to phosphorylated peptide was calculated as the ratio of coumarin emission (445 nm) to fluorescein emission (520 nm).30 Emission and excitation bandwidths were 8 nm. All incubations and reactions took place at room temperature, and no reaction mixture contained more than 5% DMSO.

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Dynamic Light Scattering (DLS). [4]
Compounds were generally dissolved to 10 mM in DMSO and diluted with filtered 50 mM KPi. All compounds were analyzed with a 3 W argon ion laser at 514.4 nm with optical systems from Brookhaven Instrument Corporation. Most of the compounds were analyzed without an incubation period; quercetin was incubated at room temperature for 30 min, over which time scattering intensity increased. The laser power and integration times were comparable for all experiments. Calculation of the mean particle diameter was performed by the cumulant analysis tool of a 400-channel BI9000AT digital autocorrelator, with the last eight channels used for baseline calculation. The detector angle was 90°. Each diameter and intensity value represents four or more independent measurements at 25 °C.

Analysis of cell viability and proliferation [2]
The cytotoxicity of PKIs was determined by using a neutral red based cytotoxicity assay. Briefly, serial two-fold dilutions of the respective drugs were prepared in MEM and 100 μl of the diluted drug were added to confluent HEL cells in a 96-well microtiter plate. After incubating the plates for 5 days at 37°C in presence of drug, the MEM was removed, the cells were washed with 400 μl PBS and incubated for 3 h with 100 μl of 0.1% neutral red in PBS. The dye solution was removed and the cells were washed again with 400 μl of PBS. In order to extract the dye 200 μl of a solution containing 50% methanol and 1% acetic acid was added and incubated for 15 min at room temperature. The OD of the neutral red dye was determined using an ELISA reader at a wavelength of 550 nm and a reference wavelength of 690 nm. The CC50 was calculated by regression analysis. For analysis of cell proliferation cells were seeded at a density of 2×103 cells/well. The cells were allowed to adhere and the number of viable cells was determined exactly as described above.

[1]. Structure-activity relationships in a series of substituted indolocarbazoles: Topoisomerase I and protein kinase C inhibition and antitumoral and antimicrobial properties J. Med. Chem. 39(22), 4471-4477 (1996).

[2]. Two indolocarbazole alkaloids with apoptosis activity from a marine-derived actinomycete Z2039-2 Arch. Pharm. Res. 30(3), 270-274 (2007).

[3]. Indolocarbazoles exhibit strong antiviral activity against human cytomegalovirus and are potent inhibitors of the pUL97 protein kinase Antiviral Res. 48(1), 49-60 (2000).

[4]. Kinase inhibitors: Not just for kinases anymore Journal of Medicinal Chemistry 46, 1478-1483 (2003).

K252c is an indolocarbazole.
6,7,12,13-tetrahydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazol-5-one has been reported in Nocardiopsis, Streptomyces longisporoflavus, and other organisms with data available.

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A series of compounds structurally related to staurosporine, rebeccamycin, and corresponding aglycones was synthesized, and their activities toward protein kinase C and topoisomerases I and II were tested together with their in vitro antitumor efficiency against murine B16 melanoma and P388 leukemia cells. Their antimicrobial activities were also examined against a Gram-negative bacterium (Escherichia coli), a yeast (Candida albicans), and three Gram-positive bacteria (Bacillus cereus, Streptomyces chartreusis, and Streptomyces griseus). To avoid side effects expected with protein kinase C inhibitors, we introduced substitution on the maleimide nitrogen and/or a sugar moiety linked to one of the indole nitrogens to obtain specific inhibitors of topoisomerase I with minimal activities on protein kinase C. As expected, these structures were inefficient on topoisomerase II, and some of them exhibited a strong activity against topoisomerase I. Generally, dechlorinated compounds were found to be more active than chlorinated analogues against both purified topoisomerase I and protein kinase C. On the other hand, opposite results were obtained in the cell antiproliferative assays. These results suggest lack of cell membrane permeability in the absence of the chlorine residue or cleavage of carbon-chlorine bonds inside the cell. [1]

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Bioassay-guided fractionation of the EtOAc extract from the fermentation broth of a marine-derived actinomycete Z(2)039-2 led to the isolation of two known indolocarbazole alkaloids, K252c (1) and arcyriaflavin A (2). 1 and 2 exhibited moderate cytotoxic activities against the K562 cell line, and induced apoptotic activities at 10 and 100 microM, respectively. This is the first report on the significant apoptosis inducing effect of indolocarbazole alkaloids against K562 cancer cells.[2]

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We have analyzed a panel of protein kinase inhibitors (PKIs) and found that some indolocarbazoles (Gö6976, K252a, K252c) proved to be highly effective inhibitors of GCV-sensitive and -resistant human cytomegalovirus (HCMV) strains, but did not show any effect against herpes simplex virus. Antiviral activity was determined by focus reduction assays (IC(50) ranging from 0.009 to 0.4 microM). Other inhibitors of serine/threonine kinases (Gö6850, H-7, roscovitine) were found to be ineffective. Virus yield at 5 days after infection was reduced by three orders of magnitude with nanomolar concentrations of the indolocarbazoles. These compounds were fully effective when added up to 24 h post infection and showed reduced activity up to 72 h post infection. Cytotoxicity assays in proliferating and non-proliferating cells demonstrated that the effective antiviral concentration of these compounds was significantly lower than either antiproliferative (IC(50)/CC(50) ranging from 6.5 to 390) or cytotoxic (IC(50)/CC(50) ranging from 72. 5 to 1000) doses. The effects of PKIs on the virus-encoded protein kinase pUL97 were studied using recombinant vaccinia viruses. Indolocarbazoles strongly inhibited both pUL97 autophosphorylation (IC(50) ranging from 0.0012 to 0.013 microM) and pUL97-dependent ganciclovir phosphorylation (IC(50) ranging from 0.05 to 0.26 microM). Other inhibitors of serine/threonine kinases showed only weak (Gö6850) or no (H-7, roscovitine) effect on these pUL97 functions, while oxoflavone tyrosine kinase inhibitors had no effect at all.[3]

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Kinase inhibitors are widely employed as biological reagents and as leads for drug design. Their use is often complicated by their lack of specificity. Although binding conserved ATP sites accounts for some of their nonspecificity, some compounds inhibit proteins not known to bind ATP. It has been found that promiscuous hits from high-throughput screening may act as aggregates. To explore whether this mechanism might explain the action of widely used nonspecific kinase inhibitors, 15 such compounds were studied. Eight of these, rottlerin, quercetin, K252c, bisindolylmaleimide I, bisindolylmaleimide IX, U0126, indirubin, and indigo, inhibited three diverse non-kinase enzymes. Inhibition was time-dependent and sensitive to enzyme concentration; by light scattering, the compounds formed particles of 100-1000 nm diameter. These observations suggest that these eight kinase inhibitors, at least at micromolar concentrations, are promiscuous and act as aggregates. Results obtained from the use of these compounds at micromolar or higher concentrations against individual enzymes should be interpreted cautiously.

C20H13N3O

311.336724042892

311.106

C, 77.16; H, 4.21; N, 13.50; O, 5.14

85753-43-1

3815

White to off-white solid powder

4.527

3

1

0

24

551

0

O=C1C2C3=C(C4=C(C=2CN1)C1C(=CC=CC=1)N4)NC1C3=CC=CC=1

MEXUTNIFSHFQRG-UHFFFAOYSA-N

InChI=1S/C20H13N3O/c24-20-17-12(9-21-20)15-10-5-1-3-7-13(10)22-18(15)19-16(17)11-6-2-4-8-14(11)23-19/h1-8,22-23H,9H2,(H,21,24)

3,13,23-triazahexacyclo[14.7.0.02,10.04,9.011,15.017,22]tricosa-1,4,6,8,10,15,17,19,21-nonaen-12-one

SD 1825; K-252c; K252C; 632-917-2; 85753-43-1; Staurosporine aglycone; staurosporinone; 6,7,12,13-tetrahydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazol-5-one; HAJ5XS5HPF; K252c; staurosporine aglycone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)