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 indole-carbazole compound. 6,7,12,13-tetrahydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5-one has been reported in Nocardia, Streptomyces, and other organisms with relevant data. A series of compounds related to astrocytocin, rebeccamycin, and their corresponding aglycones were synthesized, and their activities against protein kinase C and topoisomerases I and II were tested, as well as their in vitro antitumor activity against mouse B16 melanoma cells and P388 leukemia cells. We also examined the antibacterial activity of these compounds against Gram-negative bacteria (Escherichia coli), yeasts (Candida albicans), and three Gram-positive bacteria (Bacillus cereus, Streptomyces sarcodactylis, and Streptomyces griseus). To avoid the common side effects of protein kinase C inhibitors, we introduced substituents on the nitrogen atom of maleimide and/or on the glycosyl group attached to the nitrogen atom of indole to obtain inhibitors that are specific to topoisomerase I and have very low activity against protein kinase C. As expected, these compounds were not very effective against topoisomerase II, and some of them showed strong activity against topoisomerase I. Generally, dechlorinated compounds have higher activity against purified topoisomerase I and protein kinase C than chlorinated analogs. However, the opposite results were obtained in cell proliferation inhibition assays. These results suggest that cell membrane permeability is insufficient in the absence of chlorine residues or intracellular carbon-chlorine bond breakage. [1]

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Bioactivity-guided fractionation of the ethyl acetate extract of the fermentation broth of marine actinomycete Z(2)039-2 yielded two known indolecarbazole alkaloids, K252c (1) and arcyriaflavin A (2). Compounds 1 and 2 exhibited moderate cytotoxic activity against the K562 cell line, inducing apoptosis at concentrations of 10 μM and 100 μM, respectively. This is the first report on the significant apoptosis-inducing effect of indolecarbazole alkaloids on K562 cancer cells. [2] We analyzed a series of protein kinase inhibitors (PKIs) and found that some indolecarbazole compounds (Gö6976, K252a, K252c) were highly effective against GCV-sensitive and drug-resistant human cytomegalovirus (HCMV) strains, but ineffective against herpes simplex virus. Antiviral activity was determined by a lesion reduction assay (IC50 range of 0.009 to 0.4 μM). Other serine/threonine kinase inhibitors (Gö6850, H-7, roscovitine) were ineffective. Nanomolar concentrations of indolecarbazole compounds reduced viral yield by three orders of magnitude up to 5 days post-infection. These compounds were fully effective when added within 24 hours post-infection, but their activity decreased within 72 hours post-infection. Cytotoxicity assays in both proliferating and non-proliferating cells showed that the effective antiviral concentrations of these compounds were significantly lower than antiproliferative doses (IC50/CC50 range 6.5 to 390) or cytotoxic doses (IC50/CC50 range 72.5 to 1000). The effects of pKIs on the virus-encoded protein kinase pUL97 were investigated using recombinant vaccinia virus. Indolecarbazole compounds strongly inhibited pUL97 autophosphorylation (IC50 range 0.0012 to 0.013 μM) and pUL97-dependent ganciclovir phosphorylation (IC50 range 0.05 to 0.26 μM). Other serine/threonine kinase inhibitors showed only weak (Gö6850) or no effect on these functions of pUL97 (H-7, roscovitine), while oxoflavone tyrosine kinase inhibitors were completely ineffective. [3] Kinase inhibitors are widely used as lead compounds in biological reagents and drug design. However, their application is often limited due to their lack of specificity. While binding to conserved ATP sites can explain part of their nonspecificity, some compounds inhibit proteins known not to bind to ATP. Studies have found that nonspecific compounds obtained through high-throughput screening may act as aggregates. To investigate whether this mechanism can explain the action of widely used nonspecific kinase inhibitors, we investigated 15 such compounds. These eight compounds, namely rotegrin, quercetin, K252c, bisindolylmaleimide I, bisindolylmaleimide IX, U0126, indigo red, and indigo, inhibited three different non-kinases. The inhibition was time-dependent and sensitive to enzyme concentration; these compounds formed particles with diameters of 100–1000 nm by light scattering experiments. These observations suggest that these eight kinase inhibitors are nonspecific at least at micromolar concentrations and act as aggregates. Therefore, the results of using these compounds at micromolar or higher concentrations to inhibit specific enzymes should be interpreted with caution.

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.)