In vitro and in silico studies of interaction of synthetic 2,6,9-trisubstituted purine kinase inhibitors BPA-302, BP-21 and BP-117 with liver drug-metabolizing cytochromes P450

An evaluation of possible interactions with enzymes of drug metabolism (cytochromes P450, CYP) is an important part of studies on safety and, in general, on the properties of any drug or biologically active compound. The article is focused on the preliminary metabolic study of selected 2,6,9-trisubstituted purine kinase inhibitors with significant anticancer activities which we have developed. The compounds BP-21 and BP-117 represent strong CDK inhibitors and the compound BPA-302 was developed as selective FLT3-ITD kinase inhibitor. Here, emphasis is placed on interactions of these compounds with the nine most important forms of CYP to evaluate the possibility of inhibition of these enzymes. The possibility of their inhibitory effect was studied in vitro on selected human liver microsomal CYP enzymes. The most affected enzyme was CYP2C19. Its activity dropped to 22 % of its original value by BPA 302, to 13 % by BP-21 and to 6 % by BP-117 at the highest concentration tested (250 µmol·l(-1)). The results suggest that the metabolism of concomitantly administered drugs should not be significantly affected at lower doses. Molecular docking of BPA-302 indicated that it can bind to active site of both CYP2C19 and CYP2D6 enzymes above the heme cofactor corroborating the experimental data.

Immunogold-EM analysis reveal brefeldin a-sensitive clusters of auxin in Arabidopsis root apex cells.

Immunogold electron microscopy (EM) study of Arabidopsis root apices analyzed using specific IAA antibody and high-pressure freeze fixation technique allowed, for the first time, vizualization of subcellular localization of IAA in cells assembled intactly within plant tissues. Our quantitative analysis reveals that there is considerable portion of IAA gold particles that clusters within vesicles and membraneous compartments in all root apex cells. There are clear tissue-specific and developmental differences of clustered IAA in root apices. These findings have significant consequences for our understanding of this small molecule which is controlling plant growth, development and behavior.

Design, synthesis and biological activities of new brassinosteroid analogues with a phenyl group in the side chain.

We have prepared and studied a series of new brassinosteroid derivatives with a p-substituted phenyl group in the side chain. To obtain the best comparison between molecular docking and biological activities both types of brassinosteroids were synthesized; 6-ketones, 10 examples, and B-lactones, 8 examples. The phenyl group was introduced into the steroid skeleton by Horner-Wadsworth-Emmons. The docking studies were carried out using AutoDock Vina 1.05. Plant biological activities were established using different brassinosteroid bioassays in comparison with natural brassinosteroids. Differences in the production of the plant hormone ethylene were also observed in etiolated pea seedlings after treatment with new brassinosteroids. The most active compounds were lactone 8f and 6-oxo derivatives 8c and 9c, their biological activities were comparable or even better than naturally occurring brassinolide. Finally the cytotoxicity of the new derivatives was studied using human normal and cancer cell lines.

Anti-angiogenic effects of novel cyclin-dependent kinase inhibitors with a pyrazolo[4,3-d]pyrimidine scaffold.

BACKGROUND AND PURPOSE: Cyclin-dependent kinase 5 (CDK5) has recently emerged as an attractive target in several tumour entities. Inhibition of CDK5 has been shown to have anti-angiogenic effects in vitro and in vivo. However, potent inhibitors of CDK5, which can be applied in vivo, are still scarce. We have recently developed a new series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines that show a preference for inhibiting CDK5 and tested them in vitro and in vivo in a murine model of hepatocellular carcinoma. EXPERIMENTAL APPROACH: All compounds were initially examined for effects on proliferation of HUVECs. The most potent compounds were then tested on migration, and one of them, LGR2674, was selected for assessing effects on nuclear fragmentation, cell cycle, cell viability and metabolic activity. Furthermore, LGR2674 was tested in a tube formation assay and in vivo in a murine model of hepatocellular carcinoma, induced by s.c. injection of HUH7 cells (measurement of in vivo toxicity, tumour vascularization, tumour cell proliferation and tumour size). KEY RESULTS: LGR2674 showed an EC50 in the low nanomolar range in the proliferation and migration assays. Cytotoxic effects started at 50 nM, a concentration that did not influence the cell cycle. In vivo, LGR2674 was well tolerated and caused a clear reduction in vessel density in the tumours; also tumour cell proliferation was inhibited and tumour growth retarded. CONCLUSIONS AND IMPLICATIONS: Pyrazolo[4,3-d]pyrimidine is a novel scaffold for the development of potent CDK inhibitors with in vivo potential. Such structures are good candidates for broadening our pharmacological arsenal against various tumours.

Effect of light on growth and endogenous hormones in Chlorella minutissima (Trebouxiophyceae).

Plant growth regulators (PGRs) play an important role in mediating growth and stress responses in plants. Light influences PGRs concentrations in vascular plants. The effect of light on growth and endogenous PGR concentrations in microalgae was investigated in the present study. Chlorella minutissima MACC 360 was grown in 14:10 h light:dark (L:D), continuous dark (CD) and continuous dark with the addition of 5 g L(-1) glucose (CD + G) for 48 h. Cultures were synchronized in the L:D cultures, increasing in size during the light period and dividing during the dark period. C. minutissima cells did not increase in size or undergo cell division in CD cultures. In CD + G conditions, the cultures were no longer synchronized but did continue to increase in cell size and constantly underwent cell division although fewer cells divided than in the L:D cultures. Endogenous auxin and cytokinin concentrations increased and gibberellin concentrations decreased over time in the actively growing cultures (L:D and CD + G) but did not increase in the CD cultures. The largest increase in indole content was in the CD + G cultures while the L:D cultures had the largest cytokinin increase. Brassinosteroid concentrations decreased over time in all the cultures including those grown in CD conditions. Abscisic acid (ABA) concentrations were low and only increased in the CD cultures. These results show that endogenous PGRs were affected by the light regime and/or culture growth.

Hormone profiles in microalgae: gibberellins and brassinosteroids.

Endogenous gibberellins and brassinosteroids were quantified in 24 axenic microalgae strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae and Charophyceae microalgae strains after 4 days in culture. This is the first report of endogenous gibberellins being successfully detected in microalgae. Between 18 and 20 gibberellins were quantified in all strains with concentrations ranging from 342.7 pg mg(-1) DW in Raphidocelis subcapitata MACC 317-4746.1 pg mg(-)(1) DW in Scotiellopsis terrestris MACC 44. Slower growing strains (S. terrestris MACC 44, Gyoerffyana humicola MACC 334, Nautococcus mamillatus MACC 716 and Chlorococcum ellipsoideum MACC 712) exhibited the highest gibberellin contents while lowest levels of gibberellins were found in faster growing strains (R. subcapitata MACC 317 and Coelastrum excentrica MACC 504). In all strains, the active gibberellin detected in the highest concentration was GA6, the predominant intermediates were GA15 and GA53 and the main biosynthetic end products were GA13 and GA51. Gibberellin profiles were similar in all strains except for the presence/absence of GA12 and GA12ald. To date this is the second report of endogenous brassinosteroids in microalgae. Brassinosteroids were detected in all 24 strains with concentrations ranging from 117.3 pg mg(-)(1) DW in R. subcapitata MACC 317-977.8 pg mg(-)(1) DW in Klebsormidium flaccidum MACC 692. Two brassinosteroids, brassinolide and castasterone were determined in all the strains. Generally, brassinolide occurred in higher concentrations than castasterone.

Endogenous cytokinin profiles and their relationships to between-family differences during adventitious caulogenesis in Pinus pinea cotyledons.

Caulogenesis in mature stone pine (Pinus pinea L.) cotyledons is promoted, to varying degrees depending on genotype, by exogenous application of the cytokinin (CK) benzyladenine (BA). In the present study, endogenous CK profiles of cotyledons from open-pollinated plants and two families of stone pine with widely differing organogenic capacities were monitored during caulogenesis and linked to previously characterized BA uptake and induction phases. Changes in levels of free bases, ribosides, ribotides and glucosides of both isoprenoid and aromatic CKs were followed. Before BA application, the pool of endogenous CKs in all sets of cotyledons was dominated by isoprenoid ribotides, but 1h after BA exposure, aromatic CKs (mainly active free bases and ribosides of topolins) accounted for more than 90% of the pool. BA N-glucosides were also observed, levels of which (and topolins) rose from 2d until the end of the (six-day) culture period. The CK profiles of the two selected pine families also differed, although the general trends were similar. During the first 6h, levels of BA and meta-topolin were highest in cotyledons from the family with the strongest caulogenic responses, while levels of ribotides and aromatic glucosides were highest in cotyledons from the other family.

Frequency of BCR-ABL fusion transcripts in Serbian patients with chronic myeloid leukemia.

PURPOSE: The aim of this study was to analyze the occurrence of the most frequent BCR-ABL transcript variants (b3a2, b2a2 and e1a2) in Serbian patients with chronic myeloid leukemia (CML) and compare it with the occurrence reported in other populations. METHODS: We analyzed peripheral blood and bone marrow samples of 136 Serbian patients with CML by RT-PCR and cytogenetic methods. RESULTS: In 100 patients (73.5%) the b3a2 and in 34 (25%) the b2a2 forms of BCR-ABL were detected. One (0.75%) patient was BCR-ABL negative, but in lymphoblastic transformation he expressed the e1a2 [corrected] transcript of BCR-ABL. One (0.75%) patient displayed both b2a2 and b3a2 forms of BCR-ABL. Analysis of this group according to karyotype showed b3a2 predominance (79%) in patients with classic t(9;22); b2a2 was found in 20% and both b2a2 and b3a2 forms in 1%. In variant translocations b3a2 in 65% and b2a2 in 35% of the patients were detected. In contrast, the subgroup with normal karyotype expressed slight predominance of the b2a2 form (50%); b3a2 was found in 43% of the patients and one patient (7%) displayed e1a2. CONCLUSION: Predominance of the b3a2 form in Serbian patients with CML is in concordance with other relevant investigations, conducted mostly on Caucasian ethnic groups, but in contrast to the study performed on the Mestizo ethnic group in Ecuador. Slight predominance of the b2a2 form was also noticed among the patients with normal karyotype.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

Binding of AR to SMRT/N-CoR complex and its co-operation with PSA promoter in prostate cancer cells treated with natural histone deacetylase inhibitor NaB.

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors- (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid -hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity.The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the -following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor prote.

Antioxidant and antimicrobial properties of Moltkia petraea (Tratt.) Griseb. flower, leaf and stem infusions.

Antioxidant and antimicrobial activities as well as the quantity of phenolic substances (total phenol, flavonoid and phenolic acid contents) were determined in aqueous extracts of leaves, stems and flowers of Moltkia petraea (Tratt.) Griseb. from two mountainous localities (Sveti Jure and Snijeznica) in Croatia. In addition, the profile of phenolic acids was analyzed by UPLC-MS/MS. Antioxidant activities of all extracts in different test systems, namely the DPPH radical scavenging, reducing power assay and chelating activity, increased with extract concentration. Activity of the extracts from Snijeznica in beta-carotene-linoleic acid assay did not differ from the activity of standard, BHA. The leaf extracts from Snijeznica demonstrated superior antioxidant activity in most of the assays, while the stem extract from the same locality was the most effective Fe(2+) ion chelator. In general, the extracts from Snijeznica were more effective antioxidants than the corresponding extracts from Sveti Jure. The aqueous extracts of M. petraea did not show antimicrobial activity against bacteria and fungi tested in the diffusion and dilution assays.

Antioxidant and antimicrobial properties of Teucrium arduini L. (Lamiaceae) flower and leaf infusions (Teucrium arduini L. antioxidant capacity).

Antioxidant and antimicrobial activities, as well as total phenol (TP, Folin-Ciocalteu method) and phenolic acid (UPLC-MS/MS) contents of leaf and flower infusions of Teucrium arduini L. from six different mountainous localities in Croatia (Ucka, Vosac, Sveti Jure, Snjeznica, Vaganac, Susanj) were analysed in this study. Antioxidant capacity was evaluated using the ferric reducing/antioxidant power (FRAP) assay, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assays. The antioxidant potency composite index (ACI), giving equal weight to all three methods used to quantify antioxidant capacity, was the highest for the sample from Vosac (96.7) among flower infusions, while maximum ACI (100) was determined for the infusion from Ucka among leaf infusions. Strong positive correlation was found between the total phenols and ACI for leaf (r=0.953) and flower (r=0.977) infusions. Our results point to significantly (p<0.05) different TP content between leaf and flower infusions, as well as across localities. Leaf infusions of T. arduini from Susanj exhibited marked antibacterial activity against Staphylococcus aureus, while none of the tested infusions exhibited antimicrobial activity against gram-negative bacterial species, or the tested fungal species.

Expression of programmed cell death proteins in patients with chronic myeloid leukemia.

PURPOSE: Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease developing out of pluripotent hematopoietic stem cells that contain the fusion Bcr-Abl gene. The mechanisms that lead to these changes at molecular level are still unknown as are the mechanisms that increase the proliferative capacity of these cells. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about disease progress. In our study we analyzed the presence of mutated (mut) p53 gene and the amplification of Bax proteins in patients with CML. PATIENTS AND METHODS: This study included 30 patients with CML (23 in chronic phase, 7 in blast transformation). Using immunohistochemistry with alkaline phosphatase / anti-alkaline phosphatase (APAAP) method we analyzed the expression of cell death proteins p53 and Bax in mononuclear bone marrow cells. Polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method was used to analyze the presence of mut p53 gene in mononuclear peripheral blood cells. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to analyze the presence of Bcr-Abl in peripheral blood cells. RESULTS: High expression of Bax protein was detected in all analyzed patients, but no significant differences were noticed among them. No mut p53 gene was detected in any of the analyzed samples. Bcr-Abl b3a2 protein form was detected in all patients with variant translocations. CONCLUSION: Lack of mut p53 product in the peripheral blood and bone marrow cells in patients with CML suggests that this gene plays no important role in disease pathology. Increased level of Bax protein expression is an essential characteristic of CML cells but it is not related with the clinical stage of disease.

Spatial and temporal changes in endogenous cytokinins in developing pea roots.

Germination and seedling establishment follows a distinct pattern which is partly controlled by hormones. Roots have high levels of cytokinins. By quantifying the fluctuations in endogenous cytokinins over time, further insight may be gained into the role of cytokinins during germination and seedling establishment. Radicles were excised from sterile Pisum sativum L. seeds after 30 min and 5 h imbibition. Seedlings germinated on agar were harvested after 1, 3, 6 and 9 days. The roots were divided into the root tip, root free zone, secondary root zone and from day 6, the secondary roots. Samples were purified by various chromatographic methods and endogenous cytokinins detected by LC(+)ES-MS. Benzyladenine levels doubled after 5 h imbibition and then gradually decreased over time. Low concentrations of cis-Zeatin (cZ) type cytokinins were detected in the radicle after 30 min imbibition. After 5 h imbibition, cis-zeatin riboside-5'-monophosphate had greatly increased. The total cytokinin content of the roots increased over time with the ribotides being the predominant conjugates. From day 3 onwards, there was a gradual increase in the free bases, O-glucosides and their ribosylated forms. Mainly N ( 6 )-(2-isopentenyl)adenine (iP)-type cytokinins were detected in the root tip, whereas trans-zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins were found in the secondary roots and root zone. Cytokinin biosynthesis was only detected after day 6. Biosynthesis of iP and tZ derivatives was quite rapid, whereas biosynthesis of cZ derivatives remained at a low basal level. These fluctuations in cytokinin types and concentrations suggest the cytokinins may be synthesized from various pathways in pea roots.

Sensitivity of different cell lines to phototoxic effect of disulfonated chloroaluminium phthalocyanine.

Photodynamic therapy (PDT) is a treatment for cancer involving three key components: sensitizer, light and tissue oxygen. A sensitizer is a chemical compound that can be excited by light of a specific wavelength. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was evaluated as an inducer of photodamage on NIH3T3 (mouse fibroblasts), B16 (mouse melanoma), MCF7 (human breast adenocarcinoma) and G361 (human melanoma) cell lines. A semiconductor laser was used as a source for evocation of the photodynamic effect. We report the influence of various concentrations of the sensitizer in combination with laser irradiation on the photodamage of cells. Viability of cells was determined by means of molecular probes (Calcein AM and ethidium homodimer) for fluorescence microscopy. The quantitative changes of cell viability in relation to sensitizer concentrations and laser irradiation were proved by fluorometric measurement. We detected phototoxicity evoked by laser irradiated sensitizer in all studied cell lines. In addition, the viability studies showed that G361 melanoma cells and MCF7 breast adenocarcinoma cells were more sensitive than NIH3T3 mouse fibroblasts and B16 mouse melanoma to photodynamic damage induced by ClAlPcS(2).

Breast, colon, and prostate screening in the adult population of Croatia: does rural origin matter?

INTRODUCTION: The aim of this study was to investigate the utilization of breast, colon and prostate cancer screening in the adult Croatian population in a period without national cancer screening programs, with a special interest in respondents' rural versus urban origin. METHODS: Self-reported screening utilization was investigated in the Croatian Adult Health Survey, which collected health-related information from a representative sample of the adult Croatian population. Breast cancer screening was investigated in women aged over 40 years, while colon and prostate screening was investigated in respondents aged over 50 years. The data were analysed using binary logistic regression. RESULTS: One in five women reported breast cancer screening uptake in the year preceding the survey (22.5%), while only 4.5% reported a colon screening. A total of 6.1% men reported colon screening, while 13.7% of men reported having a prostate cancer screening. Respondents with rural origin reported all sites screening utilization less frequently than those of urban origin (breast: 14.5% vs 27.4%; prostate: 9.6% vs 16.3%; colon-men: 5.7% vs 6.3%; colon-women: 3.6% vs 5.1%; respectively). Multivariable models indicated that people with higher socio-economic status more commonly reported breast and prostate cancer screening uptake. Access to health care was the only independent variable associated with colon cancer screening in men, and the strongest variable associated with colon cancer screening in women. Rural origin was associated only with lower odds of breast screening (adjusted odds ratio 0.60 [95% confidence interval 0.48-0.74]), while in the remaining models, rural origin was not a significant predictor for cancer screening uptake. CONCLUSIONS: Opportunistic cancer screening uptake is low in the Croatian adult population, with existing socio-economic differences in breast and prostate screening, and their absence in colon cancer screening. Rural origin was significantly associated with breast screening, even after adjustment to socioeconomic status and problems in access to health care. Lack of rural origin significance in the other screening sites could be related to small sample sizes of people who reported opportunistic utilization. Overall, access to health care is the strongest cancer screening predictor, and this should have a prominent role in the development of a systematic cancer screening program on a national level.

In vitro photodynamic therapy on melanoma cell lines with phthalocyanine.

Photodynamic therapy (PDT) is a new treatment modality of tumours. The photochemical interactions of sensitizer, light, and molecular oxygen produce singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radical and superoxid anion. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was tested as an inducer of photodamage. We report the production of reactive oxygen species (ROS) and the phototoxicity of ClAlPcS(2) assessed using G361 melanoma cells. A semiconductor laser (lambda=675nm, output power 21mW) was used as a source for evocation of the photodynamic effect. ROS generation and H(2)O(2) release after PDT on G361 cells were detected using probe CM-H(2)DCFDA and recorded by luminescence spectrometer. Viability studies show, that the optimum phototoxic effect tested on G361 melanoma cells was determined in the combination of laser dose of 25Jcm(-2) and phthalocyanine ClAlPcS(2) concentration of 5microg/ml. This combination of phthalocyanine concentration and corresponding radiation dose was lethal for melanoma cells.

[Erythema nodosum]. / Erythema nodosum.

The authors describe a case of 41-year-old patient with erythema nodosum as a manifestation of colitis ulcerosa. They stress the importance of complex look at this skin disease, which can be caused by many factors. Non-specific intestinal inflammation can be a cause of the erythema nodosum in 1-2%.

Photodynamic therapy with zinc-tetra(p-sulfophenyl)porphyrin bound to cyclodextrin induces single strand breaks of cellular DNA in G361 melanoma cells.

The basis of photodynamic therapy (PDT) is the phototoxicity resulting from co-action of light, sensitizer and oxygen. In this study we demonstrate in vitro phototoxicity measurement on G361 cell lines using ZnTPPS(4) sensitizer bound to cyclodextrin hpbetaCD. We have proved its photodamage effect on cancer cell lines in the visible region of spectrum. We used the halogen lamp (24V/250W) as a source of radiation. After 24h incubation of cell cultures with 10 microM ZnTPPS(4) and 1mM cyclodextrine hpbetaCD, the cells were irradiated for 7.5 min at the total irradiation dose of 12.5 Jcm(-2). Analysis of DNA damage in the cell line after PDT was proved by comet assay and using inversion fluorescent microscope with image analysis. This treatment method gave rise to DNA damage. The used radiation dose of visible light in the absence of sensitizers does not induce DNA breaks in tumour cells. In conclusion, binding of ZnTPPS(4) sensitizer to cyclodextrin hpbetaCD may improve the efficacy of PDT for the treatment of malign melanoma.

Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor.

The study describes the protein kinase selectivity profile, as well as the binding mode of olomoucine II in the catalytic cleft of CDK2, as determined from cocrystal analysis. Apart from the main cell cycle-regulating kinase CDK2, olomoucine II exerts specificity for CDK7 and CDK9, with important functions in the regulation of RNA transcription. In vitro anticancer activity of the inhibitor in a panel of tumor cell lines shows a wide potency range with a slight preference for cells harboring a wild-type p53 gene. Cell-based assays confirmed activation of p53 protein levels and events leading to accumulation of p21(WAF1). Additionally, in olomoucine II-treated cells, Mdm2 was found to form a complex with the ribosomal protein L11, which inhibits Mdm2 ubiquitin ligase function. We conclude that perturbations in RNA synthesis may lead to activation of p53 and that this contributes to the antiproliferative potency of cyclindependent kinase inhibitors.