scholarly journals Identification of Inhibitors for a Virally Encoded Protein Kinase by 2 Different Screening Systems: In Vitro Kinase Assay and In-Cell Activity Assay

2005 ◽  
Vol 10 (1) ◽  
pp. 36-45 ◽  
Author(s):  
Helmut Mett ◽  
Kerstin Hölscher ◽  
Heidrun Degen ◽  
Christina Esdar ◽  
Birgit Felden De Neumann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Cell Activity ◽  
Kinase Assay ◽  
Activity Assay ◽  
Drug Candidates ◽  
Cellular Context ◽  
In Vitro Kinase Assay

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 μM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. ( Journal of Biomolecular Screening 2005:36-45)

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

A rapid, multiplexed kinase activity assay using 8-plex iTRAQ labeling, SPE, and MALDI-TOF/TOF MS

The Analyst ◽  
2020 ◽  
Vol 145 (3) ◽  
pp. 992-1000
Author(s):  
Yu-Ching Liu ◽  
Fuu-Jen Tsai ◽  
Chao-Jung Chen
Keyword(s):  
Kinase Activity ◽  
Kinase Assay ◽  
Activity Assay ◽  
Peptide Substrates ◽  
Itraq Labeling ◽  
Cell Lysates ◽  
Maldi Tof ◽  
Kinase Activity Assay ◽  
Tof Ms

Synthesized peptide substrates with iTRAQ labeling has been used for in vitro kinase assay using cell lysates and detected by MALDI TOF/TOF MS.

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

Use of a Homology Model of the PIM-1 Kinase To Identify Variant Flavonoids Having Selective Inhibitory Activity Against PIM-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2566-2566 ◽  
Author(s):  
Michael B. Lilly ◽  
Sheldon Holder ◽  
Marina Zemskova ◽  
Jonathan Neidigh
Keyword(s):  
Small Molecule ◽  
Kinase Inhibitors ◽  
Small Molecule Inhibitors ◽  
Homology Model ◽  
Binding Energies ◽  
Kinase Assay ◽  
Ic50 Values ◽  
In Vitro Kinase Assay ◽  
Threonine Kinases

Abstract The pim family of kinases are small cytoplasmic serine/threonine kinases implicated in the development of leukemias, lymphomas, and prostate cancer. Expression of pim-1 and pim-2 is induced by several oncogenes and growth factors. These kinases confer a survival phenotype through modulation of the expression or activity of various bcl-2 family members. To identify prototype small molecule inhibitors of pim-1 we have undertaken a series of experimental and computational studies. Central to our efforts has been the development of a homology model of the PIM-1 protein, using death-associated kinase as a template. After a series of computational refinements using the CFF forcefield, we then further modified our model through experimental approaches. The members of the pim family appear to have a unique, conserved structure to the hinge region, based on its length and the presence of two invariant prolines. This observation suggested that ATP-pocket binding compounds could be identified that would have selectivity for pim-1. We used a liquid phase, ELISA-based in vitro kinase assay to measure IC50 values for a set of 14 flavonoids, a family of known kinase inhibitors. Calculated binding energies for the test flavonoids were then compared with measured IC50 values, and the model giving the highest correlations was then used for further studies. A set of 25 additional flavonoids was then examined for binding energy to the homology model. Our studies predicted that the flavonoids quercetagetin and gossypetin could be potential pim-1 inhibitors. This hypothesis was then tested by in vitro kinase assay. Indeed these two flavonoids were found to be active pim-1 inhibitors with high-nanomolar potency. Quercetagetin was found to be competitive with ATP in the kinase assay. Furthermore the two flavonoids were seen to have a degree of selectivity for pim-1 kinase, compared with related serine-threonine kinases and a tyrosine kinase. IC50 (micromolar) of flavonoids against PIM-1 and other kinases Inhibitor PIM-1 PIM-2 cAMP-dep. kinase c-ABL quercetagetin 0.33 4.0 21 >200 gossypetin 0.430 7.1 250 >200 quercetin 2.0 ND ND 5.7 In contrast the potency and pim-1-selectivity of the related flavonoid quercetin (a promiscuous inhibitor of kinases) was much inferior. SAR analysis of the flavonoid set showed that pim-1 selectivity depended on the presence of three hydrogen bond donors on the flavonoid A ring. Quercetagetin has been tested for biologic effects on factor-dependent FDCP1 cells transfected with a cDNA for human pim-1. These test cells show prolonged, pim-1-dependent survival after removal of IL-3 from the growth medium. This phenotype was completely abolished by treatment with quercetagetin (IC50 = 3-5micromolar final concentration). Flavonoids such as quercetagetin and gossypetin may serve as guides for the development of small molecule inhibitors specific for pim family kinases. Such reagents will be useful for determining the role of constitutive pim-1 expression in the development of leukemias and lymphomas.

Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3864-3864
Author(s):  
Daniel Thomas ◽  
Joanna Woodcock ◽  
Jason A Powell ◽  
Emma F Barry ◽  
Angel F Lopez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Survival ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Cytoplasmic Domain ◽  
Kinase Assay ◽  
Serine Threonine Kinase ◽  
Protein Substrates ◽  
Threonine Kinase ◽  
Gm Csf

Abstract New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays. Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts. Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.

scholarly journals Rapid ubiquitination of Syk following GPVI activation in platelets

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3918-3924 ◽  
Author(s):  
Carol A. Dangelmaier ◽  
Patricia G. Quinter ◽  
Jianguo Jin ◽  
Alexander Y. Tsygankov ◽  
Satya P. Kunapuli ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Kinase Inhibitors ◽  
Specific Activity ◽  
High Specific Activity ◽  
Kinase Assay ◽  
Intermediate Step ◽  
Glycoprotein Vi ◽  
In Vitro Kinase Assay ◽  
Deficient Mice

AbstractSpleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl–deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor γ (FcRγ)-chain–dependent platelet activation through its interaction with Syk.

scholarly journals Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

1995 ◽  
Vol 6 (9) ◽  
pp. 1145-1158 ◽  
Author(s):  
S A Barker ◽  
K K Caldwell ◽  
A Hall ◽  
A M Martinez ◽  
J R Pfeiffer ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Kinase Activity ◽  
Receptor Internalization ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Cross Linking ◽  
Membrane Ruffling

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.

scholarly journals Protein Kinase Inhibitors - Selectivity or Toxicity?

2021 ◽  
Author(s):  
Moran Grossman ◽  
Elaine Adler
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Structural Similarity ◽  
Protein Kinase Inhibitors ◽  
Catalytic Core ◽  
Future Directions ◽  
Drug Candidates ◽  
Regulation Of Cell Cycle

Protein kinases are attractive therapeutic targets for various indications including cancer, cardiovascular, neurodegenerative and autoimmune diseases. This is due to the fact that they play key roles in the regulation of cell cycle, metabolism, cell adhesion, angiogenesis, regeneration and degeneration. Protein kinase families share a common catalytic core and hence usually display clear sequence and structural similarity. These sequence and structural similarities can lead to a lack of selectivity and off-target toxicity of drug candidates. The lack of selectivity can be beneficial but can also cause adverse toxicities which result in the discontinuation of promising drug candidates. The chapter reviews the challenges and common toxicities of protein kinase inhibitors and the latest advances in in-vitro and in-silico assays to screen for selectivity. The various methods for quantifying selectivity of kinase inhibitors and future directions including emerging more selective and safer kinase inhibitors have also been discussed.

Ki23819 (KRN383•HCl) Inhibits Kinase Activity of Wild Type and Mutant FLT3 Receptor Tyrosine Kinase In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1168-1168
Author(s):  
Yukiko Komeno ◽  
Mineo Kurokawa ◽  
Yoichi Imai ◽  
Masataka Takeshita ◽  
Tomoko Matsumura ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blotting ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Assay ◽  
Wild Type ◽  
Direct Inhibition ◽  
Flt3 Inhibitor ◽  
In Vitro Kinase Assay

Abstract [Background] FLT3 is a class III receptor tyrosine kinase which is widely expressed on hematopoietic stem/progenitor cells. Two types of constitutively active FLT3 mutations have been reported to be expressed on a subset of leukemic cells; internal tandem duplications (ITD) and kinase domain mutations. The former are associated with poor prognosis in acute myeloid leukemia (AML) patients. Although several inhibitors targeting FLT3-ITD are tested in clinical trials, their cytotoxic effects are still unsatisfactory. Innate and acquired resistance is also a problem to be solved. [Purpose] To screen a novel potent FLT3 inhibitor and characterize its in vitro activity. [Materials and Methods] MOLM13 and MV4-11 cells, human leukemia cell lines expressing FLT3-ITD, were exposed to candidate compounds for 48 hours, and cytotoxic effect was assessed by colorimetric assay. Inhibitory effect on autophosphorylation was evaluated by immunoprecipitation and Western blotting. These effects were also tested in 32D cells engineered to express wild type FLT3 (FLT3-WT) or FLT3-ITD. FLT3-WT was activated with 50 ng/ml FLT3 ligand for 15 min. Proapoptotic effect was confirmed by flow cytometry with Annexin V staining. In vitro kinase assay was performed to demonstrate direct inhibition of tyrosine kinase activity of FLT3-ITD. Inhibitory effects on downstream signaling molecules, ERK and STAT5, were assessed by Western blotting. [Results] Among candidates for VEGFR inhibitors from a library, a quinoline-urea derivative Ki23819 (KRN383•HCl) was identified to specifically inhibit proliferation and induce apoptosis to MOLM13 and MV4-11 cells. Ki23819 inhibited proliferation of MV4-11 cells more effectively than SU11248, a precedent FLT3 inhibitor (IC50 <1 nM vs 3~10 nM). Similar results were obtained when MOLM13 cells were used. Ki23819 inhibited autophosphorylation of both ligand-activated FLT3-WT and FLT3-ITD (IC50 30 nM and 3 nM, respectively), and abrogated IL-3-independent proliferation of 32D cells expressing FLT3-ITD (IC50 3~10 nM). In vitro kinase assay demonstrated direct inhibition of kinase activity of FLT3-ITD (IC50 7.8 nM). This compound also inhibited ERK and STAT5 constitutively activated by FLT3-ITD. The IC50 for inhibition of phosphorylation in 32D FLT3-ITD cells was 3 nM for both proteins, which is equivalent to that for inhibition of FLT3-ITD autophosphorylation. [Conclusion] Ki23819 is a novel and potent candidate for antileukemic agents against FLT3-ITD positive AML. In vivo activity of KRN383, the free base of Ki23819, is also to be reported in this ASH meeting (Nishiyama et al.).

PKC412 Directly Inhibits the Serine/Threonine Protein Kinase Pim-1 in Cell Lines and Acute Myeloid Leukemia Cells as a Novel Mechanism of Inhibition of Multidrug Resistance

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2655-2655
Author(s):  
Yi ngqiu Xie ◽  
Mehmet Burcu ◽  
Maria R. Baer
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Protein Kinase ◽  
Cell Lines ◽  
Drug Transport ◽  
Kinase Assay ◽  
Micromolar Concentration ◽  
Mechanism Of Inhibition ◽  
In Vitro Kinase Assay

Abstract The serine/threonine protein kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral insertion site in Moloney murine leukemia virus lymphomagenesis, is frequently overexpressed in acute myeloid leukemia (AML) and other malignancies and has been implicated in regulation of both cell cycle and apoptosis. We recently demonstrated that Pim-1 also regulates drug resistance. Pim-1 phosphorylates and activates the ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp; MDR1; ABCB1) and breast cancer resistance protein (BCRP; ABCG2), which are strongly associated with clinical drug resistance in AML and other malignancies, and Pim-1 knockdown results in inhibition of drug resistance mediated by both of these proteins. Efforts are in progress to identify and characterize clinically applicable Pim-1 inhibitors. PKC412 (n-benzoyl-staurosporine; midostaurin), a staurosporine analog originally characterized as a protein kinase C (PKC) inhibitor at nanomolar concentrations, is currently being tested as an inhibitor of fms-like tyrosine kinase 3 (FLT3) signaling in AML, with 10 micromolar trough concentrations achieved in clinical trials. Staurosporine has been shown to complex with Pim-1, and we hypothesized that PKC412 might be a clinically applicable inhibitor of Pim-1 kinase and that PKC412 inhibition of Pim-1 kinase would result in inhibition of multidrug resistance mediated by both Pgp and BCRP. Pim-1 activity was measured by an in vitro kinase assay of BAD phosphorylation, and Pim-1 expression, phosphorylation and co-immunoprecipitation were measured by Western blot analysis. Transport by Pgp and by BCRP was measured by comparing uptake of the Pgp- and BCRP-specific fluorescent substrates 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] and pheophorbide A, respectively, measured by flow cytometry, in the presence and absence of the Pgp- and BCRP-specific inhibitors PSC-833 and fumitremorgin C. PKC412 at a 5 micromolar concentration inhibited Pim-1 kinase activity in Pim-1-overexpressing multidrug resistant cell lines including HL60/VCR and 8226/Dox6, which overexpress Pgp, and 8226/MR20, which overexpresses BCRP, as well as in other cell lines with Pim-1 overexpression, including K562 and MOLM14 leukemia cells. Direct inhibition of Pim-1 kinase by PKC412 was demonstrated by the in vitro kinase assay. Moreover, PKC412 inhibition of Pim-1 auto-phosphorylation in vitro provided additional evidence that PKC412 directly inhibits Pim-1. PKC412 at a 5 micromolar concentration inhibited Pim-1 binding to both Pgp and BCRP, and markedly decreased phoshorylation of both of these proteins. PKC412 inhibited drug transport by Pgp at a range of concentrations, with maximum effect at 5 micromolar, and inhibited drug transport by BCRP at concentrations of 5 micromolar and higher. We also looked at the effect of PKC412 on Pim-1 activity in pretreatment blasts from AML patients. Pim-1 was overexpressed in 6 of 7 AML samples studied, including 3 of 3 with FLT3 internal tandem duplication, and PKC412 at a 5 micromolar concentration inhibited Pim-1 activity, as demonstrated by decreased BAD phosphorylation, in all 6 AML samples with Pim-1 overexpression. Thus PKC412 directly inhibits activity of the serine/threonine protein kinase Pim-1 at a clinically applicable concentration, and PKC412 inhibition of Pim-1 decreases its binding to, and phosphorylation of, both Pgp and BCRP and decreases drug transport by both of these proteins. Inhibition of Pim-1 by PKC412 at a 5 micromolar concentration is a novel mechanism of inhibition of multidrug resistance mediated by Pgp, in addition to its known inhibition of PKC at nanomolar concentrations, and is also a mechanism of inhibition of multidrug resistance mediated by BCRP. PKC412 is a clinically applicable inhibitor of Pim-1 kinase and PKC412 and other future Pim-1 inhibitors represent novel chemosensitizing agents, as well as novel anti-leukemic agents.

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