Hideya Komatani

Imidazopyridine derivatives as potent and selective Polo-like kinase (PLK) inhibitors

A novel class of imidazopyridine derivatives was designed as PLK1 inhibitors. Extensive SAR studies supported by molecular modeling afforded a highly potent and selective compound 36. Compound 36 demonstrated good antitumor efficacy in xenograft nude rat model.

Structures of the PKC-iota kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533-551 in the C-terminal tail and their roles in ATP binding

Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-theta, PKC-iota and PKC-betaII kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC-substrate complex has been determined. In the previously determined PKC-iota complex, residues 533-551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-iota kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0 A resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-iota are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-iota, which is different from the case of the prototypical AGC-branch kinase PKA.

Sequence-selective DNA cleavage by a topoisomerase I poison, NB-506

An indolocarbazole compound, NB‐506, inhibits the activity of topoisomerase I by stabilizing the DNA‐topoisomerase I complex (cleavable complex). NB‐506 inhibited the re‐ligation step of topoisomerase I activity more potently than camptothecin or its derivative, topotecan. A cleavage assay using an end‐labeled fragment of DNA revealed that the pattern of cleavage induced by NB‐506 was different from that induced by camptothecin. The preferred cleavage sites of NB‐506 were found to be not only T but also A or C at the 3′‐terminus of the cleaved DNA (position −1), while the DNA cleavage sites of camptothecin always had T at position −1. At the 5′‐terminus of the cleaved DNA (position +1), NB‐506 showed a preference for G, which is a feature shared in common with camptothecin. Therefore, the difference in cleavage patterns was most likely due mainly to the preferred base at position −1. Moreover, the re‐ligation rate was significantly slower at NB‐506‐selective sites, which had C at position‐1, than at camptothecin‐selective sites or at sites cleaved by both NB‐506 and camptothecin. Our data suggest that NB‐506 is an unique topoisomerase I poison and that its potent inhibition of topoisomerase I is partly dependent on retardation of re‐ligation at sites selectively induced by NB‐506. Int. J. Cancer 75:145–150, 1998.© 1998 Wiley‐Liss, Inc.

ABCG2 confers resistance to indolocarbazole compounds by ATP-dependent transport.

The ABC half-transporter, ABCG2, is known to confer resistance to chemotherapeutic agents including indolocarbazole derivatives. MCF7 cells were introduced by either wild type ABCG2 (ABCG2-482R) or mutant ABCG2 (-482T), whose amino acid at position 482 is substituted to threonine from arginine, and their cross-resistance pattern was analyzed. Although this amino acid substitution seems to affect cross-resistance patterns, both 482T- and 482R-transfectants showed strong resistance to indolocarbazoles, confirming that ABCG2 confers resistance to them. For further characterization of ABCG2-mediated transport, we investigated indolocarbazole compound A (Fig. 1) excretion in cell-free system. Compound A was actively transported in membrane vesicles prepared from one of the 482T- transfectants and its uptake was supported by hydrolysis of various nucleoside triphosphates. This transport was inhibited completely by the other indolocarbazole compound, but not by mitoxantrone, implying that the binding site of mitoxantrone or the transport mechanisms for mitoxantrone is different from those of indolocarbazoles. These results showed that ABCG2 confers resistance to indolocarbazoles by transporting them in an energy-dependent manner.

Plk3 phosphorylates topoisomerase IIα at Thr1342, a site that is not recognized by Plk1

: The Plk (polo-like kinase) family is involved in cell-cycle machinery. Despite the possible overlapping involvement of Plk1 and Plk3 in cell-cycle distribution, the precise role of each Plk might be different. To investigate mechanisms that may differentiate their physiological roles, we compared the substrate specificities of Plk1 and Plk3 using synthetic peptides. Among these substrate peptides, topoisomerase IIalpha EKT(1342)DDE-containing synthetic peptide was strongly phosphorylated by Plk3 but not by Plk1. By modulating the topoisomerase IIalpha peptide, we identified residues at positions +1, +2 and +4 as determinants of differential substrate recognition between Plk1 and Plk3. Acidic residues at positions +2 and +4 appear to be a positive determinant for Plk3 but not Plk1. Variation at position +1 appears to be tolerated by Plk3, while a hydrophobic residue at +1 is critical for Plk1 activity. The direct phosphorylation of Thr(1342) of topoisomerase IIalpha by Plk3 was demonstrated with an in vitro kinase assay, and overexpression of Plk3 induced the phosphorylation of Thr(1342) in cellular topoisomerase IIalpha. Furthermore, the physical interaction between Plk3 and topoisomerase IIalpha was also demonstrated in cells in addition to phosphorylation. These data suggest that topoisomerase IIalpha is a novel physiological substrate for Plk3 and that Plk1 and Plk3 play different roles in cell-cycle regulation.

Identification of β-catenin as a novel substrate of polo-like kinase 1

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified β-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of β-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between β-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that β-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of β-catenin in M phase.

Overexpression of Plk3 causes Morphological Change and Cell Growth Suppression in Ras Pathway-activated Cells

To unravel the growth inhibition mechanism of Polo-like kinase 3 (Plk3), the effect of overexpression of Plk3 was examined in 293T cells. Cell rounding, changes in actin organization and cellular detachment were induced by Plk3 transfection in a kinase activity-dependent manner. Although apoptosis was not observed, Plk3 overexpression suppressed cellular growth in a long-term colony-forming assay. Because both Plk3 and Ras affect F-actin organization, the effect of co-transfection of Plk3 and Ras was evaluated. Adhesion was synergistically lost by co-transfection of these two genes, compared with transfection of Plk3 alone. Furthermore, overexpression of Plk3 caused long-term growth suppression in Ras-transformed NIH3T3. Collectively, Plk3 activation might cause cytoskeleton re-organization and result in growth suppression more pronouncedly in Ras pathway-activated cells.

Expression ratio of CCND1 to CDKN2A mRNA predicts RB1 status of cultured cancer cell lines and clinical tumor samples

BackgroundThe retinoblastoma product (RB1) is frequently deregulated in various types of tumors by mutation, deletion, or inactivation through association with viral oncoproteins. The functional loss of RB1 is recognized to be one of the hallmarks that differentiate cancer cells from normal cells. Many researchers are attempting to develop anti-tumor agents that are preferentially effective against RB1-negative tumors. However, to identify patients with RB1-negative cancers, it is imperative to develop predictive biomarkers to classify RB1-positive and -negative tumors.ResultsExpression profiling of 30 cancer cell lines composed of 16 RB1-positive and 14 RB1-negative cancers was performed to find genes that are differentially expressed between the two groups, resulting in the identification of an RB1 signature with 194 genes. Among them, critical RB1 pathway components CDKN2A and CCND1 were included. We found that microarray data of the expression ratio of CCND1 and CDKN2A clearly distinguished the RB1 status of 30 cells lines. Measurement of the CCND1/CDKN2A mRNA expression ratio in additional cell lines by RT-PCR accurately predicted RB1 status (12/12 cells lines). The expression of CCND1/CDKN2A also correlated with RB1 status in xenograft tumors in vivo. Lastly, a CCND1/CDKN2A assay with clinical samples showed that uterine cervical and small cell lung cancers known to have a high prevalence of RB1-decifiency were predicted to be 100% RB1-negative, while uterine endometrial or gastric cancers were predicted to be 5-22% negative. All clinically normal tissues were 100% RB1-positive.ConclusionsWe report here that the CCND1/CDKN2A mRNA expression ratio predicts the RB1 status of cell lines in vitro and xenograft tumors and clinical tumor samples in vivo. Given the high predictive accuracy and quantitative nature of the CCND1/CDKN2A expression assay, the assay could be utilized to stratify patients for anti-tumor agents with preferential effects on either RB1-positive or -negative tumors.

Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

Abstract A274: Novel SYK inhibitors have demonstrated potent antiproliferative effects in both ABC- and GCB-DLBCL cell lines via suppression of multiple pathways downstream of the B-cell receptor.

Background: Diffuse large B-cell lymphoma (DLBCL) has two major subtypes with distinct biological and clinical characteristics: activated B-cell like (ABC) and germinal center B-cell like (GCB) lymphoma. A Bruton9s tyrosine kinase (BTK) inhibitor has shown clinical activity in a subset of ABC-DLBCL patients. Spleen tyrosine kinase (SYK) is an upstream regulator of BTK, but the effect of SYK inhibition versus BTK inhibition in DLBCL has not been elucidated. We developed potent and selective SYK inhibitors and evaluated mechanistic differences between SYK inhibition and BTK inhibition in DLBCL cell lines. Materials and Methods: GCB-DLBCL (Pfeiffer, SU-DHL-10, SU-DHL-6, SU-DHL-5, DOHH2, OCI-LY18, OCI-LY1, VAL) and ABC-DLBCL (OCI-LY10, OCI-LY3, TMD8) cell lines were cultivated under standard cell culture conditions. For the growth inhibition assay, cells were treated with compounds for 72 hr, and the number of living cells was determined by CellTiter-Glo (Promega). Cells were treated with the compounds for 24 hr and lysates were subjected to western blotting with antibodies specific for the components of PI3K, ERK, and NFkB pathways. Results: The selective SYK inhibitors, TAS-5567 and its analogue TAS-4335, inhibited SYK with IC50 values of 0.37 nM and 0.26 nM, respectively. The SYK inhibitors demonstrated antiproliferative activities in a wide range of DLBCL cell lines including both ABC and GCB subtypes (TAS-5567 IC50=298 nM in TMD8, 128 nM in SU-DHL-6, 134 nM in SU-DHL-5, and 68 nM in SU-DHL-10). On the other hand, a BTK inhibitor Ibrutinib exhibited antiproliferative activity only in the ABC subtype (IC50=2 nM in TMD8, 1468 nM in SU-DHL-6, 1240 nM in SU-DHL-5, and 1287 nM in SU-DHL-10). We further analyzed the effect of these inhibitors on BCR signaling in GCB-DLBCL cell lines SU-DHL-6, SU-DHL-5, and SU-DHL-10 and found that the SYK inhibitor but not the BTK inhibitor robustly suppressed the PI3K pathway as detected by phospho-AKT and phospho-PRAS40. In ABC-DLBCL cell line TMD8, which is known to be NFkB pathway dependent, the level of phospho-IkBα was reduced by both the SYK and BTK inhibitors. Conclusion: Our SYK inhibitors demonstrated a broad spectrum of antiproliferative efficacy in both ABC- and GCB-DLBCL cell lines, which may be explained by the blocking of multiple downstream pathways, including PI3K and NFkB signaling. Thus SYK inhibition may be a valuable approach in the treatment of heterogeneous DLBCL. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A274. Citation Format: Aki Kawagishi, Hiroki Irie, Yoshio Ogino, Hideya Komatani, Teruhiro Utsugi. Novel SYK inhibitors have demonstrated potent antiproliferative effects in both ABC- and GCB-DLBCL cell lines via suppression of multiple pathways downstream of the B-cell receptor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A274.