A Phthalimide Derivative That Inhibits Centrosomal Clustering Is Effective on Multiple Myeloma

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma

mRNA Display Selection of an Optimized MDM2-Binding Peptide That Potently Inhibits MDM2-p53 Interaction

p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins

Modulation of the human T cell response by a novel non-mitogenic anti-CD3 antibody.

The agonistic anti-human CD3ε antibody (Ab), OKT3, has been used to control acute transplant rejection. The in vivo administration of OKT3 was previously shown to induce the partial depletion of T cells and unresponsiveness (anergy) in the remaining CD4+ T cells. However, this therapy is also associated with the systemic release of several cytokines, which leads to a series of adverse side effects. We established a novel anti-human CD3ε Ab, 20-2b2, which recognized a close, but different determinant on the CD3ε molecule from that recognized by OKT3. 20-2b2 was non-mitogenic for human CD4+ T cells, could inhibit the activation of T cells in vitro, and induced T cell anergy in in vivo experiments using humanized mice. Cytokine release in humanized mice induced by the administration of 20-2b2 was significantly less than that induced by OKT3. Our results indicated that the CD3ε molecule is still an attractive, effective, and useful target for the modulation of T cell responses. The establishment of other Abs that recognize CD3ε, even though the determinant recognized by those Abs may be close to or different from that recognized by OKT3, may represent a novel approach for the development of safer Ab therapies using anti-CD3 Abs, in addition to the modification of OKT3 in terms of the induction of cytokine production

Non-stimulatory 20-2b2 induced the down-modulation of TCR expression.

<p>(A) Jurkat cells were cultured in the presence (green) or absence (red) of 20-2b2 (50% SN) at 37°C. Cells were washed and stained with the anti-TCRVβ8 Ab two hours later. Unstained Jurkat cells are shown as a blue line. (B) Jurkat cells were cultured in the presence (green) or absence (red) of 20-2b2 (50% SN) at 4°C for 20 min, and then stained with anti-TCRVβ8 Ab conjugated with FITC. (C) The PBMCs of healthy donors were cultured with the titrated amount of 20-2b2. Cell proliferation was measured two days later. Data are expressed as the mean ± SD of triplicate cultures. The proliferation of PBMCs without a stimulus was measured as 718±346 cpm. Results are representative of three independent experiments (A–C).</p

20-2b2 inhibited the activation of T cells <i>in vitro</i>.

<p>(A–C) The PBMCs of healthy donors were cultured with an anti-CD3 Ab (OKT3) (A), PHA (B), or PWM (C) in the presence (red symbols) or absence (black) of 20-2b2 (50% SN). (D) PBMCs were pre-treated with medium (black) or 20-2b2 (red) at 37°C. Cells were washed three times and cultured with PHA two hours later. Cell proliferation was measured two days later. Values are shown as the mean ± SD of triplicate cultures. Data are representative of more than three (A–C) and three (D) independent experiments. *<i>p</i><0.01, **<i>p</i><0.001 by the Student's <i>t</i>-test.</p

20-2b2 recognized CD3ε.

<p>(A, B) Jurkat (A) or J.RT3-T3.5 cells (B) were stained with anti-CD3ε Ab (OKT3) or 20-2b2 (green lines). Unstained cells are shown as red lines. (C) Jurkat cells were pre-treated with 20-2b2 (top panel) or OKT3 (bottom panel), and were then stained with the indicated Ab (yellow lines). Jurkat cells stained without the pre-treatment are shown as green lines. Negative control staining is shown as red lines. (D) Yac-1 or Yac-1 cells transfected with human CD3ε (hCD3ε-Yac-1) were stained with the anti-CD3ε Ab (OKT3) (green) or 20-2b2 (blue). Cells stained with rat IgG as a negative control are shown as red lines. (E) Cell lysates prepared from the indicated cells were separated in SDS-PAGE and blotted with 20-2b2. Arrow: the target molecule recognized by 20-2b2. (F) The lysate from Jurkat cells was immunoprecipitated (IP) with 20-2b2 or control rat IgG. Immunoprecipitates were separated by SDS-PAGE. The blot was probed (WB) with the anti-CD3ε Ab (M-20). Results are representative of three independent experiments (A-F).</p

MIP-2A Is a Novel Target of an Anilinoquinazoline Derivative for Inhibition of Tumour Cell Proliferation

<div><p>We recently identified a novel anilinoquinazoline derivative, Q15, as a potent apoptosis inducer in a panel of human cancer cell lines and determined that Q15 targets hCAP-G2, a subunit of condensin II complex, leading to abnormal cell division. However, whether the defect in normal cell division directly results in cell death remains unclear. Here, we used an mRNA display method on a microfluidic chip to search for other Q15-binding proteins. We identified an additional Q15-binding protein, MIP-2A (MBP-1 interacting protein-2A), which has been reported to interact with MBP-1, a repressor of the c-Myc promoter. Our results indicate that Q15 inhibits the interaction between MIP-2A and MBP-1 as well as the expression of c-Myc protein, thereby inducing cell death. This study suggests that the simultaneous targeting of hCAP-G2 and MIP-2A is a promising strategy for the development of antitumor drugs as a treatment for intractable tumours.</p></div

Primers used in this study.

<p>Primers used in this study.</p

Q15 increases the intranuclear localisation of MBP-1.

<p>(A) HeLa cells were transfected with FLAG-MBP-1 with or without HA-MIP-2A. After 24 h, the cells were treated with 5 µM Q15 for an additional 24 h. Immunofluorescence staining with anti-FLAG (green) and anti-HA (red) was then performed. The samples were observed using confocal microscopy. Bar; 10 µm. (B) At least 50 cells per field were counted in each of three independent experiments. The ratio of cells with MBP-1 that localised in the nucleus was quantified.</p