Delineating the active site architecture of G9a lysine methyltransferase through substrate and inhibitor binding mode analysis: a molecular dynamics study

<p>Mono- and di-methylation of the H3K9 residue in the histone tail by G9a lysine methyltransferase is associated with transcriptional suppression of genes. Here, we use molecular dynamics simulation and free energy calculations of five different modified/mutated G9a substrate peptides to elucidate the rationale behind the substrate binding to G9a. We also investigated the binding energy contribution based architecture of the active site of G9a to understand substrate and inhibitor binding. Wild-type peptide (H3K9) shows better binding affinity than mono- and di-methylated lysine (K9) and other modified peptides (K9A and R8A). Arg8 of the substrate peptide is crucial for determining the degree of conformational freedom/stability of the wild-type substrate peptide, as well as binding to G9a. Our results also suggest that the G9a active site is segregated into energy rich and low regions, and the energy rich region alone is used by the inhibitors for binding. These insights into the active site architecture should be taken into consideration in virtual screening experiments designed to discover novel inhibitors for G9a. In particular, compounds that could interact with the six residues of G9a – Asp1074, Asp1083, Leu1086, Asp1088, Tyr1154 and Phe1158 – should be preferentially tested in G9a inhibition biological assays.</p> <p>Communicated by Ramaswamy H. Sarma</p

Plasma pharmacokinetics of BPR0L075 in CD-1 mice after administration of drug at 50 mg/kg intraperitoneally.

<p>(A) LC-MS/MS analysis of BPR0L075 with chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.1 and 2.8 min, respectively. (B) The plasma concentration-time curve following BPR0L075 treatment.</p

An Antimitotic and Antivascular Agent BPR0L075 Overcomes Multidrug Resistance and Induces Mitotic Catastrophe in Paclitaxel-Resistant Ovarian Cancer Cells

<div><p>Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC₅₀ = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.</p></div

Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts

<div><p>BPR0L075, 6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1<em>H</em>-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity <em>in vitro</em>. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels <em>in vitro</em> and <em>ex vivo</em>. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.</p> </div

BPR0L075 induces concentration- and time-dependent cell cycle arrest in both parental and paclitaxel-resistant ovarian cancer cells.

<p>OVCAR-3 and OVCAR-3-TR cells were treated with ethanol vehicle or BPR0L075 (10–100 nM) for 24 hours or 10 nM BPR0L075 for indicated durations (0–48 hours), stained with propidium iodide, and analyzed by flow cytometer. The cell cycle profiles are presented as three-dimensional overlay. The X-axis shows the intensity of propidium iodide fluorescence, which indicates cellular DNA content in different cell cycle phases. The Y-axis represents the cell counts; and z-axis shows the concentration or time points of BPR0L075 treatment. 2N, cells residing in the G0–G1 phase of cell cycle; 4N, cells in the G2 phase or mitosis. BPR0L075 induces significant G2/M arrest followed by appearance of a sub-G1 population in both parental and resistant cells, with polyploid cells (arrows) only in the resistant cells. Results are representative of three independent experiments.</p

BPR0L075 induces PARP cleavage and DNA fragmentation in parental cells, but not in the paclitaxel-resistant ovarian cancer cells.

<p>Western blot analysis of the PARP, caspase-2, and caspase-3 after BPR0L075 (10–100 nM) treatment for 24 hours in the OVCAR-3/OVCAR-3-TR cells (A) and SKOV-3/SKOV-3-TR cells (B). β-actin was loading control. (C) DNA ladder assays in cells treated with 10 nM BPR0L075 for 72 hours. Genomic DNA was subjected to 1% agarose gel electrophoresis. The gel was stained with ethidium bromide and the DNA bands were visualized under an ultraviolet transilluminator. (D) Effect of caspase-3 inhibitor on BPR0L075 induced cytotoxicity. Cells were pretreated with caspase-3 inhibitor (20 µM Z-DEVD-FMK) or negative control inhibitor (20 µM Z-FA-FMK) for 24 hours before incubation with 10 nM BPR0L075 for additional 72 hours. On completion of the incubation, the cell viability was determined by SRB assay. No statistically significant difference of cytotoxicity was observed for each pair of cell lines (<i>P</i>>0.05, student <i>t</i>-test). Data represent mean ± SD.</p

Fluorescence images of mammary breast tumor response to administration of BPR0L075 at successive time points.

<p>Sequential images of a single nude mouse with orthotopic MCF7-<i>lacZ</i> and MCF7-luc-GFP-mCherry breast tumors growing in the front right (blue circle) and left mammary fat pad, respectively. Fluorescent signal (mCherry) increased over 24 hours following administration of carrier vehicle (A), but signals diminished 4, 6, and 24 hours after injection of BPR0L075 (B); (C) Variation in normalized fluorescent signal intensity for the group of three MCF7-luc-GFP-mCherry tumors in response to vehicle injection; (D) Variation in normalized fluorescent signal intensity for the group of six MCF7-luc-GFP-mCherry tumors in response to injection of BPR0L075. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.0001; ⁺ only three tumors observed at this time point.</p

Western blot analysis of the relative protein expressions in the parental and paclitaxel-resistant ovarian cancer cell lines after BPR0L075 treatment.

<p>BPR0L075 treatment (10–100 nM for 24 hours) induced changes in cyclin B1, BubR1, MPM-2, and survivin expressions in OVCAR-3/OVCAR-3-TR cells (A) and SKOV-3/SKOV-3-TR cells (B). BPR0L075 treatment (10 nM) induced changes in PARP, BCL-XL, and BCl-2 proteins at different time (0–72 hours) in OVCAR-3/OVCAR-3-TR cells (C) and SKOV-3/SKOV-3-TR cells (D). 50 µg of the protein lysate was loaded on the SDS-PAGE and experiments were repeated and representative blots are shown.</p

Histological validation of temporary vascular shutdown in MCF7-luc-GFP-mCherry mammary fat pad tumors with respect to BPR0L075 administration.

<p>(A) Tumor tissues from control mouse 4 hours after vehicle treatment showing H&E staining (original magnification ×100), mCherry fluorescence signals (×100), and luciferase expression stained with anti-luciferase antibody (red) with DAPI (blue) (magnification ×400); (B) Tumor sections from four control tumors showing vascular extent based on anti-CD31 (green) and perfusion marker Hoechst 33342 (blue); and (C) corresponding tumor sections from four tumors in mice treated with BPR0L075 (50 mg/kg i.p.) at different time points (magnification ×400). (D) Whole mount H&E section 24 hrs after administering BPR0L075 showing about 70% necrotic fraction.</p

Cytotoxicity of BPR0L075 in parental and paclitaxel-resistant ovarian cancer cells.

<p>(A) Proliferating human ovarian cancer cells (SKOV-3, OVCAR-3, and A2780-1A9) and paclitaxel-resistant sublines (SKOV-3-TR, OVCAR-3-TR, and 1A9-PTX10) were treated with BPR0L075 (red circle) or paclitaxel (blue square) at 3–15 nM concentrations for 4 days and the cytotoxicity was evaluated by SRB assay. Fraction of cell survival relative to controls represents mean ± SD of 12 determinations. (B) Summary of the P-gp expression, βIII-tubulin expression, and sensitivity of paired ovarian cancer cell lines to paclitaxel, doxorubicin, and BPR0L075 treatment. Numbers in parenthesis represents the degree of resistance (x-fold), expressed as the ratio of the IC₅₀ values as compared with the corresponding parental lines. All experiments were repeated three times.</p