Synthesis and activity of a novel Autotaxin inhibitor-Icodextrin conjugate

© Copyright 2018 American Chemical Society. Autotaxin is an extracellular phospholipase D that catalyses the hydrolysis of lysophosphatidyl choline (LPC) to generate the bioactive lipid lysophosphatidic acid (LPA). Autotaxin has been implicated in many pathological processes relevant to cancer. Intraperitoneal administration of an autotaxin inhibitor may benefit patients with ovarian cancer, however low molecular mass compounds are known to be rapidly cleared from the peritoneal cavity. Icodextrin is a polymer that is already in clinical use because it is slowly eliminated from the peritoneal cavity. Herein we report conjugation of the autotaxin inhibitor HA-155 to icodextrin. The conjugate inhibits autotaxin activity (IC50 = 0.86 ± 0.13 μg mL-1) and reduces cell migration. Conjugation of the inhibitor increased its solubility, decreased its membrane permeability and improved its intraperitoneal retention in mice. These observations demonstrate the first application of icodextrin as a covalently-bonded drug delivery platform with potential use in the treatment of ovarian cancer

The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization

<div><p>Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax.</p></div

Combinations of obatoclax with carboplatin or paclitaxel.

<p><b>A</b>. Cells were treated with obatoclax and either carboplatin or paclitaxel, combined at the ratio of the IC₅₀s determined in cell growth/survival assays with the individual drugs. After 72 hours, the surviving cell number was estimated by staining with SRB and the combination index calculated as described in the methods. The results are expressed as the combination index at fraction affected = 0.5 (mean ± S.D., n = 3–10) and were significantly different from 1.0 where indicated (*, <i>P</i> < 0.05, t-test using Welch’s correction). <b>B</b>. Scheduled combinations of obatoclax and carboplatin. Cells were exposed to: a) obatoclax for 48 hours then carboplatin for 48 hours; b) carboplatin for 48 hours, then obatoclax for 48 hours; c) carboplatin and obatoclax for 48 hours then culture medium for a further 48 hours; d) culture medium for 48 hours, then carboplatin and obatoclax for 48 hours. In each case, the cells were treated with 18 different concentrations of carboplatin and obatoclax combined at the ratio of their IC₅₀s determined in experiments with the individual drugs. Combination indices were determined at fraction affected = 0.5 mean ± S.D., n = 3–4) and were significantly different from 1.0 where indicated (*, <i>P</i> < 0.05, t-test using Welch’s correction).</p

Effect of knockdown of Beclin-1 or Atg-5 on the activity of obatoclax.

<p>Ovcar-5 or Ovcar-8 cells were transfected with siRNA SMARTpools targeting Beclin-1 or Atg-5 or non-targeting siRNA (NT) and the effect on obatoclax activity determined in cell growth assays. <b>A</b>. Knockdown of Beclin-1 and Atg-5 was confirmed by western blotting 48 hours after transfection (representative of two experiments). <b>B</b>. 24 hours after transfection of siRNA SMARTpools targetting Atg-5 and Beclin-1, obatoclax was added and the IC₅₀ of obatoclax (mean ± S.D., n = 3–4 separate transfections) determined and compared to that measured in cells transfected with a non-targeting (NT) siRNA. *, IC₅₀ significantly different from that measured in cells transfected with NT siRNA (paired t- test, P < 0.05) C. Concentration-response data (mean ± S.D., n = 3–4) from which the IC₅₀ values were determined.</p

Obatoclax localizes to lysosomes and causes their alkalinization.

<p><b>A</b>. Ovcar-5 cells expressing the acidic vesicle marker GFP-LAMP-1 (upper panel) or labelled with lysotracker blue dye (lower panel) were exposed to obatoclax for 1 hour. Obatoclax was measured by its intrinsic fluorescence. The cells were analysed by confocal microscopy and the results presented are representative of 3 experiments. <b>B</b>. Ovcar-5 Cells were labelled with lysosensor Green DND-189, exposed to 120 nM obatoclax for 1 hour. Images were captured at the indicated times. The decrease in fluorescence reflects alkalinization of the dye’s environment. The decrease in fluorescence is quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150696#pone.0150696.s005" target="_blank">S5 Fig</a>.</p

Model for the combined activity of obatoclax and chloroquine or bafilomycin.

<p>Bafilomycin blocks the vATPase reducing lysosomal H⁺ influx, while chloroquine (CQ) and obatoclax (OBX) are organic bases which become protonated and trapped in the lysosome and increase lysosomal pH. The resulting alkalinization reduces cathepsin activity as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150696#pone.0150696.ref030" target="_blank">30</a>] and the ensuing reduction in lysosomal function causes autophagosome and LC-3 accumulation.</p

Obatoclax activates caspase 3/7.

<p>Eight ovarian cancer cell lines were treated with obatoclax for 30 hours.at the indicated multiple of the IC₅₀ determined in cell proliferation assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150696#pone.0150696.t001" target="_blank">Table 1</a>) Caspase 3/7 activity was measured (mean ± S.D., n = 3–5) and is expressed as a fraction of that observed in the same cell line exposed to carboplatin (as a positive control) for the same period and at a concentration equal to 3 x its IC₅₀ in each cell line reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150696#pone.0150696.ref003" target="_blank">3</a>].</p

Obatoclax induces cleavage of PARP and accumulation of LC-3.

<p>Cells were treated with obatoclax for 48 hours at the indicated multiple of the IC₅₀ determined in cell proliferation assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150696#pone.0150696.t001" target="_blank">Table 1</a>). Lysates were prepared and PARP cleavage and accumulation of LC-3 was determined by immunoblotting. LC3-I and LC3-II were almost impossible to resolve in in most cell lines due to the very robust increase in LC3, although LC3-I (arrowed) and LC3-II could be distinguished in Ovcar-3 cells and obatoclax induced accumulation of LC3-II. The results shown are representative of 2–4 experiments per cell line.</p

Obatoclax inhibits cathepsin activity in intact cells.

<p>Ovcar-5 cells were treated with solvent or obatoclax (120 nM, representing 3 x IC₅₀ in the cell growth assay) for the indicated period. The cathepsin substrate Phe-Arg-AMC was added to the cells in serum-free medium for the last 12 hours of the incubation period. To confirm the assays measured cathepsin activity, cells were exposed to the cathepsin inhibitor E64d for the last 12 hours of the incubation period. After measuring fluorescence of the released AMC, the results were normalized to cell number by staining the cells with SRB. The results (means ± S.D., n = 4) are expressed as a fraction of the activity measured in cells treated with solvent alone and are significantly different from those measured in cells treated with solvent where indicated (“*”, t-test, P < 0.05).</p

Obatoclax promotes cell death synergistically with other agents that cause lysosomal alkalinization.

<p><b>A</b>. Ovcar-4, Ovcar-5 or Ovcar-8 cells were treated with obatoclax (“GX”, 3 x IC₅₀), 10 nM bafilomycin (“BAF”) or a combination of both drugs for 72 hours. The number of cells surviving was determined by staining with SRB and was significantly different from the number expected from the Bliss independence criterion (“expected”) where indicated (“*”, t-test, P < 0.05). <b>B</b>. Ovcar-4 or Ovcar-5 cells were exposed to obatoclax (240 nM), bafilomycin (3nM) or a combination of both drugs for 48 h and PARP cleavage assessed by western blotting. (one experiment representative of 2–3). <b>C</b>. Ovcar-5 cells were exposed to obatoclax and bafilomycin as in C, stained with annexin V and propidium iodide and analysed by flow cytometery (the result shown is representative of 4 experiments). Only 6% of the cells treated with solvent stained positive for annexin V and propidium iodide. <b>D</b>. Ovcar-4, Ovcar-5 and Ovcar-8 cells were treated with obatoclax and chloroquine combined at a ratio of their respective IC₅₀s using 18 different drug concentrations and the number of cells surviving after 3 days determined by staining with SRB. The combination index, calculated at fraction affected = 0.5, was significantly different from CI = 1.0 in all three cell lines (t- test, *, <i>P</i> < 0.05; **, <i>P</i> < 0.01)</p