Academic Drug Development: The DRIVE Model

Although there are hundreds of academic drug discovery centers open around the world, there are comparatively few academic drug development centers that contain the key core competencies needed to progress a lead compound into clinical trials. This is largely a consequence of operating in the “Valley of Death” (i.e., insufficient infrastructure, expertise, and funding). We have created an academic drug development center called DRIVE (Drug Innovation Ventures at Emory) that was designed to overcome many of the intrinsic and occasionally unintended barriers associated with academic drug development. Herein, we report a proof of concept that the DRIVE model provides a robust framework for pursuing university-based drug development, especially when the drugs in question target rare and neglected diseases

Autophagy and Apoptosis in Hepatocellular Carcinoma Induced by EF25-(GSH)₂: A Novel Curcumin Analog

<div><p>Curcumin, a spice component as well as a traditional Asian medicine, has been reported to inhibit proliferation of a variety of cancer cells but is limited in application due to its low potency and bioavailability. Here, we have assessed the therapeutic effects of a novel and water soluble curcumin analog, 3,5-bis(2-hydroxybenzylidene)tetrahydro-4<i>H</i>-pyran-4-one glutathione conjugate [EF25-(GSH)₂], on hepatoma cells. Using the MTT and colony formation assays, we determined that EF25-(GSH)₂ drastically inhibits the proliferation of hepatoma cell line HepG2 with minimal cytotoxicity for the immortalized human hepatic cell line HL-7702. Significantly, EF25-(GSH)₂ suppressed growth of HepG2 xenografts in mice with no observed toxicity to the animals. Mechanistic investigation revealed that EF25-(GSH)₂ induces autophagy by means of a biphasic mechanism. Low concentrations (<5 µmol/L) induced autophagy with reversible and moderate cytoplasmic vacuolization, while high concentrations (>10 µmol/L) triggered an arrested autophagy process with irreversible and extensive cytoplasmic vacuolization. Prolonged treatment with EF25-(GSH)₂ induced cell death through both an apoptosis-dependent and a non-apoptotic mechanism. Chloroquine, a late stage inhibitor of autophagy which promoted cytoplasmic vacuolization, led to significantly enhanced apoptosis and cytotoxicity when combined with EF25-(GSH)₂. Taken together, these data imply a fail-safe mechanism regulated by autophagy in the action of EF25-(GSH)₂, suggesting the therapeutic potential of the novel curcumin analog against hepatocellular carcinoma (HCC), while offering a novel and effective combination strategy with chloroquine for the treatment of patients with HCC.</p></div

Morphology of autophagosomes in EF25-(GSH)₂-treated HepG2 cells.

<p>HepG2 cells were treated with 20 µmol/L EF25-(GSH)₂ for 16 h and observed under transmission electron microscopy. (A) and (B), multimembranous autophagic vacuoles engulfing cytoplasmic components are indicated with black arrowheads. (C) and (D), autophagic vacuoles containing a mitochondrion are indicated with black asterisk.</p

The morphological appearance of EF25-(GSH)₂-treated HepG2 cells.

<p>(A) HepG2 cells treated with increasing concentrations of EF25-(GSH)₂ for 16 h were observed under a light microscope and representative images were visualized. EF25-(GSH)₂-treated cells underwent vacuolization, the extent of which varied when treated with different concentrations of EF25-(GSH)₂. At 20 µmol/L, apoptotic-like cell membrane blebbing was observed (arrowheads). (B) A representative transmission electron microscopy (TEM) image of untreated HepG2 cells. (C) In 5 µmol/L EF25-(GSH)₂-treated cells, most vacuolated cells regained normal morphology at 32 h post-treatment (arrows, 1-4) while some did not (arrow heads, 5 and 6). (D) Representative TEM images of cells treated with 10 µmol/L EF25-(GSH)₂ for 16 h. *, large empty vacuoles with varying size. (E) Representative TEM images of cells treated with 20 µmol/L EF25-(GSH)₂ for 16 h. *, large empty vacuoles; arrows, autophagic vacuoles.</p

Knockdown of Atg5 and Beclin-1 expression does not rescue EF25-(GSH)₂-treated HepG2 cells.

<p>(A) HepG2 cells respectively transduced with shLacZ-, shBeclin-1-C2-, shBeclin-1-C3-, shAtg5-D8- and shAtg5-D9-lentivirus were mock-, or treated with 10 µmol/L EF25-(GSH)₂ for 24 h. Cells lysates were analyzed by Western blotting with antibodies against Atg5, Beclin-1, LC3 or actin, as indicated. (B) For HepG2 cells respectively transduced with shLacZ-, shBeclin-1-C2-, shBeclin-1-C3-, shAtg5-D8- and shAtg5-D9-lentivirus, cell viability was determined by MTT assay after treatment with increasing concentrations of EF25-(GSH)₂ for 48 h. (C) HepG2 cells respectively transduced with shLacZ-, shBeclin-1-C2- and shAtg5-D8-lentivirus were treated with 10 µmol/L EF25-(GSH)₂ for 24 h and observed under the light microscope.</p

EF25-(GSH)2 inhibited proliferation of tumor cells <i>in vitro</i>.

<p>(A) The structures of curcumin, EF25 and EF25-(GSH)₂. (B) <i>a and b</i>, EF25-(GSH)₂ showed similar toxicity towards six human tumor cells (BEL-7402, HCT116, HepG2, A549, SMMC-7721 and Hela) (<i>a</i>) and the toxicity of curcumin was much lower than that of EF25-(GSH)₂ (<i>b</i>). <i>c</i>, cells were incubated with increasing doses of indicated compounds for 24-, 48-, and 72-h periods and analyzed by MTT assay. The IC₅₀ of each agent at each time period was calculated and compared using SPSS. The IC₅₀ of EF25-(GSH)₂ is much lower than that of curcumin and essentially equivalent to that of cisplatin. <i>d</i>, the cytotoxicity of EF25-(GSH)₂ to HL-7702 cells was much lower than that of cisplatin and similar to curcumin after 48-hour incubation as determined by MTT assay (*, p<0.01, **, p<0.001). (C) Cells were incubated with 0.5 µmol/L of the indicated compound for 24 h and subsequently allowed to grow into colonies (2 weeks). EF25-(GSH)₂ totally inhibited colony formation leading to clean plates, while curcumin and cisplatin did not. Results are representative of three independent experiments.</p

The apoptosis in HepG2 cells triggered by EF25-(GSH)₂ in the presence or absence of CQ/Z-VAD-FMK.

<p>(A) HepG2 cells were treated with various concentrations of EF25-(GSH)₂ for 24 h and 48 h with or without chloroquine (CQ, 100 µmol/L)/Z-VAD-FMK (30 µmol/L, pretreated for 2 h) and then analyzed for DNA content (propidium iodide, PI) and cell cycle distribution. Apoptosis was measured as the percentage of cells containing hupodiploid quantities of DNA (sub-G₁-G₀ peak). Percentage of cells within the sub-G₁-G₀ and G₂/M stages is shown for each data point. Graphs are representative of data collected from three independent experiments. (B) HepG2 cells incubated with increasing concentrations of EF25-(GSH)₂ for 48 h were stained with 4, 6-diamidino-2-phenylindole (DAPI) and examined by laser confocal microscopy. Untreated HepG2 cells showed uniformly stained nuclei, while EF25-(GSH)₂-treated cells exhibited chromatin condensation in a concentration-dependent manner. (C) Lysates from HepG2 cells incubated with increasing concentrations of EF25-(GSH)₂ for 24 or 48 h with or without chloroquine (CQ, 100 µmol/L) were analyzed by Western blotting for both full length and cleaved caspase-3 and caspase-8 expression levels.</p

The effect of Wm, CQ and Z-VAD-FMK on the cytotoxicity and morphological changes induced by EF25-(GSH)₂ in HepG2 cells.

<p>(A) Cell viability was determined by the MTT assay after treatment with increasing concentrations of EF25-(GSH)₂ for 24 h or 48 h in the absence or presence of CQ (100 µmol/L)/Wm (100 nmol/L, pretreated for 2 h)/Z-VAD-FMK (30 µmol/L, pretreated for 2 h). *, p<0.001, EF25-(GSH)₂ plus Z-VAD-FMK vs. EF25-(GSH)₂ alone. **, p<0.001, EF25-(GSH)₂ plus CQ vs. EF25-(GSH)₂ alone. (B) Representative light microscopic images of HepG2 cells treated with various concentrations of EF25-(GSH)₂ for 24 h in the absence or presence of CQ (100 µmol/L)/Z-VAD-FMK (30 µmol/L, pretreated for 2 h). (C) Representative light microscopic images of HepG2 cells treated with 10 µmol/L EF25-(GSH)₂ for 48 h in the absence or presence of Z-VAD-FMK (30 µmol/L, pretreated for 2 h).</p

Working model of the mechanisms of EF25-(GSH)₂-induced cell death in HepG2 cells.

<p>Stress induced by EF25-(GSH)₂ promotes autophagy in HepG2 cells. When treated with EF25-(GSH)₂ at concentrations of 5 µmol/L or lower, cells experienced full-scale autophagy that displayed moderate cytoplasmic vacuolization, ultimate recovery and partial rescue of cells from the resulting stress. In contrast, the protective autophagy was blocked in cells treated with EF25-(GSH)₂ at concentrations of 10 µmol/L or higher which led to massive cytoplasmic vacuolization. The latter cells arrested in the G₂/M phase succumbed to both caspase-dependent and caspase-independent cell death. EF25-(GSH)₂ treatment alone led mainly to caspase-dependent apoptotic cell death, but also to a significant proportion of caspase-independent apoptosis. The action of EF25-(GSH)₂ could be modulated by CQ (green) and Z-VAD-FMK (blue). Co-treatment of EF25-(GSH)₂ with CQ promoted autophagy blockage and cytoplasmic vacuolization, which then enhanced apoptosis for both caspase-dependent and caspase-independent mechanisms. Co-treatment of EF25-(GSH)₂ with Z-VAD-FMK inhibited caspase activation and subsequently blocked the caspase-dependent apoptotic death route. Thus, cells were trapped by cytoplasmic vacuolization and G₂/M cell cycle arrest, which eventually led to non-apoptotic cell death.</p

Characterization of XMRV pseudovirus and single-round neutralization assay.

<p>(A) Comparison of XMRV and control HIV-1 pseudoviruses in yield (p24 accumulation) and infectivity (IU/ml on TZM-bl cells). (B) Detection of antibody specificity to XMRV and HIV-1 pseudoviruses. Pseudoviruses were tested in the neutralization assay with mAb 83A25 that recognizes a shared epitope of MLV Env glycoprotein and with mAb b12 that recognizes HIV-1 Env glycoprotein. (C) Neutralization of the XMRV and HIV-1 pseudoviruses showing a broad range of sensitivity and specificity of the assay using polyclonal antibodies (anti Friend-MuLV).</p