Benzyl Ether-Linked Glucuronide Derivative of 10-Hydroxycamptothecin Designed for Selective Camptothecin-Based Anticancer Therapy

A β-glucuronidase-activated prodrug approach was applied to 10-hydroxycamptothecin, a Camptotheca alkaloid with promising antitumor activity but poor water solubility. We synthesized a glucuronide prodrug of 10-hydroxycamptothecin (7) in which glucuronic acid is connected via a self-immolative 3-nitrobenzyl ether linker to the 10-OH group of 10-hydroxycamptothecin. Compound 7 was 80 times more soluble than 10-hydroxycamptothecin in aqueous solution at pH 4.0 and was stable in human plasma. Prodrug 7 was 10- to 15-fold less toxic than the parent drug to four human tumor cell lines. In the presence of β-glucuronidase, prodrug 7 could be activated to elicit similar cytotoxicity to the parent drug in tumor cells. Enzyme kinetic studies showed that Escherichia coli β-glucuronidase had a quite low Km of 0.18 µM for compound 7 and exhibited 520 times higher catalytic efficiency for 7 than for 6 (a glucuronide prodrug of 9-aminocamptothecin). Molecular modeling studies predicted that compound 7 would have a higher binding affinity to human β-glucuronidase than compound 6. Prodrug 7 may be useful for selective cancer chemotherapy by a prodrug monotherapy (PMT) or antibody-directed enzyme prodrug therapy (ADEPT) strategy

An Improved Screening Model To Identify Inhibitors Targeting Zinc-Enhanced Amyloid Aggregation

Zinc, which is abundant in senile plaques consisting mainly of fibrillar β-amyloid (Aβ), plays a critical role in the pathogenesis of Alzheimer’s disease. Treatment with zinc chelators such as clioquinol has been used to prevent Aβ aggregation in Alzheimer’s patients; however, clioquinol produces severe side effects. A simple, easy, inexpensive, and versatile screen to identify zinc chelators for inhibition of Aβ aggregation is currently unavailable. We thus developed a high-throughput screen that identifies zinc chelators with anti-Aβ aggregation activity. The recombinant Aβ peptides, aggregated on solid-phase microplates, formed Aβ-immunopositive β-sheet-containing structures in the presence of zinc. Formation of these Aβ fibrils was specifically blocked by metal ion chelators. This screening model improves identification of zinc-enhanced Aβ fibrils and anti-Aβ aggregation mediated by zinc chelating. The convenient system could qualitatively and quantitatively assay a large sample pool for Aβ aggregation inhibition and dissolution of Aβ aggregates. This screen is practical, reliable, and versatile for comprehensive detection of amyloid fibrillation and identification of inhibitors of Aβ aggregation

Development of Membrane-Bound GM-CSF and IL-18 as an Effective Tumor Vaccine

<div><p>The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (<i>P</i><0.05) and enhanced tumor protective efficacy (<i>P</i><0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.</p></div

Schematic representation of a membrane-bound strategy for immune stimulation.

<p>(A) The establishment of genetically modified CT26 cells to express membrane-bound GM-CSF and IL-18. Co-expression of GM-CSF and IL-18 on CT26 cells induced massive accumulation of immune cells at the inoculated site and stimulated in a locally specific manner. Tumor reduction and protective effects was then assessed for evaluation of the immune stimulatory effects. (B) The membrane-bound GM-CSF and IL-18 were composed of a HA epitope tag, the cytokine gene, a Myc tag, and the B7 transmembrane domains (B7 Tm). TAA, tumor associated antigen.</p

Splenocyte proliferation assay for the bioactivity of membrane-bound IL-18 and/or GM-CSF.

<p>Splenocytes were harvested from mock-transduced CT26 immunized Balb/C mice, and were cultured with CT26/IL-18, CT26/GM-CSF, and CT26/GM-CSF/IL-18 cells for the indicated times. ATPlite luminescence assay was performed to measure the splenocytes proliferation.</p

Protective effects of membrane-bound GM-CSF and IL-18, and GM-CSF/IL-18 expressing CT26 cells.

<p>(A) Groups of BALB/c mice (n = 5) were injected s.c. in the right hind leg with 1 × 10⁶ transduced tumor cells. Ten days after tumor cell implantation, mice were challenged by s.c. injection with 5×10⁵ mock-transduced CT26 cells in the left hind leg. Tumor volume (length × width × height × 0.5) was estimated every 3 or 4 days after challenge. Independent experiments were repeated three times. (B) Tumor free survival rate was estimated simultaneously (n = 20). The tumor free survival rate of respective group is about 40% (CT26/GM-CSF/IL-18), 25% (CT26/GM-CSF), 15% (CT26), and less than 5% (CT26/IL-18 and PBS control)</p

Tumorigenicity of membrane-bound GM-CSF, IL-18, and GM-CSF/IL-18 expressing CT26 cells.

<p>Groups of (n = 5) BALB/c mice were injected with 1 ×10⁶ transduced tumor cells s.c. in the right hind leg. Tumor volume (length × width × height × 0.5) was collected from tumor-bearing mice every 3 or 4 days after injection. Independent experiments were repeated three times.</p

Protein expression of membrane-bound GM-CSF and IL-18.

<p>CT26 cells stably expressing GM-CSF (A) or IL-18 (B) were stained with mouse anti-HA antibody, followed by FITC-conjugated goat anti-mouse IgG for 30 min. Mock-transduced CT26 cells were used as a negative control (black). (C) CT26/GM-CSF/IL-18 was doubly stained by anti-GM-CSF and anti-IL-18 primary antibodies, followed by FITC and PE-conjugated secondary antibodies (Experimental details were described in Materials and Methods). The left panel is negative control, in which CT26/GM-CSF/IL-18 was stained by secondary antibody alone. The right panel is the result of double staining, in which CT26/GM-CSF/IL-18 cells was stained with first and secondary antibodies. Fluorescence intensity of membrane-bound cytokines was analyzed by flow cytometry. (D) Mock-transduced CT26, CT26/GM-CSF, CT26/IL-18, and CT26/GM-CSF/IL-18 cells were seeded into 12-well plates at the density of 5×10⁴ cells per well. The number of viable cells of was counted with the hemocytometer every twenty-four hours. Independent experiments were repeated three times.</p

Structure-Based Discovery of Triphenylmethane Derivatives as Inhibitors of Hepatitis C Virus Helicase

Hepatitis C virus nonstructural protein 3 (HCV NS3) helicase is believed to be essential for viral replication and has become an attractive target for the development of antiviral drugs. A fluorescence resonant energy transfer helicase assay was established for fast screening of putative inhibitors selected from virtual screening using the program DOCK. Soluble blue HT (1) was first identified as a novel HCV helicase inhibitor. Crystal structure of the NS3 helicase in complex with soluble blue HT shows that the inhibitor bears a significantly higher binding affinity mainly through a 4-sulfonatophenylaminophenyl group, and this is consistent with the activity assay. Subsequently, fragment-based searches were utilized to identify triphenylmethane derivatives for more potent inhibitors. Lead optimization resulted in a 3-bromo-4-hydroxyl substituted derivative 12 with an EC50 value of 2.72 μM to Ava.5/Huh-7 cells and a lower cytotoxicity to parental Huh-7 cells (CC50 = 10.5 μM), and it indeed suppressed HCV replication in the HCV replicon cells. Therefore, these inhibitors with structural novelty may serve as a useful scaffold for the discovery of new HCV NS3 helicase inhibitors

An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol–glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo