Christophe Antczak

Tumor Targeting with Antibody-Functionalized, Radiolabeled Carbon Nanotubes

Single-walled carbon nanotubes (CNT) are mechanically robust graphene cylinders with a high aspect ratio that are comprised of sp2-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. We hypothesized that novel prototype nanostructures consisting of biologics, radionuclides, fluorochromes, and CNT could be synthesized and designed to target tumor cells. Methods: Tumor-targeting CNT constructs were synthesized from sidewall-functionalized, water-soluble CNT platforms by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radiometal-ion chelates, and fluorescent probes. The constructs were characterized spectroscopically, chromatographically, and electrophoretically. The specific reactivity of these constructs was evaluated in vitro by flow cytometry and cell-based immunoreactivity assays and in vivo using biodistribution in a murine xenograft model of lymphoma. Results: A soluble, reactive CNT platform was used as the starting point to build multifunctional constructs with appended antibody, metal-ion chelate, and fluorescent chromophore moieties to effect specific targeting, to carry and deliver a radiometal-ion, and to report location, respectively. These nanoconstructs were found to be specifically reactive with the human cancer cells they were designed to target in vivo in a model of disseminated human lymphoma and in vitro by flow cytometry and cell-based immunoreactivity assays versus appropriate controls. Conclusion: The key achievement in these studies was the selective targeting of tumor in vitro and in vivo by the use of specific antibodies appended to a soluble, nanoscale CNT construct. The ability to specifically target tumor with prototype-radiolabeled or fluorescent-labeled, antibody-appended CNT constructs was encouraging and suggested further investigation of CNT as a novel delivery platform.

Live-Cell Imaging of Caspase Activation for High-Content Screening

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. (Journal of Biomolecular Screening 2009:956-969)

Synthesis of Antiproliferative Cephalotaxus Esters and Their Evaluation against Several Human Hematopoietic and Solid Tumor Cell Lines: Uncovering Differential Susceptibilities to Multidrug Resistance

Deoxyharringtonine (2), homoharringtonine (3), homodeoxyharringtonine (4), and anhydroharringtonine (5) are reported to be among the most potent members of the antileukemia alkaloids isolated from the Cephalotaxus genus. Convergent syntheses of these four natural products are described, each involving novel synthetic methods and strategies. These syntheses enabled evaluation of several advanced natural and non-natural compounds against an array of human hematopoietic and solid tumor cells. Potent cytotoxicity was observed in several cell lines previously not challenged with these alkaloids. Variations in the structure of the ester chain within this family of alkaloids confer differing activity profiles against vincristine-resistant HL-60/RV+, signalling new avenues for molecular design of these natural products to combat multi-drug resistance.

High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid—based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)—based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent. (Journal of Biomolecular Screening 2007:521-535)

A Class of Allosteric Caspase Inhibitors Identified by High-Throughput Screening

Caspase inhibition is a promising approach for treating multiple diseases. Using a reconstituted assay and high-throughput screening, we identified a group of nonpeptide caspase inhibitors. These inhibitors share common chemical scaffolds, suggesting the same mechanism of action. They can inhibit apoptosis in various cell types induced by multiple stimuli; they can also inhibit caspase-1-mediated interleukin generation in macrophages, indicating potential anti-inflammatory application. While these compounds inhibit all the tested caspases, kinetic analysis indicates they do not compete for the catalytic sites of the enzymes. The cocrystal structure of one of these compounds with caspase-7 reveals that it binds to the dimerization interface of the caspase, another common structural element shared by all active caspases. Consistently, biochemical analysis demonstrates that the compound abates caspase-8 dimerization. Based on these kinetic, biochemical, and structural analyses, we suggest that these compounds are allosteric caspase inhibitors that function through binding to the dimerization interface of caspases.

Combining integrated genomics and functional genomics to dissect the biology of a cancer‐associated, aberrant transcription factor, the ASPSCR1–TFE3 fusion oncoprotein

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1–TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1–TFE3, PRCC‐TFE3, SFPQ‐TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1–TFE3 fusion oncoprotein, defined its target promoters on a genome‐wide basis and performed a high‐throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1–TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome‐wide location analysis performed on the FU‐UR‐1 cell line, which expresses endogenous ASPSCR1–TFE3, identified 2193 genes bound by ASPSCR1–TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1–TFE3 defined a subset of 332 genes as putative up‐regulated direct targets of ASPSCR1–TFE3, including MET (a previously known target gene) and 64 genes as down‐regulated targets of ASPSCR1–TFE3. As validation of this approach to identify genuine ASPSCR1–TFE3 target genes, two up‐regulated genes bound by ASPSCR1–TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1–TFE3. As the results indicated that ASPSCR1–TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up‐regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up‐regulated direct target genes to study in high‐throughput RNAi screens, using FU‐UR‐1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1–TFE3 target genes contribute to the growth of ASPSCR1–TFE3‐positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors. Copyright

Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Cell viability assessment: toward content-rich platforms

Importance of the field: Monitoring cell viability in vitro is critical in many areas of biomedical research, and the ultimate goal in drug discovery is the ability to predict the in vivo toxicology of drug candidates based on their toxicity profile in vitro. Over the last decade, the contribution of high-throughput screening toward this goal has been tremendous, providing the ability to screen compounds in parallel against multiple cell types. However, the toxic effects of drug candidates uncovered during clinical trials are by far the main reason for their failure. Over the same period, our understanding of programmed cell death has evolved dramatically with the identification of critical control points in the cell death pathways. As a result, cell viability should no longer be characterized solely on the basis of discrete end point measurements such as membrane permeability. Areas covered in this review: This review summarizes the traditional viability assays currently commercially available, focusing on methods amenable to high density format. Assays categorized into the following classes are discussed: dye exclusion assays, DNA condensation-based assays and assays monitoring a metabolic function. What the reader will gain: We describe current approaches for assessing cell viability and, using case studies, emphasize their limitations. As an alternative, we propose the use of live, multiplexed readouts to accurately record cell death induction. Take home message: Current low-content methods based on single parameter readouts are prone to error due to the heterogeneity of cell populations and the multi-faceted nature of cell death. High-content approaches based on continuous, multiplexed readouts are becoming increasingly important for monitoring multiple markers of cell death induction simultaneously on a cell by cell basis. The use of such content-rich platforms is a necessity to predict the toxicology of drug candidates accurately.

Revisiting Old Drugs as Novel Agents for Retinoblastoma: In Vitro and In Vivo Antitumor Activity of Cardenolides

PURPOSE Intra-arterial delivery of chemotherapeutic agents offers a new and exciting opportunity for the treatment of advanced intraocular retinoblastoma. It allows local delivery of relatively high doses of chemotherapy agents while bypassing general blood circulation. For this reason, this study was undertaken to revisit some of the FDA-approved drugs for the treatment of retinoblastoma. METHODS High-throughput screening (HTS) of 2640 approved drugs and bioactive compounds resulted in the identification of cytotoxic agents with potent activity toward both the Y79 and RB355 human retinoblastoma cell lines. Subsequent profiling of the drug candidates was performed in a panel of ocular cancer cell lines. Induction of apoptosis in Y79 cells was assessed by immunofluorescence detection of activated caspase-3. Therapeutic effect was evaluated in a xenograft model of retinoblastoma. RESULTS Several FDA-approved drugs were identified that showed potent cytotoxic activity toward retinoblastoma cell lines in vitro. Among them were several cardiac glycosides, a class of cardenolides historically associated with the prevention and treatment of congestive heart failure. Caspase-3 activation studies provided an insight into the mechanism of action of cardenolides in retinoblastoma cells. When tested in a xenograft model of retinoblastoma, the cardenolide ouabain induced complete tumor regression in the treated mice. CONCLUSIONS Cardenolides were identified as a new class of antitumor agents for the treatment of retinoblastoma. Members of this class of cardiotonic drugs could be repositioned for retinoblastoma if administered locally via direct intra-arterial infusion.

Toward a Prostate Specific Antigen-Based Prostate Cancer Diagnostic Assay: Preparation of Keyhole Limpet Hemocyanin -Conjugated Normal and Transformed Prostate Specific Antigen Fragments

Prostate specific antigen (PSA) molecules secreted by cancerous and normal prostate cells differ in their N-linked glycan composition, while the peptide backbone appears to be conserved. Antibodies selectively recognizing such differentially glycosylated PSA structures could form a basis for a new diagnostic assay for prostate cancer. Twenty-amino acid PSA fragments carrying di-, tri-, and tetrabranched complex-type glycans were prepared by total synthesis and conjugated to maleimide-modified keyhole limpet hemocyanin (KLH) carrier protein through backbone Cys residues. These glycopeptide/KLH conjugates were then used for antibody generation.