Michael Scurria

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

Using caspase-3 as a model, the authors have developed a strategy for highly sensitive, homogeneous protease assays suitable for high-throughput, automated applications. The assay uses peptide-conjugated aminoluciferin as the protease substrate and a firefly luciferase that has been molecularly evolved for increased stability. By combining the proluminescent caspase-3 substrate, Z-DEVD-aminoluciferin, with a stabilized luciferase in a homogeneous format, the authors developed an assay that is significantly faster and more sensitive than fluorescent caspase-3 assays. The assay has a single-step format, in which protease cleavage of the substrate and luciferase oxidation of the aminoluciferin occurs simultaneously. Because these processes are coupled, they rapidly achieve steady state to maintain stable luminescence for several hours. Maximum sensitivity is attained when this steady state occurs; consequently, this coupled-enzyme system results in a very rapid assay. The homogeneous format inherently removes trace contamination by free aminoluciferin, resulting in extremely low background and yielding exceptionally high signal-to-noise ratios and excellent Z′ factors. Another advantage of a luminescent format is that it avoids problems of cell autofluorescence or fluorescence interference that can be associated with synthetic chemical and natural product libraries. This bioluminescent, homogeneous format should be widely applicable to other protease assays.

Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

A luminescent method to individually measure the chymotrypsin-like, trypsin-like, or caspase-like activities of the proteasome in cultured cells was developed. Each assay uses a specific luminogenic peptide substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. Luminescence is generated in a coupled-enzyme format in which proteasome cleavage of the peptide conjugated substrate generates aminoluciferin, which is a substrate for luciferase. The homogeneous method eliminates the need to prepare individual cell extracts as samples. Luminogenic proteasome substrates and buffer formulations enabled development of a single reagent addition method with adequate sensitivity for 96- and 384-well plate formats. Proteasome trypsin-like specificity was enhanced by incorporating a mixture of protease inhibitors that significantly reduce nonspecific serum and cellular backgrounds. The assays were used to determine EC(50) values for the specific proteasome inhibitors epoxomicin and bortezomib for each of the catalytic sites using a variety of cancer lines. These cell-based proteasome assays are direct, simple, and sensitive, making them ideal for high-throughput screening.

Diastereoselective Synthesis of 2′-Deoxy and 2′-O-Methyl Dinucleoside (3′ 5′)-Methylphosphonates via Alkoxymagnesium Chloride-Mediated Nucleoside Coupling

Abstract A diastereoselective dinucleoside methylphosphonate synthetic method that features coupling of diastereomerically pure 1,1,1,3,3,3-hexafluoro-2-propyl nucleoside-3′-O-methylphosphonate monomers with 3′-O-protected nucleoside monomers mediated by alkoxymagnesium chloride reagents is described. This synthetic method was found to be diastereospecific in the synthesis of selected 2′-deoxy dinucleoside methyphosphonates and diastereoselective in the synthesis of all sixteen 2′-O-methyl dinucleoside methylphosphonates.

Abstract LB-94: A non-lytic, cell-based, bioluminescent HDAC assay

Histone deacetylase (HDAC) class I and II enzymes play a critical role in the normal gene regulation events of development and homeostasis, but their dysregulation has been implicated in a variety of solid tumors and hematological malignancies. Broad spectrum inhibitors of HDAC Class I and II enzymes have been shown to achieve significant biological responses in otherwise refractory cancers. However, the underlying molecular mechanisms of these inhibitors have yet to be fully elucidated. Therefore, more sensitive and efficient tools are needed to rapidly identify and define modulators of HDAC activity. We have recently developed a one-step, homogeneous, luminescent assay for the detection of activity in a high-throughput format from multiple HDAC Class I and II isoforms, in cell culture, enriched or recombinant sources. This luciferase-containing, “glow-type” assay reagent measures HDAC activity in a proportional manner when the pro-luciferin peptide substrate becomes deacetylated and susceptible to lysine-specific cleavage by a developer enzyme contained within the reagent. This substrate is fully cell-permeable and can directly measure HDAC activity in living cells. We will present data demonstrating assay utility with attachment-dependent and suspension cell types using representative inhibitors from the hydroxamate, short-chain fatty acid, benzamide and cyclic peptide classes. Furthermore, we will correlate HDAC inhibition profiles with initiation of caspase activity and/or other cytotoxicity biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-94.