Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays.

As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1β and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway.

Homogeneous, bioluminescent proteasome assays.

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases.

A bioluminescent assay for the sensitive detection of proteases.

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min.

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.

Bioluminescent assays for ADMET.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity.

Homogeneous, bioluminescent proteasome assays.

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer . The importance of the proteasome for general cell homeostasis has been established, and the 2004 Nobel Prize for Chemistry honored the researchers that discovered the ubiquitin-proteasome pathway.

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.