Identification of molecules with therapeutic benefit has become more accessible due to the availability of large libraries of chemical and natural products. While libraries comprising hundreds of thousands of compounds are now commercially available, drug discovery and development is still a laborious, expensive, and lengthy process. In an ideal setting, it starts with target identification, followed by compound screen eventually identifying a lead compound which will be optimized for efficacy and pharmacokinetics prior to translation into patients [1]. Methods to identify new drugs from compound libraries have become more accessible due to more affordable screening devices, larger libraries and the emerging of noninvasive techniques for faster and robust assay readout, such as bioluminescence imaging (BLI) [2].

Noninvasive molecular imaging of cultured cells and living animals has opened new avenues to help understand fundamental molecular and physiological processes [3]. BLI, in particular, became a widely used laboratory technique allowing the monitoring of different biological processes in immunology [4], oncology [5], virology [6] and neuroscience [7] among many other fields. BLI helps expedite the discovery as well as the development and optimization of new drugs [8]. The sensitive and relatively quick readout of photon emission allows screening for thousands of molecules in a labor, time, and cost-effective manner [9]. In fact, BLI has been widely used to screen thousands of compounds mostly in cell-based assays for target identification, gene signaling networks as well as protein–protein interaction [2, 10, 11]. This imaging modality is also suited for multiplexing with other genomic or proteomic assays for a multiparameter profiling of the complex cellular phenotype [12].

Recently, our group has developed a small-molecule drug screening assay based on the Gaussia luciferase (Gluc) [2]. When expressed under a constitutively active promoter, Gluc activity is linear with respect to time and cell number [2, 13]. The natural secretion properties of this luciferase and its high sensitivity as well as the time and cost effectiveness of bioluminescence imaging make this reporter an ideal readout for high-throughput screening [14]. Gluc activity can be measured either from aliquots of the conditioned medium for noninvasive longitudinal studies or directly from the luciferase-expressing cells in their medium.

Glioblastoma cells lines used in this screen were genetically engineered to stably express Gluc together with the Cyan Fluorescent Protein (CFP). Both Gluc and CFP are expressed in a lentivirus vector and the latter is used to assess the transduction efficiency. For our assay we used a constitutively active promoter driving Gluc to monitor cell viability. When investigating compounds that could affect gene expression or gene regulation instead of cell viability, the constitutively active promoter can be substituted with the inducible promoter of choice. These reporters typically consist of one or multiple transcription factors binding sites linked to a minimal promoter to drive the expression of a luciferase. Such bioluminescent reporters are often used in gene-targeted drug screening. In one example, a p53-reporter vector was used to identify small molecules affecting the transcriptional activation of this gene [15]. This screen identified compounds that activate p53 transcription.

It is important to note that while Gluc was our reporter of choice for this particular type of small-molecules screening, it could be replaced with other luciferases such as Firefly, Renilla, or Cypridina (also known as Vargula luciferase, Vluc). Luciferases that use different substrates can be combined together and used in multiplexed bioluminescence based assays.

In this chapter we describe all necessary steps needed to design and perform a compound-screening assay aimed to identify potential small molecules that kill glioblastoma cells.