LAUSR

The firefly luciferase (FLuc)-based bioluminescent reaction [1] provides the basis for one of the most popular detection systems in high-throughput screening (HTS) [2], especially for cell-based (reporter gene) assays [2,3]. Bioluminescence results from the activity of FLuc, which catalyzes the ATP-dependent reaction of its natural substrate D-luciferin, a benzothiazole derivative, with oxygen that emits energy in the form of light [1]. Depending on the design of the detection system, an increase or decrease in the FLuc-dependent luminescence signal is measured. It is known that FLuc assay readouts are vulnerable to FLuc inhibition by small molecules [4,5]. FLuc inhibitors are frequently encountered and act by a variety of mechanisms including competitive, noncompetitive and anticompetitive (uncompetitive) inhibition [5]. Depending on the assay format, effects of direct FLuc inhibition can be complex and difficult to analyze. This especially applies when FLuc is used as a reporter in cell-based assays. FLuc is highly sensitive to proteolysis and only has a short half-life under cellular conditions [6]. A striking FLuc reporter assay interference effect that is counterintuitive at first sight is caused by the inhibition of FLuc, which actually results in an increase in the luminescence signal (rather than a decrease, as one might expect) [6–8]. In this case, inhibitor binding stabilizes the enzyme and protects it against degradation, which increases its half-life [6–8]. If inhibition retains baseline FLuc activity, the net effect of stabilization is an increase in the luminescence signal. Under assay conditions, increased emission of light through FLuc inhibition has been demonstrated to result from the formation of a multi-substrate adduct inhibitor (by an anticompetitive mechanism) [8]. For reporter gene assays relying on an increase in the luminescence signal relative to a control, such FLuc inhibition is highly likely to cause false positive assay readouts. In addition, various other inhibitory effects are possible, which potentially bias’s FLuc-based assays. Herein, we have addressed the question how FLuc inhibition might affect different assays with FLuc-dependent readouts and if there may be general trends that can be detected. Therefore, a systematic computer-aided analysis of public screening data was carried out.