Objective

n recent years, poor drug efficacy observed during Phase II/III clinical trials has been identified as a key cause of drug attrition in the pharmaceutical industry. Historically, efforts to find drug molecules has primarily focussed on the ability of a compound to bind to or modulate its target, often optimising the potency based on equilibrium dissociation constant (Kd) or half-maximal inhibitory concentration (IC50). Typically these values are determined in assays, either biochemical or cell based, at fixed concentrations or time points. Whilst, thermodynamic affinity observations in vitro have been shown to translate in an in vivo setting, this is not always the case. In many cases it is the length of time a drug molecule is bound to its target that will inform in vivo efficacy. From a retrospective point of view, there are a number of highly efficacious marketed drugs that demonstrate long-lasting, on-target residence time. The question arises as to how we can incorporate prospective approaches in drug discovery that allow us to identify long residence time molecules. Whilst there are a number of readily available technologies that can determine on-target residence time of molecules, these are often biochemical in nature. As such drug-target engagement and drug-target interactions are often limited to truncated proteins in the absence of native cellular components. Use of phenotypic cell-based assays to determine residual molecule binding after removal or “washout” of the drug molecules, whilst informative, are often limited by throughput, assay design and inherent deconvolution of off-target effects. A more simplistic approach to monitoring drug-target engagement and drug-target interactions has been developed by Promega which utilises bioluminescence resonance energy transfer (BRET) to monitor target engagement and on-target residence time in a cellular context. Full-length molecular targets fused to Nanoluc luciferase (NanoLuc) are expressed in intact cells and drug-target interactions monitored in the presence of a fluorescent-labelled tracer in a competitive, real time manner. Here we report on work carried out to develop and optimise this technology to deliver quantitative cellular association and dissociation rates for drug molecules against intracellular targets (bromodomains-containing proteins). Assay limitations, scalability and potential impact on drug design will also be addressed.