The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.

In vivo bioluminescence imaging has become a cornerstone technology for preclinical molecular imaging. This imaging method is based on light-emitting enzymes, luciferases, which require specific substrates for light production. When linked to a specific biological process in an animal model of human biology or disease, the enzyme-substrate interactions become biological indicators that can be studied noninvasively in living animals. Signal intensity in these animal models depends on the availability of the substrate for the reaction within living cells in intact organs. The biodistribution and clearance rates of the substrates are therefore directly related to optimal imaging times and signal intensities and ultimately determine the sensitivity of detection and predictability of the model. Modifications of d-luciferin, the substrate for the luciferases obtained from beetle, including fireflies, result in novel properties and offer opportunities for improved bioassays. For this purpose, we have synthesized a conjugate, glycine-d-aminoluciferin, and investigated its properties relative to those of d-aminoluciferin and d-luciferin. The three substrates exhibited different kinetic properties and different intracellular accumulation profiles due to differences in their molecular structure, which in turn influenced their biodistribution in animals. Glycine-d-aminoluciferin had a longer in vivo circulation time than the other two substrates. The ability to assay luciferase in vitro and in vivo using these substrates, which exhibit different pharmacokinetic and pharmacodynamic properties, will provide flexibility and improve current imaging capabilities.

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.