Delivery of small molecules to biological systems is limited by lack of specificity. “Masking” the activity of the small molecule via chemical modification and unmasking it only within target tissues could mitigate this challenge. To this end, we have developed an engineered variant of nitroreductase (NTR) that enables rapid, selective unmasking of small molecules in genetically defined cells. Using a divergent synthetic route we first synthesized a series of fluorogenic substrates based on the Oregon Green fluorophore that contain different nitroaromatic masking groups. We further developed a high-throughput screening platform to identify variants with improved activity. Multiple beneficial mutations were identified, showing an increase in k<sub>cat</sub> of seven-fold in vitro and in cultured cells. Additionally, macroengineering of NTR by generating tandem dimers and fusing to stabilizing proteins further enhances activity in cultured neurons. The combination of site-directed mutagenesis and macroengineering results in an enzyme that possesses nearly 100-fold increased activity over wild-type nitroreductase. Finally, we placed the optimized masking group on a potent NMDA receptor antagonist and found we could selectively block receptor activity in cultured NTR+ neurons without blocking co-cultured NTR- neurons. In vivo experiments using this technology are in progress. This enzyme is capable of unmasking disparate small molecules including dyes, indicators, and drugs and should find broad utility in the targeted delivery of small molecules to defined cell types.

Engineering <i>E. coli</i> nitroreductase for targeted small molecule release

251st National Meeting (2016)

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Engineering E. coli nitroreductase for targeted small molecule release