Selective esterase–ester pair for targeting small molecules with cellular specificity

Abstract

Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations. cellular imaging | microscopy | enzyme substrates | fluorophores | pharmacological agents C hemical probes are essential tools in biology for measuring and manipulating cellular properties. Optimization of the structural and electronic features of small molecules allows the fine-tuning of molecular recognition specificity for a particular cellular target. Even with high molecular specificity, however, the application of small molecules in complex biological environments is frequently limited by poor cellular specificity. The inability to target small molecules, such as imaging or pharmacological agents, to defined cellular populations can confound the evaluation and control of discrete subsets of cells within a multicellular environment. A general and efficient strategy for cell-specific targeting, combining the molecular specificity of small molecules with the cellular specificity of genetics, would allow intracellular pathways in defined cell types to be selectively probed in complex tissues.An attractive approach for general cell-specific delivery of small molecules employs selective enzyme-substrate pairs. In this strategy, compounds are masked by attachment of a standard, disposable blocking group that is stable to native cellular enzymes, but labile to a specific exogenous enzyme. Expression of such a protein in a genetically defined cell population permits unmasking of the small molecule with cellular specificity. To be useful across experimental paradigms, such a system should utilize an enzyme that unmasks molecules with high efficiency, expresses in different cell types, and exhibits low cellular toxicity. The cognate masking group must be modular, synthetically efficient, and allow molecules to diffuse passively across the cellular membrane, while also exhibiting favorable solubility and stability in aqueous solution.To date only a few enzyme-substrate pairs have been used as targeted delivery systems for small molecules, and none meet all the criteria outlined above. Strategies employing enzymes encoded by common reporter genes (1) have found some success in targeting small molecules (2-4), ...

Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach

“…However, reliable targeting of a Ca 2+ -sensitive dye to selected cells in the live mammalian brain is usually achieved by visually guided intracellular loading, such as by maneuvering a micropipette (Kitamura et al, 2008), which impedes the study of large populations of cells of a single genetic class. Emerging chemical genetic methods may combine bulk loading of an indicator dye with genetic specificity (Tian et al, 2012). An existing alternative is to use one fluorescence color channel for detecting Ca 2+ signals across a broad population of indicator labeled cells and another color channel for identifying a subset of cells that express a fluorescent genetic marker (Kerlin et al, 2010; Nimmerjahn et al, 2004; Runyan et al, 2010).…”

Integrating Optogenetic and Pharmacological Approaches to Study Neural Circuit Function: Current Applications and Future Directions

“…If optogenetic circuit analysis and RNA-seq identify a specific gene product that may perform an integral role in cellular signaling, therapeutics to inhibit or restore gene function or associated intracellular signaling modalities can then be designed. The technology for targeting genetically defined cells with small molecule drugs to restore normal functioning was demonstrated by a recent study from Tian et al (2012) that identified a specific esteresterase pair that could be used to deliver active small molecules to precisely defined cell types. The specific pair of porcine liver esterase (PLE) and a a-cyclopropyl ester was highly efficient at unmasking the small molecule and had negligible reactivity with endogenous esterases.…”

“…The underlying principle of this strategy is to mask a small molecule drug by the attachment of a cleavable blocking functional group that renders it unreactive to endogenous cellular enzymes but is easily removed by a specific exogenous enzyme Tian et al, 2012). Cell type specificity is achieved by the expression of this exogenous enzymatic protein within a target cellular population where cleavage of the prodrug will occur and its therapeutic effect may be somewhat restricted.…”

“…For this reason, investigating the transcriptome of targeted cell types within dysregulated neural circuits is likely to produce important insights into altered cellular function underlying disease. In addition, a logical extension of the pharmacogenomics effort that has been evolving in parallel is the effort in molecular genetics and nanotechnology to devise strategies for the delivery of more highly targeted, cell-specific small molecules that act precisely at the locus of dysfunction Paulo et al, 2011;Tian et al, 2012). It is within these current trends that we envision optogenetics being applied most innovatively for the identification of candidate genes and proteins within specific neuronal populations and circuits that subsequently can be targeted with cell-specific small molecule pharmacotherapies.…”

Optogenetic Brain Interfaces

“…There is recent evidence that some serotypes of AAV may cross the BBB, and could therefore be delivered via intravenous injection [231], [232]. Newer, less invasive technologies to deliver small designer drugs or molecules have been proposed using conjugated nanoparticles or specific enzyme-substrate pairs, cleaved after crossing the BBB [233], [234], [235], [182]. In addition, toolboxes of cellular and regional mini-promoters in the brain are being defined [236] that can be utilized for targeting applications.…”