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.

Fig. 1.

(A) Chemical structures of masked fluorophores 1–6. (B) Chemical structure of masked fluorophore 7.

Fig. 2.

Fluorescence microscopy and quantitative assessment of hydrolysis of compounds 1–6 catalyzed by endogenous cellular esterases. Substrates 1–6 (10 μM) were applied to Drosophila S2 cells, human embryonic kidney cells (HEK 293), human uterus carcinoma cells (HeLa), Chinese hamster ovary carcinoma cells (CHO), dissociated rat hippocampal primary neuronal culture, mouse fibroblast cells (CCL-1), and mouse cortical brain slice for 1 h and imaged live. (A) Substrate 1. (B) Substrate 2. (C) Substrate 3. (D) Substrate 4. (E) Substrate 5. (F) Substrate 6. Cultured cells were counterstained with Hoechst 33342 and imaged using wide-field fluorescence microscopy. Brain slices were imaged using two-photon microscopy. Magnification was adjusted to ensure several cells were within the imaging field. (Scale bars: 10 μm.) (G) Quantification of background-subtracted average cellular fluorescence (relative fluorescence units, RFU) after incubation with substrates 1–6. Error bars show mean ± SD.

Fig. 3.

Cell-specific unmasking of latent fluorophores by PLE in HeLa cells. (A) A 1∶1 mixture of HeLa cells with or without transfection of PLE–IRES–NLS-mCherry, followed by incubation with substrate 6 (10 μM) and Hoechst 33342 (1 μM) for 30 min. (B) A 1∶1 mixture of HeLa cells with or without transfection of PLE–IRES–GFP, followed by incubation with substrate 7 (10 μM) and Hoechst 33342 (1 μM) for 30 min. (Scale bars: 10 μm.)

Fig. 4.

Cell-type-specific esterase-mediated unmasking in dissociated hippocampal neuron–astrocyte coculture. Neuron–astrocyte coculture was transfected with PLE–IRES–NLS-mCherry driven by the neuron-specific hSYN1 promoter and incubated with substrate 6 (10 μM) or substrate 1 (10 μM) for 30 min. (A) Selective unmasking observed when incubated with substrate 6. (B) Unselective unmasking observed when incubated with substrate 1. (Scale bars: 10 μm.)

Fig. 5.

Cell-type-specific esterase-mediated unmasking in mouse brain slices. (A) Two-photon microscopic images of rat hippocampal slice culture transfected with PLE–IRES–mCherry driven by the glial-specific GFAP promoter and incubated with compound 6 (10 μM) for 1 h. (B) Two-photon microscopic images of cortical layer 2/3 in acute brain slice from mice (P14) in utero electroporated with PLE–IRES–mCherry under the CAG promoter at E16, and incubated with compound 6 (10 μM) for 1 h. (C) Two-photon microscopic images of cortical layer 2/3 in acute brain slice from mice (P35) transduced with adeno-associated viral vector for PLE–IRES–mCherry under the hSYN1 promoter, and incubated with compound 6 (10 μM) for 1 h. (Scale bars: 10 μm.) (D) Comparison of input resistance (R_m), cell capacitance (C_m), and resting membrane potential (V_m) of PLE^- and PLE⁺ neurons in acute brain slice. Error bars show mean ± SD, n.s. is not significant, p > 0.05.

Fig. 6.

Noninvasive imaging of gap junction permeability in living cells using an esterase–ester system. WB-F344 cells were first transfected with PLE–IRES–NLS-mCherry for 24 h. Transfected cells were mixed with untransfected cells and incubated at 37 °C for an additional 24 h. (A) Cells incubated with substrate 6 (10 μM) and Hoechst 33342 (1 μM) for 30 min. (B) Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) for 30 min prior to addition of compound 6 (10 μM) and Hoechst 33342 (1 μM). (Scale bars: 10 μm.)

Fig. 7.

Cell-specific pharmacology. (A) Synthesis of monastrol-CM (10). (B) Prevalence of an altered spindle phenotype in PLE^- cells and cells transfected with PLE–IRES–mCherry (PLE⁺ cells) when released from the double thymidine block to 50 μM monastrol (8), 50 μM monastrol-CM (10), or DMSO control (0.1% vol/vol) as a percentage of all mitotic cells. Error bars show mean ± SD, n.s. is not significant, p > 0.05, *** p < 0.001.