Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in β-Cells

Abstract

The loss of insulin-producing β-cells is the central pathological event in type 1 and 2 diabetes, which has led to efforts to identify molecules to promote β-cell proliferation, protection, and imaging. However, the lack of β-cell specificity of these molecules jeopardizes their therapeutic potential. A general platform for selective release of small-molecule cargoes in β-cells over other islet cells ex vivo or other cell-types in an organismal context will be immensely valuable in advancing diabetes research and therapeutic development. Here, we leverage the unusually high Zn(II) concentration in β-cells to develop a Zn(II)-based prodrug system to selectively and tracelessly deliver bioactive small molecules and fluorophores to β-cells. The Zn(II)-targeting mechanism enriches the inactive cargo in β-cells as compared to other pancreatic cells; importantly, Zn(II)-mediated hydrolysis triggers cargo activation. This prodrug system, with modular components that allow for fine-tuning selectivity, should enable the safer and more effective targeting of β-cells.

Figure 1

(A) Zn(II)-mediated unmasking of DA-ZP1 releases active fluorophore ZP1. (B) Structure of DA-FC and graph of Zn(II)-mediated fluorescence release of DA-ZP1 and DA-FC. (C) Selective unmasking of DA-ZP1 fluorescence in INS-1E cells compared to other cell types. (D) Representative images of DA-ZP1- or DA-FC-treated cells under the FITC channel (top) measuring ZP1 release, DAPI staining (middle), and the overlay (bottom). (E) Representative confocal images of dissociated human islets treated with DA-ZP1 followed by immunostaining for C-peptide. (F) Quantification of dispersed human islets treated with DA-ZP1 (n = 4). See also Figure S3. Human pancreatic donor information is available in Table S1. (G) Dispersed human islets were stained, and DA-ZP1⁺ and DA-ZP1^– cells were collected after FACS (n = 4). Representative images show C-peptide (green) and glucagon (red) staining in unsorted, DA-ZP1⁺, DA-ZP1^– cell populations. Nuclei stained with DAPI (blue).

Figure 2

(A) Structures of ZnPD1–3 and their Zn(II)-mediated unmasking of rhodamine urea fluorophore. (B) LC-MS chromatogram of ZnPD1-treated cell extracts. (C) Structures of ZnPD4 and Zn(II)-catalyzed release of BODIPY fluorophore via cascading self-immolation. (D) Selective unmasking of the ZnPD4 fluorophore in INS-1E cells compared to other cells. (E) Representative images of ZnPD4-treated cells under FITC channel (top), DAPI staining (middle), and the overlay (bottom). (F) Quantification of dispersed human islets treated with ZnPD4 (n = 3). See also Figure S7. Human pancreatic donor information is available in Table S1. (G) Dispersed human islets were stained, and BODIPY+ and BODIPY– cells were collected after FACS (n = 3). Representative images show C-peptide (green) and glucagon (red) staining in unsorted, BODIPY+, BODIPY– cell populations. Nuclei stained with DAPI (blue).

Figure 3

(A) Structures of ZnPD5 and its Zn(II)-catalyzed release of GNF-4877. (B) Reaction kinetics of ZnPD5 with different concentrations of Zn(II) as monitored by fluorescence spectroscopy. (C) Fold change in fluorescence in INS1E cells versus other cells. (D) Representative images of ZnPD5-treated cells under the FITC channel (top), DAPI staining (middle), and the overlay (bottom). (E) Representative fluorescence images of intact human islets showing β-cell selective hydrolysis of ZnPD5 under the FITC channel (green) and C-peptide (red). Intact islet cells were treated with either DMSO (top) or 150 nM of ZnPD5 (bottom). (F) Dose-dependent induction of β-cell proliferation by ZnPD5 in human islets.