Regulating miRNA-21 Biogenesis By Bifunctional Small Molecules

Abstract

We report a new strategy to regulate microRNAs (miRNAs) biogenesis by using bifunctional small molecules that consist of a pre-miRNA binding unit connected by a linker to a Dicer inhibiting unit. In this effort, fluorescence polarization-based screening was used to identify neomycin as a pre-miR-21 binding ligand. Although neomycin cannot inhibit miR-21 maturation, linking it to the RNase inhibitor 1 forms the bifunctional conjugate 7A, which inhibits the production of miR-21. We expect that this strategy will be applicable to design other molecules for miRNA regulation.

Figure 1

A schematic illustration of the new approach to regulate miRNA biogenesis by using bi-functional small molecule that target pre-miRNA.

Figure 2

Fluorescence polarization of KOF in the absence or presence of pre-miR-21 and different aminoglycosides. The error bars represent the standard error of mean (N = 3).

Figure 3

(a) The representative image of the electrophoresis analysis of Dicer-mediated pre-miR-21 cleavage in the presence of tested compounds. (b) Densitometric quantitative analysis of miR-21 levels from three independent assays as in (a). The error bars represent the standard error of mean (N = 3).

Figure 4

(a) RT-qPCR analysis of mature miR-21 expression levels. (b) The representative image of western blotting analysis of PDCD4 levels in pre-miR-21 expressing HEK293T cells with or without 7A treatment. (c) Densitometric quantitative analysis of PDCD4 levels from three independent assays as in (b). The error bars represent the standard error of mean (N = 3).

Scheme 1

(a) Structure of an influenza endo-nuclease inhibitor. (b) Synthesis of inhibitor building blocks. (c) Synthesis of bi-functional small molecules. Reagents and conditions: (a) para-methoxybenzyloxyamine, toluene, reflux; (b) trifloroacetic acid, DCM; (c) i) (Boc)₂O, Et₃N, DMF, H₂O, 60 °C; ii) TPS-Cl, pyridine, r.t.; iii) Cysteamine hyrochloride, Cs₂CO₃, DMF; (d) 6-azido-hexanoic acid, EDCI, TEA, DCM; (e) i) CuSO₄, sodium ascorbate, DMSO/H₂O; ii) trifloroacetic acid, DCM.