Myotonic dystrophy type 1 (DM1) is caused by an expanded CUG repeat (CUGexp) that sequesters muscleblind-like 1 protein (MBNL1), a protein that regulates alternative splicing. CUGexp RNA is a validated drug target for this currently untreatable disease. Herein, we develop a bioactive small molecule (1) that targets CUGexp RNA and is able to inhibit the CUGexp·MBNL1 interaction in cells that model DM1. The core of this small molecule is based on ligand 2, which was previously reported to be active in an in vitro assay. A polyamine-derivative side chain was conjugated to this core to make it aqueous-soluble and cell penetrable. In a DM1 cell model this conjugate was found to disperse CUGexp ribonuclear foci, release MBNL1, and partially reverse the mis-splicing of the insulin receptor pre-mRNA. Direct evidence for ribonuclear foci dispersion by this ligand was obtained in a live DM1 cell model using time-lapse confocal microscopy.

Fluorine-19 MRI is an emerging cellular imaging approach, enabling lucid, quantitative “hot-spot” imaging with no background signal. The utility of 19F-MRI to detect inflammation and cell therapy products in vivo could be expanded by improving the intrinsic sensitivity of the probe by molecular design. We describe a metal chelate based on a salicylidene-tris(aminomethyl)ethane core, with solubility in perfluorocarbon (PFC) oils, and a potent accelerator of the 19F longitudinal relaxation time (T1). Shortening T1 can increase the 19F image sensitivity per time and decrease the minimum number of detectable cells. We used the condensation between the tripodal ligand tris-1,1,1-(aminomethyl)ethane and salicylaldehyde to form the salicylidene-tris(aminomethyl)ethane chelating agent (SALTAME). We purified four isomers of SALTAME, elucidated structures using X-ray scattering and NMR, and identified a single isomer with high PFC solubility. Mn4+, Fe3+, Co3+, and Ga3+ cations formed stable and separable chelates with SALTAME, but only Fe3+ yielded superior T1 shortening with modest line broadening at 3 and 9.4 T. We mixed Fe3+ chelate with perfluorooctyl bromide (PFOB) to formulate a stable paramagnetic nanoemulsion imaging probe and assessed its biocompatibility in macrophages in vitro using proliferation, cytotoxicity, and phenotypic cell assays. Signal-to-noise modeling of paramagnetic PFOB shows that sensitivity enhancement of nearly 4-fold is feasible at clinical magnetic field strengths using a 19F spin-density-weighted gradient-echo pulse sequence. We demonstrate the utility of this paramagnetic nanoemulsion as an in vivo MRI probe for detecting inflammation macrophages in mice. Overall, these paramagnetic PFC compounds represent a platform for the development of sensitive 19F probes.

An expanded CUG repeat transcript (CUGexp) is the causative agent of myotonic dystrophy type 1 (DM1) by sequestering muscleblind-like 1 protein (MBNL1), a regulator of alternative splicing. Based on a ligand (1) that was previously reported to be active in an in vitro assay, we present the synthesis of a small library containing ten dimeric ligands (4-13) that differ in length, composition and attachment point of the linking chain. The oligoamino linkers gave a greater gain in affinity for CUG RNA and were more effective when compared to oligoether linkers. The most potent in vitro ligand (9) was shown to be aqueous-soluble and both cell- and nucleus-permeable displaying almost complete dispersion of MBNL1 ribonuclear foci in a DM1 cell model. Direct evidence for the bioactivity of 9 was observed in its ability to disperse ribonuclear foci in individual live DM1 model cells using time-lapse confocal fluorescence microscopy.

Further studies are warranted to determine the role of NETs as a procoagulant in human thrombosis.

Effective drug discovery and optimization can be accelerated by techniques capable of deconvoluting the complexities often present in targeted biological systems. We report a single-molecule approach to study the binding of an alternative splicing regulator, muscleblind-like 1 protein (MBNL1), to (CUG)n = 4,6 and the effect of small molecules on this interaction. Expanded CUG repeats (CUGexp) are the causative agent of myotonic dystrophy type 1 by sequestering MBNL1. MBNL1 is able to bind to the (CUG)n–inhibitor complex, indicating that the inhibition is not a straightforward competitive process. A simple ligand, highly selective for CUGexp, was used to design a new dimeric ligand that binds to (CUG)n almost 50-fold more tightly and is more effective in destabilizing MBNL1–(CUG)4. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions.