Identification of Ligands for the Tau Exon 10 Splicing Regulatory Element RNA by Using Dynamic Combinatorial Chemistry

Abstract

We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC(50)): 2-58 μM), as determined by fluorescence titration experiments. Importantly, most of them are able to stabilize both the wild-type and the +3 and +14 mutated sequences associated with the development of FTDP-17 without producing a significant change in the overall structure of the RNA (as analyzed by circular dichroism (CD) spectroscopy), which is a key factor for recognition by the splicing regulatory machinery. A good correlation has been found between the affinity of the ligands for the target and their ability to stabilize the RNA secondary structure.

Exploring the effect of aminoglycoside guanidinylation on ligands for Tau exon 10 splicing regulatory element RNA

“…The ends of the chains were modified with 2'-O-methylribonucleosides (denoted by an asterisk). b) Structure and peptide sequence of the building blocks used in the DCC experiments in our previous work 8 and in the present study.…”

“…On the basis of the DCC results, we planned the synthesis of the guanidinylated analogues of three acridine-neamine compounds previously identified as Tau RNA ligands: Acr1-Nea, Acr2-Nea and Acr2-Nea2. 8 The structures of the parent ligands as well as of their guanidinylated analogues (Acr1-NeaG4, Acr2-NeaG4 and Acr2-Nea2G4, respectively) are shown in Scheme 3. For comparison purposes, the length and the type of linkage between the aminoglycoside and the acridine moieties were the same as those of the non-guanidinylated ligands (e.g.…”

“…6 We have recently described the usefulness of this approach in combination with dynamic combinatorial chemistry (DCC) 7 to identify ligands for the stem-loop structure located at the exon 10-5' intron junction of Tau pre-mRNA. 8 In vitro and in vivo experiments had previously demonstrated that this stem-loop structure is an important regulatory element in pre-mRNA splicing since the extent of exon 10 inclusion is inversely proportional to its stability. 9 The fact that mutations found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) diminish the stability of this stem-loop structure, led to the search for small molecules that selectively bind and stabilize to this structure, in particular the mutated variants.…”

“…9 The fact that mutations found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) diminish the stability of this stem-loop structure, led to the search for small molecules that selectively bind and stabilize to this structure, in particular the mutated variants. 8,10 Such compounds are expected to restore the physiological balance of Tau protein isoforms generated upon alternative splicing of the tau gene and, consequently, the tauopathy to be treated.…”

“…8 The most promising RNA-3 templated ligands combined a small aminoglycoside, neamine (Nea or Nea2 in Scheme 1),…”