Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing

Abstract

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Small molecule recognition of disease-relevant RNA structures

“…118 Additional structure-activity relationships (SAR) were used to optimize MTX's ability to decrease exon 10 inclusion, leading to compounds with enhanced affinity for tau pre-mRNA and increased potency for reducing the levels of 4R tau (Table S1, ESI †). 119 More recently, Chen et al 120 reported that stabilizing the SRE by small molecule binding to the A bulge present in the SRE structure could inhibit recognition by U1 snRNP and promote exon 10 exclusion in wild-type (WT) and DDPAC tau. Tanimoto score-based similarity searching using a previously reported Inforna hit 121 as a query identified A-1 as a modulator of the 4R/3R tau ratio ( Fig.…”

“…Notably, this assertion is bolstered by studies that direct the splicing outcome of MAPT exon 10 (tau), another IDP. 120 10. Targeted cleavage of diseasecausing RNAs using bleomycin A5-conjugates…”

RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays

“…Besides ASO-based tau expression inhibitors, drug molecules that induce the exon 10 skipping in the mutant MAPT would also be of potential therapeutic interest for restoring the aberrant splice pattern for FTDP-17 [ 9 ]. ASOs bound to either the 5′ or 3′ splice site of exon 10 were demonstrated to induce exon 10 skipping [ 76 ].…”

“…NMR experiments demonstrated that mitoxantrone stabilizes the stem-loop structure through intercalating into the stem base [ 145 ]. Several other small-molecule splicing modifiers were identified by the Disney group via structure-based design [ 9 , 79 ] or phenotypic screening (e.g., “compound 9” and “compound 2” from the original report [ 9 ] ( Figure 8 b). Compound 9 was shown to stabilize an internal bulge of the stem-loop structure and reduce exon 10 splicing [ 9 ].…”

“…Some genes encoded in eukaryotic organelles (e.g., mitochondria [ 1 ]) and in prokaryotic cells [ 2 ] must undergo spliceosome-independent RNA splicing before translation. In the past two decades, gene- or exon-specific splicing modifiers have been developed for the treatment of several human disease states, including spinal muscular atrophy (SMA) [ 3 , 4 ], Duchenne muscular dystrophy (DMD) [ 5 , 6 , 7 ], and influenza virus infection [ 8 ], as well as pre-clinical drug development for diseases such as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) [ 9 ], yeast infection [ 10 ], and familial dysautonomia [ 11 ]. Instead of a traditional protein-targeting drug modality, these compounds act through direct binding to the pre-mRNAs.…”