Yuquan Tong’s research while affiliated with The Scripps Research Institute and other places
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Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by the aberrant alternative pre-mRNA splicing of microtubule-associated protein tau ( MAPT ) exon 10, the inclusion of which encodes for a toxic tau protein harboring four microtube domains (4R tau). Here, we describe the design of an RNA-targeted small molecule that thermodynamically stabilizes the structure of a pre-mRNA splicing regulator element in the MAPT pre-mRNA exon 10-intron junction to reduce the inclusion of exon 10 and hence 4R tau abundance. Structure-based drug design was used to obtain compounds that form a network of specific interactions to the RNA including multiple interactions between a one nucleotide A-bulge and the Hoogsteen face of a closing GC base pair, the latter of which was enabled by the design of base triple interactions. A battery of assays revealed that the compound binds the target in vitro and in cells and affects pre-mRNA splicing in various cellular models including primary neurons from a human tau (htau) knock-in mouse model. The orally bioavailable compound was administered per os ( p.o. ), where treatment diminished exon 10 inclusion, and reduced the 4R tau protein isoform. Further, the molecule mitigated cellular pathologies and behavioral phenotypes observed in the htau transgenic mouse model. This study provides a potentially general pipeline to design compounds that target RNAs and affect disease pathways and deliver compounds that have oral bioavailability and blood-brain barrier penetrance.
c-MYC (MYC) is an important oncogene in multiple myeloma (MM), driving MM cell proliferation, metabolism, and survival. However, MYC has been challenging to therapeutically target because of its protein structure lacking well-defined binding sites. In this study, we investigated a novel Ribonuclease Targeting Chimera (MYC-RiboTAC) to degrade MYC mRNA in MM cells. This dimeric small molecule binds both the Internal Ribosomal Entry Site (IRES) of MYC mRNA and RNase L. RNase L then dimerizes into its active conformation and degrades the MYC mRNA.
First, by immunohistochemistry (IHC) analysis of MM tumor microarrays from 24 MM patients. we confirmed significant expression of RNase L in all patients compared to healthy donors (n=11). Notably, RNase L was selectively expressed by plasma cells in the bone marrow. Based on RNA-seq and Western blot analyses of MYC and RNase L expression, to test MYC-RiboTAC, we selected AMO1, H929, R8226, MOLP8, MM1S, OPM2 (MYC+/RNase L+), KMS12BM (MYC+/RNase L-), and U266 (MYC-/RNase L-) MM cell lines. MYC-RiboTAC significantly reduced MYC mRNA levels in cell lines expressing both MYC and RNase L, with the most substantial reduction observed in OPM2 (~75%) and similar reductions in R8226, MOLP8, MM1S, AMO1, and H929 cells (~35%). The compound exerted more pronounced effects at the protein level, downregulating MYC protein in all cell lines expressing MYC and RNase L, with no effect on RNase L negative KMS12BM cell line. Additionally, single-molecule RNA FISH to detect MYC mRNA and immunofluorescence to detect MYC protein confirmed that MYC-RiboTAC significantly reduced both MYC mRNA (located in the cytosol) and MYC protein (located in the nucleus). Transcriptomic analysis in AMO1 cells further supported the selective action of MYC-RiboTAC on the MYC pathway. We further demonstrated the RNase L-dependent effect of MYC-RiboTAC by i) RNase L knockout in AMO1 cells, which abrogated the downregulation of MYC by MYC-RiboTAC, and ii) ectopic expression of RNase L in KMS12BM cells, which restored the MYC downregulating activity of MYC-RiboTAC.
MYC-RiboTAC inhibited growth in all six MM cell lines that expressed both MYC and RNase L but had no impact on the growth of KMS12BM and U266 cell lines. It suppressed colony formation in AMO1 and H929 cells in a dose-dependent manner. Its anti-MM activity was not abrogated when MM cells were co-cultured with HS5 stromal cells. Additionally, MYC-RiboTAC showed synergistic effects with clinically active drugs such as Lenalidomide, Pomalidomide, and Melphalan, which were found to upregulate RNase L and potentially enhance MYC-RiboTAC efficacy. The essential role of RNase L was further confirmed by treating MM tumor cells from three patients with MYC-RiboTAC, which reduced MYC protein levels only in the RNase L-expressing patient. Moreover, the compound reduced the viability of CD138+ cells from an MM patient without affecting the viability of CD138- cells and normal cells such as PBMCs or human fibroblasts.
We performed a DMPK study in mice and found that daily intraperitoneal administration of MYC-RiboTAC at 30 mg/kg, achieved an active concentration of 6 µM in the blood and a half-life of 5.2 hours. In H929 xenograft mouse model a significant reduction (~64% p<0.05) in tumor growth was observed in MYC-RiboTAC treated animals compared to the vehicle-treated group, with no overt toxicity. Western blot analysis of retrieved tumors from mice revealed significantly lower MYC protein levels.
In conclusion, we report functional effects of a first RNA-targeting small molecules in MM and establish MYC-RiboTAC as a potential therapeutic molecule targeting MYC. This study opens new frontiers in drug discovery for targets long considered undruggable.
RNA plays important roles in regulating both health and disease biology in all kingdoms of life. Notably, RNA can form intricate three‐dimensional structures, and their biological functions are dependent on these structures. Targeting the structured regions of RNA with small molecules has gained increasing attention over the past decade, because it provides both chemical probes to study fundamental biology processes and lead medicines for diseases with unmet medical needs. Recent advances in RNA structure prediction and determination and RNA biology have accelerated the rational design and development of RNA‐targeted small molecules to modulate disease pathology. However, challenges remain in advancing RNA‐targeted small molecules towards clinical applications. This review summarizes strategies to study RNA structures, to identify small molecules recognizing these structures, and to augment the functionality of RNA‐binding small molecules. We focus on recent advances in developing RNA‐targeted small molecules as potential therapeutics in a variety of diseases, encompassing different modes of actions and targeting strategies. Furthermore, we present the current gaps between early‐stage discovery of RNA‐binding small molecules and their clinical applications, as well as a roadmap to overcome these challenges in the near future.
Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we present a protocol for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells. We describe steps for compound treatment, cross-linking, RNA extraction, fragmentation, and pull-down. We then detail procedures for RNA sequencing and data analysis.
For complete details on the use and execution of this protocol, please refer to Tong et al.¹
Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we describe the procedures for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells, identifying both the targets bound by the small molecule and the sites of binding therein. For complete details on the use and execution of this protocol, please refer to 1.
α-Synuclein is an important drug target for the treatment of Parkinson’s disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5′ untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5′ UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient–derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.
... Furthermore, the binding sites can be formed by several protein molecules at their interaction interface (Ferré et al., 2014;Wang et al., 2018a), opening another opportunity for proximity-induced drug discovery (Békés et al., 2022;Dewey et al., 2023;Liu and Ciulli, 2023;Tan et al., 2024). While proteins are the most common pharmacological targets, nucleic acids, particularly RNAs, are gaining increasing interest in structure-based drug design (Chen et al., 2024;Tong et al., 2024). RNA plays a vital role in gene regulation and information transfer, making it an appealing target for drug development (Warner et al., 2018). ...
... The formation of a covalent bond between an RNA and a small molecule binder enables direct target engagement and identification of the binding site, as often employed in the protein-targeting field [ 22 , 23 ]. Dubbed Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP), an RNA binder is appended with a cross-linking module, such as chlorambucil [ 16 , 20 , 21 ] or photoreactive diazirine [24][25][26], and a purification tag. The latter enables the pull-down of probe-modified transcripts with streptavidin beads or by direct copper-catalyzed azide-alkyne cycloaddition (CuAAC) [ 27 , 28 ] to azide-functionalized beads. ...
... Administration of iron chelators reduce the mRNA expression of α-syn in HEK293 cells (Febbraro et al. 2012). Research by Tong et al. identified Synucleozid-2.0, a small molecule that targets the IRE region of α-syn mRNA, promoting its degradation and reducing α-syn levels (Tong and Zhang 2024). These findings suggest that iron overload facilitates IRP dissociation from α-syn mRNA, enhancing α-syn translation and promoting its aggregation, which worsens PD pathology. ...
... To date, numerous small molecules that selectively bind to target RNAs and suppress their expression or function have been reported for disease treatment owing to high stability, good biocompatibility, and fast metabolism [21][22][23][24][25][26] . For instance, Disney and co-workers pioneered the ribonuclease targeting chimeras (RiboTACs) that consists of an RNA-binding small molecule and a ribonuclease recruiter for selective degradation of target RNAs by recruiting the RNase L without activation of the immune system [27][28][29][30][31] . The emergence of this RiboTACs technology as an RNAi, which overcomes the undruggability of traditional RNA targets, has recently drawn tremendous attention and opened up an avenue for accurate therapy of various diseases [32][33][34] . ...
... [84] (b) Overview of the binders linked to a bleomycin unit utilized to target different RNAs and their K d , highlighted in red. References for Bl-2, Bl-3, Bl-4, Bl-5 and Bl-6 are, [93,46,46,94,95] and, [96] respectively. ...
... Thus, positional reactivity could be a general strategy for improving the selectivity of binding small molecules, as observed for targeted degradation approaches, either directly 100 or via enzymatic recruitment. 101 was not certified by peer review) is the author/funder. All rights reserved. ...
... Other polyGA-specific antibodies have been used in HEK293 cells expressing GA 175 -GFP [9] and in transgenic BAC C9ORF72 mice [37]. A lead molecule screened from ReFRAME small molecule library [38] was further optimized to emerge as a facilitator of degradation of polyGP in patient-derived induced pluripotent stem cells [39]. Since inhibition of GA DPR protein aggregation could provide a therapeutic strategy for C9 ALS-FTD, we have selected specific high affinity aptamers and investigated their role in inhibition of aggregation of polyGA DPRs. ...
... Developing scoring methods that generalize beyond the available training data is especially important for the next generation of drugs. Recent advances in drug discovery have expanded interest beyond the traditional druggable proteome, targeting intrinsically disordered proteins [38], protein-protein interactions [33], and RNA tertiary structures [12] as promising avenues for nextgeneration therapeutics. The ability to efficiently identify small-molecule binders for these novel targets could greatly accelerate the development of new treatments. ...
... The formation of a covalent bond between an RNA and a small molecule binder enables direct target engagement and identification of the binding site, as often employed in the protein-targeting field [ 22 , 23 ]. Dubbed Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP), an RNA binder is appended with a cross-linking module, such as chlorambucil [ 16 , 20 , 21 ] or photoreactive diazirine [24][25][26], and a purification tag. The latter enables the pull-down of probe-modified transcripts with streptavidin beads or by direct copper-catalyzed azide-alkyne cycloaddition (CuAAC) [ 27 , 28 ] to azide-functionalized beads. ...
... As mentioned above, DNA-encoded library (DEL) technology has been applied to the OBOC format 28 and binding screens have been reported in which ligand-displaying beads have been isolated using fluorescence-activated cell sorting. [33][34][35][36][37] Thus, this assay should be immediately transferable to screening OBOC DELs for molecular glues and E3 ligase ligands. These studies are underway. ...
