The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G 4 C 2 RNA repeat [r(G 4 C 2 ) exp ] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G 4 C 2 ) exp . The compound was able to selectively bind r(G 4 C 2 ) exp 's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patientderived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G 4 C 2 ) exp . In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G 4 C 2 ) exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.
Genetically defined amyotrophic lateral
sclerosis (ALS) and frontotemporal
dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide
GGGGCC repeat expansions [r(G4C2)exp] within the C9orf72 gene. In these diseases, neuronal
loss occurs through an interplay of deleterious phenotypes, including
r(G4C2)exp RNA gain-of-function mechanisms.
Herein, we identified a benzimidazole derivative, CB096, that specifically
binds to a repeating 1 × 1 GG internal loop structure, 5′CGG/3′GGC, that is formed
when r(G4C2)exp folds. Structure–activity
relationship (SAR) studies and molecular dynamics (MD) simulations
were used to define the molecular interactions formed between CB096
and r(G4C2)exp that results in the
rescue of disease-associated pathways. Overall, this study reveals
a unique structural feature within r(G4C2)exp that can be exploited for the development of lead medicines
and chemical probes.
A solid-phase DNA-encoded library (DEL) was studied for
binding
the RNA repeat expansion r(CUG)exp, the
causative agent of the most common form of adult-onset muscular dystrophy,
myotonic dystrophy type 1 (DM1). A variety of uncharged and novel
RNA binders were identified to selectively bind r(CUG)exp by using a two-color flow cytometry screen. The cellular
activity of one binder was augmented by attaching it with a module
that directly cleaves r(CUG)exp. In DM1
patient-derived muscle cells, the compound specifically bound r(CUG)exp and allele-specifically eliminated r(CUG)exp, improving disease-associated defects.
The approaches herein can be used to identify and optimize ligands
and bind RNA that can be further augmented for functionality including
degradation.
The hexanucleotide repeat expansion
GGGGCC [r(G4C2)exp] within intron
1 of C9orf72 causes genetically defined amyotrophic
lateral sclerosis and frontotemporal
dementia, collectively named c9ALS/FTD. , the repeat expansion causes
neurodegeneration via deleterious phenotypes stemming from r(G4C2)exp RNA gain- and loss-of-function
mechanisms. The r(G4C2)exp RNA folds
into both a hairpin structure with repeating 1 × 1 nucleotide
GG internal loops and a G-quadruplex structure. Here, we report the
identification of a small molecule (CB253) that selectively binds
the hairpin form of r(G4C2)exp. Interestingly,
the small molecule binds to a previously unobserved conformation in
which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed
by a series of binding and structural studies. NMR and molecular dynamics
simulations suggest that the r(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal
loops through the process of strand slippage. We provide experimental
evidence that CB253 binding indeed shifts the equilibrium toward the
2 × 2 GG internal loop conformation, inhibiting mechanisms that
drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation
formation of stress granules and defective nucleocytoplasmic transport
in various cellular models of c9ALS/FTD.
Since conventional computers are straining to handle the increased size and sophistication of non-numeric processing (data management, information retrieval, artificial intelligence), a new class of non-numeric architectures is evolving. The segment sequential architecture is one of these. Further development of this architecture requires new techniques for multiple cell operation and intercell communication to handle control and search operations. This paper describes such techniques for instruction fetching, operand recall, string, set and tree context searching, and pointer transfer. It is expected that combinations of these techniques will appear in future architectures that are needed for non-numeric processing.
A hexanucleotide repeat expansion in intron 1 of the
C9orf72
gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G
4
C
2
)
exp
, causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat proteins (DPRs), and sequestration of RNA-binding proteins (RBPs) in RNA foci. Here, we describe a small molecule that potently and selectively interacts with r(G
4
C
2
)
exp
and mitigates disease pathologies in spinal neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) and in two c9ALS/FTD mouse models. These studies reveal a mode of action whereby a small molecule diminishes intron retention caused by the r(G
4
C
2
)
exp
and allows the liberated intron to be eliminated by the nuclear RNA exosome, a multi-subunit degradation complex. Our findings highlight the complexity of mechanisms available to RNA-binding small molecules to alleviate disease pathologies and establishes a pipeline for the design of brain penetrant small molecules targeting RNA with novel modes of action in vivo.
The
interrogation and manipulation of biological systems by small
molecules is a powerful approach in chemical biology. Ideal compounds
selectively engage a target and mediate a downstream phenotypic response.
Although historically small molecule drug discovery has focused on
proteins and enzymes, targeting RNA is an attractive therapeutic alternative,
as many disease-causing or -associated RNAs have been identified through
genome-wide association studies. As the field of RNA chemical biology
emerges, the systematic evaluation of target validation and modulation
of target-associated pathways is of paramount importance. In this
Review, through an examination of case studies, we outline the experimental
characterization, including methods and tools, to evaluate comprehensively
the impact of small molecules that target RNA on cellular phenotype.
Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG) exp , harbored in the 3′ untranslated region (3′ UTR) of dystrophia myotonica protein kinase (DMPK) mRNA and drives disease through a gain-offunction mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG) exp in vitro, affording perimidin-2-amine diazirine (1) that bound to r(CUG) exp . The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1. Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG) exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites.
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