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Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1.

ABSTRACT: Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3' untranslated region (3'UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1 and therefore many therapeutic efforts are focused on recovering its normal activity. For that purpose, herein we present pyrido[2,3-d]pyrimidin-7-(8H)-ones, a privileged scaffold showing remarkable biological activity against many targets involved in human disorders including cancer and viral diseases. Their combination with a flexible linker meets the requirements to stabilise DM1 toxic transcripts, and therefore, enabling the release of MBNL1. Therefore, a set of novel pyrido[2,3-d]pyrimidin-7-(8H)-ones derivatives (1a-e) were obtained using click chemistry. 1a exerted over 20% MBNL1 recovery on DM1 toxic RNA activity in primary cell biology studies using patient-derived myoblasts. 1a promising anti DM1 activity may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1.

SUBMITTER: Ondono R

PROVIDER: S-EPMC7753043 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1.

Ondono Raul R Lirio Ángel Á Elvira Carlos C Álvarez-Marimon Elena E Provenzano Claudia C Cardinali Beatrice B Pérez-Alonso Manuel M Perálvarez-Marín Alex A Borrell José I JI Falcone Germana G Estrada-Tejedor Roger R

Computational and structural biotechnology journal 20201206

Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3' untranslated region (3'UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind ...[more]

PMID: 33363709

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Project description:The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3' untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)(400) repeat tract was positioned 3' of the termination codon and 5' of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.

Project description:BackgroundExperimental studies of gene expression have identified some of the individual molecular components and elementary reactions that comprise and control cellular behavior. Given our current understanding of gene expression, and the goals of biotechnology research, both scientists and engineers would benefit from detailed simulators that can explicitly compute genome-wide expression levels as a function of individual molecular events, including the activities and interactions of molecules on DNA at single base pair resolution. However, for practical reasons including computational tractability, available simulators have not been able to represent genome-scale models of gene expression at this level of detail.ResultsHere we develop a simulator, TABASCO http://openwetware.org/wiki/TABASCO, which enables the precise representation of individual molecules and events in gene expression for genome-scale systems. We use a single molecule computational engine to track individual molecules interacting with and along nucleic acid polymers at single base resolution. Tabasco uses logical rules to automatically update and delimit the set of species and reactions that comprise a system during simulation, thereby avoiding the need for a priori specification of all possible combinations of molecules and reaction events. We confirm that single molecule, base-pair resolved simulation using TABASCO (Tabasco) can accurately compute gene expression dynamics and, moving beyond previous simulators, provide for the direct representation of intermolecular events such as polymerase collisions and promoter occlusion. We demonstrate the computational capacity of Tabasco by simulating the entirety of gene expression during bacteriophage T7 infection; for reference, the 39,937 base pair T7 genome encodes 56 genes that are transcribed by two types of RNA polymerases active across 22 promoters.ConclusionTabasco enables genome-scale simulation of transcription and translation at individual molecule and single base-pair resolution. By directly representing the position and activity of individual molecules on DNA, Tabasco can directly test the effects of detailed molecular processes on system-wide gene expression. Tabasco would also be useful for studying the complex regulatory mechanisms controlling eukaryotic gene expression. The computational engine underlying Tabasco could also be adapted to represent other types of processive systems in which individual reaction events are organized across a single spatial dimension (e.g., polysaccharide synthesis).

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Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1.

Publications

Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1.

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