RESUMEN
The muscleblind-like protein family (MBNL) plays a prominent role in the regulation of alternative splicing. Consequently, the loss of MBNL function resulting from sequestration by RNA hairpins triggers the development of a neuromuscular disease called myotonic dystrophy (DM). Despite the sequence and structural similarities between the four zinc-finger domains that form MBNL1, recent studies have revealed that the four binding domains have differentiated splicing activity. The dynamic behaviors of MBNL1 ZnFs were simulated using conventional molecular dynamics (cMD) and steered molecular dynamics (sMD) simulations of a structural model of MBNL1 protein to provide insights into the binding selectivity of the four zinc-finger (ZnF) domains toward the GpC steps in YGCY RNA sequence. In accordance with previous studies, our results suggest that both global and local residue fluctuations on each domain have great impacts on triggering alternative splicing, indicating that local motions in RNA-binding domains could modulate their affinity and specificity. In addition, all four ZnF domains provide a distinct RNA-binding environment in terms of structural sampling and mobility that may be involved in the differentiated MBNL1 splicing events reported in the literature.
Asunto(s)
Empalme Alternativo , Distrofia Miotónica , Humanos , Simulación de Dinámica Molecular , ARN/genética , ARN/metabolismo , Empalme del ARN , Distrofia Miotónica/genética , Zinc/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Myotonic dystrophy type 1 (DM1) is a rare multisystemic disorder associated with an expansion of CUG repeats in mutant DMPK (dystrophia myotonica protein kinase) transcripts; the main effect of these expansions is the induction of pre-mRNA splicing defects by sequestering muscleblind-like family proteins (e.g. MBNL1). Disruption of the CUG repeats and the MBNL1 protein complex has been established as the best therapeutic approach for DM1, hence two main strategies have been proposed: targeted degradation of mutant DMPK transcripts and the development of CUG-binding molecules that prevent MBNL1 sequestration. Herein, suitable CUG-binding small molecules were selected using in silico approaches such as scaffold analysis, similarity searching, and druggability analysis. We used polarization assays to confirm the CUG repeat binding in vitro for a number of candidate compounds, and went on to evaluate the biological activity of the two with the strongest affinity for CUG repeats (which we refer to as compounds 1-2 and 2-5) in DM1 mutant cells and Drosophila DM1 models with an impaired locomotion phenotype. In particular, 1-2 and 2-5 enhanced the levels of free MBNL1 in patient-derived myoblasts in vitro and greatly improved DM1 fly locomotion in climbing assays. This work provides new computational approaches for rational large-scale virtual screens of molecules that selectively recognize CUG structures. Moreover, it contributes valuable knowledge regarding two compounds with desirable biological activity in DM1 models.
Asunto(s)
Anabolizantes/farmacología , Benzamidinas/farmacología , Proteínas de Drosophila/antagonistas & inhibidores , Distrofia Miotónica/tratamiento farmacológico , Proteína Quinasa de Distrofia Miotónica/antagonistas & inhibidores , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Empalme Alternativo , Anabolizantes/química , Animales , Benzamidinas/química , Modelos Animales de Enfermedad , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Descubrimiento de Drogas , Humanos , Locomoción/efectos de los fármacos , Simulación del Acoplamiento Molecular , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Mioblastos/patología , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica/química , Proteína Quinasa de Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/metabolismo , Cultivo Primario de Células , Pirimidinas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Expansión de Repetición de Trinucleótido/efectos de los fármacosRESUMEN
RNA is an important drug target, but current approaches to identify bioactive small molecules have been engineered primarily for protein targets. Moreover, the identification of small molecules that bind a specific RNA target with sufficient potency remains a challenge. Computer-aided drug design (CADD) and, in particular, ligand-based drug design provide a myriad of tools to identify rapidly new chemical entities for modulating a target based on previous knowledge of active compounds without relying on a ligand complex. Herein we describe pharmacophore virtual screening based on previously reported active molecules that target the toxic RNA that causes myotonic dystrophy type 1 (DM1). DM1-associated defects are caused by sequestration of muscleblind-like 1 protein (MBNL1), an alternative splicing regulator, by expanded CUG repeats (r(CUG)exp). Several small molecules have been found to disrupt the MBNL1-r(CUG)exp complex, ameliorating DM1 defects. Our pharmacophore model identified a number of potential lead compounds from which we selected 11 compounds to evaluate. Of the 11 compounds, several improved DM1 defects both in vitro and in cells.
Asunto(s)
Distrofia Miotónica/tratamiento farmacológico , ARN/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Empalme Alternativo/efectos de los fármacos , Animales , Diseño Asistido por Computadora , Proteínas de Unión al ADN/metabolismo , Diseño de Fármacos , Humanos , Ratones , Terapia Molecular Dirigida , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The design and selection of a combinatorial library of pyrido[2,3-d]pyrimidin-7(8H)-ones (4) has allowed the synthesis of 121 compounds, using known and new synthetic methodologies, and the evaluation of the inhibitory activity against hepatitis C virus (HCV) genotype 1b replicon. Among these compounds, 21{4,10} and 24{2,10} presented very high activities [EC50 = 0.027 µM (CC50 = 5.3 µM) and EC50 = 0.034 µM (CC50 = 13.5 µM), respectively] and high selectivity indexes, 196 and 397. These values are similar to the EC50 reported for sofosbuvir (2) (0.048 µM) using a similar methodological approach and the same virus subtype. 21{4,10} and 24{2,10} are obtained through shorter synthetic itineraries than sofosbuvir and 24{2,10} is achiral contrary to sofosbuvir which presents 4 stereogenic centers. In silico studies suggest that 21{4,10} and 24{2,10} inhibits NS5B polymerase through allosteric site binding.
Asunto(s)
Hepacivirus/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Simulación del Acoplamiento Molecular , Pirimidinas/síntesis químicaRESUMEN
Non-coding RNAs play a pivotal role in a number of diseases promoting an aberrant sequestration of nuclear RNA-binding proteins. In the particular case of myotonic dystrophy type 1 (DM1), a multisystemic autosomal dominant disease, the formation of large non-coding CUG repeats set up long-tract hairpins able to bind muscleblind-like proteins (MBNL), which trigger the deregulation of several splicing events such as cardiac troponin T (cTNT) and insulin receptor's, among others. Evidence suggests that conformational changes in RNA are determinant for the recognition and binding of splicing proteins, molecular modeling simulations can attempt to shed light on the structural diversity of CUG repeats and to understand their pathogenic mechanisms. Molecular dynamics (MD) are widely used to obtain accurate results at atomistic level, despite being very time consuming, and they contrast with fast but simplified coarse-grained methods such as Elastic Network Model (ENM). In this paper, we assess the application of ENM (traditionally applied on proteins) for studying the conformational space of CUG repeats and compare it to conventional and accelerated MD conformational sampling. Overall, the results provided here reveal that ANM can provide useful insights into dynamic rCUG structures at a global level, and that their dynamics depend on both backbone and nucleobase fluctuations. On the other hand, ANM fail to describe local U-U dynamics of the rCUG system, which require more computationally expensive methods such as MD. Given that several limitations are inherent to both methods, we discuss here the usefulness of the current theoretical approaches for studying highly dynamic RNA systems such as CUG trinucleotide repeat overexpansions.
Asunto(s)
Simulación de Dinámica Molecular , ARN no Traducido/química , Expansión de Repetición de Trinucleótido , Humanos , Distrofia Miotónica/genética , Conformación de Ácido Nucleico , ARN no Traducido/genéticaRESUMEN
Spinocerebellar ataxia type 10 (SCA10) is caused by a pentanucleotide repeat expansion of r(AUUCU) within intron 9 of the ATXN10 pre-mRNA. The RNA causes disease by a gain-of-function mechanism in which it inactivates proteins involved in RNA biogenesis. Spectroscopic studies showed that r(AUUCU) repeats form a hairpin structure; however, there were no high-resolution structural models prior to this work. Herein, we report the first crystal structure of model r(AUUCU) repeats refined to 2.8 Å and analysis of the structure via molecular dynamics simulations. The r(AUUCU) tracts adopt an overall A-form geometry in which 3 × 3 nucleotide (5')UCU(3')/(3')UCU(5') internal loops are closed by AU pairs. Helical parameters of the refined structure as well as the corresponding electron density map on the crystallographic model reflect dynamic features of the internal loop. The computational analyses captured dynamic motion of the loop closing pairs, which can form single-stranded conformations with relatively low energies. Overall, the results presented here suggest the possibility for r(AUUCU) repeats to form metastable A-from structures, which can rearrange into single-stranded conformations and attract proteins such as heterogeneous nuclear ribonucleoprotein K (hnRNP K). The information presented here may aid in the rational design of therapeutics targeting this RNA.