RESUMEN
Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.
Asunto(s)
Compuestos Azo/metabolismo , Atrofia Muscular Espinal/genética , Pirimidinas/metabolismo , Precursores del ARN/genética , Empalme del ARN , Compuestos Azo/química , Compuestos Azo/uso terapéutico , Secuencia de Bases , Sitios de Unión/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Exones/genética , Cinética , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Estructura Molecular , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/metabolismo , Mutación , Fármacos Neuromusculares/química , Fármacos Neuromusculares/metabolismo , Fármacos Neuromusculares/uso terapéutico , Conformación de Ácido Nucleico , Pirimidinas/química , Pirimidinas/uso terapéutico , Precursores del ARN/química , Precursores del ARN/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.
