Despite interest in developing therapeutics that leverage binding pockets in structured RNAs-whose dysregulation leads to diseases-such drug discovery efforts are limited. Here, we have used a small molecule microarray (SMM) screen to find inhibitors of a large ribozyme: the Methanobrevibacter smithii RNase P RNA (Msm RPR, ∼300 nt). The ribonucleoprotein form of RNase P, which catalyzes the 5'-maturation of precursor tRNAs, is a suitable drug target as it is essential, structurally diverse across life domains, and present in low copy. From an SMM screen of 7,300 compounds followed by selectivity profiling, we identified 48 hits that bound specifically to the Msm RPR-the catalytic subunit in Msm (archaeal) RNase P. When we tested these hits in precursor-tRNA cleavage assays, we discovered that the drug-like M1, a diaryl-piperidine, inhibits Msm RPR (KI, 17 ± 1 μM) but not a structurally related archaeal RPR, and binds to Msm RPR with a KD(app) of 8 ± 3 μM. Structure-activity relationship analyses performed with synthesized analogs pinpointed groups in M1 that are important for its ability to inhibit Msm RPR. Overall, the SMM method offers prospects for advancing RNA druggability by identifying new privileged scaffolds/chemotypes that bind large, structured RNAs.
Alphaviruses are globally distributed, vector-borne RNA viruses with high outbreak potential and no clinical interventions, posing a significant global health threat. Previously, the production and packaging of both viral capped and noncapped genomic RNAs (cgRNA and ncgRNA) during infection was reported. Studies have linked ncgRNA production to viral infectivity and pathogenesis, but its precise role remains unclear. To define the benefits of ncgRNAs, pure populations of capped and noncapped Sindbis virus (SINV) gRNAs were synthesized and transfected into host cells. The data showed that mixtures of cgRNAs and ncgRNAs had higher infectivity compared to pure cgRNAs, with mixtures containing low cgRNA proportions exceeding linear infectivity expectations. This enhancement depended on co-delivery of cgRNAs and ncgRNAs to the same cell and required the noncapped RNAs to be viral in origin. Contrary to the initial hypothesis that the ncgRNAs serve as replication templates, the cgRNAs were preferentially replicated. Further analysis revealed that viral gene expression, viral RNA (vRNA) synthesis and particle production were enhanced in the presence of ncgRNAs, which function to promote cgRNA translation early in infection. Our findings highlight the importance of ncgRNAs in alphaviral infection, showing they enhance cgRNA functions and significantly contribute to viral infectivity.
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