Abstract
Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the “hit” compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.
Highlights
MicroRNAs are small non-coding RNAs that regulate gene expression and are known to play a role in various cellular functions, such as development and differentiation [1,2,3]; miRNAs do not function by themselves but bind to certain proteins to carry out their functions
Adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), guanine monophosphate (GMP), and adenosine were purchased from Sigma-Aldrich
Since the probability of acquisition of “hits” might have been improved by selection based on multiple options, both the substructure search focusing on the phosphate group and structure-based screening were conducted
Summary
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are known to play a role in various cellular functions, such as development and differentiation [1,2,3]; miRNAs do not function by themselves but bind to certain proteins to carry out their functions. The pri-miRNA is further cleaved by Drosha and DiGeorge syndrome critical region 8 (DGCR8) to produce pre-miRNAs. Following transportation to the cytoplasm by Exportin-5, pre-miRNA is cleaved by the RNase III enzyme Dicer, generating double-stranded RNA (miRNA/miRNA duplex). Following transportation to the cytoplasm by Exportin-5, pre-miRNA is cleaved by the RNase III enzyme Dicer, generating double-stranded RNA (miRNA/miRNA duplex) This doublestranded RNA is incorporated into Argonaute (AGO), followed by removal of the passenger strand to form the RNA-induced silencing complex (RISC), which suppresses gene expression [4,5]. AGO proteins are composed of four domains, namely, the amino-terminal domain (N-domain), the middle (MID) domain, the Piwi-Argonaute-Zwille (PAZ) domain, and the P-element-induced wimpy testis (PIWI) domain. Each miRNA recognizes 7-8 nucleotides of the target mRNA, such that one miRNA can recognize a large number of mRNAs (~100) and regulate various functions in vivo
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