Drug target miRNAs: chances and challenges Marco F. Schmidt  Trends in Biotechnology  Volume 32, Issue 11, Pages 578-585 (November 2014) DOI: 10.1016/j.tibtech.2014.09.002 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 Structure and mechanism of the locked nucleic acid (LNA)-based drug Miravirsen (simplified) [12]. (A) Structure of Miravirsen. Abbreviations: P-thio, phosphorothioate; A, adenosine; dA, 2′-deoxyadenosine; dT, 2′-deoxythymidine; T, thymidine; G, guanosine; dC, 2′-deoxycytidine; mC, 5-methylcytidine. (B) Hepatitis C virus (HCV) is either protected by the Argonaute 2 protein (AGO2) in the absence of drugs (left) or allowed to degrade in the presence of Miravirsen (right). Trends in Biotechnology 2014 32, 578-585DOI: (10.1016/j.tibtech.2014.09.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 Cellular screening assays based on luciferase expression for identification of miRNA small molecule modulators. Target miRNA downregulates luciferase activity when paired with their specific binding sequence. Abbreviations: HCV, hepatitis C virus; m7G, 5-terminal cap. Trends in Biotechnology 2014 32, 578-585DOI: (10.1016/j.tibtech.2014.09.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 Enhancement (left) or inhibition (right) of DICER-mediated miRNA maturation induced by small molecules. Abbreviations: PACT, protein activator of the interferon induced protein kinase; MW, molecular weight; TRBP, HIV transactivating response RNA-binding protein-2. Trends in Biotechnology 2014 32, 578-585DOI: (10.1016/j.tibtech.2014.09.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 Crystal structures of Argonaute 2 (AGO2) proteins. (A) Crystal structure of the human AGO2 with its five domains: MID (brown), PAZ (light green), PIWI (grey), L (yellow), and N (blue) and a bound miRNA (red) [29,30]. (B) Structure of the catalytically inactive AGO2 protein bound to guide DNA (red) and RNA substrate (green) [33]. Panels (A) and (B) adapted, with permission, from [38]. Trends in Biotechnology 2014 32, 578-585DOI: (10.1016/j.tibtech.2014.09.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 5 Targeting Argonaute 2 (AGO2) protein to modulate the function of miRNAs. (A) Small molecule inhibitor of RNA-induced silencing complex (RISC) loading (violet) blocks generally every miRNA. (B) Binding of substrate mRNA occurs in two steps: Firstly, mRNA binds a rate-dependent step to the 5′-end (seed region) of miRNA. Then, full binding to the miRNA/AGO2 complex occurs. Next, the target mRNA is cleaved by AGO2 [34–36]. (C) Accordingly, short, octamer locked nucleic acids (LNAs; orange), so-called ‘tiny LNAs’, inhibit miRNA totally only by blocking the seed region of miRNA [37]. (D) Analogously, an anti-miR-AGO blocks miRNA's seed region (blue part of the inhibitor) and then binds additionally in the active site of AGO2 (red part of the inhibitor), thus inhibiting the silencing of target mRNA more effectively. [38,39]. Panels (B) and (D) adapted, with permission, from [38]. Abbreviations: AAA, poly(A) tail; m7G, 5-terminal cap. Trends in Biotechnology 2014 32, 578-585DOI: (10.1016/j.tibtech.2014.09.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure I Steps of gene silencing. Abbreviations: AAA, poly(A) tail; AGO2, Argonaute 2 protein; DGCR8, a microprocessor complex subunit; GW182, glycine(g)-tryptophan(w) repeat-containing protein of 182kDa; PABP, poly(A)binding protein; PACT, protein activator of the interferon induced protein kinase; PAN2 and PAN3, poly(A) nucleases 2 and 3; RNA pol, RNA polymerase; TRBP, HIV transactivating response RNA-binding protein-2. Trends in Biotechnology 2014 32, 578-585DOI: (10.1016/j.tibtech.2014.09.002) Copyright © 2014 Elsevier Ltd Terms and Conditions