The density of the upper bands corresponding to the NS3-ssRNA complex, which represents NS3 RNA binding activity, decreased dose-dependently in the presence of both hal3 and suvanine
November 1, 2021
The density of the upper bands corresponding to the NS3-ssRNA complex, which represents NS3 RNA binding activity, decreased dose-dependently in the presence of both hal3 and suvanine. values of 8, 8, and 14 M, and 7, 3, and 34 M, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV CX-4945 (Silmitasertib) NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 M. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an conversation with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes. family of positive-stranded RNA viruses. The viral genome contains a single open reading frame encoding a polyprotein that is processed by virus-encoded and host cellular proteases into structural and nonstructural proteins. The structural proteins (core protein [C], and the envelope glycoproteins E1 and E2) build up the virus particle, whereas the nonstructural proteins p7 and NS2 support particle assembly without being incorporated into the viral particles [7,8]. The remaining nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) form a complex with CX-4945 (Silmitasertib) viral RNA to support viral replication . NS3 is usually a multifunctional enzyme with serine protease and NTPase/helicase domains at the and shows the control reaction in the absence of NS3. The inhibitory effects of hal3 and suvanine were confirmed using a gel-based helicase assay. The helicase activity was calculated as the ratio of the signal intensity derived from single-stranded (ssRNA) in the sample made up of the inhibitor to the control sample (lacking the inhibitor but made up of DMSO vehicle). Similar to the results of the fluorescence helicase CX-4945 (Silmitasertib) assay, hal3 and suvanine inhibited helicase-catalyzed RNA unwinding in a dose-dependent manner (Physique 2C,D). Therefore, these data clearly indicate that hal3 and suvanine exert inhibitory effects. Hal3 and suvanine were identified in 1988  and 1985 , respectively. CX-4945 (Silmitasertib) They have comparable distinguishing structural features of a sulfated side chain and a furan moiety at the terminus of the molecule (Physique 1). Although some bioactivities for hal3 and suvanine have been reported, this report is the first that identifies these compounds as helicase inhibitors. In addition, bioactive effects of hal3 alone have not been reported. A mixture of halisulfates 2C5 (hal3 and its analogues) showed antimicrobial activity against contains the control reaction without NS3. Lanes (A) and (B) show the ATP hydrolysis reaction with poly(U) RNA at increasing concentrations (0C100 M) of hal3 and suvanine, respectively. As RNA binding is required for NS3 helicase activity, the effects of hal3 and suvanine on NS3 RNA binding activity were examined by gel mobility shift assay (Physique 4). As a control, the non-specific binding of ssRNA to bovine serum albumin SIGLEC6 (BSA) was assessed (lane 2). The density of the upper bands corresponding to the NS3-ssRNA complex, which represents NS3 RNA binding activity, decreased dose-dependently in the presence of both hal3 and suvanine. RNA binding activity was calculated as the ratio of the signal intensity derived from the NS3-ssRNA complex in the sample made up of the inhibitor to that in the control sample (lacking the inhibitor but made up of DMSO vehicle). The IC50 values of hal3 and suvanine were calculated to be 8 and 3 M, respectively. The data presented in Physique 2 and Physique 4 reveal that this NS3 helicase and RNA binding activities decrease at comparable inhibitor concentration ranges for hal3 and suvanine, suggesting that this inhibition of NS3 helicase by these compounds is associated with RNA binding activity. Open in a separate window Physique 4 Effects of hal3 and suvanine on NS3 RNA binding activity, assessed by autoradiography of a gel mobility shift assay using 32P-labeled ssRNA. Lanes and contain control reactions consisting of heat-denatured ssRNA and 300 nM BSA instead of NS3, respectively. Lanes (A) and (B) show the RNA binding reaction with increasing concentrations (0?100 M) of hal3 and suvanine, respectively. It was reported that this helicase activity of NS3 is usually interdependently CX-4945 (Silmitasertib) linked to its serine protease activity [23,24,25]. Therefore, we examined the effects of hal3 and suvanine on NS3 serine protease activity using a fluorescence serine protease assay (Physique 5). Serine protease activity decreased in a dose-dependent manner in the presence of hal3 and suvanine, with IC50 values of 14 and 34 M, respectively. Although the inhibition of the serine protease activity seems to be rather modest compared with that of the ATPase and RNA binding activities (Physique 3 and Physique 4), the inhibition of NS3 helicase by hal3 and suvanine.