The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution.Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.crystallography | hepatitis C virus inhibitor | RNA structure I nfection with hepatitis C virus (HCV), which affects over 170 million individuals worldwide, is a leading cause of liver failure and hepatocellular carcinoma (1). Until earlier this year, when two protease inhibitors were approved as the first direct antiviral drugs for the treatment of HCV infection (2), the standard anti-HCV therapy consisted of an immunostimulatory regimen of pegylated interferon-α and the nucleoside analog ribavirin, which suffered from low efficacy as well as serious side effects (3). The prevalence of preexisting drug-resistance mutations in HCV quasispecies due to the low fidelity of the viral RNA-dependent RNA polymerase (NS5B) creates an urgent need for combination therapy with unique antiviral agents directed at distinct HCV targets (4).Among the potential targets for HCV inhibitors, the 5′ untranslated region (UTR) of the viral RNA genome stands out for its high sequence conservation within virus clinical isolates (5), which exceeds the conservation of the HCV protein reading frames. The HCV 5′ UTR harbors an internal ribosome entry site (IRES) which recruits host cell 40S ribosomal subunits and ultimately initiates translation of virus proteins via a 5′ cap-independent mechanism (6, 7). The function of the IRES relies on a structured RNA element, which contains several independently folding domains (Fig. 1A) (8, 9). The three-dimensional structure of the subdomain IIa target was previously determined in our laboratory revealing an overall bent architecture around an RNA internal loop (Fig. 1B) (10), in agreement with NMR analyses of the full domain II (11) and cryoelectron microscopy studies of 13). The L-shaped conformation of subdomain IIa directs the apical hairpin loop IIb toward the ribosomal E site in proximity of the active site. Ribosomal association of domain II induces a conformational change in the 40S head and closes the mRNA binding cleft. Both, the correct positioning of the viral mRNA initiation codon as well as the joining of the ribosomal subunits to form functional 80S units depend critically on the L-shaped architecture of the domain II (7...

The internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5′ UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide and small molecule ligands. Emphasis will be given to the IRES subdomain IIa which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.

Short stereoselective syntheses of various cyclitols, including the derivatives of conduritol B, conduritol F, myo-inositol and chiro-inositol, have been accomplished. The key steps in the syntheses are a ring-closing metathesis process and a diastereodivergent organometallic addition to a D-xylosederived alde-hyde. Keywordsconduritol; inositol; Grignard; stereochemistry model; chelation; Felkin-Anh; cyclophellitol; latent symmetryThe realization that inositols (hexahydroxycyclohexanes), conduritols (tetrahydroxycyclohexenes) and their numerous derivatives play important biological roles has made their study an important endeavor in health-related sciences. 1 Thus, myo-inositol phosphates have been intensively investigated for their role in intracellular signal transduction and calcium mobilization. 1d,2 Both myo-and chiro-inositols have been studied as components of inositolphosphoglycans (IPGs), believed to be important in insulin signaling. 3 It was discovered that various conduritols act as modulators of insulin release 4 and possess antifeedant, antibiotic, anticancer, and growth-regulating activities. 5 Conduritol epoxides, and more prominently fungal metabolite cyclophellitol, are potent glycosidase inhibitors and are under investigation as inhibitors of HIV infection and cancer metastasis. 6 These and related research activities have generated considerable synthetic effort directed at developing practical preparations of the numerous biologically important cyclitols and their derivatives. Commercially available, inexpensive myo-inositol has been a common starting point in the syntheses of myo-inositol derivatives, 7 while significantly more expensive naturally occurring methylated chiro-inositols, pinitol and quebrachitol, have been utilized to prepare cyclitols with D-and L-chiro-inositol stereochemical configurations respectively. 8 However, labor-intensive selective hydroxyl protection/deprotection strategies and the necessity of optical resolution of racemic myo-inositol derivatives have led to utilization of chiral pool starting materials for cyclitol syntheses. Of these, carbohydrates represent a logical choice due to their availability in optically pure form and stereochemically complex oxygenation patterns that can be relayed to their target destinations in the desired cyclitols. Thus, D-glucose, 9 D-galactose, 10 D-mannitol, 11 and L-iditol, 12 among others, have served as starting points in efficient syntheses of cyclitol derivatives. One of our research groups has recently reported an enantiodiver-gent synthesis of (+)-and (-)-cyclophellitol from D-xylose. 13 Utilizing the latent plane of symmetry present in the starting carbohydrate, aldehydes 1 and ent-1 were prepared as key intermediates for this enantiodivergent strategy (Scheme 1). In this paper we show that this chemistry has a much broader scope and provide a full account of an investigation resulting in the development of synthetic pathways to diverse cyclitols starting from D-xylose and utilizing a ring-closing metathesis ...

Methods for the enantioselective conversion of D-xylose to differentially protected myo-inositol and L-chiro-inositol have been developed. The key transformation is a highly diastereoselective intramolecular SmI2-promoted pinacol coupling. The stereoselectivity was extremely dependent on the conditions, suggesting a change in mechanism. Preliminary mechanistic experiments and possible explanations for this behavior are discussed.

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