“…It is noteworthy that the pathway of IFN inhibition of viral replication occurs via an IRES-dependent mechanism (11). Both the IRES structure and the mechanism of HCV translation initiation (e.g., 12, 13) have been the subject of intense research in recent years as a therapeutic target (14)(15)(16)(17). As an example, the synthetic steroid mifepristone specifically inhibits in vitro translation initiation from the HCV IRES (18).…”
The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >1013 different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.
“…It is noteworthy that the pathway of IFN inhibition of viral replication occurs via an IRES-dependent mechanism (11). Both the IRES structure and the mechanism of HCV translation initiation (e.g., 12, 13) have been the subject of intense research in recent years as a therapeutic target (14)(15)(16)(17). As an example, the synthetic steroid mifepristone specifically inhibits in vitro translation initiation from the HCV IRES (18).…”
The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >1013 different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.
“…[12][13][14][15][16][17] HIV inhibitors bind to a 3-nucleotide bulge or a hairpin in transactivating response (TAR) RNA. [18,19] Viral RNAs from HIV [18][19][20][21][22] and hepatitis C [23,24] as well as catalytic RNAs such as group I introns and RNase P RNAs [25][26][27] have also been targets for small molecules. In the vast majority of cases, smallmolecule RNA inhibitors were identified by screening combinatorial libraries using a traditional medicinal chemistry approach.…”
Herein, we describe our initial steps towards identifying the RNA secondary structure motifs that are recognized by small molecules. We selected members of an RNA 3x3 internal loop motif library that bind kanamycin A, an RNA-binding aminoglycoside antibiotic, by using only one round of selection. A small internal-loop library was chosen because members are likely to be present in other larger, biologically relevant RNAs. We have identified several internal loops of various size and base composition that kanamycin A prefers to bind. The highest affinity structures are two 5'-UU/3'-CU 2x2 internal loops closed by AU pairs. Binding is specific for the selected internal loops with the highest affinities, since binding to the RNA cassette used to display the library or to DNA is >150-fold weaker. Enzymatic mapping experiments also confirm binding of kanamycin A to the internal loops. This method lays the foundation for finding RNA secondary structure elements that bind small molecules and for interrogating factors affecting RNA-ligand interactions. Information from these and subsequent studies will: 1) facilitate the rational and modular design of drugs or probes that bind target RNAs with high affinity, provided the secondary structure of the target is known and 2) give insight into the potential bystander RNAs that aminoglycosides bind.
“…We developed an optimum two-step approach based partially on previous synthetic work [31,32]. In short, the synthesis of DMPITC was achieved by treating the commercially available 2-amino-4,6-dichloropyrimidine with excess sodium metal in methanol, followed by a nucleophilic substitution with thiophosgene in dichloromethane.…”
Section: Synthesis Of [D 0 ]-/[D 6 ]-Dmpitcmentioning
confidence: 99%
“…The synthetic procedures of the 'light' and 'heavy' DMPITC were optimized based partly on previous published papers [31,32]. 2-Amino-4,6-dichloropyrimidine (500 mg, 2.5 mmol), dissolved in 10 mL of anhydrous methanol, was added drop wise to a solution of 200 mg of sodium metal in 20 mL of anhydrous methanol.…”
Section: Syntheses Of [D 0 ]-/[D 6 ]-Dmpitcmentioning
A multifunctional isothiocyanate-based isotope labeling reagent, [d 0 ]-/[d 6]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b 1 ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b 1 ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsindigested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research.
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