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US 8,932,818 B2Granted
Screening assays for compounds that modulate programmed ribosomal frameshifting
AssigneePTC Therapeutics, Inc.
InventorsSergey V. Paushkin, Nikolai A. Naryshkin, Ellen Welch
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Abstract
The present invention relates to compounds that modulate ribosomal frameshifting and nucleic acid constructs for use in methods for identifying or validation of compounds that modulate ribosomal frameshifting. In particular, the present invention relates to the use of nucleic acid constructs to identify or validate compounds capable of modulating the efficiency of programmed ribosomal frameshifting and the use of compounds that modulate the efficiency of programmed ribosomal frameshifting to inhibit the replication or infectivity of viruses that employ programmed ribosomal frameshifting.
Claims16
A method for screening compounds for a compound that modulates the efficiency of viral programmed ribosomal frameshifting comprising:
- (a) contacting a compound with a host cell containing an mRNA transcript encoded by a nucleic acid construct, wherein said nucleic acid construct comprises, in 5′ to 3′ order:
- (i) a start codon;
- (ii) the nucleic acid residues of exon 7 of SMN, wherein a single guanine residue is inserted after the 48th nucleotide residue from the 5′ end of exon 7 of SMN;
- (iii) the nucleic acid residues of intron 7 of SMN or a fragment thereof, wherein the fragment of the nucleic acid residues of intron 7 comprises any number of nucleotides of intron 7 of SMN required for a functional, minimum intron;
- (iv) a fragment of the nucleic acid residues of exon 8 of SMN;
- and (v) a reporter gene coding sequence lacking a start codon, wherein:
- (A) the reporter gene coding sequence is fused to the fragment of the nucleic acid residues of exon 8 of SMN such that the first codon of the reporter gene coding sequence and the start codon are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct;
- and (B) the production of the mRNA transcript generates a stop codon upstream from the reporter gene coding sequence in the region of the mRNA transcript that corresponds to the fragment of the nucleic acid residues of exon 8 of SMN;
- and (C) the start codon and the stop codon upstream from the reporter gene coding sequence in the mRNA transcript are in the same contiguous open reading frame;
- and
- (b) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase of 1.5-fold or more in the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a compound when compared to (i) a previously determined reference range for a negative control, (ii) the activity or amount of the fusion protein translated from the mRNA transcript in the absence of the compound, or (iii) the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a negative control indicates that the compound modulates the efficiency of programmed ribosomal frameshifting;
- and
- (c) contacting a compound that increases the activity or amount of the fusion protein translated from the mRNA transcript with a cell containing a virus that employs programmed ribosomal frameshifting and assaying the ability of the compound to decrease viral replication, wherein a decrease in viral replication of at least 1.5 log 10 in the presence of the compound indicates that a compound that modulates the efficiency of viral programmed ribosomal frameshifting is identified.
10 more elements
A method for screening compounds for a compound that modulates the efficiency of viral programmed ribosomal frameshifting comprising:
- (a) contacting a compound with a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct, wherein said nucleic acid construct comprises, in 5′ to 3′ order:
- (i) a start codon;
- (ii) the nucleic acid residues of exon 7 of SMN, wherein a single guanine residue is inserted after the 48th nucleotide residue from the 5′ end of exon 7 of SMN;
- (iii) the nucleic acid residues of intron 7 of SMN or a fragment thereof, wherein the fragment of the nucleic acid residues of intron 7 comprises any number of nucleotides of intron 7 of SMN required for a functional, minimum intron;
- (iv) a fragment of the nucleic acid residues of exon 8 of SMN;
- and (v) a reporter gene coding sequence lacking a start codon, wherein:
- (A) the reporter gene coding sequence is fused to the fragment of the nucleic acid residues of exon 8 of SMN such that the first codon of the reporter gene coding sequence and the start codon are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct;
- and (B) the production of the mRNA transcript generates a stop codon upstream from the reporter gene coding sequence in the region of the mRNA transcript that corresponds to the fragment of the nucleic acid residues of exon 8 of SMN;
- and (C) the start codon and the stop codon upstream from the reporter gene coding sequence in the mRNA transcript are in the same contiguous open reading frame;
- and
- (b) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase of 1.5-fold or more in the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a compound when compared to (i) a previously determined reference range for a negative control, (ii) the activity or amount of the fusion protein translated from the mRNA transcript in the absence of the compound, or (iii) the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a negative control indicates that the compound modulates the efficiency of programmed ribosomal frameshifting;
- and
- (c) contacting a compound that increases the activity or amount of the fusion protein translated from the mRNA transcript with a cell containing a virus that employs programmed ribosomal frameshifting and assaying the ability of the compound to decrease viral replication, wherein a decrease in viral replication of at least 1.5 log 10 in the presence of the compound indicates that a compound that modulates the efficiency of viral programmed ribosomal frameshifting is identified.
10 more elements
Dependencies are inferred from the claim text.