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US 8,283,116 B1Granted
Methods of screening for compounds for treating spinal muscular atrophy using SMN mRNA translation regulation
AssigneePTC Therapeutics, Inc.
InventorsAnuradha Bhattacharyya, Bansri S. Furia, Meenal Patel, Sergey V. Paushkin, Love Volkova
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Abstract
The present invention provides compounds and assays for the identification and validation of compounds for use in the treatment of spinal muscular atrophy (SMA), in which said compounds up-regulate the post-transcriptional expression of SMN1 or SMN2.
Claims25
A method for human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the untranslated regions (UTRs) of human SMN1 mRNA or human SMN2 mRNA, the method comprising:
- (a) contacting a compound with a first host cell engineered to express a first reporter protein translated from a first mRNA transcript comprising a first reporter gene coding sequence operably linked to a first 5′-UTR fragment of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:4 or 5 and a first 3′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:2 or 3, or a fragment thereof, wherein the first 5′-UTR fragment is upstream of the first reporter gene coding sequence and the first 3′-UTR or a fragment thereof is downstream of the first reporter gene coding sequence, and wherein the first reporter protein is not SMN;
- (b) contacting the compound with a second host cell engineered to express a second reporter protein translated from a second mRNA transcript comprising the first reporter gene coding sequence operably linked to a second 5′-UTR or a fragment thereof and a second 3′-UTR or a fragment thereof of a mRNA different from the 5′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof and the 3′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof, wherein the second 5′-UTR is upstream of the first reporter gene coding sequence and the second 3′ UTR is downstream of the first reporter gene coding sequence;
- and
- (c) detecting the amount or activity of the first and second reporter proteins (i) an alteration in the amount or activity of the first reporter protein in the presence of the compound relative to the amount or activity in the absence of the compound or the presence of a negative control, and (ii) no alteration in or not a substantially altered amount or activity of the second reporter protein in the presence of the compound relative to the amount or activity of the second reporter protein in the absence of the compound or the presence of the negative control indicates that the compound modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the UTRs of human SMN1 mRNA or human SMN2 mRNA.
1 more element
A method for screening for a compound that modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the untranslated regions (UTRs) of human SMN1 or human SMN2 mRNA, the method comprising:
- (a) contacting a compound with a first composition comprising a first cell-free extract and a first mRNA transcript comprising a first reporter gene coding sequence operably linked to a first 5′-UTR fragment of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:
- 4 or 5 and a first 3′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:2 or 3, or a fragment thereof, wherein the first 5′-UTR fragment is upstream of the first reporter gene coding sequence and the first 3′-UTR or a fragment thereof is downstream of the first reporter gene coding sequence, and wherein the first reporter protein is not SMN;
- (b) contacting the compound with a second composition comprising a second cell-free extract and a second mRNA transcript comprising the first reporter gene coding sequence operably linked to a second 5′-UTR or a fragment thereof and a second 3′-UTR or a fragment thereof of a mRNA different from the 5′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof and the 3′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof, wherein the second 5′-UTR is upstream of the first reporter gene coding sequence and the second 3′ UTR is downstream of the first reporter gene coding sequence, and wherein the first reporter protein is not SMN;
- and
- (c) detecting the amount or activity of the first and second reporter proteins translated from the first and second mRNA transcripts, respectively, wherein (i) an alteration in the amount or activity of the first reporter protein in the presence of the compound relative to the amount or activity in the absence of the compound or the presence of a negative control, and (ii) no alteration in or not a substantially altered amount or activity of the second reporter protein in the presence of the compound relative to the amount or activity of the second reporter protein in the absence of the compound or the presence of the negative control indicates that the compound modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the UTRs of human SMN1 mRNA or human SMN2 mRNA.
2 more elements
A method for screening for a compound that modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the untranslated regions (UTRs) of human SMN1 mRNA or human SMN2 mRNA, the method comprising:
- (a) contacting a compound with a first host cell engineered to express a first reporter protein translated from a first mRNA transcript comprising a first reporter gene coding sequence operably linked to a modified 5′-UTR of human SMN and the 3′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:
- 2 or 3, or a fragment thereof, wherein the modified 5′-UTR is upstream of the first reporter gene coding sequence and the 3′-UTR or a fragment thereof is downstream of the first reporter gene coding sequence, wherein the first reporter protein is not SMN, and wherein the modified 5′-UTR of human SMN is encoded by (1) the nucleotide sequence of SEQ ID NO:1 which has a mutation that mutates or eliminates the ATG site at nucleotide positions 7-9, or (2) the nucleotide sequence of SEQ ID NO:
- 1 which has a mutation that deletes the uORF at nucleotide positions 7-33;
- (b) contacting the compound with a second host cell expressing a second reporter protein translated from a second mRNA transcript comprising a first reporter gene coding sequence operably linked to a second 5′-UTR or a fragment thereof and a second 3′-UTR or a fragment thereof of a mRNA different from the modified 5′-UTR of human SMN and said 3′-UTR of human SMN1 or human SMN2 or a fragment thereof, wherein the second 5′-UTR is upstream of the first reporter gene coding sequence and the second 3′ UTR is downstream of the first reporter gene coding sequence;
- and
- (c) detecting the amount or activity of the first and second reporter proteins, wherein (i) an alteration in the amount or activity of the first reporter protein in the presence of the compound relative to the amount or activity in the absence of the compound or the presence of a negative control, and (ii) no alteration in or not a substantially altered amount or activity of the second reporter protein in the presence of the compound relative to the amount or activity of the second reporter protein in the absence of the compound or the presence of the negative control indicates that the compound modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the UTRs of human SMN1 mRNA or human SMN2 mRNA.
3 more elements
A method for screening for a compound that modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the untranslated regions (UTRs) of human SMN1 or human SMN2 mRNA comprising:
- (a) contacting a compound with a first composition comprising a first cell-free extract and a first mRNA transcript comprising a first reporter gene coding sequence operably linked to a modified 5′-UTR of human SMN and a 3′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:2 or 3, or a fragment thereof, wherein the modified 5′-UTR is upstream of the first reporter gene coding sequence and the 3′-UTR or a fragment thereof is downstream of the first reporter gene coding sequence, and wherein the modified 5′-UTR of human SMN is encoded by (1) the nucleotide sequence of SEQ ID NO:1 which has a mutation that mutates or eliminates the ATG site at nucleotide positions 7-9, or (2) the nucleotide sequence of SEQ ID NO:
- 1 which has a mutation that deletes the uORF at nucleotide positions 7-33;
- (b) contacting the compound with a second composition comprising a second cell-free extract and a second mRNA transcript comprising the first reporter gene coding sequence operably linked to a second 5′-UTR or a fragment thereof and a second 3′-UTR or a fragment thereof of a mRNA different from the modified 5′-UTR of human SMN and said 3′-UTR of human SMN1 or human SMN2 or a fragment thereof, wherein the second 5′-UTR is upstream of the first reporter gene coding sequence and the second 3′ UTR is downstream of the first reporter gene coding sequence;
- and
- (c) detecting the amount or activity of the first and second reporter proteins translated from the first and second mRNA transcripts, respectively, wherein (i) an alteration in the amount or activity of the first reporter protein in the presence of the compound relative to the amount or activity in the absence of the compound or the presence of a negative control, and (ii) no alteration in or not a substantially altered amount or activity of the second reporter protein in the presence of the compound relative to the amount or activity of the second reporter protein in the absence of the compound or the presence of the negative control indicates that the compound modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the UTRs of human SMN1 mRNA or human SMN2 mRNA.
2 more elements
A method for human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the untranslated regions (UTRs) of human SMN1 mRNA or human SMN2 mRNA, the method comprising:
- a) contacting a compound with a first host cell engineered to express a first reporter protein translated from a first mRNA transcript comprising a first reporter gene coding sequence operably linked to a first 5′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:1 or a fragment thereof and a first 3′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:2 or 3, or a fragment thereof, wherein the first 5′-UTR or a fragment thereof is upstream of the first reporter gene coding sequence and the first 3′-UTR or a fragment thereof is downstream of the first reporter gene coding sequence, and wherein the first reporter protein is not SMN, and wherein the compound is (E)-N-((5-bromothiophen-2-yl)methylene)-4-(6-methylbenzo[d]thiazol-2-yl)aniline, 3,4,4-trimethyl-1-phenylpent-1-yn-3-ol, 2-amino-3-(2-(4-chlorophenyl)-2-oxoethyl)thiazol-3-ium, or N-cyclohexyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
- (b) contacting the compound with a second host cell engineered to express a second reporter protein translated from a second mRNA transcript comprising the first reporter gene coding sequence operably linked to a second 5′-UTR or a fragment thereof and a second 3′-UTR or a fragment thereof of a mRNA different from the 5′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof and the 3′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof, wherein the second 5′-UTR is upstream of the first reporter gene coding sequence and the second 3′ UTR is downstream of the first reporter gene coding sequence;
- and
- (c) detecting the amount or activity of the first and second reporter proteins (i) an alteration in the amount or activity of the first reporter protein in the presence of the compound relative to the amount or activity in the absence of the compound or the presence of a negative control, and (ii) no alteration in or not a substantially altered amount or activity of the second reporter protein in the presence of the compound relative to the amount or activity of the second reporter protein in the absence of the compound or the presence of the negative control indicates that the compound modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the UTRs of human SMN1 mRNA or human SMN2 mRNA.
1 more element
A method for screening for a compound that modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the untranslated regions (UTRs) of human SMN1 or human SMN2 mRNA, the method comprising:
- (a) contacting a compound with a first composition comprising a first cell-free extract and a first mRNA transcript comprising a first reporter gene coding sequence operably linked to a first 5′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:
- 1 or a fragment thereof and a first 3′-UTR of human SMN1 or human SMN2 encoded by the nucleotide sequence of SEQ ID NO:2 or 3, or a fragment thereof, wherein the first 5′-UTR or a fragment thereof is upstream of the first reporter gene coding sequence and the first 3′-UTR or a fragment thereof is downstream of the first reporter gene coding sequence, and wherein the first reporter protein is not SMN, and wherein the compound is (E)-N-((5-bromothiophen-2-yl)methylene)-4-(6-methylbenzo[d]thiazol-2-yl)aniline, 3,4,4-trimethyl-1-phenylpent-1-yn-3-ol, 2-amino-3-(2-(4-chlorophenyl)-2-oxoethyl)thiazol-3-ium, or N-cyclohexyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
- (b) contacting the compound with a second composition comprising a second cell-free extract and a second mRNA transcript comprising the first reporter gene coding sequence operably linked to a second 5′-UTR or a fragment thereof and a second 3′-UTR or a fragment thereof of a mRNA different from the 5′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof and the 3′-UTR of human SMN1 mRNA or human SMN2 mRNA or a fragment thereof, wherein the second 5′-UTR is upstream of the first reporter gene coding sequence and the second 3′ UTR is downstream of the first reporter gene coding sequence, and wherein the first reporter protein is not SMN;
- and
- (c) detecting the amount or activity of the first and second reporter proteins translated from the first and second mRNA transcripts, respectively, wherein (i) an alteration in the amount or activity of the first reporter protein in the presence of the compound relative to the amount or activity in the absence of the compound or the presence of a negative control, and (ii) no alteration in or not a substantially altered amount or activity of the second reporter protein in the presence of the compound relative to the amount or activity of the second reporter protein in the absence of the compound or the presence of the negative control indicates that the compound modulates human SMN1 mRNA or human SMN2 mRNA translation that is regulated by the UTRs of human SMN1 mRNA or human SMN2 mRNA.
2 more elements
Dependencies are inferred from the claim text.