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Pharmacodynamic assays to facilitate preclinical and clinical development of pre-mRNA splicing modulatory drug candidates.

Shi Y, Joyner AS, Shadrick W, Palacios G, Lagisetti C, Potter PM, Sambucetti LC, Stamm S, Webb TR - Pharmacol Res Perspect (2015)

Bottom Line: We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression.Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments.The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, SRI International Menlo Park, California, 94025.

ABSTRACT
The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

No MeSH data available.


Related in: MedlinePlus

Schematic overview of the splicing reaction. (A) Exons are shown as boxes, introns as lines. The spliceosome recognizes the 5′ and 3′ splice sites and the branchpoint (indicted by the “A” in the intron). Splicing results in the joining of the exons and removal of the intron (dotted arrow). Splicing starts with the early (E) complex formation that contains U1 snRNP recognizing the 5′ splice site through RNA:RNA interaction. The entry of U2 snRNP marks the formation of complex A that forms complex B after the entry of U4/U6/U5 snRNPs. The B complex is activated through the exit of U4 and U1, leading to rearrangements of the U2/U5/U6 snRNPs that allows catalysis in complex C. The catalysis results in the joining of exons and the release of the former intron as a lariat. After the splicing reaction, these snRNPs dissociate from the postspliceosomal complex and the lariat is degraded. (B) Recognition of the branchpoint sequence through U2 snRNP in the A complex. U2 snRNA binds the RNA surrounding the branchpoint adenosine and leads to a “bulging out” of the adenosine, which is contacted by the U2 component p14. In addition, the U2 component SF3B1 contacts the U2AF protein, located at the 3′ splice site. In this arrangement, the 5′ splice site, the branchpoint and the 3′ splice site are recognized by the spliceosome. Sudemycin D6 is a promising cancer drug that binds to SF3B1, the structure is shown as an insert.
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fig01: Schematic overview of the splicing reaction. (A) Exons are shown as boxes, introns as lines. The spliceosome recognizes the 5′ and 3′ splice sites and the branchpoint (indicted by the “A” in the intron). Splicing results in the joining of the exons and removal of the intron (dotted arrow). Splicing starts with the early (E) complex formation that contains U1 snRNP recognizing the 5′ splice site through RNA:RNA interaction. The entry of U2 snRNP marks the formation of complex A that forms complex B after the entry of U4/U6/U5 snRNPs. The B complex is activated through the exit of U4 and U1, leading to rearrangements of the U2/U5/U6 snRNPs that allows catalysis in complex C. The catalysis results in the joining of exons and the release of the former intron as a lariat. After the splicing reaction, these snRNPs dissociate from the postspliceosomal complex and the lariat is degraded. (B) Recognition of the branchpoint sequence through U2 snRNP in the A complex. U2 snRNA binds the RNA surrounding the branchpoint adenosine and leads to a “bulging out” of the adenosine, which is contacted by the U2 component p14. In addition, the U2 component SF3B1 contacts the U2AF protein, located at the 3′ splice site. In this arrangement, the 5′ splice site, the branchpoint and the 3′ splice site are recognized by the spliceosome. Sudemycin D6 is a promising cancer drug that binds to SF3B1, the structure is shown as an insert.

Mentions: Exons are defined by the 5′ splice site, the 3′ splice site, and the branch point. The spliceosome recognizes these elements and then assembles, in a stepwise manner, onto the nascent pre-mRNA (see Fig.1). First, the U1 snRNP binds to the 5′ splice site thereby forming the “early (E) complex.” This is then followed by the binding of splicing factor 1 (SF1) to the branch point, which in turn facilitates the binding of the U2AF factor (U2 auxiliary factor) on the 3′ splice site. Upon the stabilization of U2 snRNP binding, SF1 is displaced by the SF3 complex, which results in an interaction between U2AF65 and SF3B1 that are components of the U2AF and SF3B complexes, respectively. The U2 snRNP participates in an RNA:RNA interaction with the branch point, which leads to the recognition of the branchpoint adenosine. Through the exchange and recruitment of other factors, the “A complex” is transformed into the spliceosomal “B complex” that removes an intron and joins the exons by a trans-esterification reaction. The intron then undergoes debranching and is subsequently degraded (Kramer 1996).


Pharmacodynamic assays to facilitate preclinical and clinical development of pre-mRNA splicing modulatory drug candidates.

Shi Y, Joyner AS, Shadrick W, Palacios G, Lagisetti C, Potter PM, Sambucetti LC, Stamm S, Webb TR - Pharmacol Res Perspect (2015)

Schematic overview of the splicing reaction. (A) Exons are shown as boxes, introns as lines. The spliceosome recognizes the 5′ and 3′ splice sites and the branchpoint (indicted by the “A” in the intron). Splicing results in the joining of the exons and removal of the intron (dotted arrow). Splicing starts with the early (E) complex formation that contains U1 snRNP recognizing the 5′ splice site through RNA:RNA interaction. The entry of U2 snRNP marks the formation of complex A that forms complex B after the entry of U4/U6/U5 snRNPs. The B complex is activated through the exit of U4 and U1, leading to rearrangements of the U2/U5/U6 snRNPs that allows catalysis in complex C. The catalysis results in the joining of exons and the release of the former intron as a lariat. After the splicing reaction, these snRNPs dissociate from the postspliceosomal complex and the lariat is degraded. (B) Recognition of the branchpoint sequence through U2 snRNP in the A complex. U2 snRNA binds the RNA surrounding the branchpoint adenosine and leads to a “bulging out” of the adenosine, which is contacted by the U2 component p14. In addition, the U2 component SF3B1 contacts the U2AF protein, located at the 3′ splice site. In this arrangement, the 5′ splice site, the branchpoint and the 3′ splice site are recognized by the spliceosome. Sudemycin D6 is a promising cancer drug that binds to SF3B1, the structure is shown as an insert.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492733&req=5

fig01: Schematic overview of the splicing reaction. (A) Exons are shown as boxes, introns as lines. The spliceosome recognizes the 5′ and 3′ splice sites and the branchpoint (indicted by the “A” in the intron). Splicing results in the joining of the exons and removal of the intron (dotted arrow). Splicing starts with the early (E) complex formation that contains U1 snRNP recognizing the 5′ splice site through RNA:RNA interaction. The entry of U2 snRNP marks the formation of complex A that forms complex B after the entry of U4/U6/U5 snRNPs. The B complex is activated through the exit of U4 and U1, leading to rearrangements of the U2/U5/U6 snRNPs that allows catalysis in complex C. The catalysis results in the joining of exons and the release of the former intron as a lariat. After the splicing reaction, these snRNPs dissociate from the postspliceosomal complex and the lariat is degraded. (B) Recognition of the branchpoint sequence through U2 snRNP in the A complex. U2 snRNA binds the RNA surrounding the branchpoint adenosine and leads to a “bulging out” of the adenosine, which is contacted by the U2 component p14. In addition, the U2 component SF3B1 contacts the U2AF protein, located at the 3′ splice site. In this arrangement, the 5′ splice site, the branchpoint and the 3′ splice site are recognized by the spliceosome. Sudemycin D6 is a promising cancer drug that binds to SF3B1, the structure is shown as an insert.
Mentions: Exons are defined by the 5′ splice site, the 3′ splice site, and the branch point. The spliceosome recognizes these elements and then assembles, in a stepwise manner, onto the nascent pre-mRNA (see Fig.1). First, the U1 snRNP binds to the 5′ splice site thereby forming the “early (E) complex.” This is then followed by the binding of splicing factor 1 (SF1) to the branch point, which in turn facilitates the binding of the U2AF factor (U2 auxiliary factor) on the 3′ splice site. Upon the stabilization of U2 snRNP binding, SF1 is displaced by the SF3 complex, which results in an interaction between U2AF65 and SF3B1 that are components of the U2AF and SF3B complexes, respectively. The U2 snRNP participates in an RNA:RNA interaction with the branch point, which leads to the recognition of the branchpoint adenosine. Through the exchange and recruitment of other factors, the “A complex” is transformed into the spliceosomal “B complex” that removes an intron and joins the exons by a trans-esterification reaction. The intron then undergoes debranching and is subsequently degraded (Kramer 1996).

Bottom Line: We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression.Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments.The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, SRI International Menlo Park, California, 94025.

ABSTRACT
The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

No MeSH data available.


Related in: MedlinePlus