Limits...
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

Alternative splicing changes from sudemycin treatment of human blood. (A) Freshly drawn human blood samples were treated with 1 μmol/L SD6 and incubated for 0–12 h. The splicing patterns were analyzed by end-point RT-PCR, as previously described (Convertini et al. 2014). The structure of the RT-PCR products is indicated schematically next to the gels. Numbers indicate the sizes of the RT-PCR products. (B) Quantification of splicing changes in four human subjects. The shading of each bar indicates the time of drug treatment; Off-white: 0 h; Gray: 6 h; Black: 12 h. The error bars indicate standard errors between individuals, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492733&req=5

fig05: Alternative splicing changes from sudemycin treatment of human blood. (A) Freshly drawn human blood samples were treated with 1 μmol/L SD6 and incubated for 0–12 h. The splicing patterns were analyzed by end-point RT-PCR, as previously described (Convertini et al. 2014). The structure of the RT-PCR products is indicated schematically next to the gels. Numbers indicate the sizes of the RT-PCR products. (B) Quantification of splicing changes in four human subjects. The shading of each bar indicates the time of drug treatment; Off-white: 0 h; Gray: 6 h; Black: 12 h. The error bars indicate standard errors between individuals, **P < 0.01, ***P < 0.001.

Mentions: As seen in Figure5, we found that DUSP11, RPp30, SRRM1, and PAPOLG genes were highly expressed in primary human lymphocytes and exhibited alternative splicing changes after SD6 treatment. Exon-skipping was found in all four genes at both 6-h and 12-h time points (Fig.5A). We further quantified the percentage of exon skipping of those genes in treated blood samples of four human subjects and found that the alternative splicing changes were statistically significant with P-values <0.01 or <0.001 (Fig.5B).


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)

Alternative splicing changes from sudemycin treatment of human blood. (A) Freshly drawn human blood samples were treated with 1 μmol/L SD6 and incubated for 0–12 h. The splicing patterns were analyzed by end-point RT-PCR, as previously described (Convertini et al. 2014). The structure of the RT-PCR products is indicated schematically next to the gels. Numbers indicate the sizes of the RT-PCR products. (B) Quantification of splicing changes in four human subjects. The shading of each bar indicates the time of drug treatment; Off-white: 0 h; Gray: 6 h; Black: 12 h. The error bars indicate standard errors between individuals, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Alternative splicing changes from sudemycin treatment of human blood. (A) Freshly drawn human blood samples were treated with 1 μmol/L SD6 and incubated for 0–12 h. The splicing patterns were analyzed by end-point RT-PCR, as previously described (Convertini et al. 2014). The structure of the RT-PCR products is indicated schematically next to the gels. Numbers indicate the sizes of the RT-PCR products. (B) Quantification of splicing changes in four human subjects. The shading of each bar indicates the time of drug treatment; Off-white: 0 h; Gray: 6 h; Black: 12 h. The error bars indicate standard errors between individuals, **P < 0.01, ***P < 0.001.
Mentions: As seen in Figure5, we found that DUSP11, RPp30, SRRM1, and PAPOLG genes were highly expressed in primary human lymphocytes and exhibited alternative splicing changes after SD6 treatment. Exon-skipping was found in all four genes at both 6-h and 12-h time points (Fig.5A). We further quantified the percentage of exon skipping of those genes in treated blood samples of four human subjects and found that the alternative splicing changes were statistically significant with P-values <0.01 or <0.001 (Fig.5B).

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