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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

SD6 Dose response, orthogonal RT-PCR assay, and assay validation. (A, B, and C) SK-MEL-2/Luc-MDM2 stable cells were plated at a density of 5000/well in 96-well plates and incubated overnight at 37°C in 5% CO2. The following day, cells were treated with serial dilutions of SD6 for 2, 4, 6, 8 (A), 48 (B) hours, or Herboxidiene or Pladienolide B (C) for 4 h, ONE-Glo reagents (Promega) were added to measure the luciferase activity. (D) RT-PCR was performed in Rh18 cells. PCR products (in bp) are indicated by the arrows; 1604 bp represents the full-length MDM2 transcript. 1001 and 767 bp represents alternative MDM2 splicing. Ubiquitin transcripts (165 bp) were used as a control.
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fig03: SD6 Dose response, orthogonal RT-PCR assay, and assay validation. (A, B, and C) SK-MEL-2/Luc-MDM2 stable cells were plated at a density of 5000/well in 96-well plates and incubated overnight at 37°C in 5% CO2. The following day, cells were treated with serial dilutions of SD6 for 2, 4, 6, 8 (A), 48 (B) hours, or Herboxidiene or Pladienolide B (C) for 4 h, ONE-Glo reagents (Promega) were added to measure the luciferase activity. (D) RT-PCR was performed in Rh18 cells. PCR products (in bp) are indicated by the arrows; 1604 bp represents the full-length MDM2 transcript. 1001 and 767 bp represents alternative MDM2 splicing. Ubiquitin transcripts (165 bp) were used as a control.

Mentions: In order to develop an in vivo pharmacodynamic assay with broad applications we established an SK-MEL-2 cell line stably expressing the same Luc-MDM2 reporter (SK-MEL-2/Luc-MDM2) as described above. No co-expression of RL-CMV was needed in this model as the cells were stably transfected. We conducted dose-response and time-course assessments of this novel stable reporter cell line and observed increasing amounts of luciferase activity with increasing concentrations of SD6 and exposure time in vitro. Strong luciferase activity was obtained with 4–8 h incubation time (Fig.3A); however, incubation times up to 48 h produced even higher luciferase activity (500-fold increase from baseline at 48 h, Fig.3B). Incubation times of 4 h or more produced large dynamic ranges (about 30-fold increase from baseline) in vitro, such that 4 h was chosen as the time point for further in vitro assays. Maximum luciferase activity was obtained at approximately 10 μmol/L at every time point. Furthermore, we confirmed the potent exon-skipping activity of two other SF3B1 targeted agents (pladienolide B and herboxidiene) (Kotake et al. 2007; Hasegawa et al. 2011; Lagisetti et al. 2014) in this stable cell line (Fig.3C).


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)

SD6 Dose response, orthogonal RT-PCR assay, and assay validation. (A, B, and C) SK-MEL-2/Luc-MDM2 stable cells were plated at a density of 5000/well in 96-well plates and incubated overnight at 37°C in 5% CO2. The following day, cells were treated with serial dilutions of SD6 for 2, 4, 6, 8 (A), 48 (B) hours, or Herboxidiene or Pladienolide B (C) for 4 h, ONE-Glo reagents (Promega) were added to measure the luciferase activity. (D) RT-PCR was performed in Rh18 cells. PCR products (in bp) are indicated by the arrows; 1604 bp represents the full-length MDM2 transcript. 1001 and 767 bp represents alternative MDM2 splicing. Ubiquitin transcripts (165 bp) were used as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: SD6 Dose response, orthogonal RT-PCR assay, and assay validation. (A, B, and C) SK-MEL-2/Luc-MDM2 stable cells were plated at a density of 5000/well in 96-well plates and incubated overnight at 37°C in 5% CO2. The following day, cells were treated with serial dilutions of SD6 for 2, 4, 6, 8 (A), 48 (B) hours, or Herboxidiene or Pladienolide B (C) for 4 h, ONE-Glo reagents (Promega) were added to measure the luciferase activity. (D) RT-PCR was performed in Rh18 cells. PCR products (in bp) are indicated by the arrows; 1604 bp represents the full-length MDM2 transcript. 1001 and 767 bp represents alternative MDM2 splicing. Ubiquitin transcripts (165 bp) were used as a control.
Mentions: In order to develop an in vivo pharmacodynamic assay with broad applications we established an SK-MEL-2 cell line stably expressing the same Luc-MDM2 reporter (SK-MEL-2/Luc-MDM2) as described above. No co-expression of RL-CMV was needed in this model as the cells were stably transfected. We conducted dose-response and time-course assessments of this novel stable reporter cell line and observed increasing amounts of luciferase activity with increasing concentrations of SD6 and exposure time in vitro. Strong luciferase activity was obtained with 4–8 h incubation time (Fig.3A); however, incubation times up to 48 h produced even higher luciferase activity (500-fold increase from baseline at 48 h, Fig.3B). Incubation times of 4 h or more produced large dynamic ranges (about 30-fold increase from baseline) in vitro, such that 4 h was chosen as the time point for further in vitro assays. Maximum luciferase activity was obtained at approximately 10 μmol/L at every time point. Furthermore, we confirmed the potent exon-skipping activity of two other SF3B1 targeted agents (pladienolide B and herboxidiene) (Kotake et al. 2007; Hasegawa et al. 2011; Lagisetti et al. 2014) in this stable cell line (Fig.3C).

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