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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

Characterization of the exon-skipping reporter. (A) The Luc-MDM2 construct contains parts of an MDM2 minigene sequence (intronic and exonic sequences) within the luciferase ORF in the pGL4.51 vector. (B) SK-MEL-2 cells transfected with the RL-CMV plasmid alone (RL) or Luc-MDM2 and RL-CMV plasmids (Luc + RL) and treated with either 0.5% DMSO or different concentrations of sudemycin D1 for 4 h. Dose response curve of sudemycin D1 in Luc-MDM2/RL-CMV dual reporter assay. SK-MEL-2 cells were co-transfected with Luc-MDM2 and RL-CMV plasmids and treated with indicated sudemycin D1 concentrations for 4 h. RLU, relative luminescence units; FF/Ren, firefly/Renilla
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fig02: Characterization of the exon-skipping reporter. (A) The Luc-MDM2 construct contains parts of an MDM2 minigene sequence (intronic and exonic sequences) within the luciferase ORF in the pGL4.51 vector. (B) SK-MEL-2 cells transfected with the RL-CMV plasmid alone (RL) or Luc-MDM2 and RL-CMV plasmids (Luc + RL) and treated with either 0.5% DMSO or different concentrations of sudemycin D1 for 4 h. Dose response curve of sudemycin D1 in Luc-MDM2/RL-CMV dual reporter assay. SK-MEL-2 cells were co-transfected with Luc-MDM2 and RL-CMV plasmids and treated with indicated sudemycin D1 concentrations for 4 h. RLU, relative luminescence units; FF/Ren, firefly/Renilla

Mentions: In order to facilitate the development of spliceosome modulators we sought to design a reporter assay to rapidly report the exon-skipping activity in the context of an intact cell. We selected the proto-oncogene MDM2 as the basis for our reporter because its alternative splicing in cancer is well characterized and we have previously reported a PCR-based assay that is highly specific for inhibitors of SF3B1 (Fan et al. 2011). MDM2 is an E3 ubiquitin ligase and chief negative regulator of the tumor suppressor protein p53 and it is encoded by 12 exons (Wu et al. 1993; Bartel et al. 2002). A splice variant, containing only exons 3 and 12, leads to the stabilization of p53, resulting in cell cycle arrest and/or apoptosis (Evans et al. 2001). Our previous studies on MDM2 minigenes (Singh et al. 2009) revealed that treatment with sudemycins induces alternative splicing such that exons 4–11 of MDM2 are skipped (Fan et al. 2011). On the basis of these observations, we designed an exon-skipping Luc-MDM2 reporter (Fig.2A); that requires skipping MDM2 exons 4, 10, and 11, inserted into the firefly luciferase (Luc) gene, to produce full-length luciferase transcript and bioluminescence.


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)

Characterization of the exon-skipping reporter. (A) The Luc-MDM2 construct contains parts of an MDM2 minigene sequence (intronic and exonic sequences) within the luciferase ORF in the pGL4.51 vector. (B) SK-MEL-2 cells transfected with the RL-CMV plasmid alone (RL) or Luc-MDM2 and RL-CMV plasmids (Luc + RL) and treated with either 0.5% DMSO or different concentrations of sudemycin D1 for 4 h. Dose response curve of sudemycin D1 in Luc-MDM2/RL-CMV dual reporter assay. SK-MEL-2 cells were co-transfected with Luc-MDM2 and RL-CMV plasmids and treated with indicated sudemycin D1 concentrations for 4 h. RLU, relative luminescence units; FF/Ren, firefly/Renilla
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Characterization of the exon-skipping reporter. (A) The Luc-MDM2 construct contains parts of an MDM2 minigene sequence (intronic and exonic sequences) within the luciferase ORF in the pGL4.51 vector. (B) SK-MEL-2 cells transfected with the RL-CMV plasmid alone (RL) or Luc-MDM2 and RL-CMV plasmids (Luc + RL) and treated with either 0.5% DMSO or different concentrations of sudemycin D1 for 4 h. Dose response curve of sudemycin D1 in Luc-MDM2/RL-CMV dual reporter assay. SK-MEL-2 cells were co-transfected with Luc-MDM2 and RL-CMV plasmids and treated with indicated sudemycin D1 concentrations for 4 h. RLU, relative luminescence units; FF/Ren, firefly/Renilla
Mentions: In order to facilitate the development of spliceosome modulators we sought to design a reporter assay to rapidly report the exon-skipping activity in the context of an intact cell. We selected the proto-oncogene MDM2 as the basis for our reporter because its alternative splicing in cancer is well characterized and we have previously reported a PCR-based assay that is highly specific for inhibitors of SF3B1 (Fan et al. 2011). MDM2 is an E3 ubiquitin ligase and chief negative regulator of the tumor suppressor protein p53 and it is encoded by 12 exons (Wu et al. 1993; Bartel et al. 2002). A splice variant, containing only exons 3 and 12, leads to the stabilization of p53, resulting in cell cycle arrest and/or apoptosis (Evans et al. 2001). Our previous studies on MDM2 minigenes (Singh et al. 2009) revealed that treatment with sudemycins induces alternative splicing such that exons 4–11 of MDM2 are skipped (Fan et al. 2011). On the basis of these observations, we designed an exon-skipping Luc-MDM2 reporter (Fig.2A); that requires skipping MDM2 exons 4, 10, and 11, inserted into the firefly luciferase (Luc) gene, to produce full-length luciferase transcript and bioluminescence.

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