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

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.

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

“…The lack of a close correlation between the MDM2 triple exon skipping and CLK inhibition and biochemical activity may be due to the fact that inhibitors of CLKs do not efficiently inhibit this unusual type of alternative splicing modulation, which has only been previously reported for SF3B1 modulators. 22, 24, 38 The use of the RT PCR assay as a confirmatory counterscreen for MDM2 exon skipping in Rh-18 cells validates this splicing effect in full length MDM2 pre-mRNA in a different cell background. 22 Using this screening approach we have identified compounds that are dual CLK2/CDK1 inhibitors (such as 1 ) that we optimized to yield more specific CLK inhibitors (such as SRI-29329, 8 ).…”

“…38 As can be seen by inspection of Table 1, replacement of the N7 nitrogen with CH completely abrogated all activity at 10 μM (compound 11 ), as did acetylation of the basic primary amine (compound 12 ), and dramatically reduced activity by replacement of the valanol group of 2 with an unsubstituted methyl ether (compound 13 ). The absolute and relative stereochemistry of the cyclohexane diamine also affected the activity.…”

“…22 Based on these observations, we designed an exon-skipping MDM2-Luc reporter (see Figure 2). 38 The principal strategy in the design of this reporter is that, when expressed, cellular luminescence is dependent on the production of a correctly spliced full-length luciferase transcript due to the drug induced skipping of MDM2 cassette exons 4, 10, and 11, which interrupt the luciferase gene in this construct in the absence of a splicing modulator drug (see Figure 2). We have previously reported the use of this construct as a reporter for the real time pharmacodynamics of SF3B1 targeted agents in vivo , via a stable reporter cell line.…”

“…We have previously reported the use of this construct as a reporter for the real time pharmacodynamics of SF3B1 targeted agents in vivo , via a stable reporter cell line. 38 Using a stable cell line expressing this reporter, luminescent signals are generated in a dose-dependent fashion in the presence of splicing modulators (such as the sudemycins, herboxidiene and pladienolide B). 38 We have used this assay to screen a small molecule library (composed of 830 known drugs and 4,359 bioactive compounds) 39 and identified several active ‘hit’ compounds.…”

“…38 Using a stable cell line expressing this reporter, luminescent signals are generated in a dose-dependent fashion in the presence of splicing modulators (such as the sudemycins, herboxidiene and pladienolide B). 38 We have used this assay to screen a small molecule library (composed of 830 known drugs and 4,359 bioactive compounds) 39 and identified several active ‘hit’ compounds. These hits were then subjected to dose-response assays in the MDM2-Luc exon-skipping assay and those actives were then confirmed in an orthogonal PCR-based MDM2 alternative splicing assay in Rh-18 cells (see Supporting Information).…”