CX-5461, a selective inhibitor of rRNA synthesis, suppresses Pol I-driven transcription of rRNA.

CX-5461 demonstrates the capacity to inhibit cell proliferation in various cell lines, notably HCT-116 (EC50=167 nM), A375 (EC50=58 nM), and MIA PaCa-2 (EC50=74 nM). In solid tumor cells, CX-5461 induces autophagy or senescence rather than apoptosis. For instance, in melanoma A375 cells and pancreatic cancer MIA PaCa-2 cells, its effects are quantified with Pol I IC50 values of 113 nM and 54 nM, respectively, while showing significantly less effect on Pol II (Pol II IC50 > 25 μM for A375 and ~25 mM for MIA PaCa-2). Furthermore, in HCT-116 cells, CX-5461 selectively inhibits rRNA synthesis, with an observed Pol I IC50 of 142 nM and minimal impact on Pol II (IC50 > 25 μM).

In a mouse model of xenograft human solid tumors, oral administration of CX-5461 (50 mg/kg) demonstrated antitumor activity against solid tumors.

Pol I and Pol II Transcription Assay: Two short-lived RNA transcripts (half-lives ~20-30 minutes), one produced by Pol I and another by Pol II, are quantitated by qRT-PCR as a measure of CX-5461-related effects on transcription. The 45S pre-rRNA served as the Pol I transcript and the mRNA for the protooncogene c-myc served as the comparator Pol II transcript. Both Pol I and Pol II transcription are known to be affected by general cellular stress. To minimize the potential effects of such stress, cells are exposed to test agents for only a short period of time (2 hours). This is sufficient time for these transcripts to be reduced by greater than 90% if CX-5461 affects their synthesis.

Cells are plated on 96-well plates and treated the next day with dose response of CX-5461 for 96 hours. Cell viability is determined using Alamar Blue and CyQUANT assays(Only for Reference)