Click chemistry enables evaluation of the molecular mechanism of the 2nd generation RNA polymerase I inhibitor, PMR-116

Despite the overwhelming evidence of dysregulated RNA Polymerase I (Pol I) transcription in cancer, only 1 selective Pol I transcription inhibitor, CX5461, has entered clinical trials (haematological and solid cancers). While promising, CX-5461 is associated with additional activities (e.g., Top2a inhibition), which possibly contribute to its efficacy, toxicity profiles and acquired resistance mechanisms.

To address the need
for new, improved Pol I inhibitors a series of orally available 2nd generation selective inhibitors were developed with
improved toxicology, tissue distribution (penetration of the blood brain barrier), lower plasma protein binding compared to CX-5461. Preliminary studies on the 2nd Pol I inhibitor, PMR-116, has demonstrated
improved survival administrated at a maximal tolerated dose in murine models of acute myeloid leukemia and B-cell lymphoma. PMR-116 is a selective inhibitor of Pol I
transcription, with ~200x more selectivity for Pol I vs Pol II, however the cellular targets and underlying molecular mechanisms are still unknown.

To evaluate the molecular mechanism chemical tagging approaches will be employed to identify the in situ localization and cellular protein/DNA targets of PMR-116 at the rDNA. Click chemistry with simple (unobtrusive) alkynes to attach biotin to PMR-116 at sites that don’t adversely impact on activity were synthesised, tested and will be used to treat human AML cell lines. Nuclear extracts from treated cells will be generated and the protein complexes affinity purified on streptavidin coated beads, direct digestion on the beads with the resultant mix of peptides analysed by standard shotgun LC-MS/MS (compared to unlabeled PMR-116). To identify sites of drug occupancy on chromatin Chem-seqwill be perform using Bio-PMR-116 to map genome-wide drug-chromatin target interactions.

The success of novel therapeutic compounds rests on characterizing of their mechanisms of action and is not onlycritical to develop better clinical compounds, but will
likely further expand our knowledge on the mechanism of Pol I transcription dysregulation in cancer.