Quarfloxin (CX-3543) disrupts the Nucleolin/ rDNA quadruplex complexes, inhibits the elongation by RNA Polymerase I and exhibits potent antitumor activity in models of cancer

3301 RNA Polymerase I (Pol I) is a critical component of the Ribosome Biogenesis pathway and can be regulated with small molecules at the initiation step, elongation step or at its catalytic site. The initiation step of Pol I dependents on proteins such as SL1 and UBF. Another nucleolar protein, Nucleolin, binds to the rDNA gene and modulates Pol I transcription elongation. rDNA contains multiple GC-rich sequences which can form quadruplex secondary structures. Such structures were shown to possess sub-nanomolar affinities for Nucleolin. Small molecule drug Quarfloxin (CX-3543) disrupts the Nucleolin/ rDNA quadruplex complexes in biochemical assays as well as in cells and consistent with Pol I elongation inhibition has been found to suppress rRNA synthesis. The compound also showed strong antitumor effect in multiple cell lines and demonstrates good efficacy in xenograft models of cancer.
 Using a novel search algorithm we identified 14 potential quadruplex sites in the non-template strand of each human rRNA gene. The DNA oligonucleotides corresponding to those sites were synthesized and their quadruplex nature was confirmed by CD spectrophotometry. Eleven of these quadruplexes bound to Nucleolin with Kd’s of under 10 nM. These Nucleolin/ quadruplex complexes were disrupted by Quarfloxin, a compound designed to bind G-quadruplexes, with Ki’s in the range of 0.15 to 1.0 uM as measured by electrophoretic mobility shift assay. Treatment with Quarfloxin resulted in dissociation of Nucleolin from rDNA quadruplexes as well as redistribution of Nucleolin from nucleoli into nucleoplasm as monitored by ChIP and immunofluorescence microscopy. Nuclear run-on experiment demonstrated that Quarfloxin inhibited the elongation by RNA Polymerase I in isolated nuclei with IC50 = 3.3 uM. This translated in the inhibition of rRNA synthesis in HCT-116 cells with IC50 = 4.3 uM as measured by qRT-PCR. This inhibition was specific for RNA Polymerase I as Quarfloxin failed to affect the synthesis of c-myc mRNA by RNA Polymerase II at these concentrations. Quarfloxin triggered apoptosis and inhibited multiple tumor cell lines in Alamar Blue cell viability assay typically with an average IC50 of approximately 1.0 uM. The molecule also demonstrated antitumor activity in multiple Xenograft models of human cancer when given IV, for instance in the Mia PaCa-2 model of pancreatic cancer with a T/C of 42%.
 Compounds that bind rDNA Quadruplexes can inhibit rRNA synthesis. Quarfloxin disrupts Nucleolin/ rDNA quadruplex complexes, selectively inhibits rRNA synthesis, and possesses broad spectrum cell proliferation inhibition potency and antitumor activity in Xenograft models of cancer.