Novel Small-Molecule Human G-CSF Receptor Agonist Stimulates Neutrophil Counts In Monkeys and Displays Anti-Proliferative Effects On Tumor Cells Mediated By a Reduction Of Intracellular Iron

Previously we reported on the discovery of a series of novel, non-peptidyl, small molecules that selectively activate the human granulocyte colony stimulating factor receptor (hG-CSFR) in a manner distinct from G-CSF and similar to the mechanism of small-molecule human thrombopoietin receptor (hTPOR) agonists [Marschke KB, et al. ASH 53rd Annual Meeting Abstracts, Blood 2011 118(21):1453, Abstract 3391]. The lead compound, LG7455, activates STAT5 and STAT3 signal transduction and induces G-CSF-dependent hematopoietic cell growth, but not TPO-dependent or erythropoietin-dependent cell growth. LG7455 promotes the differentiation of human bone marrow cells into granulocytes, significantly increasing expression of the granulocyte-specific marker CD15 (FUT4). The G-CSF-like effect of LG7455 requires the expression of human or monkey G-CSFR, and a histidine in the transmembrane domain is needed for activity, similar to what has been found for small-molecule hTPOR agonists, such as eltrombopag. LG7455 increases the binding of GCSF in a manner consistent with allosteric receptor modulation. To test the ability of LG7455 to stimulate neutrophil counts in vivo, cynomolgus monkeys were dosed intravenously (IV) 3 times per day (TID) to increase LG7455 exposure. Increases in peripheral blood neutrophils were observed in male cynomolgus monkeys with LG7455 (2 mg/kg/dose, TID). Because of the potential for LG7455 to interact with metal ions, their role in the activity of LG7455 was investigated. The ability of LG7455 to activate luciferase expression in cells transiently transfected with an hG-CSFR expression vector and a STAT5-responsive luciferase reporter was dependent on the addition of zinc (II) ion (up to 30 µM) to the cell culture media. The addition of Zn(II) also significantly enhanced the efficacy (63% vs. 23% relative to rhG-CSF) and potency ( EC₅₀ of 28 nM vs. 300 nM) by which LG7455 stimulated the growth of UT-7 cells made dependent on G-CSF by stable transfection of hGCSFR (UTP-hGCSFR). LG7455 did not activate luciferase expression or stimulate growth of cells incubated in the presence of additional iron (III), copper (II), magnesium (II) or calcium (II) ions. Similar to the antitumor effect of the well-described iron chelator salicylaldehyde isonicotinyl hydrazine (SIH), LG7455 inhibited the growth of a selection of human tumor cell lines, including the myeloid leukemia cell lines HL-60 and Kasumi-1, the lung carcinoma cell line A549, the embryonic kidney cell line HEK293, and the hepatocellular carcinoma cell line HepG2 (IC₅₀’s of 5.0, 12.1, 6.1, 1.4 and 1.0 µM, respectively). SIH did not stimulate but instead inhibited the growth of UTP-hGCSFR cells. The anti-proliferative effect of LG7455 on HL-60 cells was accompanied by an increase in apoptosis as determined by Annexin V/propidium iodide staining and FACS analysis. The addition of excess Fe(III) to the tumor cells was able to overcome the anti-proliferative effect of LG7455 in a concentration-dependent manner, indicating that a reduction in intracellular iron plays a role in the effect. The addition of Zn(II) and Cu(II) were less efficient at blocking the anti-proliferative effect of LG7455 than Fe(III). Addition of Mg(II) or Ca(II) had no effect. LG7455 increased the fluorescence of HL-60 cells loaded with calcein-AM, a fluorescent dye quenched when bound to iron, further supporting that a reduction of free intracellular iron occurs in cells treated with LG7455. Lastly, as has been shown with other iron chelators with anti-proliferative activity, LG7455 increased intracellular reactive oxygen species (ROS) formation in HL-60 cells loaded with the cell-permeant ROS indicator, CM-H₂-DCFDA, and evaluated by flow cytometry. Further optimization of the LG7455 series may provide a novel, orally-available adjuvant anticancer therapy that is also a supportive care agent to treat neutropenia in patients receiving bone-marrow suppressive treatments.