Novel Brain-Penetrant, Small-Molecule Tubulin Destabilizers for the Treatment of Glioblastoma

Abstract

Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide (TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (C_max of 7807 ng/mL (20 μM), T_max at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC₅₀s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC₅₀ values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.

Keywords: brain cancers; glioblastoma; microtubules; small molecules; tubulin destabilizer.

Figure 1

RGN3067 inhibits the viability of glioblastoma cell lines. (A,B) Representative dose–response curves of cell viability after treatment with RGN3067 and colchicine (positive control). U87 (A) and LN-18 (B) cells were exposed to compounds in dose response using an 8-point half-log dilution series (4.5 nM–10 µM). Cell viability was assessed after 72 h using the alamarBlue assay. Data were normalized to DMSO-treated cells. IC₅₀ curves were fitted with a nonlinear regression model (Absolute IC₅₀) using GraphPad v10. The mean absolute IC₅₀ values from at least three independent experiments are shown in Supplementary Table S3.

Figure 2

RGN3067 inhibits tubulin polymerization in vitro and triggers G2/M arrest in glioblastoma cells. (A,B) Representative tubulin polymerization plots and data quantification. DMSO, RGN3067 (5 μM), colchicine (5 μM), and paclitaxel (3 μM) were co-incubated with porcine tubulin (20 μM) in general tubulin buffer containing 15% glycerol and 1 mM GTP. Fluorescence was detected every min for 160 min (EX: 350/EM: 435) at 37 °C using a CLARIOstar microplate reader. **** p < 0.0001. (C) Immunofluorescence staining of β-tubulin in U87 glioblastoma cells. Cells were treated with DMSO, RGN3067 (0.5 μM), and colchicine (20 nM) and stained for β-tubulin-III (green) and the Hoechst 33342 (blue) nuclear marker. Images were captured using an Operetta confocal microscope at 40× magnification. Scale bar, 10 μm. (D) Representative histograms of cell cycle analysis of U87 cells treated with DMSO, RGN3067 (5 μM), and colchicine (20 nM) for 48 h and stained with propidium iodide (PI). Data was collected with a BD FACS Canto II Flow Cytometer and analyzed using the BD FACSDiva software. (E) The percentage of cells in G1, S, and G2/M phases from the histograms in (D). Data are plotted as mean ± SD from at least three independent experiments. Analysis between groups in (B) was performed using a one-way ANOVA with Tukey’s multiple comparisons test.

Figure 3

RGN3067 binds to the colchicine binding site in β-tubulin. (A) Fluorescence-based colchicine competitive binding assay. Porcine tubulin (20 μM) was incubated with DMSO, RGN3067 (50 µM), and the colchicine binding site inhibitor nocodazole (NOC, 50 µM) for 45 min at 37 °C in tubulin buffer containing 0.2 mM GTP, followed by incubation with colchicine (10 μM final) for 45 min. Fluorescence of the colchicine–tubulin complex was measured using a CLARIOstar Plus microplate reader (EX: 380 nm/EM: 435 nm). (B) Quantification of the fluorescence-based colchicine competitive binding assay. *** p < 0.001, **** p < 0.0001. (C) EBI competition assay in MCF-7 breast cancer cells. Cells were incubated with RGN3067 (50 µM) and colchicine (50 μM) for 2 h, followed by EBI (100 μM) for 2 h. Total proteins were lysed and subjected to Western blot analysis for β-tubulin. The β-tubulin adduct formed by EBI is detectable as a second immunoreacting band of β-tubulin. GAPDH was employed as the loading control. (D) Quantification of the Western blot in (C). The stacked bar graph shows the percentage of native β-tubulin (black) vs. EBI-β-tubulin adduct (white) of total tubulin in cells treated with compounds. Data are plotted as mean ± SD from at least three independent experiments. Analysis between groups in (B) was performed using a one-way ANOVA with Tukey’s multiple comparison test.

Figure 4

RGN3067 induces reversible effects on cell viability. (A–C) Representative IC₅₀ curves for the reversibility experiments. U87 cells were treated with RGN3067 (A) and control compounds colchicine (COL, B), and sabizabulin (SAB, C) in dose response using an 8-point half-log dilution series (4.5 nM–10 µM). Compounds were washed out with medium after 6 h or allowed to remain in the wells. Cell viability was detected after 72 h with the alamarBlue assay. IC₅₀ curves were fitted with a nonlinear regression model (Absolute IC₅₀) using GraphPad v10. The mean absolute IC₅₀ values from at least three independent experiments are shown in Supplementary Table S4.

Figure 5

Pharmacokinetic evaluation of RGN3067. (A) Mean plasma concentration of RGN3067 after 100 mg/kg oral administration in normal CD-1 mice (n = 3). (B) Mean plasma and brain levels of RGN3067 with 100 mg/kg oral dose in CD-1 mice (n = 3). Data from (A,B) are shown as mean ± SD. Raw values are shown in Supplementary Tables S5 and S6.

Figure 6

Effect of RGN3067 on a SC GBM12 xenograft model in mice. (A) MTD determination of doses up to 200 mg/kg of RGN3067 twice daily for 5 days + post drug for 5 days (n = 5). (B) RGN3067 shows activity against the PDX HGG cell lines GBM12, GBM15, GBM39, and GBM43 after a 144 h incubation with the compound. RGN3067 was tested using a 13-point half-log dilution series (0.2 nM–100 µM). (C,D) Mice (4–6 weeks old, n = 8 for treated vs. non-treated) were inoculated with patient-derived GBM12 cells, and tumors were allowed to grow to a volume of 150–250 mm³. Mice were then treated with 100 mg/kg of RGN3067 BID for 5 days, followed by 2 days of no treatment, for 4 cycles. Mice were euthanized when the tumor volume reached 2000 mm³. (C) Tumor sizes were measured twice per week until tumors were harvested. (D) No significant toxicity or weight loss was observed. Data in (A,C,D) are shown as mean ± SEM. Multiple unpaired t-tests were used in (C), and statistical significance was determined using the Holm–Šídák method. * p < 0.05; ** p < 0.01; *** p < 0.001.