Biomedicines | Free Full-Text | Novel Brain-Penetrant, Small-Molecule Tubulin Destabilizers for the Treatment of Glioblastoma

2.1. Cell Culture

U87 glioblastoma (ATCC HTB-14; Manassas, VA, USA) cells were grown in DMEM/10% FCS, LN-18 glioblastoma cells (ATCC CRL-2610; Manassas, VA, USA) were grown in DMEM/5% FCS, HMC3 microglial cells (ATCC CRL-3304; Manassas, VA, USA) were grown in EMEM/10% FCS, and MCF-7 mammary carcinoma cells (ATCC HTB-22; Manassas, VA, USA) were grown in RPMI/10% FCS. Patient-derived, low-passage, genetically heterogeneous primary cells GBM12, GBM15, GBM39 and GBM43 (established in Mayo Clinic Hospital) were cultured in serum-free DMEM/F12 (Cytiva HyClone; Logan, UT, USA) with 20 ng/mL EGF, 20 ng/mL FGF, 2% B12, 1% N2, and 1% gentamycin solution. Cells were cultured at 37 °C in 5% CO₂.

2.2. Cell Viability Assay

Cells (100 µL/well) were seeded in 96-well tissue culture plates (Greiner Bio-One 650090; Kremsmünster, Austria). The plating density (cells/well) for each cell line was as follows: LN-18 (3000), U87 (4000), and HMC3 (5000). After overnight incubation, compounds were added to the cells (50 µL at 3× concentration, final 0.1% DMSO), and plates were incubated for 72 h. Cell viability was assessed by the addition of 25 µL of alamarBlue for a final concentration of 100 µM final concentration (Resazurin redox reagent, Sigma-Aldrich R7017; St. Louis, MO, USA). After a 4 h incubation, fluorescence was read on a CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany) at EX 570/EM 590 nm.

Short-term cultures of patient-derived cells GBM12, GBM15, GBM39 and GBM43 (used with permission Mayo Foundation) were seeded (1000 cells/well) in solid white Corning 384-well plates (Corning, NY, USA) and cultured overnight. Cells were treated with DMSO or RGN3067 in a 13-point dose response with a 1:3 serial dilution from an initial concentration of 100 μM. After 144 h, cell viability was measured using CellTiter-Glo (Promega, Madison, WI, USA). Data were normalized to DMSO control, and absolute IC₅₀ values were calculated using GraphPad Prism 10 (San Diego, CA, USA).

2.3. Cell Cycle Analysis

Cells (400,000) were seeded into T25 tissue culture flasks and treated with compound for 24 h prior to harvesting with TrypLE (Thermo Fisher Scientific 12605036; Waltham, MA, USA). Cells were washed in ice-cold PBS, resuspended in 1 mL of ice-cold 70% ethanol, and fixed overnight at −20 °C. Cells were centrifuged and resuspended in PBS containing RNAse A at a final concentration of 0.5 mg/mL (MilliporeSigma 10109142001; Burlington, MA, USA) and propidium iodide at a final concentration of 0.04 mg/mL (Sigma-Aldrich P4170, St. Louis, MO, USA) for 1 h at 37 °C. Nuclear staining was analyzed using a BD FACSCanto II flow cytometer (BD Biosciences; Franklin Lakes, NJ, USA) to determine cell cycle distribution. Cell cycle analysis was performed using the BD FACSDiva software v8.0.

2.4. Immunofluorescent Staining of Cellular β-Tubulin

U87 cells (4000/well) were plated in black 96-well plates (Perkin Elmer 6055302; Waltham, MA, USA) and grown overnight. Cells were treated with compounds for 24 h, fixed with 4% paraformaldehyde for 20 min, and permeabilized with FoxP3 permeabilization buffer (eBioscience/Thermo Fisher Scientific 00-5523-00; Waltham, MA, USA) for 10 min at RT. Cells were incubated with an anti-TUBB3 (Tuj1) antibody (1:1000; STEMCELL Technologies 60052; Vancouver, BC, Canada) labeled with Hoechst 33342 dye (1:2000; Thermo Fisher Scientific H3570; Waltham, MA, USA). Images (40×) were taken with an automated high-content imaging microscope (Operetta, Perkin Elmer; Waltham, MA, USA).

2.5. Tubulin Polymerization Assay

Tubulin polymerization was evaluated by monitoring the incorporation of a fluorescent reporter into MTs during polymerization. The tubulin polymerization assay kit (Cytoskeleton, Inc. BK011P; Denver, CO, USA) was used according to the manufacturer’s instructions. Briefly, the tubulin reaction mix was kept on ice until immediately prior to use. The compound (5 µL/well) was added to a half-area 96-well plate (Corning Costar 3686; Corning, NY, USA) and incubated for 1 min in the CLARIOstar Plus plate reader (BMG Labtech; Ortenberg, Germany) at 37 °C before the addition of 50 µL of the tubulin reaction mix/well. Fluorescence was read every min for 160 min at EX 350/EM 435 nm.

2.6. Colchicine Competitive Binding Assay

Porcine tubulin (20 µM, Cytoskeleton, Inc., T240; Denver, CO, USA) was incubated with compounds for 45 min at 37 °C in 80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 0.2 mM GTP prior to the addition of colchicine for a 10 µM final concentration (Selleckchem S2284; Houston, TX, USA). After a 45 min incubation, the fluorescence of the colchicine–tubulin complex was measured at EX 380/EM 435 nm with the CLARIOstar Plus plate reader (BMG Labtech; Ortenberg, Germany).

2.7. Colchicine-Binding Site Cellular Assay

N,N′-ethylene-bis (iodoacetamide) (EBI) cross-links cysteines 239 and 354 in the colchicine binding pocket of β-tubulin, the formation of which can be inhibited by colchicine binding site molecules. A total of 1.5 × 10⁶ MCF-7 cells were seeded overnight in a T25 flask and pre-incubated with compounds for 2 h, then treated with EBI (Abcam ab144980; Cambridge, MA, USA) for 1.5 h at 37 °C. Cells were washed with PBS and lysed for 30 min at 4 °C in RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich P8340; St. Louis, MO, USA). Lysates were collected by centrifugation (12,000× g, 4 °C, 30 min) and stored at −80 °C until detection of the EBI: β-tubulin adduct by Western blot. Briefly, 20 µg of each sample was loaded on a 10% Mini-Protean TGX SDS gel (Bio-Rad 4561034; Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes (iBlot dry blotting system, Invitrogen; Waltham, MA, USA) and blocked for 1 h post-transfer (Intercept blocking buffer, Li-COR Biosciences 927-80001; Lincoln, NE, USA). Primary antibodies were incubated overnight at 4 °C (mouse anti-β-tubulin, Sigma-Aldrich T4026, St. Louis, MO, USA; rabbit anti-GAPDH, Cell Signaling Technology 14C10, Danvers, MA, USA) in blocking buffer/0.2% Tween 20. Membranes were washed twice (PBS/0.1% Tween 20) and incubated for 1 h at RT with secondary antibodies (IRDye 680 anti-mouse, Li-COR Biosciences 926-68072; IRDye 800 anti-rabbit, Li-COR Bioscience 926-32211; Lincoln, NE, USA) in blocking buffer/0.1% Tween 20. After three washes, membranes were dried for 2 h prior to imaging on the Odyssey CLx Imager (Li-COR Biosciences; Lincoln, NE, USA). Band intensities were quantified by densitometric analysis using Fiji/ImageJ software (NIH) v2.3.0.

2.8. Reversibility Assay

U87 cells were treated with increasing concentrations (up to 10 μM) of RGN3067 and the control compounds colchicine (Selleckchem S2284; Houston, TX, USA) and sabizabulin (MedChem Express HY-120599; Monmouth Junction, NJ, USA) and cultured for 6 h. Compounds were either washed out or left in the medium. Cells were then cultured for an additional 72 h and cell viability was examined using CellTiter-Glo (Promega, Madison, WI, USA).

2.9. Compound Kinetic Solubility

Kinetic solubility was determined in phosphate buffer. The compound (4 µL, 10 mM DMSO) was added to the buffer (396 μL 100 mM phosphate buffer, final 1% DMSO) and shaken at 1000 rpm at RT for 1 h. Samples were filtered (Millipore MSSLBPC10; Burlington, MA, USA), the primary filtrate was discarded, and the subsequent filtrate was collected. The supernatant solution was diluted 10× with DMSO. Test samples (10 µL) and working standard curve samples were added to the stop solution (100 μL, containing internal standard) and water/0.1% FA (100 μL). Samples were analyzed by LC-MS/MS. Albendazole, propranolol, and 4,5-diphenylimidazole were used as positive controls.

2.10. Compound Microsomal Stability

Compounds (5 µL, 20 mM DMSO) were added to 195 µL of methanol to obtain a 500 µM spiking solution. Spiking solution (1.5 μL) and liver microsomes (18.75 μL, 20 mg/mL) were added to Buffer C (479.75 μL) on ice. Plates were pre-incubated at 37 °C for 5 min. At 0 min, spiking/microsome solution (30 μL) was added to the precipitation solution (400 μL) and NADPH stock solution (6 mM in Buffer C, 0.1 M PBS, pH 7.4) on ice. At additional time points, spiking/microsome solution (180 μL) was added to the assay plates, and the reaction initiated with the NADPH stock solution (90 μL, 6 mM). Plates were sealed and incubated at 37 °C. At 5, 15, 30 and 60 min time points, aliquots of the microsomal incubation system (45 μL) were removed and added to precipitation solution (400 μL). Supernatants were evaluated by LC-MS/MS. Ketanserin was used as a positive control. All reactions were performed in duplicate.

2.11. Plasma Protein Binding Studies

Compounds (5 µM/0.2% DMSO) were tested in dialysis chambers. Blank dialysis buffer was added to the receiver side (100 μL), and plasma (100 μL) with test and reference compounds was added to the donor side of the chamber and incubated at 37 °C on an orbital shaker. Plasma was precipitated at 0 and 5 h. Aliquots from donor and receiver sides of the dialysis apparatus (25 μL) were added to sample preparation plates and mixed with aliquots of the opposite matrix (25 μL, blank buffer to plasma and vice versa). Samples were quenched with stop solution (400 μL) containing internal standard, vortexed at 600 rpm for 10 min, and centrifuged at 4000 rpm for 10 min. Supernatants were analyzed by LC-MS. Quinidine and warfarin were used as positive controls.

2.12. Cellular Permeability of RGN3067

Cellular permeability was tested using the MDR1-MDCK (multidrug-resistant-1, Madin–Darby canine kidney) cell line. Donor solution was added to a 96-well plate (2 μL, 2 mM compound; 6 μL DMSO in 1992 μL of Hanks’ balanced salt solution [HBSS] buffer) and shaken at 1000 rpm for 10 min (final 0.4% DMSO). Transepithelial electrical resistance (TEER) values were used as a quality control check for monolayer integrity. At 3–5 days post-seeding, each MDCK II cell monolayer was tested to ensure the TEER value was ≥ 83 Ω⋅cm². The compound donor working solution was centrifuged (4000 rpm for 5 min) to remove any precipitated compound prior to loading donor chambers. Apical and basolateral plates were warmed at 37 °C for about 5 min prior to the initiation of transport (37 °C, 90 min).

Luciferin marker Lucifer Yellow (150 μL, 50 μM Lucifer Yellow in HBSS, pH 7.4) was added to the apical compartment, and transfer buffer (600 μL) was added to the receiver plate (basolateral compartment) and incubated at 37 °C for 60 min. Apical Lucifer Yellow concentrations for LYT0 and LYT60 were measured by a fluorometer (EX 485/EM 535 nm). Donor and receiver samples were analyzed by LC-MS. Metoprolol, enalapril, and quinidine were used as positive controls. The efflux ratio was calculated as the ratio of apparent permeability (basal-to-apical) and apparent permeability (apical-to-basal).

2.13. Pharmacokinetics of RGN3067

Pharmacokinetic studies were carried out by DMPK at WuXi AppTec, Inc. (Cranbury, NJ, USA). The pharmacokinetics of RGN3067 were evaluated in three male CD-1 mice. RGN3067 formulated as a 10 mg/mL solution (20% DMSO, 80% PEG400: PVP 95:5) was administered by oral gavage at a dose of 100 mg/kg. Plasma samples were drawn at 0.5, 1, 2, 4, 6, 10 and 24 h. Samples were analyzed by LC-MS/MS. A calibration curve was established using Waters ACQUITY UPLC BEH C18 column and formic acid in water and formic acid in acetonitrile as mobile phase. Tolbutamide and labetalol were used as internal standards for LC-MS/MS analysis. Compound was detected using electrospray ionization (ESI) positive detection. Pharmacokinetic parameters were calculated using the PO-noncompartmental linear model in Phoenix WinNonlin software v8.3 (Certara, Princeton, NJ, USA).

2.14. Brain Pharmacokinetics of RGN3067

Brain pharmacokinetic studies were carried out by DMPK at WuXi AppTec, Inc. (Cranbury, NJ, USA). The brain penetrance of RGN3067 was evaluated in three male CD-1 mice. RGN3067 was formulated as a 10 mg/mL solution (20% DMSO, 80% PEG400: PVP 95:5) and administered by oral gavage at a dose of 100 mg/kg. Plasma and brain samples were collected at 1, 4, and 8 h. Samples were analyzed by LC-MS/MS. A calibration curve was established using Waters ACQUITY UPLC BEH C18 column and formic acid in water and formic acid in acetonitrile as mobile phase. Tolbutamide and labetalol were used as internal standards for LC-MS/MS analysis. Compound was detected using electrospray ionization (ESI) positive detection. Pharmacokinetic parameters were calculated using the PO-noncompartmental linear model in Phoenix WinNonlin software v8.3 (Certara, Princeton, NJ, USA).

2.15. Maximum Tolerated Dose Study with RGN3067

RGN3067 was administered to athymic female mice (4–6 weeks old) twice daily for 5 days by oral gavage at doses of 25, 50, 100 or 200 mg/kg (BID; 6 mice per group, including vehicle control). One mouse from each group was euthanized for PK analysis at 6 h post-initial dose. The weight and physical states of the animals were monitored during the 5 days of treatment and for 5 days following drug administration. The maximum tolerated dose was defined as the dose at which none of the mice within the cohort lost more than 20% body weight during the 10-day study and was used for subsequent efficacy testing.

2.16. The Efficacy of RGN3067 in a PDX Model of GB

After tumors reached 100–150 mm³, mice were dosed with RGN3067 solution (43 µL, 100 mg/kg) or vehicle control twice daily by oral gavage (total dose was 200 mg/kg). Mice were dosed using cycles of 5 days on/2 days off, for a total of four cycles. Mice were euthanized once the tumor reached 2000 mm³.

2.17. Metabolite Profiling of RGN3067 in Hepatocytes

Metabolite profiling was carried out by WuXi DMPK (Nanjing, China). RGN3067 (10 μM) was profiled in CD-1 mice (BioIVT, Westbury, NY, USA), SD rats (BioIVT), beagle dogs (BioIVT), cynomolgus monkeys (Research Institute for Liver Diseases [RILD], Shanghai, China), and human cryopreserved hepatocytes (BioIVT). The positive control was 7-ethoxycoumarin (30 μM). Compounds were added to hepatocytes (400 µL, 1 × 10⁶/mL), and reactions were carried out at 37 °C in 5% CO₂/saturated humidity for 120 min. Reactions were stopped by the addition of ice-cold ACN (400 μL). Samples were shaken at 300 rpm for 10 min, then centrifuged at 13,000× g for 10 min. Supernatants were evaporated under a stream of N₂. Residue from the RGN3067 incubation was reconstituted with ACN: H₂O (200 μL, v:v, 3:7), followed by centrifugation at 14,000 rpm for 10 min, and the supernatant was used for qualitative and semi-quantitative analysis using LC-UV/HRMS. A Shimadzu UFLC-20A with autosampler was used for chromatographic separation. Detection and structure were determined using a Thermo Q-Extractive mass spectrometer in full MS/MS² scan mode and Xcalibur Data Acquisition and Interpretation Software v4.2.

2.18. Data Processing

All statistical analyses were performed using GraphPad Prism v10 (San Diego, CA, USA). Absolute IC₅₀ values were determined by a nonlinear regression model. Tests to determine significance are detailed in the relevant figure and table legends. Briefly, the Shapiro–Wilk test was used for testing the normality of the data. Unpaired t-tests or one-way ANOVAs were used to determine the statistical significance of treatment conditions. Asterisks denote significance (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001).