Fused Imidazopyrazine-Based Tubulin Polymerization Inhibitors Inhibit Neuroblastoma Cell Function

Abstract

Targeting the colchicine binding site on tubulin is a promising approach for cancer treatment to overcome the limitations of current tubulin polymerization inhibitors. New classes of colchicine binding site inhibitors (CBSIs) are continually being uncovered; however, balancing metabolic stability and cellular potency remains an issue that needs to be resolved. Therefore, we designed and synthesized a series of novel fused imidazopyridine and -pyrazine CBSIs and evaluated their cellular activity, metabolic stability, and tubulin-binding properties. Evidence shows that the imidazo[1,2-a]pyrazine series are effective against neuroblastoma cell lines marked by MYCN amplification. Further assessment shows that a combination of an imidazo[1,2-a]pyrazine core with a trimethoxyphenyl ring D results in the highest cellular activity and binding characteristics compared with a dichloromethoxyphenyl or difluoromethoxyphenyl ring D. However, the metabolic stability of compounds with a dichloromethoxyphenyl or difluoromethoxyphenyl ring D is significantly higher than that of those containing a trimethoxyphenyl ring D, suggesting that improved metabolic stability is achieved with a moderate impact on potency.

Figure 1

(A) Chemical structures of different classes of reported CBSIs. (B) Design and structure–activity relationship (SAR) of target compounds.

Scheme 1. Synthesis of Imidazo[1,2-a]pyridine Analogues

Scheme 2. Synthesis of Reverse Imidazopyridine Derivative

Scheme 3. Synthesis of Imidazo[1,2-a]pyrazine Derivatives

Figure 2

Compounds inhibit tubulin polymerization in vitro. In vitro tubulin polymerization was monitored in the absence of any compound (“standard”) and with the addition of 5 μM paclitaxel, 5 μM colchicine, or 5 μM of the indicated compound 4a, 4h, 4j, or 4k. The same curves generated from the standard, paclitaxel, and colchicine reactions are superimposed on all four graphs for comparison with each of the indicated compounds. All compounds tested inhibit microtubule formation as compared with the standard and colchicine reactions.

Figure 3

Cell cycle progression is impaired with compound 4h. (A) Representative cell cycle profile images of Kelly cells treated with DMSO, 100 nM colchicine, or 100 nM compound 4h for 24 h. (B) Quantification of cell cycle phase distributions for cells treated as in (A) (n = 3 biological replicates, error bars are standard error, ****P < 0.0001, *P = 0.018, **P < 0.002 using unpaired t test, two-tailed). (C) Representative cell cycle profile images of Kelly cells treated with DMSO or 500 nM compound 4j for 24 h. (D) Quantification of cell cycle phase distributions for cells treated as in (C) (n = 3 biological replicates, error bars are standard error). (E) Representative cell cycle profile images of CHP-134 cells treated with DMSO, 100 nM colchicine, or 100 nM compound 4h for 24 h. (F) Quantification of cell cycle phase distributions for cells treated as in (E) (n = 3 biological replicates, error bars are standard error, **P = 0.0089, *P < 0.019 using unpaired t test, two-tailed).

Figure 4

Compounds cause induction of mitotic protein markers. Western blots of protein lysates collected following treatment of indicated cells with DMSO, 100 nM colchicine, 100 nM compound 4h, or 500 nM compounds 4j and 4k for 24 h. GAPDH is used as a loading control.

Figure 5

Impact of compound treatment on intracellular tubulin. Representative immunostaining images of Kelly cells treated with DMSO, 100 nM colchicine, 100 nM compound 4h, or 500 nM compounds 4j and 4k for 24 h. Nuclei are stained with DAPI in blue, and α-tubulin is stained with Alexa-488 in green. Scale bar = 20 μm.

Figure 6

Molecular docking. (A) Docking of 4b (brown) at the colchicine binding site of tubulin (PDB: 6X1F). The α-tubulin monomer is shown in cyan and the β-tubulin monomer in green. Native ligand (SB-216/5m) is shown in pink. (B) Amino acid residues within 5 Å of the docked ligand 4b. (C) Docked pose of 4k (yellow) at the colchicine binding site. (D) Docked pose of 4h (gold) at the colchicine binding site. (E) Docked pose of 4j (purple) at the colchicine binding site. (F) Binding surface (hydrophobic) around the ligand 4k. Hydrogen bonding is shown by purple dotted lines.

Figure 7

RMSD and binding energy vs time plots for 4h and 4k. (A) RMSD vs simulation time plot for protein backbone atoms with 4h as ligand in red, movement of 4h in its binding site in green, protein backbone atoms with 4k as ligand in purple, and movement of 4k in its binding site in orange. (B) Binding energy of protein–ligand interactions of the colchicine binding site of tubulin with 4h and 4k vs time. Binding energy was calculated by analyzing the MD trajectory using the BoundaryFast method in YASARA. Based on the YASARA algorithm, higher binding energy values mean stronger binding.