Heterocyclic-Fused Pyrimidines as Novel Tubulin Polymerization Inhibitors Targeting the Colchicine Binding Site: Structural Basis and Antitumor Efficacy

Abstract

We report the design, synthesis, and biological evaluation of heterocyclic-fused pyrimidines as tubulin polymerization inhibitors targeting the colchicine binding site with significantly improved therapeutic index. Additionally, for the first time, we report high-resolution X-ray crystal structures for the best compounds in this scaffold, 4a, 4b, 6a, and 8b. These structures not only confirm their direct binding to the colchicine site in tubulin and reveal their detailed molecular interactions but also contrast the previously published proposed binding mode. Compounds 4a and 6a significantly inhibited tumor growth in an A375 melanoma xenograft model and were accompanied by elevated levels of apoptosis and disruption of tumor vasculature. Finally, we demonstrated that compound 4a significantly overcame clinically relevant multidrug resistance in a paclitaxel resistant PC-3/TxR prostate cancer xenograft model. Collectively, these studies provide preclinical and structural proof of concept to support the continued development of this scaffold as a new generation of tubulin inhibitors.

Figure 1

Recently developed VDAs that target the colchicine binding site. Three are currently in clinical trials (1a, 1b, and 1c), and three reported derivatives of verubulin are in preclinical development (1d, 1e, and 1f).

Figure 2

New analogues inhibit tubulin polymerization. (A) Polymerization of purified tubulin in a cell-free assay. Tubulin (3.33 mg/mL) was exposed to vehicle control or compounds at the indicated concentrations (n = 2). Absorbance at 340 nm was monitored at 37 °C every minute for 50 min. (B) Microtubules of WM164 cells. (C) Effect on microtubules following 18 h treatment with 100 nM docetaxel or (D) 4a. Immunofluorescence is visualized by α-tubulin primary antibody and Alexa Fluor 647 secondary antibody via confocal microscopy. Scale bar = 20 μM.

Figure 3

X-ray crystal structures of tubulin-RB3-SLD-TTL proteins in complex with 4a, 4b, 6a, and 8b. Panel A shows the overall high-resolution crystal structure of the tubulin-RB3-SLD-TTL complex with GTP, GDP, and ligand indicated by the arrow. Panel B shows superimposed unliganded (gray), colchicine bound (violet), and 4a bound (gold) tubulin complexes showing the different loop conformations resulting from binding of ligands. Panels C–F show complexes with 4a (panel C, resolution 2.3 Å), 4b (panel D, resolution 2.5 Å), 6a (panel E, resolution 2.7 Å), and 8b (panel F, resolution 2.6 Å). The tubulin α-monomer is shown in green, and the β-monomer is shown in cyan for panels C–F.

Figure 4

Diagrammatic representation of the binding poses for 4a revealed by X-ray crystallography (A) and by prediction using crystal structures of different CBSI (colchicine binding site inhibitors) scaffolds (B). Note that the predicted pose is incorrectly 180° flipped relative to the binding poses revealed by X-ray analysis. The red dot in panel A stands for a water molecule.

Figure 5

4a inhibits cell migration in a wound healing assay. (A) Representative images of wound closure after 18–22 h by A375 (top) or RPMI7951 (bottom) cells after treatment with 5 or 25 nM concentrations of 4a, colchicine, or untreated control. (B) Percentage of the wound closed for A375 cells or RPMI7951 cells (n = 3). Area of the wound channel was calculated using ImageJ software. Statistical analysis was performed by Dunnett’s multiple comparison test, comparing each treatment group to the control group: (****) P < 0.0001, (***) P < 0.001, (**) P < 0.01, (*) P < 0.05.

Figure 6

4a and 6a inhibit tumor growth in vivo. (A) A375 xenograft model in nude mice. Graph represents mean tumor volume ± SEM (n = 8). (B) Individual tumor final volumes. Bar graph represents the mean tumor volume for each group ± 95% confidence interval. (C) Tumor weights ± SEM. (D) Mouse body weights in the xenograft model. Graph represents mean body weight change as a percentage compared to initial weight ± SEM values. Statistical significance was determined by ANOVA analysis followed by Dunnett’s multiple comparison test: (****) P < 0.0001, (***) P < 0.001 for the treatment group compared with the corresponding results of control group.

Figure 7

Histological analysis of tumors. (A) Representative images of tumor samples for treatment groups by hematoxylin and eosin (H&E) at 100× magnification. (B) IHC showing cleaved caspase 3 expression, indicative of apoptosis. (C) CD31 expression showing microvessel density. Treatment groups display greater vessel disruption compared to the control group.

Figure 8

4a escapes paclitaxel resistance in vivo. (A) Prostate cancer xenograft models in nude mice for PC-3 tumors and (B) paclitaxel resistant PC-3/TxR tumors. Graph represents mean tumor volume ± SEM (n = 7). (C) Individual PC-3/TxR tumor final volumes. Bar graph represents the mean tumor volume for each group ± SEM. (D) PC-3/TxR tumor weights ± SEM. (E) Representative images of PC-3/TxR tumors. (F) Mouse body weights in the PC-3/TxR xenograft model. Graph represents mean body weight change as a percentage compared to initial weight ± SEM values. Statistical significance was determined by ANOVA analysis followed by Dunnett’s multiple comparison test final tumor volumes and weights: (****) P < 0.0001, (**) P < 0.01, (*) P < 0.05 for the treatment group compared with the corresponded results of control group.

Scheme 1

Design and Synthesis of Ring A Modified Heterocyclic-Fused Pyrimidine Analogues

Scheme 2

Synthesis of Ring A Modified 1H-Pyrazolopyrimidine Analogue

Scheme 3

Synthesis of Ring B Modified Pyridopyrimidine Analogues

Scheme 4

Synthesis of Ring B and Ring C Modified Pyridopyrimidine Analogues