The Discovery of Novel BCR-ABL Tyrosine Kinase Inhibitors Using a Pharmacophore Modeling and Virtual Screening Approach

Abstract

Chronic myelogenous leukemia (CML) typically results from a reciprocal translocation between chromosomes 9 and 22 to produce the bcr-abl oncogene that when translated, yields the p210 BCR-ABL protein in more than 90% of all CML patients. This protein has constitutive tyrosine kinase activity that activates numerous downstream pathways that ultimately produces uncontrolled myeloid proliferation. Although the use of the BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have increased the overall survival of CML patients, their use is limited by drug resistance and severe adverse effects. Therefore, there is the need to develop novel compounds that can overcome these problems that limit the use of these drugs. Therefore, in this study, we sought to find novel compounds using Hypogen and Hiphip pharmacophore models based on the structures of clinically approved BCR-ABL TKIs. We also used optimal pharmacophore models such as three-dimensional queries to screen the ZINC database to search for potential BCR-ABL inhibitors. The hit compounds were further screened using Lipinski's rule of five, ADMET and molecular docking, and the efficacy of the hit compounds was evaluated. Our in vitro results indicated that compound ZINC21710815 significantly inhibited the proliferation of K562, BaF3/WT, and BaF3/T315I leukemia cells by inducing cell cycle arrest. The compound ZINC21710815 decreased the expression of p-BCR-ABL, STAT5, and Crkl and produced apoptosis and autophagy. Our results suggest that ZINC21710815 may be a potential BCR-ABL inhibitor that should undergo in vivo evaluation.

Keywords: BCR-ABL; CML; ZINC21710815; apoptosis; autophagy; pharmacophore model.

FIGURE 1

The flow chart for the virtual screening process.

FIGURE 2

(A) 3D spatial relationship and geometric parameters of Hypogen1. (B) Hypogen1 mapping with the most active compound. (C) Hypogen 1 mapping with the least active compound.

FIGURE 3

Correlation between the experimented and predicted efficacies of the training set and the test set based on the Hypogen1 model.

FIGURE 4

The difference in cost between the initial hypothesis and 19 random hypotheses. The 95% confidence level was selected to validate the Fischer randomization test results (Hypogen1).

FIGURE 5

(A) The Hiphop1 pharmacophore model. (B) Hiphop1 mapped with the most active compound.

FIGURE 6

Cytotoxic efficacy of compounds in K562 leukemia cells. Cells were incubated with various concentrations of IM, ZINC36617838, ZINC30201139, ZINC65008391, ZINC45895251, ZINC36617852, or ZINC36617849 for 72 h. Cell viability was determined using the MTT assay. The results are represented as the mean ± SD.

FIGURE 7

The work flow chart of structure optimization and its screening process.

FIGURE 8

The binding modes of three compounds and imatinib with the BCR-ABL protein (PDB: 1IEP): hydrogen bond (green), pi-pi (yellow). (A) ZINC20617585; (B) ZINC36617889; (C) ZINC21710815; and (D) Imatinib (IM). The effect of these compounds on the proliferation of (E) K562, (F) BaF3, (G) BaF3/T315I leukemia cells, and (H) CCC-HEL-1 cells. The cells were incubated with various concentrations of imatinib (IM), ZINC21710815, ZINC36617889, or ZINC20617585 for 72 h. The viability of the cells was assessed using the MTT assay. The results are represented as the mean ± S.D.

FIGURE 9

The effect of ZINC21710815 on the cell cycle in K562, BaF3/WT and BaF3/T315I leukemia cells. (A) K562 leukemia cells were incubated with various concentrations of ZINC21710815 for 24 h at a density of 1 × 10⁶ cells/well. (B) BaF3 leukemia cells were incubated with various concentrations of ZINC21710815 for 24 h at a density of 5 × 10⁵ cells/well. (C) BaF3/T315I leukemia cells were incubated with various concentrations of ZINC21710815 for 24 h at a density of 5 × 10⁵ cells/well. (D) The relative percentages of the cell cycles for K562, BaF3/WT, and BaF3/T315I leukemia cells were calculated using ModFit LT software. The results are represented as the mean ± SD.

FIGURE 10

ZINC21710815 induces apoptosis and autophagy in CML cells. (A) K562, BaF3/WT, and BaF3/T315I cells were incubated with various concentration of ZINC21710815 for 24 h at a density of 5 × 10⁵ cells/well, stained using Annexin V-FITC and PT and analyzed by FlowJo. (B) Caspase-3 protein expression in BaF3/WT and BaF3/T315I cells incubated with 0 (vehicle), 0.1, or 1 μM of ZINC21710815 for 48 h. LC3 (C) and Beclin1 (D) protein expression in BaF3/WT and BaF3/T315I cells incubated with 0 (vehicle), 0.1, or 1 μM of ZINC21710815 for 48 h.

FIGURE 11

BaF3/WT leukemia cells were incubated with 0 (vehicle), 0.1, or 1 μM for 48 h with either imatinib and ZINC21710815 the levels of BCR-ABL and phosphorylated BCR-ABL (A), and the downstream proteins p-Crkl (B), p-STAT5 (C) were analyzed using western blotting. The results are represented as the mean ± SD.