Objectives:
Triple-negative breast cancer (TNBC) poses a significant clinical challenge due to its high heterogeneity and lack of effective therapeutic targets. DKK1 is closely associated with TNBC proliferation, metastasis and microenvironment regulation, representing a potential novel therapeutic target. Given that the DKK1-LRP5/6 binding interface lacks classical small-molecule binding pockets, this study will employ DNA-encoded library (DEL) technology to design and synthesize a series of novel DKK1-targeting small-molecule inhibitors. We aim to elucidate their structure-activity relationships and ultimately develop high-affinity, drug-like DKK1 PPI small-molecule inhibitors, providing new therapeutic strategies and potential drug candidates for TNBC treatment.
Methods:
Employ DNA-encoded library technology to screen candidate small molecules with potential affinity for DKK1 protein and identify lead compounds.
(2) Perform structure-based drug design to optimize lead compounds, using bio-layer interferometry (BLI) and MTT assays to evaluate binding constants to DKK1 and anti-proliferative activities, thereby elucidating structure-activity relationships.
(3) Utilize molecular docking to predict binding modes between optimized compounds and DKK1, validating target specificity.
(4) Assess anti-proliferative effects of selected compounds on TNBC cells through colony formation assays, EdU incorporation assays, and Ki67 immunofluorescence staining.
(5) Evaluate anti-migratory effects of selected compounds on TNBC cells using scratch wound healing assays, Transwell migration assays, and Snail immunofluorescence staining.
(6) Determine apoptosis-inducing capacity of selected compounds in TNBC cells by flow cytometry and Western blot analysis.
Results:
Using DNA-encoded library technology, we obtained 38 small-molecule compounds exhibiting binding affinity to DKK1, including 16 benzimidazole derivatives, 13 3-phenylpropanamides, and 9 peptide-based compounds.
Based on molecular docking results, we selected benzimidazole derivatives as lead compounds for structural optimization, synthesizing 120 derivatives whose structures were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry.
(3) Anti-TNBC activity screening revealed compound 19g exhibited optimal anti-proliferative effects against BT-549 (IC50 = 2.77 µM), MDA-MB-231 (IC50 = 3.41 µM), MCF-7 (IC50 = 7.50 µM), and 4T1 (IC50 = 3.81 µM) cells.
(4) Bio-layer interferometry demonstrated compound 19g showed strongest DKK1 binding with equilibrium dissociation constant (KD) = 2.727 × 10⁻⁶ M; association rate (ka) = 4177 M⁻¹s⁻¹; and dissociation rate (kdis) = 1.139 × 10⁻² s⁻¹.
(5) Analysis of the anti-TNBC cell proliferation activity results of 120 compounds and the parameter results of their binding ability to DKK1 protein revealed that when the substituent at position 9 of the benzimidazole core is a saturated nitrogen-containing heterocycle, the activity is superior to that of an aromatic heterocycle; when the N-substituent of the amide is a saturated cycloalkyl group, it is better than an alkyl chain; when the substituent at position 8 is 4'-methyl-[2,2'-bipyridin]-4-yl, it significantly contributes to the anti-TNBC cell proliferation activity. The structure-activity relationship was preliminarily elucidated.
(6) Molecular docking results showed that the benzimidazole core forms a stable π-π stacking interaction with residue HIS204 and a π bond with residue LYS211. The piperidine ring, cyclobutyl group, and 4'-methyl-[2,2'-bipyridin]-4-yl group form stable π bonds with residues PRO212, ARG236, and GLU185, respectively. Moreover, the cyclobutylamide, benzimidazole core, and ethylpiperidine of compound 19g face the internal cavity of the DKK1 protein and occupy the binding site of LRP6, which serves as the structural basis for the superior activity of this class of compounds. The strong binding ability of this class of compounds to the DKK1 protein was confirmed at the molecular level.
(7) The results of the colony formation assay, EdU assay, and Ki67 immunofluorescence staining assay demonstrated that compound 19g significantly inhibited the proliferation of TNBC cells in a concentration-dependent manner.
(8) The results of the scratch wound healing assay, Transwell assay, and Snail immunofluorescence staining assay demonstrated that compound 19g significantly inhibited the migration of TNBC cells in a concentration-dependent manner.
(9) The results of Annexin-V-PI double staining and Western blot assays indicated that compound 19g induced the apoptosis of TNBC cells in a concentration-dependent manner.
Conclusions:
In conclusion, in this study, lead compounds were screened out by utilizing the technology of DNA-encoded chemical library and molecular docking. Subsequently, based on the structure-based drug design approach, the lead compounds were structurally optimized. A total of 120 DKK1 inhibitors with benzimidazole as the core were designed and synthesized. Anti-TNBC activity experiments of these compounds and tests of their binding ability to DKK1 protein were carried out. The optimized compound 19g was screened out, and the structure-activity relationship was preliminarily elucidated. The molecular docking results indicated that compound 19g exerted its activity by binding to the internal cavity of the DKK1 protein. In vitro pharmacological experiments showed that compound 19g had significant anti-proliferative, anti-migratory, and apoptosis-inducing effects on TNBC cells.
This study provides a candidate drug and a theoretical basis for the treatment of TNBC with PPI inhibitors of DKK1, and it has important potential application value.
谷歌
火狐
