Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP‐dependent peri‐centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP–tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP–tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de‐clustering, prolonged multipolar mitosis, and cell death. 3D‐organotypic invasion assays reveal that CCB02 has broad anti‐invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)‐resistant EGFR‐mutant non‐small‐cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug‐resistant cancers exhibiting high incidence of centrosome amplification.
Herein, we report the discovery and characterization of a novel class of PAK4 inhibitors with a quinazoline scaffold. Based on the shape and chemical composition of the ATP-binding pocket of PAKs, we chose a 2,4-diaminoquinazoline series of inhibitors as a starting point. Guided by X-ray crystallography and a structure-based drug design (SBDD) approach, a series of novel 4-aminoquinazoline-2-carboxamide PAK4 inhibitors were designed and synthesized. The inhibitors' selectivity, therapeutic potency, and pharmaceutical properties were optimized. One of the best compounds, 31 (CZh226), showed remarkable PAK4 selectivity (346-fold vs PAK1) and favorable kinase selectivity profile. Moreover, this compound potently inhibited the migration and invasion of A549 tumor cells by regulating the PAK4-directed downstream signaling pathways in vitro. Taken together, these data support the further development of 31 as a lead compound for PAK4-targeted anticancer drug discovery and as a valuable research probe for the further biological investigation of group II PAKs.
Histone recognition by ‘reader’ modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as “K4me3-R8me2a”, and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 “K4me3-K9me3/2” bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nM between Spindlin1 and H3 “K4me3-K9me3” peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 “K4me3-K9me3/2” bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions.
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