Discovery of a Covalent Inhibitor Selectively Targeting the Autophosphorylation Site of c-Src Kinase.

Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain (R)-LW-Srci-8 with nearly 75-fold improved potency (IC50 = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C-F···C═O interactions that may contribute to tight binding. The kinact and Ki values validated the improved binding affinity and decreased warhead reactivity of (R)-LW-Srci-8 for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by (R)-LW-Srci-8. Intriguingly, (R)-LW-Srci-8 preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.

Deciphering Biophysical Modulation in Ovarian Cancer Cells.

It has been long known that the oncogenic extracellular environment plays an indispensable role in developing and nurturing cancer cell progression and in resistance to standard treatments. However, by how much the biophysical components of tumour extracellular environment contribute to these processes is uncertain. In particular, the topographic environment is scarcely explored. The biophysical modulation of cell behaviour is primarily facilitated through mechanotransduction-associated mechanisms, including focal adhesion and Rho/ROCK signalling. Dysregulation of these pathways is commonly observed in ovarian cancer and, therefore, biophysical modulation of these mechanisms may be of great importance to ovarian cancer development and progression. In this work, aspects of the biophysical environment were explored using a bioimprinting technique. The study showed that topography-mediated substrate sensing delayed cell attachment, however, cell-cell interactions overrode the effect of topography in some cell lines, such as OVCAR-5. Also, 3D topographical cues were shown to modulate the inhibition of focal adhesion and Rho signalling, which resulted in higher MAPK activity in cells on the bioimprints. It was revealed that c-Src is vital to the biophysical modulation of cell proliferation and inhibition of c-Src could downregulate biophysically modulated MAPK activity. This study provides evidence that the biophysical extracellular environment affects key intracellular mechanisms associated with tumourigenicity in ovarian cancer cells.

Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase.

BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

Crosstalks of the PTPIP51 interactome revealed in Her2 amplified breast cancer cells by the novel small molecule LDC3/Dynarrestin.

LDC3/Dynarrestin, an aminothiazole derivative, is a recently developed small molecule, which binds protein tyrosine phosphatase interacting protein 51 (PTPIP51). PTPIP51 interacts with various proteins regulating different signaling pathways leading to proliferation and migration. Her2 positive breast cancer cells (SKBR3) express high levels of PTPIP51. Therefore, we investigated the effects of LDC3/Dynarrestin on PTPIP51 and its interactome with 12 different proteins of various signal pathways including the interaction with dynein in SKBR3 cells. The localization and semi-quantification of PTPIP51 protein and the Tyr176 phosphorylated PTPIP51 protein were evaluated. Protein-protein-interactions were assessed by Duolink proximity ligation assays. Interactions and the activation of signal transduction hubs were examined with immunoblots. LDC3/Dynarrestin led to an increased PTPIP51 tyrosine 176 phosphorylation status while the overall amount of PTPIP51 remained unaffected. These findings are paralleled by an enhanced interaction of PTPIP51 with its crucial kinase c-Src and a reduced interaction with the counteracting phosphatase PTP1B. Furthermore, the treatment results in a significantly augmented interaction of PTPIP51/14-3-3ß and PTPIP51/Raf1, the link to the MAPK pathway. Under the influence of LDC3/Dynarrestin, the activity of the MAPK pathway rose in a concentration-dependent manner as indicated by RTK assays and immunoblots. The novel small molecule stabilizes the RelA/IκB/PTPIP51 interactome and can abolish the effects caused by TNFα stimulation. Moreover, LDC3/Dynarrestin completely blocked the Akt signaling, which is essential for tumor growth. The data were compared to the recently described interactome of PTPIP51 in LDC3/Dynarrestin treated non-cancerous keratinocyte cells (HaCaT). Differences were identified exclusively for the mitochondrial-associated ER-membranes (MAM) interactions and phospho-regulation related interactome of PTPIP51.LDC3/Dynarrestin gives the opportunity/possibility to influence the MAPK signaling, NFkB signaling and probably calcium homeostasis in breast cancer cells by affecting the PTPIP51 interactome.

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.