High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM.

With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant kinact/Ki, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire-MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract kinact/Ki in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of kinact/Ki values from single digit to 3 × 105 M-1 s-1.

Cereblon Promotes the Ubiquitination and Proteasomal Degradation of Interleukin Enhancer-Binding Factor 2.

Interleukin enhancer-binding factor 2 (ILF2) forms a heterodimer with interleukin enhancer-binding factor 3 (ILF3) via double-stranded RNA-binding motif and zinc finger associated domain and thus regulates gene expression and cancer cell growth. However, how ILF2 is degraded in cells remains elusive. In this work, using stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomics, we find that ILF2 is downregulated in cells expressing cereblon (CRBN). Using affinity purification and immunoblotting analysis, we demonstrate that CRBN interacts with ILF2 and functions as a substrate receptor of the cullin-4 RING E3 ligase complex. Biochemical experiments disclose that CRBN expression reduces ILF2 protein level and this reduction is diminished when the proteasome is inhibited. Upon protein synthesis inhibition, the degradation of ILF2 is enhanced by CRBN. Moreover, CRBN promotes the ubiquitination of ILF2 and thus results in the ubiquitin-mediated proteasomal degradation. Analyses of previously identified post-translational modification sites and the crystal structure of ILF2 discover the potential ubiquitination sites on ILF2. Through mutagenesis and biochemical experiments, we further reveal that the K45R mutation completely abolishes the effect of CRBN on ILF2, suggesting that this is the key residue responsible for its ubiquitination. Taken together, we identify an E3 ligase that regulates ILF2 and uncover a molecular pathway for its degradation. This work might be helpful to elucidate the molecular mechanism by which CRBN regulates diverse cellular functions.

[Inhibition of PI3K/AKT signaling pathway promotes the nuclear translocation of B7-H4].

OBJECTIVE: To investigate the regulatory effect of phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway on the subcellular distribution of negative co-stimulatory molecule B7-H4. METHODS: The HEK293 cells transfected stably with B7-H4, named B7-H4/HEK293, were treated with the PI3K/AKT specific inhibitor LY294002 and/or the nuclear export inhibitor leptomycin B (LMB). The subcellular localization of B7-H4 in B7-H4/HEK293 was observed by immunofluorescence and confocal laser scanning microscopy (CLSM), and the expression levels of B7-H4 in membrane, cytoplasm and nuclear were detected by Western blotting. RESULTS: CLSM showed that LY294002 effectively induced the nuclear translocation of B7-H4 when compared with vehicle group. When the B7-H4/HEK293 cells were treated with LY294002 and LMB, more B7-H4 was translocated into nuclear. Western blotting demonstrated that after the PI3K/AKT signal pathway was inhibited by LY294002 for 24 hours, the levels of B7-H4 in cell membrane and cytoplasm decreased significantly (P<0.05), while the expression in nuclear increased significantly (P<0.05). CONCLUSION: The PI3K/AKT signal pathway might inhibit the nuclear translocation of B7-H4.