Subtle Structural Differences Affect the Inhibitory Potency of RGD-Containing Cyclic Peptide Inhibitors Targeting SPSB Proteins.

The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible nitric oxide synthase (iNOS), and recruit an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in the proteasomal degradation of iNOS. Inhibitors that can disrupt the endogenous SPSB-iNOS interactions could be used to augment cellular NO production, and may have antimicrobial and anticancer activities. We previously reported the rational design of a cyclic peptide inhibitor, cR8, cyclo(RGDINNNV), which bound to SPSB2 with moderate affinity. We, therefore, sought to develop SPSB inhibitors with higher affinity. Here, we show that cyclic peptides cR7, cyclo(RGDINNN), and cR9, cyclo(RGDINNNVE), have ~6.5-fold and ~2-fold, respectively, higher SPSB2-bindng affinities than cR8. We determined high-resolution crystal structures of the SPSB2-cR7 and SPSB2-cR9 complexes, which enabled a good understanding of the structure-activity relationships for these cyclic peptide inhibitors. Moreover, we show that these cyclic peptides displace full-length iNOS from SPSB2, SPSB1, and SPSB4, and that their inhibitory potencies correlate well with their SPSB2-binding affinities. The strongest inhibition was observed for cR7 against all three iNOS-binding SPSB proteins.

Structural basis for the regulation of inducible nitric oxide synthase by the SPRY domain-containing SOCS box protein SPSB2, an E3 ubiquitin ligase.

Relatively high concentration of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in response to a variety of stimuli is a source of reactive nitrogen species, an important weapon of host innate immune defense. The SPRY domain-containing SOCS box protein 2 (SPSB2) is an E3 ubiquitin ligase that regulates the lifetime of iNOS. SPSB2 interacts with the N-terminal region of iNOS via a binding site on the SPRY domain of SPSB2, and recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, leading to its proteasomal degradation. Although critical residues for the SPSB2-iNOS interaction have been identified, structural basis for the interaction remains to be explicitly determined. In this study, we have determined a crystal structure of the N-terminal region of iNOS in complex with the SPRY domain of SPSB2 at 1.24 Å resolution. We have resolved the roles of some flanking residues, whose contribution to the SPSB2-iNOS interaction was structurally unclear previously. Furthermore, we have evaluated the effects of SPSB2 inhibitors on NO production using transient transfection and cell-penetrating peptide approaches, and found that such inhibitors can elevate NO production in RAW264.7 macrophages. These results thus provide a useful basis for the development of potent SPSB2 inhibitors as well as recruiting ligands for proteolysis targeting chimera (PROTAC) design.

Crystal structure of the SPRY domain of human SPSB2 in the apo state.

The SPRY domain-containing SOCS box protein 2 (SPSB2) is one of four mammalian SPSB proteins that are characterized by a C-terminal SOCS box and a central SPRY/B30.2 domain. SPSB2 interacts with inducible nitric oxide synthase (iNOS) via the SPRY domain and polyubiquitinates iNOS, resulting in its proteasomal degradation. Inhibitors that can disrupt SPSB2-iNOS interaction and augment NO production may serve as novel anti-infective and anticancer agents. The previously determined murine SPSB2 structure may not reflect the true apo conformation of the iNOS-binding site. Here, the crystal structure of human SPSB2 SPRY domain in the apo state is reported at a resolution of 1.9 Å. Comparison of the apo and ligand-bound structures reveals that the iNOS-binding site is highly preformed and that major conformational changes do not occur upon ligand binding. Moreover, the C-terminal His6 tag of the recombinant protein binds to a shallow pocket adjacent to the iNOS-binding site on a crystallographically related SPSB2 molecule. These findings may help in structure-based and fragment-based SPSB2 inhibitor design in the future.

Crystal structure of SPSB2 in complex with a rational designed RGD-containing cyclic peptide inhibitor of SPSB2-iNOS interaction.

SPRY domain-containing SOCS box protein 2 (SPSB2) is a negative regulator of inducible nitric oxide synthase (iNOS) that modulates the lifetime of iNOS and thus the levels of nitric oxide (NO) production. Inhibitors that can disrupt the endogenous SPSB2-iNOS interaction and augment NO production have potential as novel antimicrobial and anticancer drugs. In this study, we have designed a cyclic peptide (cR8), containing an RGD motif and the SPSB2 binding motif (DINNNV). ITC and chemical shift perturbation showed that cR8 binds to the iNOS binding site on SPSB2 with a Kd of 671 nM, and saturation transfer difference NMR showed that cR8 binds to αvß3 integrin-expressing cells. Moreover, we determined the crystal structure of SPSB2 in complex with cR8, at a resolution of 1.34 Å. cR8 forms extensive hydrogen bonding with SPSB2 residues, but loss of an intramolecular hydrogen bond that is present in SPSB2-bound iNOS peptide may destabilize the bound conformation of cR8 and lead to a gentle reduction in SPSB2 binding affinity. These results serve as a useful basis for designing site-directed SPSB2 inhibitors in the future.

[Cannabinoid WIN55, 212-2 inhibits proliferation, invasion and migration of human SMMC-7721 hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effects of WIN55, 212-2 (WIN) on the proliferation, invasion and migration of SMMC-7721 hepatocellular carcinoma cells and its underlying mechanisms. METHODS: SMMC-7721 cells were treated with (0, 1, 5, 10, 20) µmol/L WIN, and cell viability was determined by CCK-8 assay. The morphological changes of the cells were observed under a fluorescence microscope with Hoechst33258 staining. Cell apoptosis was measured by flow cytometry combined with annexin V-FITC/PI staining. The expression levels of apoptosis-related proteins P53, P21, Bcl-2 and Bax, and the phosphorylated AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were analyzed by Western blotting. Transwell(TM) invasion assay was used to detect cell invasion ability. Would healing assay was performed to test cell migration ability. The expression level of matrix metalloproteinase 14 (MMP-14) was evaluated by Western blotting. RESULTS: WIN inhibited the proliferation of SMMC-7721 cells and induced cell apoptosis in a dose-dependent manner. After treatment with WIN, the cell nucleus concentrated and broken, indicating obvious cell apoptosis. Western blotting exhibited an up-regulation in the protein expression of P53, P21 and Bax. And the anti-apoptotic protein Bcl-2 was repressed. The expression levels of AKT, p-AKT and p-ERK were down-regulated, whereas the expression of total ERK was not obviously changed. Compared with control group, there was a significant inhibition of cell invasion and migration abilities when SMMC-7721 cells were treated with WIN. The expression level of MMP-14 decreased as well. CONCLUSION: WIN can inhibit the proliferation of SMMC-7721 cells and induce cell apoptosis. The mechanism is associated with the activation of P53 and the inhibition of AKT, p-AKT and p-ERK. WIN can inhibit the invasion and migration of SMMC-7721 cells through down-regulating the protein expression of MMP-14.