Jui-Wen Huang

From the Cyclooxygenase-2 Inhibitor Celecoxib to a Novel Class of 3-Phosphoinositide-Dependent Protein Kinase-1 Inhibitors

The blockade of Akt activation through the inhibition of 3-phosphoinositide-dependent kinase-1 (PDK-1) represents a major signaling mechanism whereby celecoxib mediates apoptosis. Celecoxib, however, is a weak PDK-1 inhibitor (IC50, 48 μm), requiring at least 30 μm to exhibit discernable effects on the growth of tumor cells in vitro. Here, we report the structure-based optimization of celecoxib to develop PDK-1 inhibitors with greater potency in enzyme inhibition and growth inhibition. Kinetics of PDK-1 inhibition by celecoxib with respect to ATP suggest that celecoxib derivatives inhibit PDK-1 by competing with ATP for binding, a mechanism reminiscent to that of many kinase inhibitors. Structure-activity analysis together with molecular modeling was used to generate compounds that were tested for their potency in inhibiting PDK-1 kinase activity and in inducing apoptosis in PC-3 prostate cancer cells. Docking of potent compounds into the ATP-binding site of PDK-1 was performed for lead optimization, leading to two compounds, OSU-03012 and OSU-03013, with IC50 values in PDK-1 inhibition and apoptosis induction in the low μm range. Exposure of PC-3 cells to these agents led to Akt dephosphorylation and inhibition of p70 S6 kinase activity. Moreover, overexpression of constitutively active forms of PDK-1 and Akt partially protected OSU-03012-induced apoptosis. Screening in a panel of 60 cell lines and more extensive testing in PC-3 cells indicated that the mean concentration for total growth inhibition was ∼3 μm for both agents. Considering the conserved role of PDK-1/Akt signaling in promoting tumorigenesis, these celecoxib analogs are of translational relevance for cancer prevention and therapy.

Signal transducer and activator of transcription 3 is a major kinase-independent target of sorafenib in hepatocellular carcinoma

BACKGROUND & AIMS Recently, we reported that sorafenib sensitizes hepatocellular carcinoma (HCC) cells to TRAIL through the inhibition of signal transducer and activator of transcription 3 (STAT3). Here, we report that sorafenib inhibits HCC via a kinase-independent mechanism: SHP-1 dependent STAT3 inactivation. METHODS SC-1 is a sorafenib derivative that closely resembles sorafenib structurally but with no kinase inhibition activity. HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib or SC-1 and apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with Huh-7 xenografts. RESULTS SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis in all tested HCC cell lines. SC-1 down-regulated phosphorylation of phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested HCC cells. Expression of STAT3-driven genes, including Cyclin D1 and Survivin, was also repressed by SC-1. Luciferase reporter assay confirmed the inhibition of transcriptional activity of STAT3 in both sorafenib-treated and SC-1-treated cells. Ectopic expression of STAT3 in PLC5 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 up-regulated SHP-1 activity. Knockdown of SHP-1, but not SHP-2 or PTP-1B, by small interference RNA reduced apoptosis induced by SC-1. Finally, SC-1 reduced Huh-7 tumor growth significantly in vivo, which was associated with down-regulation of p-STAT3 and up-regulation of SHP-1 activity. CONCLUSIONS STAT3 is a major kinase-independent target of sorafenib in HCC.

α-Tocopheryl Succinate Induces Apoptosis in Prostate Cancer Cells in Part through Inhibition of Bcl-xL/Bcl-2 Function

Although the antitumor effect of α-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of Bcl-xL/Bcl-2 function represents a major pathway whereby α-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that α-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC50 of 26 μm, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of α-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of α-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.

Blockade of STAT3 activation by sorafenib derivatives through enhancing SHP-1 phosphatase activity.

Previously, we demonstrated that the multiple kinase inhibitor sorafenib mediates the repression of phospho-STAT3 in hepatocellular carcinoma cells. In this study, we used this kinase-independent mechanism as a molecular basis to use sorafenib as scaffold to develop a novel class of SHP-1-activating agents. The proof of principle of this premise was provided by SC-1, which on replacement of N-methylpicolinamide by a phenylcyano group showed abolished kinase activity while retaining phospho-STAT3 repressive activity. Structural optimization of SC-1 led to compound 6, which repressed phospho-STAT3 through SHP-1 activation and inhibited PLC5 cell proliferation at sub-micromolar potency. In light of the pivotal role of phospho-STAT3 in promoting tumorigenesis and drug resistance, this novel SHP-1-activating agent may have therapeutic relevance in cancer therapy.

Discovery of novel src homology region 2 domain‐containing phosphatase 1 agonists from sorafenib for the treatment of hepatocellular carcinoma

Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma (HCC). Here, we report that Src homology region 2 (SH2) domain‐containing phosphatase 1 (SHP‐1) is a major target of sorafenib and generates a series of sorafenib derivatives to search for potent SHP‐1 agonists that may act as better anti‐HCC agents than sorafenib. Sorafenib increases SHP‐1 activity by direct interaction and impairs the association between the N‐SH2 domain and the catalytic protein tyrosine phosphatase domain of SHP‐1. Deletion of the N‐SH2 domain (dN1) or point mutation (D61A) of SHP‐1 abolished the effect of sorafenib on SHP‐1, phosphorylated signal transducer and activator of transcription 3 (p‐STAT3), and apoptosis, suggesting that sorafenib may affect SHP‐1 by triggering a conformational switch relieving its autoinhibition. Molecular docking of SHP‐1/sorafenib complex confirmed our findings in HCC cells. Furthermore, novel sorafenib derivatives SC‐43 and SC‐40 displayed more potent anti‐HCC activity than sorafenib, as measured by enhanced SHP‐1 activity, inhibition of p‐STAT3, and induction of apoptosis. SC‐43 induced substantial apoptosis in sorafenib‐resistant cells and showed better survival benefits than sorafenib in orthotopic HCC tumors. Conclusion: In this study, we identified SHP‐1 as a major target of sorafenib. SC‐43 and SC‐40, potent SHP‐1 agonists, showed better anti‐HCC effects than sorafenib in vitro and in vivo. Further clinical investigation is warranted. (Hepatology 2014;58:190–201)

Fragment-based design of small molecule X-linked inhibitor of apoptosis protein inhibitors.

We report on a general structure- and NMR-based approach to derive druglike small molecule inhibitors of protein-protein interactions in a rapid and efficient manner. We demonstrate the utility of the approach by deriving novel and effective SMAC mimetics targeting the antiapoptotic protein X-linked inhibitor of apoptosis protein (XIAP). The XIAP baculovirus IAP repeat 3 (Bir3) domain binds directly to the N-terminal of caspase-9, thus inhibiting programmed cell death. It has been shown that in the cell this interaction can be displaced by the protein second mitochondrial activator of caspases (SMAC) and that its N-terminal tetrapeptide region (NH2-AVPI, Ala-Val-Pro-Ile) is responsible for this activity. However, because of their limited cell permeability, synthetic SMAC peptides are inefficient when tested in cultured cells, limiting their use as potential chemical tools or drug candidates against cancer cells. Hence, as an application, we report on the derivation of novel, selective, druglike, cell permeable SMAC mimics with cellular activity.

A novel obatoclax derivative, SC-2001, induces apoptosis in hepatocellular carcinoma cells through SHP-1-dependent STAT3 inactivation

We investigated the effects of a novel compound, SC-2001, on hepatocellular carcinoma (HCC). SC-2001, which is structurally related to the Mcl-1 inhibitor obatoclax, showed better antitumor effects than obatoclax in HCC cell lines, including HepG2, PLC5 and Huh-7. Like obatoclax, SC-2001 inhibited the protein-protein interactions between Mcl-1 and Bak. However, SC-2001 downregulated the protein levels of Mcl-1 by reducing its transcription whereas obatoclax had no significant effect on Mcl-1 expression. As Mcl-1 is regulated by signal transducers and activators of transcription 3 (STAT3), we found that SC-2001 downregulated the phosphorylation of STAT3 (Tyr 705) and subsequently inhibited transcriptional activities of STAT3 in a dose-dependent manner. In addition to Mcl-1, STAT3-regulated proteins, including survivin and cyclin D1, were also repressed by SC-2001. Notably, SC-2001 reduced IL-6-induced STAT3 activation in HepG2 and PLC5 cells. Ectopic expression of STAT3 abolished the prominent apoptotic death in SC-2001-treated PLC5 cells, indicating that STAT3 is indispensable in mediating the effects of SC-2001. Importantly, SC-2001 enhanced the expression of SHP1, a negative regulator of STAT3. Inhibition of SHP-1 by either specific inhibitor or small interference RNA reduced the apoptotic effects of SC-2001, indicating that SHP-1 plays a key role in mediating SC2001-induced cell death. SC-2001 enhanced the activity of SHP-1 in all tested HCC cells including HepG2, PLC5 and Huh-7. Finally, SC-2001 reduced PLC5 tumor growth, downregulated p-STAT3 and upregulated SHP-1 expression and activity in vivo. In conclusion, our results suggest that SC-2001 induces apoptosis in HCC, and that this effect is mediated through SHP-1-dependent STAT3 inactivation.

Sorafenib derivatives induce apoptosis through inhibition of STAT3 independent of Raf.

STAT3 is a transcription factor that modulates survival-directed transcription. It is persistently activated in many human cancers. Literature has shown that sorafenib, Raf kinase inhibitor, reduces Phospho-STAT3 and induces cell death. A series of sorafenib derivatives were synthesized as new inhibitors for STAT3. Urea, sulfonamide, and carboxamide linkers brought out different SARs from the end of sorafenib. Urea and carboxamide linked derivatives showed greater inhibition against STAT3 activity than sulfonamide linked derivatives. In particular, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophenoxy)phenyl)urea (1), a urea linker, was as potent as sorafenib in reducing P-STAT3 level and cell death but no inhibition for Raf activity. Such result provides a new lead for the design of STAT3 inhibitors.

Development of erlotinib derivatives as CIP2A-ablating agents independent of EGFR activity

Cancerous inhibitor of PP2A (CIP2A) is a novel human oncoprotein that inhibits PP2A, contributing to tumor aggressiveness in various cancers. Several studies have shown that downregulation of CIP2A by small molecules reduces PP2A-dependent phosphorylation of Akt and induces cell death. Here, a series of mono- and di-substituted quinazoline and pyrimidine derivatives based on the skeleton of erlotinib (an EGFR inhibitor) were synthesized and their bioactivities against hepatocellular carcinoma were evaluated. The di-substituted quinazoline and pyrimidine derivatives were more potent inhibitors of cancer-cell proliferation than the mono-substituted derivatives. In particular, compound 1 with chloride at position 2 of quinazoline was as potent as erlotinib in inducing cell death but no inhibition for EGFR activity. Further assays confirmed a correlation between cell death, and CIP2A and Akt inhibition by these derivatives. Among all the derivatives, compounds 19 and 22 showed the most potent antiproliferative activities and the strongest inhibition of CIP2A and p-Akt expression.

Nintedanib (BIBF-1120) inhibits hepatocellular carcinoma growth independent of angiokinase activity

BACKGROUND & AIMS Nintedanib, a triple angiokinase inhibitor, is currently being evaluated against advanced HCC in phase I/II clinical trials. Here, we report the underlying molecular mechanism by which nintedanib (BIBF-1120) induces an anti-HCC effect. METHODS To further elucidate whether the effect of nintedanib on SHP-1 is dependent on its angiokinase inhibition activity, we developed a novel kinase-independent derivative of nintedanib, ΔN. HCC cell lines were treated with nintedanib or its derivative (ΔN) and apoptosis, signal transduction, and phosphatase activity were analyzed. Purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants were used to investigate the potential effect of nintedanib on SHP-1. In vivo efficacy was determined in nude mice with HCC subcutaneous xenografts (n⩾8 mice). RESULTS Nintedanib induced anti-proliferation in HCC cell lines by targeting STAT3. Ectopic STAT3 abolished nintedanib-mediated apoptosis in HCC cells. Nintedanib further activated SHP-1 in purified SHP-1 proteins suggesting that nintedanib directly affects SHP-1 for STAT3 inhibition. HCC cells or recombinant SHP-1 proteins expressing deletion of N-SH2 domain or D61A mutants restored the activity of nintedanib suggesting that the auto-inhibition structure of SHP-1 was relieved by nintedanib. Although ΔN only retained the backbone of nintedanib without kinase activity, ΔN still induced substantial anti-HCC activity in vitro and in vivo by targeting STAT3. CONCLUSIONS Nintedanib induced significant anti-HCC activity independent of angiokinase inhibition activity in a preclinical HCC model by relieving autoinhibition of SHP-1. Our findings provide new mechanistic insight into the inhibition of HCC growth by nintedanib.