Naohisa Ogo

Identification of a New Series of STAT3 Inhibitors by Virtual Screening

The signal transducer and activator of transcription 3 (STAT3) is considered to be an attractive therapeutic target for oncology drug development. We identified a N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarboxamide derivative, STX-0119, as a novel STAT3 dimerization inhibitor by a virtual screen using a customized version of the DOCK4 program with the crystal structure of STAT3. In addition, we used in vitro cell-based assays such as the luciferase reporter gene assay and the fluorescence resonance energy transfer-based STAT3 dimerization assay. STX-0119 selectively abrogated the DNA binding activity of STAT3 and suppressed the expression of STAT3-regulated oncoproteins such as c-myc and survivin in cancer cells. In contrast, a truncated inactive analogue, STX-0872, did not exhibit those activities. Oral administration of STX-0119 effectively abrogated the growth of human lymphoma cells in a SCC-3 subcutaneous xenograft model without visible toxicity. Structure-activity relationships of STX-0119 derivatives were investigated using the docking model of the STAT3-SH2 domain/STX-0119.

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma.

Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Antitumor activity of a novel small molecule STAT3 inhibitor against a human lymphoma cell line with high STAT3 activation.

Signal transducer and activator of transcription (STAT)3, a member of a family of DNA-binding molecules mediating numerous physiological and oncogenic signaling pathways, is a novel target in cancer cells which show high phosphorylation of STAT3. Recently, we identified a novel small-molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. We investigated the mechanisms responsible for the antitumor activity in vitro and in vivo through numerous biochemical and biological assays. Specifically, the effects of STX-0119 on target genes (c-myc, cyclin D1, survivin) and apoptosis induction were analyzed in tumors treated with STX-0119 in vivo. STX-0119 showed strong growth-inhibitory activity against a broad range of hematological cancer cell lines, particularly lymphomas. STX-0119 suppressed the growth of SCC3 cells, a human lymphoma cell line with highly activated STAT3, through apoptosis and down-regulation of STAT3 targets such as c-myc, cyclin D1, survivin and Bcl-xL. Notably, Tyr-705-phosphorylated STAT3 up-regulation was not significantly suppressed by STX-0119, as opposed to other STAT3 inhibitors. STX-0119 demonstrated potent antitumor effects in vivo in SCC3-bearing nude mice by way of the down-regulation of STAT3 target genes and induction of apoptosis in the tumors. Thus, STX-0119 may be a new type of STAT3 inhibitor exhibiting strong antitumor activity.

Identification of a small-molecule inhibitor of the interaction between Survivin and Smac/DIABLO

The protein Survivin is selectively overexpressed in a variety of cancers, but not in normal tissues. It has been reported to be involved in cell survival and cell division. However, the molecular mechanisms involved in its function are not clear, although several binding partner proteins have been proposed to date. Here, we report the identification of a novel small molecule Survivin antagonist, which disrupts the Survivin-Smac/DIABLO interaction in cells. In order to identify Survivin-directed antagonists, we developed a high-throughput screening system based on AlphaScreen technology, which allows the identification of small molecules with the ability to inhibit the interaction of Survivin with Smac/DIABLO or INCENP in vitro. We screened chemical libraries, generated in-house, using this system and identified a 5-deazaflavin analog (compound 1) as a hit compound that selectively inhibited the interaction of Survivin with Smac/DIABLO but not INCENP. In cultured cells, compound 1 abrogated the formation of the complex between Survivin and Smac/DIABLO. In addition, this compound was able to sensitize cultured cells to doxorubicin-mediated DNA damage stress and synergistically enhance apoptotic cell death. Thus, the small-molecule inhibitor described here may serve as a proof-of-principle agent for discriminating between the multiple functions of Survivin.

Bis(hetero)aryl derivatives as unique kinesin spindle protein inhibitors.

Synthesis of 4-(4-tert-butylphenyl)pyridine analogues as kinesin spindle protein (KSP) inhibitors, SAR, cytotoxicity and mitotic arrest in HeLa cells are described. Interestingly, PVZB1194 showed potent KSP inhibition only in the presence of microtubules and distinct KSP localization from a known KSP inhibitor S-trytylcysteine analogue in mitosis. The observations would have resulted from a different molecular mechanism of KSP inhibition and suggest a novel biological regulation for KSP in mitosis.

Structural basis of new allosteric inhibition in Kinesin spindle protein eg5

Kinesin spindle protein Eg5 is a target for anticancer therapies, and small molecule inhibitors of its ATPase activity have been developed. We herein report for the first time the crystal structure of and biochemical studies on the Eg5 motor domain in complex with a new type of allosteric inhibitor. The biphenyl-type inhibitor PVZB1194 binds to the α4/α6 allosteric pocket 15 Å from the ATP-binding pocket, which differs from conventional allosteric inhibitors that bind to the allosteric L5/α2/α3 pocket of Eg5. Binding of the inhibitor is involved in the neck-linker conformation and also causes conformational changes around the ATP-binding pocket through Tyr104 to affect the interaction of ATP with the pocket. This structure provides useful information for the development of novel types of allosteric drugs as well as a novel insight into the molecular mechanism responsible for regulating the motor activity of kinesins.

S-trityl-L-cysteine derivative induces caspase-independent cell death in K562 human chronic myeloid leukemia cell line.

Effect of CF(3)-STLC, a potent kinesin spindle protein (KSP) inhibitor, on K562 human CML cell line was investigated. Treatment with CF(3)-STLC induced mitotic arrest of the cell cycle with the appearance of characteristic monoastral spindles, subsequent apoptotic cell death and cleavage of PARP-1, caspase-3, and 4E-BP1. The wide ranging caspase inhibitor z-VAD fmk prevented the cleavage of caspase-3 and 4E-BP1, but failed to attenuate PARP-1 cleavage or cell death triggered by CF(3)-STLC. These results suggest that CF(3)-STLC can induce apoptotic cell death in a caspase-independent manner, and may work effectively as an anti-cancer agent for hematological malignancies.

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood–brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach.

Identification of novel kynurenine production-inhibiting benzenesulfonamide derivatives in cancer cells

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is known to be a key regulator of human immune responses including cancer immune tolerance. Therefore, abrogation of Kyn production from cancer cells by small molecules may be a promising approach to anticancer therapy. Indeed, several small molecule inhibitors of indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the catabolism of Trp to Kyn, exert antitumor effects in animal models. We screened our chemical libraries using a cell-based Kyn production assay to identify a new type of small molecules that regulate Kyn production, and for the first time identified a benzenesulfonamide derivative (compound 1) as a hit with the ability to inhibit Kyn production in interferon-γ (IFN-γ)-stimulated A431 and HeLa cells. Unlike the previously identified S-benzylisothiourea derivative, compound 2, compound 1 had little effect on the enzymatic activity of recombinant human IDO in vitro but suppressed the expression of IDO at the mRNA level in cells. Furthermore, compound 1 suppressed STAT1-dependent transcriptional activity and DNA binding, whereas no decrement in either the expression or phosphorylation level of STAT1 was observed. The inhibition of IDO expression by several benzenesulfonamide derivatives is associated with the suppression of STAT1. Thus, compound 1 and its analogs might be useful for analyzing the regulation of IDO activation, and STAT1-targeting could be an alternative to the IDO-directed approach for the regulation of Kyn levels by small molecules in the tumor microenvironment.