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.

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.

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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.

The signal transducer and activator of transcription 3 (STAT3) signaling pathway is a key mediator of cancer cell proliferation, survival and invasion. Aberrant STAT3 has been demonstrated in various malignant cancers. YHO-1701 is a novel quinolinecarboxamide derivative generated from STX-0119. Here, we examined the effect of YHO-1701 on STAT3 and evaluated antitumor activity of YHO-1701 as a single agent and in combination. YHO-1701 inhibited STAT3-SH2 binding to phospho-Tyr peptide selectively and more potently than STX-0119 in biochemical assays. Molecular docking studies with STAT3 suggested more stable interaction of YHO-1701 with the SH2 domain. YHO-1701 exhibited approximately 10-fold stronger activity than STX-0119 in abrogating the STAT3 signaling pathway of human oral cancer cell line SAS. YHO-1701 also blocked multi-step events by inhibiting STAT3 dimerization and suppressed STAT3 promoter activity. As expected, YHO-1701 exerted strong antiproliferative activity against human cancer cell lines addicted to STAT3 signaling. Orally administered YHO-1701 showed statistically significant antitumor effects with long exposure to high levels of YHO-1701 at tumor sites in SAS xenograft models. Moreover, combination regimen with sorafenib led to significantly stronger antitumor activity.In addition, the suppression level of survivin (a downstream target) was superior for the combination as compared with monotherapy groups within tumor tissues. Thus, YHO-1701 had a favorable specificity for STAT3 and pharmacokinetics after oral treatment; it also contributed to the enhanced antitumor activity of sorafenib. The evidence presented here provides justification using for this approach in future clinical settings. K E Y W O R D Scombination therapy, SH2 domain, signaling pathway, sorafenib, STAT3 inhibitor | 1775 NISHISAKA et Al.