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Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell growth, survival, and metastasis. STAT3 signaling is constitutively activated in various types of hematologic or solid malignancies. YHO-1701 has been developed as an orally available STAT3 inhibitor. Herein, YHO-1701 in combination with molecular-targeted agents was evaluated. Additive or synergistic effects were observed in a broad spectrum of "combination treatment + cell line" pairs. Of particular interest was the synergistic effect observed when YHO-1701 was combined with imatinib or dasatinib [breakpoint cluster region-abelson (BCR-ABL) inhibitors], osimertinib [epidermal growth factor receptor (EGFR) inhibitor], crizotinib, alectinib, or ceritinib [anaplastic lymphoma kinase (ALK) inhibitors]. The results further showed a close relationship between these synergistic effects and the cellular levels of the key molecules involved in the target pathways for YHO-1701 and each combination drug. The combination of YHO-1701 with alectinib resulted in significantly greater antitumor activity without exhibiting body weight loss in an NCI-H2228 [echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion] xenograft mouse model. Our results strongly suggest that the logical strategy in combination with the novel STAT3 inhibitor YHO-1701 and other mechanistically different targeted agents, could be a promising approach in future clinical settings. [ABSTRACT FROM AUTHOR]

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6 , which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC 50  = 0.34 µM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment. [ABSTRACT FROM AUTHOR]

STAT3 is considered to be a key molecule to mediating tumor-induced immunosuppression in various manners at tumor sites, by acting through immune-regulatory cytokines derived from the tumor cells. Specific anti-STAT3 inhibitors have been developed using nude mouse models transplanted with human tumor cells. However, mouse systems cannot accurately represent the human immune response induced by STAT3 inhibitors, and more humanized therapeutic model based on human immune cells and tumors are needed. In the present study, an immune-deficient NOG mouse with the deletion of both MHC-class I and class II genes, an MHC-double knockout mouse (dKO-NOG), was developed and used to establish humanized immunotherapeutic model. We investigated the immunological effect of the STAT3 inhibitor STX-0119 against TMZ-resistant (TMZ-R) U87 glioma tumors by using humanized dKO-NOG mice. We compared the anti-tumor effects of STX-0119 between the nude and humanized dKO-NOG mouse models. An in vivo study using the nude mouse model showed that STX-0119 inhibited the growth of TMZR U87 tumors, but accumulation of tumor-infiltrating lymphocytes (TILs) were not promoted compared with the control levels. In contrast, STX-0119 inhibited tumor growth more rapidly and strongly in humanized dKO-NOG mice than in nude mice, and a large amount of TILs, mainly consisting of CD8 + T cells and macrophages, were found in the tumors. These results suggest that STX-0119 has anti-tumor activity occurring through the promotion of TIL accumulation at the tumor site and that humanized dKO-NOG mouse system may be a powerful tool to evaluate the effects of STAT3 inhibitors on human systems. [ABSTRACT FROM AUTHOR]

Bendamustine (BENDA), which bears the bis(2-chloroethyl)amino moiety, is an alkylating agent that stops the growth of cancer cells by binding to DNA and interfering with its replication. However, the mechanism of action underlying its excellent clinical efficacy remains unclear. In this work, we report that BENDA inhibits signal transducer and activator of transcription 3 (STAT3). In an AlphaScreen-based biochemical assay using recombinant human STAT3, binding of STAT3–Src homology 2 (SH2) to the phosphotyrosine (pTyr, pY) peptide was inhibited by BENDA but not by the inactive metabolite dihydroxy bendamustine (HP2). When a single point mutation of C550A or C712A was introduced into recombinant human STAT3, its sensitivity to BENDA was substantially reduced, suggesting that these cysteine residues are important for BENDA to inhibit STAT3. Furthermore, BENDA suppressed the function of cellular STAT3 as a transcriptional activator in a human breast cancer cell line, MDA-MB-468, with constitutively activated STAT3. A competitive pull-down assay using biotinylated BENDA (Bio-BENDA) revealed that BENDA bound tightly to cellular STAT3, presumably through covalent bonds. Therefore, our results suggest that the anticancer effects of BENDA may be associated, at least in part, with its inhibitory effect on the SH2 domain of STAT3. [ABSTRACT FROM AUTHOR]

A set of new sulfurated drug hybrids, mainly derived from caffeic and ferulic acids and rosmaricine, has been synthesized and their ability to inhibit both STAT3 and NF-κB transcription factors have been evaluated. Results showed that most of the new hybrid compounds were able to strongly and selectively bind to STAT3, whereas the parent drugs were devoid of this ability at the tested concentrations. Some of them were also able to inhibit the NF-κB transcriptional activity in HCT-116 cell line and inhibited HCT-116 cell proliferationin vitrowith IC50in micromolar range, thus suggesting a potential anticancer activity. Taken together, our study described the identification of new derivatives with dual STAT3/NF-κB inhibitory activity, which may represent hit compounds for developing multi-target anticancer agents. [ABSTRACT FROM PUBLISHER]

With the aim to discover new STAT3 direct inhibitors, potentially useful as anticancer agents, a set of methanethiosulfonate drug hybrids were synthesized. Thein vitrotests showed that all the thiosulfonic compounds were able to strongly and selectively bind STAT3-SH2 domain, whereas the parent drugs were completely devoid of this ability. In addition, some of them showed a moderate antiproliferative activity on HCT-116 cancer cell line. These results suggest that methanethiosulfonate moiety can be considered a useful scaffold in the preparation of new direct STAT3 inhibitors. Interestingly, an unusual kind of organo-sulfur derivative, endowed with valuable antiproliferative activity, was occasionally isolated. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell growth regulation and survival. An aberrant STAT3 activation and/or expression is implied in various solid and blood tumors as well as in other pathologies like rheumatoid arthritis and pulmonary fibrosis, thus making the search for STAT3 inhibitors a growing field of study. With the aim of identifying new inhibitors of STAT3 dimerization, we screened a database including more than 1 320 000 commercially available compounds using a receptor-based pharmacophore model comprising the key protein–protein interactions identified in the STAT3 dimer and refining the search through docking and molecular dynamic simulations studies. STAT3 binding assays revealed a significant STAT3 inhibitory activity and selectivity versus Grb2 for one of the four top-scored compounds, thus verifying the reliability of the virtual screening workflow. Moreover, such compound could already be considered as a lead for the development of new and more potent STAT3 dimerization inhibitors. [ABSTRACT FROM PUBLISHER]

Three new ureido-pyridazinone derivatives, which are structurally related to the known STAT3 inhibitor AVS-0288, were designed by taking into account the structure-activity relationships determined for several ureido-oxadiazole derivatives previously studied by our group. Their synthesis was first attempted through suitable 5-aminopyridazinone intermediates ( 6a and 6b), which molecular structures were confirmed by means of X-ray diffraction data on 6a. Amine functionalization was unsuccessful, therefore, an alternative method was devised. Dual-luciferase and AlphaScreen-based assays were used to test their activity. The obtained data were rationalized on the basis of a modeling study, which focused our attention on the geometrical preferences of the ureido moiety. Computational results seem to indicate that both the 1,2,5-oxadiazole ring and the extended ZZ arrangement are essential and probably act in a synergistic way to confer significant activity against STAT3. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) plays key roles in cancer cell proliferation, invasion, and immunosuppression. In many human cancer cells, STAT3 is hyperactivated, which leads to tumor progression and drug resistance, and therefore STAT3 and its modulators are considered effective drug targets. However, the complex regulatory mechanisms of STAT3 have made it difficult to develop potent anticancer drugs that suppress its activity. Here, we report serum and glucocorticoid-regulated kinase 1 (SGK1) as a novel regulator of STAT3 signaling and an effective target for combination therapy with Janus kinase (JAK) inhibitors. We screened small molecules using a gain-of-function mutant of STAT3 resistant to JAK inhibition and found that an SGK1 inhibitor suppressed the constitutive activation of STAT3. Importantly, our results revealed that SGK1 also mediated the activation of wild-type STAT3. Further examination suggested that the tuberous sclerosis complex 2 and mammalian target of rapamycin signaling pathway were involved in STAT3 activation by SGK1. Finally, we demonstrated that SGK1 inhibition enhanced the inhibitory effect of a JAK inhibitor on STAT3 phosphorylation and cancer cell proliferation. Our findings provide new insights into the molecular mechanisms of STAT3 activation and suggest SGK1 as a potential target for STAT3-targeted combination cancer therapy. • Compound screening identifies an SGK1 inhibitor as an inhibitor of STAT3. • SGK1 is a positive regulator of STAT3. • The TSC2/mTOR signaling pathway is involved in STAT3 activation by SGK1. • SGK1 and JAK inhibition synergistically suppresses cancer cell proliferation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]