AID	Name	Type	Probe	Comment
862	Primary cell-based high throughput screening assay to measure STAT3 inhibition	screening		Primary screen (STAT3 inhibition)
920	Primary cell-based high throughput screening assay to measure STAT1 inhibition	screening		Primary screen (STAT1 inhibition)
1265	Confirmation cell-based high throughput screening assay to measure STAT3 inhibition	screening		Confirmation screen (STAT3 inhibition)
1308	Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure NF-kappaB inhibition.	screening		Counterscreen (NFkB inhibition)
1317	Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure STAT1 inhibition	screening		Counterscreen (STAT1 inhibition)
1399	Dose response cell-based assay to measure STAT3 inhibition	confirmatory		Dose response (STAT3 inhibition)
1411	Dose response counterscreen assay for STAT3 inhibitors: cell-based high throughput assay to measure STAT1 inhibition	confirmatory		Dose response counterscreen (STAT1 inhibition)
1806	Summary of probe development efforts to identify inhibitors of signal transducer and activator of transcription 3 (STAT3)	summary	2	Summary AID (STAT3)

Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frank, Dana Farber Cancer Institute
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank, Dana Farber Cancer Institute
External Assay ID: STAT3_INH_PROBES_LATE STAGE

Name: Late stage results from the probe development efforts to identify inhibitors of signal transducer and activator of transcription 3 (STAT3).

Description:

The signal transducer and activator of transcription (STAT) family of transcription factors transduce signals from a variety of extracellular stimuli and are important mediators of inflammation, cell survival, differentiation, and proliferation (1, 2). STATs are activated in response to growth factors, cytokines, and G-CSF binding to cell surface receptor tyrosine kinases (1-3). In resting cells STATs are inactive in the cytoplasm. In response to stimuli, STATs are phosphorylated by the Janus-activated kinases (Jaks), which induces STAT dimerization and nuclear translocation, where STATs bind to specific enhancer elements in target genes (2). Although structurally similar, the seven STAT family member (STATs 1, 2, 3, 4, 5a, 5b, and 6) possess diverse biological roles (2). For example, STAT1 activation is pro-inflammatory and anti-proliferative, while STAT3 activation is anti-inflammatory and pro-apoptotic (2). STAT1 is largely responsible for mediating the effects of IFN- ?, while STAT3 is predominantly involved in IL-6 signaling (4). STAT1 induces expression of genes that inhibit the cell cycle, and thus STAT1 is considered to have tumor suppressor properties (5). Studies show that STAT3 is activated in a majority of breast and prostate cancers, and that STAT3 inhibition using RNA interference or a dominant negative leads to reduced cell proliferation, survival, and wound healing (1, 4, 6). Blocking STAT3 interaction with the epidermal growth factor receptor (EGFR) using peptide aptamers has been shown to reduce tumor growth (7). Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective modulators of STAT3 activity may lead to pharmacological tools for cancer, wound healing, and inflammatory diseases.

Summary of Probe Development Effort:

Following primary HTS in singlicate to identify STAT3 inhibitors (AID 862), confirmation of hit activity in triplicate (AID 1265), counterscreening in triplicate against NFkB (AID 1308) and STAT3 (AID 1317) to determine selectivity, followed by titration assays to determine compound potency (AID 1399) and selectivity (AID 1411), a compound (SID24825594) belonging to the thienopyrimidine scaffold was identified as a candidate for probe development. This compound does not share structural similarities with the known STAT3 inhibitor and state of the art, nifuroxazide (SID 11532872). The probe candidate was purchase in powder form (SID 87326012), along with analogs belonging to the thienopyrimidine scaffold, which were either purchased in powder form, re-ordered from the MLSMR in liquid form, or synthesized by the SRIMSC. These compounds were tested in dose response assays against STAT3 and STAT1, as well as additional counterscreening assays to determine cytotoxicity, and qPCR assays by the assay provider. The probe candidate (SID24825594/SID 87326012) confirmed activity and is claimed as a potent, selective, and non-toxic STAT3 inhibitor probe.

This probe (SID24825594 liquid/SID 87326012 powder) is inactive against the related STAT1 and NF?B anti-targets. In contrast, the probe is an improvement over the nonselective prior art compound, nifuroxazide, which inhibits both STAT3 and STAT1. Additionally, qPCR experiments run by the assay provider confirm that the probe downregulates transcription of the oncogenes BCL3, BCL6 and BCLX, supporting the physiologic relevance of the probe. The probe does not exhibit cytotoxicity in the parental cell line used for the STAT3 assay (HT-1080).

Probe selection:

The probe was identified from the STAT3 uHTS campaign. It was selected as a starting point for medicinal chemistry because of its positive results in the BCL qPCR assays. The SRIMSC probe development effort resulted in analogs that exhibited better potency against STAT3 than the probe. However, despite improved potency, these compounds showed greater inhibition in the STAT1 antitarget IC50 assay, making them less attractive probe candidates.

Recommendations for the scientific use of this probe:

The probe is useful to study cellular STAT3 signaling, and its effect on oncogene transcription. Its minimal effect on STAT1 and NF?B activities can be exploited in cell-signaling pathway analysis. Giving the qPCR results, it is also useful as a pharmacological tool in physiologically relevant readouts.

The above probe development effort resulted in the identification of one probe. A probe report has been published (http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html). Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs below.

References:

1. Alvarez JV, Febbo PG, Ramaswamy S, Loda M, Richardson A, Frank DA. Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res. 2005 Jun 15;65(12):5054-62.
2. Schindler C, Levy DE, Decker T. JAK-STAT signaling: from interferons to cytokines. J Biol Chem. 2007 Jul 13;282(28):20059-63.
3. Germain D, Frank DA. Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res. 2007 Oct 1;13(19):5665-9.
4. Levy DE, Darnell JE Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002 Sep;3(9):651-62.
5. Battle TE, Wierda WG, Rassenti LZ, Zahrieh D, Neuberg D, Kipps TJ, Frank DA. In vivo activation of signal transducer and activator of transcription 1 after CD154 gene therapy for chronic lymphocytic leukemia is associated with clinical and immunologic response. Clin Cancer Res. 2003 Jun;9(6):2166-72.
6. Takeda, K. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S.1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell- specific Stat3-deficient mice. J. Immunol. 161:4652-4660.
7. Buerger C, Nagel-Wolfrum K, Kunz C, Wittig I, Butz K, Hoppe-Seyler F, Groner B. Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells. J Biol Chem. 2003 Sep 26;278(39):37610-21.
8. Nelson EA, Walker SR, Kepich A, Gashin LB, Hideshima T, Ikeda H, Chauhan D, Anderson KC, Frank DA. Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3. Blood. 2008 Dec 15;112(13):5095-102.

Keywords:

Late stage, probes, probe, STAT3, signal transducer and activator of transcription 3, acute-phase response factor, APRF, inhibitor, inhibition, U3A, transcription factor, luciferase, luminescence, reporter, dose response, counterscreen, 1536, HTS, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assays

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		STAT3 Inhibition	7	13	STAT3 [Homo sapiens] [gi:13272532]		Taxonomy id: 9606 Gene id: 6774
2		STAT1 Inhibition		20	signal transducer and activator of transcription 1 isoform alpha [Homo sapiens] [gi:6274552]		Taxonomy id: 9606 Gene id: 6772
3		Cytotoxicity		1			Taxonomy id: 9606
4		Quantitative PCR	1				Taxonomy id: 9606

---

§ Panel component ID.

Please see AIDs 862, 920, 1265, 1308, 1317, 1399, 1411, Summary AID 1806 and below for protocols performed in this probe development effort.

STAT3 Inhibition Assay (Assay 1):

The purpose of this assay is to determine the STAT3 inhibition potency of powder compounds. Inhibition of STAT3 transcription was measured using a human U3A fibrosarcoma cell line that stably expresses a human STAT3::luciferase construct. This cell line is deficient in STAT1. In this assay, test compounds were screened for their ability to prevent or reduce IL-6-mediated STAT3::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with IL-6 to activate STAT3 transcription. Changes in STAT3::luciferase activity were monitored by measuring luminescence. As designed, a STAT3 antagonist will block IL-6-mediated STAT3 transcription, thus preventing or reducing the activation of the luciferase reporter gene, and decreasing luminescence. Compounds were tested in a 10-point, 1:3 dilution series in triplicate, starting at a nominal test concentration of 55.7 micromolar.

STAT1 Inhibition Counterscreen Assay (Assay 2):

The purpose of this assay is to determine the STAT1 inhibition potency of powder compounds. This assay also serves as a counterscreen for assay 1 to determine whether compounds were nonselective due to inhibition of STAT1. Inhibition of STAT1 activity was measured using a murine NIH 3T3 fibroblast cell line cell line that stably expresses a STAT1::luciferase construct. In these assays, test compounds were screened for their ability to prevent or reduce IFN-gamma-mediated STAT1::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with IFN-gamma. Changes in STAT1::luciferase activity were monitored by measuring luminescence. As designed, a STAT1 antagonist will block IFN-gamma-mediated STAT1 transcription, thus preventing or reducing the activation of the luciferase reporter gene, and decreasing luminescence. Compounds were tested in a 10-point, 1:3 dilution series in triplicate, starting at a nominal test concentration of 55.7 micromolar.

Cytotoxicity Counterscreen Assay (Assay 3):

The purpose of this assay is to determine the cytotoxicity of compounds identified as probe candidates. This assay also serves as a counterscreen to determine whether compounds were inhibiting STAT3::luciferase activity due to cytotoxicity. This assay employs the CellTiter-Glo luminescent reagent, which contains luciferase to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. As designed, cytotoxic compounds will reduce viable cell numbers and ATP levels, resulting in decreased well luminescence. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 56 micromolar.

Quantitative PCR Assay (Assay 4):

The purpose of this assay is to determine whether compounds of interest can inhibit the expression of the BCL STAT3 target genes. The assay protocol has been described (8). Briefly, cells were incubated with either compound or vehicle, and RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA). cDNA was generated using the Taqman reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using SYBR green master mix (Applied Biosystems) on a model 7500 real time PCR system (Applied Biosystems). Data are expressed as the mean fold change plus or minus SE of 3 replicates. Compounds were tested at 10 micromolar. The reference gene was actin. The assay was repeated at least 3 times.

Probes were identified. Not all compounds were tested in all biological assays, as indicated.

The following compounds were not tested in the Cytotoxicity and qPCR assays due to lower STAT3 target activity and/or nonselectivity, compared to the probe candidate SID/ 24825594/ SID 87326012: 81080223, 81080222, 81080221, 85145925, 57288075, 57288040, 57287832, 57288038, 57288032, 57287833, 57288037, 57288042, 57287838, 57287843, 57288036, 57288041, 57288035, 85281103, 85281104.

Grant Number: 1 X01 MH079826-01