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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.
The transcription factor STAT3 (signal transducer and activator of transcription 3) mediates the effects of growth factors and cytokines by regulating gene expression. In many human cancers, including breast and prostate, STAT3 is constitutively active. This leads to increased expression of genes regulating survival and proliferation, and drives the malignant behavior of these cells. As a result, the identification of novel compounds that selectively inhibit STAT3 activity may lead to useful tools to reduce cancer-associated cell proliferation, inflammation, and chemotherapeutic resistance. In this report we describe the identification of a potent and selective STAT3 inhibitor through the use of high throughput screening, synthetic medicinal chemistry, and molecular assays. The novel inhibitor (PubChem CID-2100018) belongs to the thienopyrimidine scaffold and is assigned probe number ML116 within the NIH Molecular Libraries Probe Production Center Network. Probe ML116 exhibits a STAT3 IC50 value of approximately 4 micromolar and does not inhibit the STAT1, STAT5, or NFkB signaling pathways (IC50 values greater than 56 micromolar), as determined using cell-based luciferase reporter assays. Quantitative PCR experiments demonstrated that probe ML116 inhibits transcription of the STAT3-regulated gene BCL3. In contrast, the gene A20 which is regulated by the unrelated transcription factor NF-kB, was not inhibited, further supporting the STAT3 selectivity of this probe. Whereas this compound can induce loss of viability of breast cancer cell lines such as MDA-MB-468 cells that are dependent on STAT3 activity, it displays essentially no toxicity to STAT3-independent SKBR3 breast cancer cells. Similar results were found in ovarian cancer cell lines and multiple myeloma cell lines. Due to the central role of aberrant STAT3 signaling in cancer pathogenesis, this compound may provide a useful starting point for the development of chemical scaffolds to block STAT3 signaling for cancer therapy.
Assigned Assay Grant #: 1 X01 MH079826-01
Screening Center Name & PI: Scripps Research Institute Molecular Screening Center (SRIMSC); Hugh Rosen
Chemistry Center Name & PI: SRIMSC; Hugh Rosen
Assay Submitter & Institution: David Frank, Dana Farber Cancer Institute
PubChem Summary Bioassay Identifier (AID): 1806
Probe Structure & Characteristics: ML116
| STAT3 Inhibitor Probe: MLS 001007458 Thienopyrimidine Scaffold: 4-(4-benzylpiperidin-1-yl) thieno [2,3-d]pyrimidine | ||||||
|---|---|---|---|---|---|---|
| CID/ML | Target Name | IC50 (nM) [SID, AID] | Anti- target | IC50 (μM) [SID, AID] | Fold Selective | Secondary Assays: IC50 (nM) [SID, AID] |
| CID-2100018/ML116 | STAT3 | 4200 nM [SID-24825594, AID-2078] Active | STAT1 | > 56 μM (23.52% INH) [SID-24825594, AID-2078] Inactive | >13.3 fold selective | NFkB Counterscreen (3X%INH): 12.47%INH [SID-24825594, AID-1308] Inactive HT-1080 Cytotoxicity Counterscreen: [SID-24825594, AID-2078] Non-toxic (81.2% viability at 56 μM) BCL3 QPCR Assay: [SID-24825594, AID-2078] Active |
The probe compound CID-2100018, belonging to the thienopyrimidine scaffold, is presented as a selective inhibitor of STAT3. This probe is inactive against the related STAT1 and NFκB anti-targets. In contrast, the prior art compound, nifuroxazide, is nonselective, inhibiting 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).
Recommendations for scientific use of the probe (ML116)
Limitations in state of the art. As prescribed in the original CPDP, the chief goal for this probe development project was to find a selective inhibitor of the signal transducer and activator of transcription 3 (STAT3). In response to this goal, the probe compound ML116 (CID-2100018), belonging to the thienopyrimidine scaffold, is claimed as a potent and selective inhibitor of STAT3. In contrast to the prior art compounds nifuroxazide and Stattic, probe ML116 has a low IC50 for STAT3 and is inactive against the related STAT1 and NFκB anti-targets. As expected for a biologically relevant STAT3 inhibitor, QPCR experiments performed by the assay provider confirm that the probe inhibits transcription of the STAT3-regulated oncogene BCL3. In addition, the probe does not exhibit cytotoxicity in the parental cell lines used for the STAT3 assay (HT-1080), indicating that the inhibitory action was not the result off general off-target cytotoxicity or inhibition of cell growth.
Since STAT3 is downstream of several receptor and non-receptor tyrosine (Tyr) kinases, inhibitors that target the upstream acting Tyr kinase have been developed that are active against their respective targets. For example, compounds such as AG490 and WP1066 inhibit STAT3 activity through targeted disruption of the upstream kinase, JAK2. Unfortunately, these compounds do not target STAT3 directly, and thus induce off-target effects or their impact is not sufficiently potent to STAT3 signaling [1]. Indeed, AG490 does not offer consistent antitumor effect in vivo, which led to the development of the more potent and blood-brain barrier penetrant WP compound [2]. Additional studies are needed to validate the action of these compounds in humans.
Compounds that directly inhibit STAT3 are also reported [3]. For example, S3I-201 suppresses Stat3-dependent but not Stat3-independent transcriptional activity. Unfortunately, this compound was tested at significantly higher doses (> 30 –100 μM) than the current probe, so its activity may be the result of off-target action. Additional studies are needed.
The current probe ML116 was identified from the STAT3 MLSCN HTS 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 dose response assay, making them less attractive probe candidates due to non-selectivity. The probe is useful to study cellular STAT3 signaling, and its effect on oncogene transcription. Its minimal effect on STAT1 and NFkB activities can be exploited in cell-signaling pathway analysis. Given the qPCR results, probe ML116 is also useful as a pharmacological tool in physiologically relevant readouts.
In summary, probe ML116 addresses the lack of STAT3 selectivity and potency of the current state of the art.
Probe Applications. Studies show that the signal transducer and activator of transcription 3 (STAT3) is activated in 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, 3, 4, 5). Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective inhibitors of STAT3 activity may lead to pharmacological tools for cancer, wound healing, and inflammatory diseases.
The probe is useful to study cellular STAT3 signaling, and its effect on oncogene transcription. Its minimal effect on STAT1 and NFκB 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.
Expected end-users of the probe in the research community. Probe ML116 can be used by academic researchers studying cell biology, molecular biology, and tumor biology, breast cancer biology in particular. In addition, the identified probe will be useful for any researcher working in the field of drug discovery.
Relevant biology of the probe. As presented in the original peer reviewed grant application, a suitable target for molecular therapies of cancer must malfunction commonly in a cancer cell, be necessary for the continued maintenance of the cancer, and be dispensable in normal cells. In recent years, much evidence has indicated that members of the STAT family of transcription factors fulfill these criteria. STAT3 is activated in a majority of breast and prostate cancers, and inhibition of this protein through the use of dominant inhibitory forms of RNA interference inhibits cell proliferation and survival [4]. Studies showing that signal transducer and activator of transcription 3 (STAT3) is activated in breast and prostate cancers, that genetic inhibition of STAT3 reduces cell proliferation, survival, and wound healing [4–7]. As a result, the identification of selective STAT3 modulators may provide useful tools for exploring STAT3 biology. In particular, several lines of evidence indicate a crucial role for STAT3 proteins in selectively inducing and maintaining a pro-carcinogenic inflammatory environment, both at the initiation of malignant transformation and during cancer progression [8]. Studies show that STAT3 is activated in breast and prostate cancers, and that STAT3 inhibition using RNA interference or a dominant negative leads to reduced cell proliferation, survival, and wound healing ([4, 6, 9, 10]. In addition, reports have demonstrated that disrupting STAT3-EGFR interactions reduces tumor growth [9], which suggests that STAT3 signaling has broad cellular effects. Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective inhibitors of STAT3 activity may lead to pharmacological tools for cancer, wound healing, and inflammatory diseases.
1. Introduction
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 [4, 7]. STATs are activated in response to growth factors, cytokines, and G-CSF binding to cell surface receptor tyrosine kinases [4, 5, 7]. 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 [7]. Although structurally similar, the seven STAT family member (STATs 1, 2, 3, 4, 5a, 5b, and 6) possess diverse biological roles [7]. For example, STAT1 activation is pro-inflammatory and anti-proliferative, while STAT3 activation is anti-inflammatory and pro-apoptotic [7]. STAT1 is largely responsible for mediating the effects of IFN-γ while STAT3 is predominantly involved in IL-6 signaling [6]. STAT1 induces expression of genes that inhibit the cell cycle, and thus STAT1 is considered to have tumor suppressor properties [11]. 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 [4, 6, 9]. Blocking STAT3 interaction with the epidermal growth factor receptor (EGFR) using peptide aptamers has been shown to reduce tumor growth [12]. 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. A common goal of the HTS screens is to identify compounds which act selectively on either STAT1 or STAT3. Compounds will be considered selective based upon their ability to modulate only one member of the STAT family. Compounds of interest should exhibit high potency and selectivity. Test compounds that do not affect STAT1::luciferase or NFkB::luciferase activities will be considered selective activators of STAT3. The novel probe identified here met these criteria.
2. Materials and Methods
Descriptions of assays follow the summary tables.
PubChem BioAssay Table
| AID | Assay Name | Assay Type | Target | Powder Sample | Compound Concentration |
|---|---|---|---|---|---|
| 862 | Primary cell-based high throughput screening assay to measure STAT3 inhibition. | Primary (1X%INH) | STAT3 | No | 5.7 μM |
| 920 | Primary cell-based high throughput screening assay to measure STAT1 inhibition. | Primary (1X%INH) | STAT1 | No | 5.7 μM |
| 1265 | Confirmation cell-based high throughput screening assay to measure STAT3 inhibition. | Confirmation (3X%INH) | STAT3 | No | 5.7 μM |
| 1308 | Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure NF-kappaB inhibition. | Counterscreen (3X%INH) | NFκB | No | 5.7 μM |
| 1317 | Counterscreen assay for STAT3 inhibitors: Cell-based high throughput assay to measure STAT1 inhibition. | Counterscreen (3X %INH) | STAT1 | No | 5.7 μM |
| 1399 | Dose response cell-based assay to measure STAT3 inhibition. | Dose Response (3X IC50) | STAT3 | No | 10-point, 1:3 dilution starting at 55.7 μM |
| 1411 | Dose response counterscreen assay for STAT3 inhibitors: cell-based high throughput assay to measure STAT1 inhibition. | Dose Response Counterscreen (3X IC50) | STAT1 | No | 10-point, 1:3 dilution starting at 55.7 μM |
| 1806 | Summary of probe development efforts to identify inhibitors of signal transducer and activator of transcription 3 (STAT3). | Summary | STAT3 | No | N/A |
| 2078 | Late stage results from the probe development effort to identify inhibitors of signal transducer and activator of transcription 3 (STAT3). | Late Stage | STAT3 | Yes | Various |
Table of Assay Rationale and Screening Statistics
| AID | Assay Rationale | Assay Description | Z′ | S:B |
|---|---|---|---|---|
| 862 | Identify compounds that inhibit STAT3. | A human U3A fibrosarcoma cell line (deficient in STAT1) stably expressing a human STAT3-luciferase construct is incubated with test compounds and IL-6. Well luminescence is read after 6 hours. | 0.84 | 6.22 |
| 920 | Identify compounds that inhibit STAT1. | A murine NIH 3T3 fibroblast cell line stably expressing STAT1-luciferase is incubated with test compounds and IFNγ. Well luminescence is read after 6 hours. | 0.81 | 11.00 |
| 1265 | Confirm activity of compounds active in AID-862. | Same as AID-862 except that compounds are tested in triplicate. | 0.88 | 8.04 |
| 1308 | Determine whether compounds active in AID-862 were nonselective due to inhibition of NFκB. | A human HEK 293T cell line stably expressing a human NFκB::luciferase construct is incubated with test compounds in the presence of TNFα. Well luminescence is read after 6 hours. Compounds are tested in triplicate. | 0.87 | 17.60 |
| 1317 | Determine whether compounds active in AID-862 were nonselective due to inhibition of STAT1. | Same as AID-920 except that compounds are tested in triplicate. | 0.87 | 7.29 |
| 1399 | Determine STAT3 inhibition dose response curves for compounds active inAID-862. | Same as AID-862 except that compounds are tested in triplicate using a 10-point dilution series starting at 55.7 μM. | 0.82 | 6.88 |
| 1411 | Determine STAT1 inhibition dose response curves for compounds active in AID-862. | Same as AID-920 except that compounds are tested in triplicate using a 10-point dilution series starting at 55.7 μM. | 0.86 | 14.58 |
| 1806 | Summarize probe development efforts. | N/A | N/A | N/A |
| 2078 | Describe the assays performed during the probe development effort. | STAT3 Inhibition Assays, STAT1 Inhibition Counterscreen Assays, NFkB Inhibition Counterscreen Assay as above, plus additional assays to determine compound cytotoxicity. | N/A | N/A |
2.1. Assays
(Click on the hyperlinks to obtain itemized protocols directly from PubChem)
STAT3 Inhibition Assays (PubChem AIDs 862, 1265, and 1399, 2078)
The purpose of these assays is to determine the STAT3 percent inhibition values and dose response curves for MLSCN compounds. In these assays STAT3 inhibition was measured using a human U3A fibrosarcoma cell line that stably expresses a human STAT3::luciferase construct. This cell line is deficient in STAT1. 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 well luminescence. As designed, a STAT3 inhibitor will block IL-6-mediated STAT3 transcription, thus reducing transcription of the luciferase reporter gene, leading to decreased well luminescence. Compounds were tested at a concentration of 5.5 μM in singlicate (AID-862) or in triplicate (AID-1265) and in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 55.7 μM for liquid samples (AID-1399) and powder samples (AID-2078).
STAT1 Inhibition Counterscreens (PubChem AIDs 920, 1317, 1411, and 2078)
The purpose of these assays is to determine the STAT1 percent inhibition values and dose response curves for MLSCN compounds. Inhibition of STAT1 activity was measured using a murine NIH 3T3 fibroblast cell line cell line that stably expresses a STAT1::luciferase construct. In the primary assay approximately 195,000 test compounds from the MLSCN library were screened for their ability to prevent or reduce IFN-gamma-mediated STAT1::luciferase reporter activity. Cells were exposed to test compounds from the MLSCN library, followed by treatment with IFN-gamma. Changes in STAT1::luciferase activity were monitored by measuring well 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 at a concentration of 5.5 μM in singlicate (AID-920) or in triplicate (AID-1317) and in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 55.7 μM for liquids (AID-1411) and powder samples (AID-2078).
NFkB Inhibition Counterscreen (AID-1308)
The purpose of this assay is to determine whether a subset of compounds identified as active in a previous set of experiments entitled, “Primary cell-based high throughput screening assay to measure STAT3 inhibition” (PubChem AID-862) were nonselective STAT3 inhibitors due to inhibition of NF-kappaB. The compounds selected for testing in this AID met at least the two following criteria: 1) they were declared active in AID-862; and 2) they were declared inactive in a previous set of experiments entitled, “Primary cell-based high throughput screening assay to measure STAT1 inhibition” (PubChem AID-920).
In this assay inhibition of NF-kB transcription was measured using a human HEK 293T cell line that stably expresses a human NF-kB::luciferase construct. Test compounds were screened for their ability to prevent or reduce TNF-alpha-mediated induction of NF-kB::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with TNF-alpha to activate NF-kB transcription. Changes in NF-kB::luciferase activity were monitored by measuring luminescence. An inhibitor will block TNF-alpha-mediated NF-kB transcription, thus preventing or reducing the activation of the luciferase reporter gene, and decreasing well luminescence. As designed, test compounds that inhibit NF-kB activity are considered non-selective inhibitors. Compounds were tested in triplicate at a single concentration of 5.7 micromolar.
BCL3 QPCR Activation Counterscreens (AID-2078)
The purpose of this assay is to determine whether compounds of interest can inhibit the expression of the STAT3 target gene BCL3. The assay protocol has been described [10]. 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.
Cytotoxicity Counterscreen (AID-2078)
The purpose of this assay is to determine the cytotoxicity of compounds identified as STAT3 inhibitor probe candidates. This assay also serves as a counterscreen to identify false positives and non-selective compounds active in previous STAT3::luciferase inhibition assays due to cellular toxicity or inhibition of cell proliferation. 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.
2.2. Probe Chemical Characterization
Synthetic route. The STAT3 inhibitor probe ML116was synthesized by the nucleophilic aromatic substitution sequence summarized in Scheme 1.
Scheme 1
Reagents and conditions: (a) DIPEA, iPrOH, 90 °C, 2h.
A number of analogs of the probe (1a) were synthesized according to this procedure. Data for these compounds are provided in Table 1 below, along with data for a selection of analogs that were obtained from a commercial source. These results show that substitution of the thiophene ring of the thienopyrimidine scaffold leads, in general, to a substantial loss of STAT3 inhibition activity (see compounds 1f – 1o). In contrast, substitution of the terminal aryl ring or modifications of the spacer “X” connecting the aryl ring to the piperdine ring are tolerated. In particular, replacement of X = CH2 in 1a by a ketone group (X = CO) in 1b leads to a 5-fold increase in STAT3 inhibition in the primary assay, however this change led to a greater off-target activity for 1b compared to 1a. Off target activity also increased for analogs 1c–1e.
Table 1
Substituted thienopyrimidines as STAT3 inhibitors.
Probe chemical structure including stereochemistry. Separation of diastereomers (if necessary). The probe is achiral—there is no stereochemistry for this compound. The probe’s chemical structure is shown below.
Structure verification with 1H NMR and LCMS results. The title compound was obtained as a pale yellow solid (119 mg, 87%; purity: >99% by analytical HPLC). 1H-NMR (400 MHz, d6-DMSO) δ 8.37 (s, 1H), 7.59 (d, J = 6.1 Hz, 1H), 7.54, (d, J = 6.2 Hz, 1H), 7.31–7.27 (m, 2H), 7.20-7.18 (m, 3H), 4.57 (d, J = 13.3 Hz, 2H), 3.12-3.05 (m, 2H), 2.54 (d, J = 7.2 Hz, 2H), 1.91-1.85 (m, 1H), 1.72-1.69 (m, 2H), 1.29-1.20 (m, 2H); 13C-NMR (100 MHz, d6-DMSO) δ 168.55, 157.74, 152.56, 140.04, 129.00, 128.14, 125.80, 121.98, 121.53, 115.58, 46.69, 42.09, 37.42, 31.57; MS (m/z): 310.2 [M+1]+.
Solubility. The solubility of the probe was measured in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4) at room temperature (23°C). The solubility was found to be 1.3 μM.
Stability. The stability of the probe was measured at room temperature (23ºC) in PBS (no antioxidants or other protectants; DMSO concentration below 0.1%). The stability, represented by the probe’s half-life, was found to be > 48 hours. Below is a graph showing the loss of compound with time over a 48 hour period with a minimum of 6 time points. The table indicates the percent of compound remaining at the end of the 48 hours.
| Probe ML 116 (SR01000051757) Stability in PBS Buffer (pH 7.4) | ||
|---|---|---|
| Sample concentration: 1 μM | ||
| Storage condition: microfuge tube on lab benchtop | ||
| Time (hr) | % remaining | In (%remaining) |
| 0 | 100 | 4.61 |
| 1 | 87 | 4.47 |
| 2 | 103 | 4.63 |
| 4 | 104 | 4.64 |
| 8 | 105 | 4.65 |
| 24 | 124 | 4.82 |
| 48 | 121 | 4.80 |
The probe was measured for its ability to form glutathione adducts. At concentrations of 100 μM reduced GSH, 10 μM probe does not appear to be a Michael acceptor [13, 14].
CID, SID and ML# of the probe and five related analog samples submitted to the SMR collection
| Compound | SR Number | MLS | CID | SID |
|---|---|---|---|---|
| Probe ML116 | SR-01000051757 | MLS001007458 | 2100018 | 24825594 (liquid); 87326012 (powder) |
| Analog 1 | SR-03000000949 | MLS002473459 | 41724711 | 81080223 |
| Analog 2 | SR-03000000948 | MLS002473458 | 42640809 | 81080222 |
| Analog 3 | SR-03000000947 | MLS002473457 | 42640808 | 81080221 |
| Analog 4 | SR-03000001009 | MLS002473641 | 44141938 | 85145925 |
| Analog 5 | SR-01000309219 | MLS000736205 | 1478199 | 24824606 |
2.3. Probe Preparation
Procedure for synthesis of probe 4-(4-benzylpiperidin-1-yl)thieno[2,3-d]pyrimidine (1a). A pressure vial was charged with 4-chlorothieno[2,3-d]pyrimidine (75 mg, 0.44 mmol), 4-benzylpiperidine (78 μl, 0.44 mmol), DIEA (153 μl, 0.92 mmol) and 2-propanol (2 mL). The vial was sealed and the contents were stirred at 90 ºC for 2 hours. The vessel was cooled to room temperature and saturated aqueous ammonium chloride was added. The aqueous phase was extracted with dichloromethane and the combined organic extracts dried over sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by silica gel chromatography.
3. Results
3.1. Summary of Screening Results
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), compounds were identified as possible candidates for probe development. Additional STAT3 and STAT1 IC50 assays were performed, along with determinations of the effect of potential probe(s) on cell viability. Probes were identified (AID-2078). Compared to previously described compounds, the compounds described herein offer greatly improved STAT3 selectivity and potency. The identified probe is potent, selective, and non-toxic. The novel probe compound reported here acts to inhibit STAT3 activity mediated by exposure to the known inflammatory cytokine, interleukin-6 (IL-6). This probe acts in part by reducing the transcriptional activity of the STAT3 promoter. This activity appears selective to the STAT3 signaling pathway, as no inhibition of the STAT1 pathway, NFκB pathway, or cytotoxicity was detected.
Table 2uHTS campaign summary and results
| Step | Screen type | Target | Number of compounds tested | Selection criteria | Number of selected compounds | PubChem AIDc | Assay statistics | |
|---|---|---|---|---|---|---|---|---|
| Z′ | S/B | |||||||
| 1 | Primary screen | STAT3 | 194698 | %Inha>43.4% | 1723 | 862 | 0.84 | 6.22 |
| 2 | Confirmation | STAT3 | 1215 | %Inhb>43.4% | 544 | 1265 | 0.88 | 8.04 |
| 3a | Selectivity | NF-kB | 544 | Inactive in both the NF-κB and STAT1 assays | 105 | 1308 | 0.87 | 17.60 |
| 3b | STAT1 | 105 | 87 | 1317 | 0.87 | 7.29 | ||
| 4a | Titration | STAT3 | 122 | Active in the STAT3 assay and inactive in the STAT1 assay | 116 | 1399 | 0.82 | 6.88 |
| 4b | STAT1 | 1411 | 0.86 | 14.58 | ||||
- a
The primary screen hit-cutoff was calculated at the average percent activation of all test compounds plus three times the standard deviation.
- b
The hit-cutoff calculated for the primary run was also applied to the confirmation run.
- c
PubChem AIDs are accessible on-line at http://www
.ncbi.nlm.nih .gov/sites/entrez?db =pcassay&term=xxxx, where xxxx represents the PubChem AID number listed in the table.
3.2. Dose Response Curves for Probe
Dose-response curves of STAT3 inhibitor probe ML116 in the STAT3 and STAT1 cell-based assays.
Ten-point, 1:3 serial dilution of probe compound ML116 (SID-24825594) were tested in triplicate in both the STAT3 (circles) and STAT1 (squares) inhibition assays at a starting nominal concentration of 55.7 μM using the protocols described in the technical section of this report (see AIDs 1399 and 1411, respectively). Error bars represent the standard deviation of three separate experiments.
3.3. Scaffold/Moiety Chemical Liabilities
Four scaffolds identified from the STAT3 inhibitor HTS campaign were deemed appropriate for initial follow-up. Structures of the lead compounds in the four scaffold series are presented below. The MLSMR was interrogated to identify analogs and structurally related compounds present in the screening deck, to begin to assemble a SAR picture. Additional analogs were identified and purchased from commercial sources testing. During this analysis, structures in the thiourea series (SID-22401616 and 22401426) were de-emphasized owing to concerns about the reactivity and stability of molecules in this structural class. Therefore, the SAR by purchase efforts focused on the three series headed by compounds SID-24825594, 7971575 and4262010. SID-24825594 and 4262010 were also synthesized in order to confirm structures and activity.
The potency of the selected hits was below 2 μM in the original HTS screen. The activity of SID-971575 could not be confirmed upon re-testing of the purchased fresh powder. The remaining three hits were found to be equally potent; however, SID-22401426 and SID-4262010 inhibited purified Luciferase activity and were consequently not considered as potential probes. Therefore, SAR efforts focused on the SID-24825594 scaffold series. SAR efforts concentrated on changing substituents on the thienopyrimidine core skeleton, and on the nature of the benzyl side chain. We found increased potency by inserting a ketone in the side chain (as in compounds SID-81080223 and 85145925). However, SID-24825594 was selected as the probe because unlike the more potent analogs it has a desirable biological activity profile (please see title page of this report and corresponding probe table for details). The more potent analogs also exhibited a greater level of off-target activity than for SID-24825594.
Analogs were synthesized or purchased in powder form or re-ordered from the MLSMR in liquid form and tested in dose response assays against both STAT3 and STAT1. Results for these compounds are summarized in the SAR table below and in AID-2078.
Ultimately, based on the efforts summarized above, a compound belonging to the thienopyrimidine scaffold was identified as a probe: SID-24825594/CID-2100018. This compound does not share structural similarities with the known STAT3 inhibitor, nifuroxazide.
3.4. SAR Tables
SAR Table 1STAT3 Inhibitor SAR Table (Thienopyrimidine Scaffold)
| Compound Information | Screening Assays | Probe Development (AIDs 1806 and 2078) | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Compound | Scripps ID | Structure | CID | SID | MLS ID | Vendor | Vendor Catalog ID | STAT3 Primary (AID-862: %INH) | STAT1 Primary (AID-920: %INH) | STAT3 Confirmation (AID-1265: %INH) | NFkB (3X Counterscreen (AID-1308: %INH) | STAT1 (3X Counterscreen (AID-1317: %INH) | STAT3 Dose Response (AID-1399: IC50, μM) | STAT1 Dose Response Counterscreen (AID-1411: IC50, μM) | STAT3 IC50 (μM) | STAT1 IC50 (μM) | Cytotoxicity Assay | qPCR Assay |
| STAT3 Inhibitor PROBE | SR- 01000051757 |
 | 2100018 | 24825594 | MLS 001007458 | Enamine | T5234097 | Active (83.2) | Inactive (25.97) | Active (78.9) | Inactive (12.47) | Inactive (7.67) | Active (1.61) | Inactive (>55.7) | Active (4.2) | Inactive (>56) | Non-toxic (81.2% viability at 56 μM) | Active |
| Analog 1 | SR- 03000000949 |
 | 41724711 | 81080223 | MLS002473459 | TSRI | SR- 03000000949 | These compounds were not in the MLSMR collection | Active (0.839) | Inactive (>56) | See below# | |||||||
| Analog 2 | SR- 03000000948 |
 | 42640809 | 81080222 | MLS002473458 | TSRI | SR- 03000000948 | Active (1.69) | Inactive (>56) | |||||||||
| Analog 3 | SR- 03000000947 |
 | 42640808 | 81080221 | MLS002473457 | TSRI | SR- 03000000947 | Active (2.53) | Inactive (>56) | |||||||||
| Analog 4 | SR- 03000001009 |
 | 44141938 | 85145925 | MLS002473641 | TSRI | SR- 03000001009 | Active (0.837) | Inactive (>56) | |||||||||
| Analog 5 | SR- 01000309219 |
 | 1478199 | 24824606 | MLS000736205 | Key Organics | 4X-0840 | Inactive (−2.9) | Inactive (−3.7) | See below* | ||||||||
- #
These compounds were not tested due to higher off-target activity, compared to probe.
- *
This compound was not tested due to inactivity in the Primary screen.
- ^
This compound was not tested due to non-selectivity as determined by the Assay Provider (PMID 18824601).
SAR Table 2Additional synthesized and purchased analogs
| Compound Information | Screening Assays | Probe Development (AIDs 1806 and 2078) | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Compound | SR-Number | Structure | CID | SID | MLS ID | Vendor | Vendor Catalog ID | STAT3 Primary (AID-862: %INH) | STAT1 Primary (AID-920: %INH) | STAT3 Confirmation (AID-1265: %INH) | NFkB (3X Counterscreen (AID-1308: %INH) | STAT1 (3X Counterscreen (AID-1317: %INH) | STAT3 Dose Response (AID-1399: IC50, μM) | STAT1 Dose Response Counterscreen (AID-1411: IC50, μM) | STAT3 IC50 (μM) | STAT1 IC50 (μM) | Cytotoxicity Assay | qPCR Assay |
| Analog 6 | SR- 03000000876 |
 | 675430 | 57288075 | None | Princeton BioMolecular | OSSK_852766 | These compounds were not in the MLSMR collection | Inactive (20) | Inactive (>56) | See below† | |||||||
| Analog 7 | SR- 03000000838 |
 | 16582798 | 57288040 | None | UkrOrgSyn thesis | PB221597938 | Inactive (5.21) | Inactive (>56) | |||||||||
| Analog 8 | SR- 01000307211 |
 | 1478208 | 57287832 | MLS000720742 | Key Organics | 4X-0850 | Inactive (24.5%) | Inactive (22.17%) | See below* | Inactive (13.7) | Inactive (>56) | ||||||
| Analog 9 | SR- 03000000836 |
 | 2012886 | 57288038 | None | Enamine | T5751803 | These compounds were not in the MLSMR collection | Inactive (16.7) | Inactive (>56) | ||||||||
| Analog 10 | SR- 03000000830 |
 | 2238888 | 57288032 | None | Enamine | ZT- 2781134 | Inactive (>18.6 ) | Inactive (>56) | |||||||||
| Analog 11 | SR- 01000307258 |
 | 1478193 | 57287833 | None | Key Organics | 4X-0832 | Inactive (>18.6 ) | Inactive (>56) | |||||||||
| Analog 12 | SR- 03000000835 |
 | 25181300 | 57288037 | None | Enamine | Z128135924 | Inactive (>18.6 ) | Inactive (>56) | |||||||||
| Analog 13 | SR- 03000000840 |
 | 2241376 | 57288042 | None | Enamine | T5890287 | Inactive (>56) | Inactive (>56) | |||||||||
- *
This compound was not tested due to inactivity in the Primary screen.
- †
These compounds were not tested due to lower target potency, compared to probe.
SAR Table 3Additional synthesized and purchased analogs
| Compound Information | Screening Assays | Probe Development (AIDs 1806 and 2078) | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Compound | SR-Number | Structure | CID | SID | MLS ID | Vendor | Vendor Catalog ID | STAT3 Primary (AID-862: %INH) | STAT1 Primary (AID-920: %INH) | STAT3 Confirmation (AID-1265: %INH) | NFkB (3X Counterscreen (AID-1308: %INH) | STAT1 (3X Counterscreen (AID-1317: %INH) | STAT3 Dose Response (AID-1399: IC50, μM) | STAT1 Dose Response Counterscreen (AID-1411: IC50, μM) | STAT3 IC50 (μM) | STAT1 IC50 (μM) | Cytotoxicity Assay | qPCR Assay |
| Analog 14 | SR- 01000410943 |
 | 403054 | 57287838 | None | Innovapharm Ltd. | STT- 00083650 | These compounds were not in the MLSMR collection. | Inactive (>56) | Inactive (>56) | See below † | |||||||
| Analog 15 | SR- 01000468078 |
 | 22330557 | 57287843 | None | ChemDiv | 3909-8202 | Inactive (>56) | Inactive (>56) | |||||||||
| Analog 16 | SR- 03000000834 |
 | 2461091 | 57288036 | None | Enamine | T0518-5756 | Inactive (>56) | Inactive (>56) | |||||||||
| Analog 17 | SR- 03000000839 |
 | 25181302 | 57288041 | None | Enamine | T5949401 | Inactive (>56) | Inactive (>56) | |||||||||
| Analog 18 | SR- 03000000833 |
 | 2383960 | 57288035 | None | Enamine | T0513-7859 | Inactive (>56) | Inactive (>56) | |||||||||
| Analog 19 | SR- 03000000961 |
 | 17415585 | 85281103 | None | Enamine | T5746186 | Inactive (>18.6 ) | Inactive (>56) | |||||||||
| Analog 20 | SR- 03000000966 |
 | 24620995 | 85281104 | None | Enamine | T6328260 | Active (1.84) | Inactive (>56) | |||||||||
- †
These compounds were not tested due to lower target potency, compared to probe.
3.5. Cellular Activity
The probe (ML116/CID-2100018/SID-24825594/SR01000051757/ST3-01) was tested in a variety of cell-based assays performed by the SRIMSC and assay provider. These assays were performed to determine the probe’s selectivity for STAT3 in comparison to the related STAT1, STAT5, and NFkB signaling pathways. The results of these studies demonstrated that the probe does not inhibit STAT1, STAT5 or NFkB signaling pathways. In addition, cytotoxicity assays reveal that the selective STAT3 inhibition is not due to cytotoxicity. Whereas this probe can induce loss of viability of breast cancer cell lines dependent on STAT3 activity (such as MDA-MB-468 cells), it displays essentially no toxicity to STAT3-independent SKBR3 breast cancer cells (see figure below). Similar results have been found in ovarian cancer cell lines and multiple myeloma cell lines. Consequently, ML116 is a selective and potent STAT3 inhibitor probe.
3.6. Profiling Assays
In addition to the above cellular assays, we have also obtained profiling results for the probe at 10 μM using the NCI-60 DTP Human Tumor Cell Line Screen (Background and Methods can be found at http://dtp.nci.nih.gov/branches/btb/ivclsp.html). This profiling screen examined the effect of the probe at a single high concentration (10 μM) on cellular growth of 60 well-characterized cell lines during 48 hours of exposure (significantly longer than the 6–8 hour exposure used in our cell-based reporter assays). Despite the use of this concentration which is several orders of magnitude above the EC50 of this probe, the mean cell growth among all 60 cell lines was 71% of that seen in untreated cells, as shown in the figure below. Importantly, probe ML116 shows inhibition of growth of all blood cancer cell lines tested (red bars), and 4 of 5 breast cancer cell lines tested (pink bars). Thus, it is clear that this probe is a potent inhibitor of cancer cell growth at the dose range utilized in these experiments.
4. Discussion
4.1. Comparison to existing art and how the new probe is an improvement
As shown in the table below, the current probe ML116 addresses the lack of STAT3 selectivity of the prior art compounds. Although several STAT3-specific agents are promising, none are in clinical development, mostly because of drug delivery and stability issues [15]. In contrast, several JAK inhibitors that are orally available and ATP-competitive are in clinical development for myeloproliferative disorders.
Comparative Activity Table: Probe and Prior Art Compounds
| Compound | Structure | CID | CAS # | STAT3 IC50 | STAT1 IC50 | PubChem Activity Profile | Selectivity | PMID |
|---|---|---|---|---|---|---|---|---|
| Probe ML116 |
 | 2100018 | None | 4.2 μM | Inactive (>56 μM) | 17 of 270 (6.3%) | STAT3 Selective | Pending |
| Prior art Stattic |
 | 2779853 | 19983-44-9 | >4–6 μM | Active (4–6 μM) | 3 of 44 (6.8%) | Non- selective | 20576164 (see Fig 4B) |
| Prior art Nifuroxazide (oral nitrofuran antibiotic) |
 | 5337997 | 965-52-6 | 3 μM (IC50 = 8.34 μM when tested by Scripps) | Active against STAT1 and STAT5 | 2 of 20 (10%) | Non- selective | 18824601 |
4.2. Mechanism of Action Studies
In addition to the cellular and profiling assays above, it was important to determine whether the STAT3 inhibitor probe ML116 could block expression of relevant STAT3 target genes. Thus, the assay provider performed quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to examine the mRNA expression of a panel of STAT3 regulated genes. These assays are described [10]. 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 the standard error (SE) of 3 replicates, normalized to cells treated with vehicle (DMSO) alone. Each assay was repeated at least 3 times. The results (see figure below) show that in the STAT3-dependent OVCAR8 ovarian cancer cell line, the probe (SID-24825594/CID-2100018/ST3-01) inhibits expression of four STAT3-regulated genes (Bclx, cyclin D1, Mcl1, and Smad7), consistent with its ability to block STAT3 signaling. Importantly, gene A20 which is regulated by the unrelated transcription factor, NF-kB, is not inhibited, further supporting the STAT3 selectivity of this probe. Similar results have been found in STAT3-dependent breast cancer and multiple myeloma cell lines, indicating that the STAT3 inhibitory effect is not cell type specific. It should be noted that any given gene may be regulated by multiple transcription factors, and thus patterns of changes of gene expression are more informative than responses of any individual gene.
Given the evidence that constitutive STAT3 activation in a tumor cell drives the continued expression of genes that promote survival, it would be expected that inhibition of STAT3 would sensitize cells to undergoing apoptosis. To test this hypothesis, the effect of probe SID-24825594/CID-2100018/ST3-01 was evaluated in a BH3 profiling assay that assesses the effect of an agent on the activity of modulators of mitochondrial permeability (see figure above). When STAT3-dependent MDA-MB-468 cells were treated with the probe for 16 hours, enhanced permeabilization of the mitochondrial outer membrane potential (MOMP) was detected with peptide mimics of PUMA or BMF, compared to vehicle treated cells. This result indicates that the probe can recapitulate the biological effects consistent with its targeted inhibition of STAT3 function.
4.3. Planned Future Studies
As indicated by the assay provider, future studies with the probe will fall into three spheres: structure-activity analysis, identification of the molecular mechanism of action, and elucidation of biological effects. First, through an ongoing synthetic chemistry effort we will make structural analogues of the identified probe that will initially be tested for inhibition of STAT3-dependent gene transcription in the luciferase reporter system and subsequently with endogenous STAT3 target genes. This will provide key insights into structure-function relationships of this probe.
Second, through a series of experiments analyzing STAT3 phosphorylation, nuclear localization, DNA binding, and co-activator recruitment, we will identify the direct molecular mechanism(s) by which the probe is able to block STAT3-dependent gene expression. We will also make use of our ability to perform large scale RNA interference-based screens to identify direct molecular targets by which the probe exerts its effects.
Finally, we will examine the biological effects of the probe and analogues in both cell culture and animal models. Among the cell-based phenotypes to be explored will be cell cycle distribution, colony formation, migration, invasion, and apoptotic sensitization (both alone and in combination with clinically relevant anti-cancer therapies). The activity of the probe (and active analogues) will also be determined in STAT3-dependent tumor models in mice. This will include both xenografts of human tumors introduced into immunodeficient mice, as well as transgenic murine models that phenocopy human tumors. Where appropriate pharmacokinetic studies also will be performed to optimize dosing. For all models, tumor will be recovered so that inhibition of STAT3 function in situ can be determined as a mechanism-specific pharmacodynamic marker. The underlying hypothesis for potential medical applications is that a STAT3 inhibitor would inhibit cancer cell proliferation and survival, while causing little toxicity to normal cells. Such a molecule could represent a targeted therapy with a high therapeutic index, which may also synergize with other anti-cancer modalities.
5. References
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- PMCPubMed Central citations
- PubChem BioAssay for Chemical ProbePubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter
- PubChem SubstanceRelated PubChem Substances
- PubMedLinks to PubMed
- Modulators of STAT Transcription Factors for the Targeted Therapy of Cancer (STA...Modulators of STAT Transcription Factors for the Targeted Therapy of Cancer (STAT3 Inhibitors) - Probe Reports from the NIH Molecular Libraries Program
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