High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains

Araujo, Elvin D. de; Manaswiyoungkul, Pimyupa; Israelian, Johan; Park, Jisung; Yuen, Karen K. Y.; Farhangi, Shiva; Berger-Becvar, Angelika; Abu-Jazar, Lubna; Gunning, Patrick T.

doi:10.1016/j.jpba.2017.04.052

Cited by 15 publications

(11 citation statements)

References 34 publications

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“…These hits were optimized to generate the STAT5B inhibitor, 13a, through a structure-guided approach (Figure 3o). Studies with 13a have shown it can effectively reduce pYSTAT5B levels in cellulo, and follow up thermal stability studies showed an inhibitor-induced reduction in STAT5 stability and the potential to block de novo phosphorylation [28,93,94]. Additional studies extended this work to demonstrate AML cell viability inhibition with AC-4-130, a further improved lead compound (Figure 3p) [95].…”

Section: Stat5 Inhibitorsmentioning

confidence: 71%

Direct Targeting Options for STAT3 and STAT5 in Cancer

Orlova

Wagner

Araujo

et al. 2019

Cancers

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Signal transducer and activator of transcription (STAT)3 and STAT5 are important transcription factors that are able to mediate or even drive cancer progression through hyperactivation or gain-of-function mutations. Mutated STAT3 is mainly associated with large granular lymphocytic T-cell leukemia, whereas mutated STAT5B is associated with T-cell prolymphocytic leukemia, T-cell acute lymphoblastic leukemia and γδ T-cell-derived lymphomas. Hyperactive STAT3 and STAT5 are also implicated in various hematopoietic and solid malignancies, such as chronic and acute myeloid leukemia, melanoma or prostate cancer. Classical understanding of STAT functions is linked to their phosphorylated parallel dimer conformation, in which they induce gene transcription. However, the functions of STAT proteins are not limited to their phosphorylated dimerization form. In this review, we discuss the functions and the roles of unphosphorylated STAT3/5 in the context of chromatin remodeling, as well as the impact of STAT5 oligomerization on differential gene expression in hematopoietic neoplasms. The central involvement of STAT3/5 in cancer has made these molecules attractive targets for small-molecule drug development, but currently there are no direct STAT3/5 inhibitors of clinical grade available. We summarize the development of inhibitors against the SH2 domains of STAT3/5 and discuss their applicability as cancer therapeutics.

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Section: Stat5 Inhibitorsmentioning

confidence: 71%

Direct Targeting Options for STAT3 and STAT5 in Cancer

Orlova

Wagner

Araujo

et al. 2019

Cancers

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“…The Tm of STAT3 Similar to these thermal stability assays, another thermofluorescence assay was recently reported for STAT proteins. 25…”

Section: Discussionmentioning

confidence: 99%

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

Desroses

Busker

Astorga‐Wells

et al. 2018

Journal of Pharmaceutical and Biomedical Analysis

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STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided.

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“…It seems that this SEAP reporter gene assay is a viable approach to the discovery of small molecules interfering with type-I IFN action, especially via the JAK-STAT pathway. It provides an alternative to other screening systems such as thermofluor- 13 and fluorescence resonance energy transfer (FRET)-based 34 techniques.…”

Section: Discussionmentioning

confidence: 99%

“…High-throughput screening (HTS) efforts in identifying JAK and STAT inhibitors have been made 13 , 14 , 15 , 16 . Nonetheless, up to now there is only one reported small molecule inhibitor with clinical efficacy for this pathway 17 .…”

Section: Introductionmentioning

confidence: 99%

High-throughput screening for small molecule inhibitors of the type-I interferon signaling pathway

Yuliantie

Dai

Yang

et al. 2018

Acta Pharmaceutica Sinica B

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Interferons (IFNs) are cytokines with fundamental roles in resistance to infections, cancer and other diseases. Type-I IFNs, interferon α (IFN-α) and interferon β (IFN-β), act through a shared receptor complex (IFNAR) comprised of IFNAR1 and IFNAR2 subunits. Binding of type-I IFN to IFNAR1 will robustly activate Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Aberrant activation of the type-I IFN response results in a spectrum of disorders called interferonopathies. The purpose of this research is to develop an assay for high-throughput screening (HTS) of small molecule inhibitors of the type-I IFN signaling pathway. Inhibition of type-I IFN signaling can be beneficial in terms of therapeutic use and understanding the underlying mechanism of action. We report here a HTS campaign with the secreted embryonic alkaline phosphatase (SEAP) reporter gene assay against 32,000 compounds which yielded 25 confirmed hits. These compounds were subsequently characterized for their cytotoxicity, effects on STAT phosphorylation and activities in IFN regulatory factor (IRF) transcription.

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High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains

Cited by 15 publications

References 34 publications

Direct Targeting Options for STAT3 and STAT5 in Cancer

Direct Targeting Options for STAT3 and STAT5 in Cancer

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

High-throughput screening for small molecule inhibitors of the type-I interferon signaling pathway

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