Novel STAT3 small-molecule inhibitors identified by structure-based virtual ligand screening incorporating SH2 domain flexibility.

Efforts to develop STAT3 inhibitors have focused on its SH2 domain starting with short phosphotyrosylated peptides based on STAT3 binding motifs, e.g. pY905LPQTV within gp130. Despite binding to STAT3 with high affinity, issues regarding stability, bioavailability, and membrane permeability of these peptides, as well as peptidomimetics such as CJ-887, have limited their further clinical development and led to interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors, identified using structure-based virtual ligand screening (SB-VLS); while having favorable drug-like properties, suffer from weak binding affinities, possibly due to the high flexibility of the target domain. We conducted molecular dynamic (MD) simulations of the SH2 domain in complex with CJ-887, and used an averaged structure from this MD trajectory as an "induced-active site" receptor model for SB-VLS of 110,000 compounds within the SPEC database. Screening was followed by re-docking and re-scoring of the top 30% of hits, selection for hit compounds that directly interact with pY + 0 binding pocket residues R609 and S613, and testing for STAT3 targeting in vitro, which identified two lead hits with good activity and favorable drug-like properties. Unlike most small-molecule STAT3 inhibitors previously identified, which contain negatively-charged moieties that mediate binding to the pY + 0 binding pocket, these compounds are uncharged and likely will serve as better candidates for anti-STAT3 drug development. IMPLICATIONS: SB-VLS, using an averaged structure from molecular dynamics (MD) simulations of STAT3 SH2 domain in a complex with CJ-887, a known peptidomimetic binder, identify two highly potent, neutral, low-molecular weight STAT3-inhibitors with favorable drug-like properties.

Targeting STAT3 anti-apoptosis pathways with organic and hybrid organic-inorganic inhibitors.

Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3 has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven challenging. This paper describes synthetic studies and biological assays for a naphthalene sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation in cellulo and induce apoptosis in AML cells. We describe two different approaches to inhibitor design: first, variation of substituents on the naphthalene sulfonamide core allows improvements in anti-STAT activity and creates a more thorough understanding of anti-STAT SAR. Second, a novel approach involving hybrid sulfonamide-rhodium(ii) conjugates tests our ability to use cooperative organic-inorganic binding for drug development, and to use SAR studies to inform metal conjugate design. Both approaches have produced compounds with improved binding potency. In vivo and in cellulo experiments further demonstrate that these approaches can also lead to improved activity in living cells, and that compound 3aa slows disease progression in a xenograft model of AML.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer.

OBJECTIVE: Lung cancer is the leading cause of cancer death in both men and women. Non-small cell lung cancer (NSCLC) has an overall 5-year survival rate of 15%. While aberrant STAT3 activation has previously been observed in NSCLC, the scope of its contribution is uncertain and agents that target STAT3 for treatment are not available clinically. METHODS: We determined levels of activated STAT3 (STAT3 phosphorylated on Y705, pSTAT3) and the two major isoforms of STAT3 (α and ß) in protein extracts of 8 NSCLC cell lines, as well as the effects of targeting STAT3 in vitro and in vivo in NSCLC cells using short hairpin (sh) RNA and two novel small-molecule STAT3 inhibitors, C188-9 and piperlongumine (PL). RESULTS: Levels of pSTAT3, STAT3α, and STATß were increased in 7 of 8 NSCLC cell lines. Of note, levels of pSTAT3 were tightly correlated with levels of STAT3ß, but not STAT3α. Targeting of STAT3 in A549 cells using shRNA decreased tSTAT3 by 75%; this was accompanied by a 47-78% reduction in anchorage-dependent and anchorage-independent growth and a 28-45% reduction in mRNA levels for anti-apoptotic STAT3 gene targets. C188-9 and PL (@30 µM) each reduced pSTAT3 levels in all NSCLC cell lines tested by ≥50%, reduced anti-apoptotic protein mRNA levels by 25-60%, and reduced both anchorage-dependent and anchorage-independent growth of NSCLC cell lines with IC50 values ranging from 3.06 to 52.44 µM and 0.86 to 11.66 µM, respectively. Treatment of nude mice bearing A549 tumor xenografts with C188-9 or PL blocked tumor growth and reduced levels of pSTAT3 and mRNA encoding anti-apoptotic proteins. CONCLUSION: STAT3 is essential for growth of NSCLC cell lines and tumors and its targeting using C188-9 or PL may be a useful strategy for treatment.

Monoclonal Antibodies Specific for STAT3ß Reveal Its Contribution to Constitutive STAT3 Phosphorylation in Breast Cancer.

Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3ß, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p) Stat3ß homodimers are more stable, bind DNA more avidly, are less susceptible to dephosphorylation, and exhibit distinct intracellular dynamics, most notably markedly prolonged nuclear retention, compared to pStat3α homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3ß acted as a dominant-negative of Stat3α in transformation assays; however, studies with mouse strains deficient in one or the other isoform indicated distinct contributions of Stat3 isoforms to inflammation. Current immunological reagents cannot differentiate Stat3ß proteins derived from alternative splicing vs. proteolytic cleavage of Stat3α. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3ß (CT7) and do not cross-react with Stat3α. Immunoblotting studies revealed that levels of Stat3ß protein, but not Stat3α, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3ß may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3ß from proteolytic Stat3ß and Stat3α will provide new insights into the contribution of Stat3ß vs. Stat3α to oncogenesis, as well as other biological and pathological processes.

Synthetic and biological studies of phaeosphaerides.

The signal transducer and activator of transcription 3 (STAT3) has been validated as a suitable target for cancer therapy. Recent evidence by our group and others has shown that phaeosphaerides act as inhibitors of the STAT3 pathway. An efficient synthetic sequence to phaeosphaeride 1a has been previously disclosed. In this work, the first total synthesis of (±)-phaeosphaeride B (1d) and the unnatural phaeosphaeride 1b is reported. Additionally, the biological activities of 1a and 1b were investigated. (6S,7S,8S)-1a and (6R,7S,8S)-1b inhibited granulocyte colony-stimulating factor (GCSF)-stimulated phosphorylation of STAT1, STAT3, and STAT5 and IL-6-stimulated nuclear translocation of STAT3 alpha. In an SPR-based assay, (6S,7S,8S)-1a and (6R,7S,8S)-1b showed minimal ability to inhibit binding of STAT3 to its immobilized phosphotyrosylpeptide ligand (IC50 > 100 µM). Thus, (6S,7S,8S)-1a and (6R,7S,8S)-1b are likely upstream inhibitors of a kinase in the STAT signaling pathway and do not act through the inhibition of STAT3 dimerization by the blocking of the SH2 binding domain.

Crayfish mechanoreceptor neuron prevents photoinduced apoptosis of satellite glial cells.

Interactions between neurons and glia play a key role in the development, functioning and survival of the nervous system. However, the influence of neurons on glial cells has received less attention than the role of glia in supporting neural functions. We here investigated the role of isolated crayfish stretch receptor neuron in the death of satellite glial cells under photodynamic impact. After staining with aluminum phthalocyanine photosens, the neuronal cell body was locally irradiated with a focused beam of He-Ne (633 nm, 200 W/cm2) or semiconductor laser (650 nm, 50 W/cm2). This rapidly abolished neuronal activity. The whole preparation was then subjected to total laser irradiation with lower intensity (633 nm, 0.3 W/cm2), which induced death of glial cells. Double staining of the preparation with propidium iodide and Hoechst 33342 in the following 6-7h allowed the visualization of necrotic, apoptotic and alive cells. Previous neuron inactivation with the focused laser beam was found to increase photodynamically-induced apoptosis but not necrosis of satellite glial cells enwrapping the axon. Therefore, the intact neuronal cell body protected satellite glial cells against photoinduced apoptosis. Altogether the data indicate that mechanoreceptor neurons release some signaling molecules involved in the prevention of glial apoptosis. This may provide integrity of the stretch receptor organ and its resistance to injurious factors.

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment.

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.

Photodynamic effect of hypericin and a water-soluble derivative on isolated crayfish neuron and surrounding glial cells.

Hypericin (Hyp) has been proposed as a fluorochrome for fluorescence diagnostics and as a photosensitizer for photodynamic therapy of cancer. However, its insolubility in water is a serious drawback. A novel water-soluble hypericin derivative (Hyp-S) has been constructed, using polyvinylpyrrolidone as a carrier. We used the crayfish stretch receptor, consisting of receptor neuron and satellite glial cells, for comparison of the photodynamic effects of Hyp and Hyp-S. Hyp-S was more toxic in the dark than Hyp and inactivated the neurons at concentrations exceeding 4 microM while Hyp was toxic to the neurons only at the concentrations larger than 20 microM. Electrophysiological investigations revealed polyphasic neuron responses to photosensitization with Hyp as well as with Hyp-S (1 microM concentration, 30 min incubation; irradiation with filtered light from a lamp with an emission maximum near 600 nm and an intensity of 0.2 W/cm2). In the concentration range 1-4 microM Hyp-S was more phototoxic than Hyp. Fluorescence microscopy showed that both sensitizers were predominately localized in the glial envelope surrounding the neuron. A minor fraction of hypericin was found in the neuron perinuclear area rich in cytoplasm organelles. This suggests the potential application of Hyp and Hyp-S for visualization and selective photodynamic treatment of malignant gliomas.

Photodynamic inactivation of isolated crayfish neuron requires protein kinase C, PI 3-kinase and Ca2+.

Involvement of some signalling pathways in response to photodynamic therapy (PDT) of sulfonated aluminium phthalocyanine Photosens has been studied in isolated nerve cell. Neurone photosensitisation with 10(-7) M Photosens gradually inhibited firing and irreversibly abolished neuronal activity. Activation of protein kinase C (PKC) by phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) precipitated PDT-induced abolition of neurone activity and caused nucleus swelling and impairment of the nucleus border. Elevation of cytosolic Ca(2+) concentration by ionomycin or thapsigargin also reduced neurone lifetime. In contrast, the PKC inhibitors staurosporine, hypericin or chelerythrine as well as the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002 increased neurone lifetime. These results showed that PKC, PI 3-kinase and Ca(2+) are involved in PDT-induced neurone inactivation and following death.