Design, synthesis, and biological evaluation of naphthoylamide derivatives as inhibitors of STAT3 phosphorylation.

The phosphorylation of STAT3 plays a critical physiological role in the proliferation of rectal cancer. Hence, inhibiting STAT3 phosphorylation is an effective anticancer approach. In this work, we designed a novel 5-R'-1-naphthylmethylamide scaffold as a small molecule inhibitor of STAT3 phosphorylation. The results showed that 3D and 4D have exceptional inhibitory ability against three different colorectal cancer (CRC) cell lines, and can induce apoptosis of CRC cells by inhibiting STAT3 phosphorylation, while having no killing effect on normal human cells. 3D and 4D can inhibit STAT3 phosphorylation in a time- and concentration-dependent manner, and also inhibit the nuclear translocation of interleukin (IL)-6-induced STAT3. In the in vivo tumor model research, 4D significantly reduced the tumor volume of mice and had no drug toxicity on other organ tissues. Furthermore, molecular docking studies revealed that 3D and 4D had greater binding free energy when interacting with the STAT3 SH2 structural domain, and could establish H-π interaction modes. Dynamic simulation studies indicated that both compounds were able to bind tightly to STAT3.

Novel STAG3 variant associated with primary ovarian insufficiency and non-obstructive azoospermia in an Iranian consanguineous family.

Non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) present the most severe forms of male and female infertility. In the last decade, the increasing use of whole exome sequencing (WES) in genomics studies of these conditions has led to the introduction of a number of novel genes and variants especially in meiotic genes with restricted expression to gonads. In this study, exome sequencing of a consanguineous Iranian family with one POI and two NOA cases in three siblings showed that all three patients were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 (NM_001282717.1:c.1942G > A: p.Ala648Thr; NM_001282717.1:c.1951_1953del: p. Leu652del). Both variants occur within a short proximity of each other affecting the relatively conserved armadillo-type fold superfamily feature. STAG3 is a specific meiotic cohesin complex component that interacts with the α-kleisin subunit through this feature. Protein homology modeling indicated that the in-frame deletion destabilizes kleisin biding by STAG3. Although the missense variant did not seem to affect the binding significantly, protein homology modeling suggests that it further destabilizes kleisin binding when in double homozygous state with the deletion. Our findings are in line with several other studies having associated deleterious variants affecting this region with male and female infertility in humans and mouse models. This is the first report associating an in-frame STAG3 variant with NOA and POI in a single family. SUMMARY SENTENCE: A patient with primary ovarian failure and her two brothers with non-obstructive azoospermia were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 that potentially disrupt the protein's meiotic functions.

Structural exploration with AlphaFold2-generated STAT3α structure reveals selective elements in STAT3α-GRIM-19 interactions involved in negative regulation.

STAT3, an important transcription factor constitutively activated in cancers, is bound specifically by GRIM-19 and this interaction inhibits STAT3-dependent gene expression. GRIM-19 is therefore, considered as an inhibitor of STAT3 and may be an effective anti-cancer therapeutic target. While STAT3 exists in a dimeric form in the cytoplasm and nucleus, it is mostly present in a monomeric form in the mitochondria. Although GRIM-19-binding domains of STAT3 have been identified in independent experiments, yet the identified domains are not the same, and hence, discrepancies exist. Human STAT3-GRIM-19 complex has not been crystallised yet. Dictated by fundamental biophysical principles, the binding region, interactions and effects of hotspot mutations can provide us a clue to the negative regulatory mechanisms of GRIM-19. Prompted by the very nature of STAT3 being a challenging molecule, and to understand the structural basis of binding and interactions in STAT3α-GRIM-19 complex, we performed homology modelling and ab-initio modelling with evolutionary information using I-TASSER and avant-garde AlphaFold2, respectively, to generate monomeric, and subsequently, dimeric STAT3α structures. The dimeric form of STAT3α structure was observed to potentially exist in an anti-parallel orientation of monomers. We demonstrate that during the interactions with both unphosphorylated and phosphorylated STAT3α, the NTD of GRIM-19 binds most strongly to the NTD of STAT3α, in direct contrast to the earlier works. Key arginine residues at positions 57, 58 and 68 of GRIM-19 are mainly involved in the hydrogen-bonded interactions. An intriguing feature of these arginine residues is that these display a consistent interaction pattern across unphosphorylated and phosphorylated monomers as well as unphosphorylated dimers in STAT3α-GRIM-19 complexes. MD studies verified the stability of these complexes. Analysing the binding affinity and stability through free energy changes upon mutation, we found GRIM-19 mutations Y33P and Q61L and among GRIM-19 arginines, R68P and R57M, to be one of the top-most major and minor disruptors of binding, respectively. The proportionate increase in average change in binding affinity upon mutation was inclined more towards GRIM-19 mutants, leading to the surmise that GRIM-19 may play a greater role in the complex formation. These studies propound a novel structural perspective of STAT3α-GRIM-19 binding and inhibitory mechanisms in both the monomeric and dimeric forms of STAT3α as compared to that observed from the earlier experiments, these experimental observations being inconsistent among each other.

Discovery of Novel Polycyclic Polyprenylated Acylphloroglucinols from the Fruits of Garcinia xanthochymus as Antitumor Agents by Suppressing the STAT3 Signaling.

Pharmacologic studies have revealed that polycyclic polyprenylated acylphloroglucinols (PPAPs) collectively exhibit a broad range of biological activities, including antineoplastic potential. Here, six new PPAPs, named garcixanthochymones F-K (3, 5, 7, 8, 11, and 15), together with nine known analogues were isolated from the fruits of Garcinia xanthochymus. Their structures were elucidated based on the spectroscopic data, including UV, HRESIMS, and NMR, and quantum chemical calculations. All the isolated PPAPs were tested for anti-proliferative activity against four human tumor cell lines, including SGC7901, A549, HepG2, and MCF-7. Most of the PPAPs possessed high anti-proliferative activity with IC50 values in the range of 0.89 to 36.98 µM, and significant apoptosis was observed in MCF-7 cells exposed to compounds 2 and 5. Besides, docking results showed that compounds 2 and 5 could strongly combine with the Src homology 2 (SH2) domain of STAT3 via hydrogen bond and hydrophobic interaction, which is one of the key oncogenes and crucial therapeutic targets. Furthermore, compounds 2 and 5 efficiently downregulated the expression of p-STAT3Tyr705 and pivotal effector proteins involved in oncogenic signaling pathways of MCF-7 cells.

The anti-parallel dimer binding interface in STAT3 transcription factor is required for the inactivation of cytokine-mediated signal transduction.

Signal transducer and activator of transcription 3 (STAT3) gain-of-function mutations have been widely reported in patients with tumors and haematological malignancies. However, the molecular mechanisms of these pathogenic mutations remain largely uninvestigated. In this study, we have extensively characterized two STAT3 missense mutations, namely a valine-to-alanine exchange in the amino-terminal region (V77A) and a phenylalanine-to-alanine substitution (F174A) in the coiled-coil domain. The two mutants displayed elevated levels of tyrosine phosphorylation, premature nuclear accumulation, and differential transcriptional responses following stimulation of cells with interleukin-6 and interferon-É£. In line with their hyper-phosphorylated status, a greater fraction of V77A and F174A proteins was bound to DNA on high-affinity binding sites termed sis-inducible elements (SIE) as compared to the wild-type (WT) protein. Unexpectedly, these STAT3 variants displayed similar kinetics using in vitro kinase and dephosphorylation assays performed with recombinant Janus kinase 2 (JAK2) and Tc45 phosphatase, respectively. This indicates that the two mutations neither affected the susceptibility of STAT3 to the enzymatic activity of the inactivating tyrosine phosphatase nor to the activating kinase. However, experiments triggering intracellular dephosphorylation by the addition of the tyrosine-kinase inhibitor staurosporine to cytokine-pretreated cells showed that the two mutants partially resisted dephosphorylation. From these data, we propose that the F174A missense mutation hinders the exchange from a parallel to an anti-parallel dimer conformation, thereby increasing the ratio of tyrosine-phosphorylated molecules bound to DNA and enhancing gene-dependent transcription. Our data point to the physiological importance of the anti-parallel dimer conformation in the inactivation of the cytokine-induced STAT3 signalling pathway.

A Michael Acceptor Analogue, SKSI-0412, Down-Regulates Inflammation and Proliferation Factors through Suppressing Signal Transducer and Activator of Transcription 3 Signaling in IL-17A-Induced Human Keratinocyte.

The activation of signal transducer and activator of transcription 3 (STAT3), as well as up-regulation of cytokines and growth factors to promote STAT3 activation, have been found in the epidermis of psoriatic lesions. Recently, a series of synthetic compounds possessing the Michael acceptor have been reported as STAT3 inhibitors by covalently binding to cysteine of STAT3. We synthesized a Michael acceptor analog, SKSI-0412, and confirmed the binding affinity between STAT3 and SKSI-0412. We hypothesized that the SKSI-0412 can inhibit interleukin (IL)-17A-induced inflammation in keratinocytes. The introduction of IL-17A increased the phosphorylation of STAT3 in keratinocytes, whereas the inactivation of STAT3 by SKSI-0412 reduced IL-17A-induced STAT3 phosphorylation and IκBζ expression. In addition, human ß defensin-2 and S100A7, which are regulated by IκBζ, were significantly decreased with SKSI-0412 administration. We also confirmed that SKSI-0412 regulates cell proliferation, which is the major phenotype of psoriasis. Based on these results, we suggest targeting STAT3 with SKSI-0412 as a novel therapeutic strategy to regulate IL-17A-induced psoriatic inflammation in keratinocytes.

Biomedical Applications of a Novel Class of High-Affinity Peptides.

Most therapeutic peptides available on the market today are naturally occurring hormones or protein fragments that were serendipitously discovered to possess therapeutic effects. However, the limited repertoire of available natural resources presents difficulties for the development of new peptide drug candidates. Traditional peptides possess several shortcomings that must be addressed for biomedical applications, including relatively low affinity or specificity toward biological targets compared to antibody- and protein scaffold-based affinity molecules, poor in vivo stability owing to rapid enzymatic degradation, and rapid clearance from circulation owing to their small size. Going forward, it will be increasingly important for scientists to develop novel classes of high-affinity and -specificity peptides against desired targets that mitigate these limitations while remaining compatible with pharmaceutical manufacturing processes. Recently, several highly constrained, artificial cyclic peptides have emerged as platforms capable of generating high-affinity peptide binders against various disease-associated protein targets by combining with phage or mRNA display method, some of which have entered clinical trials. In contrast, although linear peptides are relatively easy to synthesize cost-effectively and modify site-specifically at either N- or C-termini compared to cyclic peptides, there have been few linear peptide-based platforms that can provide high-affinity and -specificity peptide binders.In this Account, we describe the creation and development of a novel class of high-affinity peptides, termed "aptide"-from the Latin word "aptus" meaning "to fit" and "peptide"-and summarize their biomedical applications. In the first part, we consider the design and creation of aptides, with a focus on their unique structural features and binding mode, and address screening and identification of target protein-specific aptides. We also discuss advantages of the aptide platform over ordinary linear peptides lacking preorganized structures in terms of the affinity and specificity of identified peptide binders against target molecules. In the second part, we describe the potential biomedical applications of various target-specific aptides, ranging from imaging and therapy to theranostics, according to the types of aptides and diseases. We show that certain aptides can not only bind to a target protein but also inhibit its biological function, thereby showing potential as therapeutics per se. Further, aptides specific for cancer-associated protein antigens can be used as escort molecules or targeting ligands for delivery of chemotherapeutics, cytokine proteins, and nanomedicines, such as liposomes and magnetic particles, to tumors, thereby substantially improving therapeutic effects. Finally, we present a strategy capable of overcoming the critical issue of short blood circulation time associated with most peptides by constructing a hybrid system between an aptide and a hapten cotinine-specific antibody.

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.

Downregulation of SHANK-associated RH domain-interacting protein elevates interleukin-33 expression by stimulating the Janus kinase 2/signal transducer and activator of transcription signalling pathway in HaCaT cells.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease in which T-helper type 2 (Th2) immune responses are dominant. SH3 and multiple ankyrin repeat domains (SHANK)-associated RH domain-interacting protein (SHARPIN) is expressed at low levels in AD, resulting in the upregulation of the signal transducer and activator of transcription (STAT)3 protein and the Th2 cytokine, interleukin (IL)-33. However, the roles of SHARPIN in AD are not yet fully elucidated. AIM: To evaluate the signalling interactions of SHARPIN and IL-33 in order to improve understanding of AD pathogenesis. METHODS: Western blotting was used to detect the Janus kinase (JAK)/STAT signalling proteins and IL-33 protein in HaCaT cells to determine the key proteins mediating the interaction between SHARPIN and IL-33. The findings were validated by immunofluorescence and immunohistochemical staining. Chromatin immunoprecipitation assays were used to evaluate the activity of STAT3 at the IL-33 promoter. RESULTS: We found that phosphorylated (p)JAK2 and pSTAT3 were upregulated in SHARPIN-knockdown HaCaT cells. Subsequent chromatin immunoprecipitation assays revealed that STAT3 binds to the IL-33 promoter to mediate IL-33 expression. Moreover, SHARPIN-mediated expression of IL-33 was reduced after treatment of HaCaT cells with the JAK/STAT inhibitor ruxolitinib. STAT3 and IL-33 expression levels were higher in AD skin lesion tissues than in normal skin tissues. CONCLUSION: These findings suggest that SHARPIN modulates inflammation in HaCaT cells by inhibiting JAK/STAT signalling, supporting the application of SHARPIN as a potential therapeutic target for AD.

Potent antitumor effects of cell-penetrating peptides targeting STAT3 axis.

To date, there are no inhibitors that directly and specifically target activated STAT3 and c-Myc in the clinic. Although peptide-based inhibitors can selectively block activated targets, their clinical usage is limited because of low cell penetration and/or serum stability. Here, we generated cell-penetrating acetylated (acet.) STAT3, c-Myc, and Gp130 targeting peptides by attaching phosphorothioated (PS) polymer backbone to peptides. The cell-penetrating peptides efficiently penetrated cells and inhibited activation of the intended targets and their downstream genes. Locally or systemically treating tumor-bearing mice with PS-acet.-STAT3 peptide at low concentrations effectively blocked STAT3 in vivo, resulting in significant antitumor effects in 2 human xenograft models. Moreover, PS-acet.-STAT3 peptide penetrated and activated splenic CD8+ T cells in vitro. Treating immune-competent mice bearing mouse melanoma with PS-acet.-STAT3 peptide inhibited STAT3 in tumor-infiltrating T cells, downregulating tumor-infiltrating CD4+ T regulatory cells while activating CD8+ T effector cells. Similarly, systemic injections of the cell-penetrating c-Myc and Gp130 peptides prevented pancreatic tumor growth and induced antitumor immune responses. Taken together, we have developed therapeutic peptides that effectively and specifically block challenging cancer targets, resulting in antitumor effects through both direct tumor cell killing and indirectly through antitumor immune responses.

Clinical and Immunological Heterogeneity in Japanese Patients with Gain-of-Function Variants in STAT3.

PURPOSE: Germline loss-of-function variants in the signal transducer and activator of transcription 3 (STAT3) gene result in autosomal dominant hyper IgE syndrome, whereas somatic gain-of-function (GOF) variants in STAT3 are associated with some malignancies. In addition, germline GOF variants in STAT3 are linked to disorders involving autoimmunity and lymphoproliferation. In this study, we describe five Japanese families with germline GOF variants in STAT3, including three novel variants. We also present the clinical and immunological characteristics of these patients. METHODS: Eight patients from five families were enrolled in this study. We performed genetic and immunological analyses, and collected the associated clinical information. RESULTS: We identified five heterozygous variants in STAT3 using whole-exome sequencing and target gene sequencing. Two of these (E286G and T716M) were previously reported and three (K348E, E415G, and G618A) were novel. A STAT3 reporter assay revealed that all of the variants were GOF. However, the immunological and clinical characteristics among the patients were highly variable. CONCLUSION: Patients with STAT3 GOF variants exhibited clinical and immunological heterogeneity with incomplete penetrance.

Novel anti-cancer candidates from a combinatorial peptide library.

STAT3 is attractive target for development of anti-cancer therapeutics as it is implicated in nearly all forms of human tumors. To identify novel leads, we screened a combinatorial peptide library displayed on the surface of M13 bacteriophage. After three rounds of biopanning, a dodecapeptide with the YYVSWPPDMMHY sequence was found to be enriched by 36% while another with a short consensus motif was displayed in 20% of the phages. Binding analysis by isothermal titration calorimetry shows the most displayed peptide interacted with a Kd of 1.79 µM, which on modification of its structure to mimic the natural binding partners of STAT3 brought the affinity to high nanomolar range (Kd  = 500 nM). Using a panel of tumor cell lines, we show that the peptides prevented the proliferation of triple-negative breast cancer cells with a moderate activity (GI50  = 50 µM). Furthermore, gene expression analysis shows the peptide reduced the expression of oncoproteins critical for tumor cell proliferation, angiogenesis, and metastasis. To find novel STAT3-interacting proteins, we searched the non-redundant sequences of the National Center for Biotechnology Information database which allowed us to identify potential binding partners of the protein. In sum, our data show the identified agents could serve as useful therapeutics candidates for further development.

STAT3 and its targeting inhibitors in osteosarcoma.

Signal transducer and activator of transcription 3 (STAT3) is one of seven STAT family members involved with the regulation of cellular growth, differentiation and survival. STAT proteins are conserved among eukaryotes and are important for biological functions of embryogenesis, immunity, haematopoiesis and cell migration. STAT3 is widely expressed and located in the cytoplasm in an inactive form. STAT3 is rapidly and transiently activated by tyrosine phosphorylation by a range of signalling pathways, including cytokines from the IL-6 family and growth factors, such as EGF and PDGF. STAT3 activation and subsequent dimer formation initiates nuclear translocation of STAT3 for the regulation of target gene transcription. Four STAT3 isoforms have been identified, which have distinct biological functions. STAT3 is considered a proto-oncogene and constitutive activation of STAT3 is implicated in the development of various cancers, including multiple myeloma, leukaemia and lymphomas. In this review, we focus on recent progress on STAT3 and osteosarcoma (OS). Notably, STAT3 is overexpressed and associated with the poor prognosis of OS. Constitutive activation of STAT3 in OS appears to upregulate the expression of target oncogenes, leading to OS cell transformation, proliferation, tumour formation, invasion, metastasis, immune evasion and drug resistance. Taken together, STAT3 is a target for cancer therapy, and STAT3 inhibitors represent potential therapeutic candidates for the treatment of OS.

Stat3 oxidation-dependent regulation of gene expression impacts on developmental processes and involves cooperation with Hif-1α.

Reactive oxygen species are bona fide intracellular second messengers that influence cell metabolism and aging by mechanisms that are incompletely resolved. Mitochondria generate superoxide that is dis-mutated to hydrogen peroxide, which in turn oxidises cysteine-based enzymes such as phosphatases, peroxiredoxins and redox-sensitive transcription factors to modulate their activity. Signal Transducer and Activator of Transcription 3 (Stat3) has been shown to participate in an oxidative relay with peroxiredoxin II but the impact of Stat3 oxidation on target gene expression and its biological consequences remain to be established. Thus, we created murine embryonic fibroblasts (MEFs) that express either WT-Stat3 or a redox-insensitive mutant of Stat3 (Stat3-C3S). The Stat3-C3S cells differed from WT-Stat3 cells in morphology, proliferation and resistance to oxidative stress; in response to cytokine stimulation, they displayed elevated Stat3 tyrosine phosphorylation and Socs3 expression, implying that Stat3-C3S is insensitive to oxidative inhibition. Comparative analysis of global gene expression in WT-Stat3 and Stat3-C3S cells revealed differential expression (DE) of genes both under basal conditions and during oxidative stress. Using differential gene regulation pattern analysis, we identified 199 genes clustered into 10 distinct patterns that were selectively responsive to Stat3 oxidation. GO term analysis identified down-regulated genes to be enriched for tissue/organ development and morphogenesis and up-regulated genes to be enriched for cell-cell adhesion, immune responses and transport related processes. Although most DE gene promoters contain consensus Stat3 inducible elements (SIEs), our chromatin immunoprecipitation (ChIP) and ChIP-seq analyses did not detect Stat3 binding at these sites in control or oxidant-stimulated cells, suggesting that oxidised Stat3 regulates these genes indirectly. Our further computational analysis revealed enrichment of hypoxia response elements (HREs) within DE gene promoters, implying a role for Hif-1. Experimental validation revealed that efficient stabilisation of Hif-1α in response to oxidative stress or hypoxia required an oxidation-competent Stat3 and that depletion of Hif-1α suppressed the inducible expression of Kcnb1, a representative DE gene. Our data suggest that Stat3 and Hif-1α cooperate to regulate genes involved in immune functions and developmental processes in response to oxidative stress.

Polysulfide and Hydrogen Sulfide Ameliorate Cisplatin-Induced Nephrotoxicity and Renal Inflammation through Persulfidating STAT3 and IKKß.

Cisplatin, a widely used chemotherapy for the treatment of various tumors, is clinically limited due to its extensive nephrotoxicity. Inflammatory response in tubular cells is a driving force for cisplatin-induced nephrotoxicity. The plant-derived agents are widely used to relieve cisplatin-induced renal dysfunction in preclinical studies. Polysulfide and hydrogen sulfide (H2S) are ubiquitously expressed in garlic, and both of them are documented as potential agents for preventing and treating inflammatory disorders. This study was designed to determine whether polysulfide and H2S could attenuate cisplatin nephrotoxicity through suppression of inflammatory factors. In renal proximal tubular cells, we found that sodium tetrasulfide (Na2S4), a polysulfide donor, and sodium hydrosulfide (NaHS) and GYY4137, two H2S donors, ameliorated cisplatin-caused renal toxicity through suppression of the massive production of inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2). Mechanistically, the anti-inflammatory actions of Na2S4 and H2S may be mediated by persulfidation of signal transducer and activator of transcription 3 (STAT3) and inhibitor kappa B kinase ß (IKKß), followed by decreased phosphorylation of STAT3 and IKKß. Moreover, the nuclear translocation of nuclear transcription factor kappa B (NF-κB), and phosphorylation and degradation of nuclear factor kappa B inhibitor protein alpha (IκBα) induced by cisplatin, were also mitigated by both polysulfide and H2S. In mice, after treatment with polysulfide and H2S donors, cisplatin-associated renal dysfunction was strikingly ameliorated, as evidenced by measurement of serum blood urea nitrogen (BUN) and creatinine levels, renal morphology, and the expression of renal inflammatory factors. Our present work suggests that polysulfide and H2S could afford protection against cisplatin nephrotoxicity, possibly via persulfidating STAT3 and IKKß and inhibiting NF-κB-mediated inflammatory cascade. Our results might shed light on the potential benefits of garlic-derived polysulfide and H2S in chemotherapy-induced renal damage.

A STAT3 palmitoylation cycle promotes TH17 differentiation and colitis.

Cysteine palmitoylation (S-palmitoylation) is a reversible post-translational modification that is installed by the DHHC family of palmitoyltransferases and is reversed by several acyl protein thioesterases1,2. Although thousands of human proteins are known to undergo S-palmitoylation, how this modification is regulated to modulate specific biological functions is poorly understood. Here we report that the key T helper 17 (TH17) cell differentiation stimulator, STAT33,4, is subject to reversible S-palmitoylation on cysteine 108. DHHC7 palmitoylates STAT3 and promotes its membrane recruitment and phosphorylation. Acyl protein thioesterase 2 (APT2, also known as LYPLA2) depalmitoylates phosphorylated STAT3 (p-STAT3) and enables it to translocate to the nucleus. This palmitoylation-depalmitoylation cycle enhances STAT3 activation and promotes TH17 cell differentiation; perturbation of either palmitoylation or depalmitoylation negatively affects TH17 cell differentiation. Overactivation of TH17 cells is associated with several inflammatory diseases, including inflammatory bowel disease (IBD). In a mouse model, pharmacological inhibition of APT2 or knockout of Zdhhc7-which encodes DHHC7-relieves the symptoms of IBD. Our study reveals not only a potential therapeutic strategy for the treatment of IBD but also a model through which S-palmitoylation regulates cell signalling, which might be broadly applicable for understanding the signalling functions of numerous S-palmitoylation events.

Activating mutations of STAT3: Impact on human growth.

The signal transducer and activator of transcription (STAT) 3 is the most ubiquitous member of the STAT family and fulfills fundamental functions in immune and non-immune cells. Mutations in the STAT3 gene lead to different human diseases. Germline STAT3 activating or gain-of-function (GOF) mutations result in early-onset multiorgan autoimmunity, lymphoproliferation, recurrent infections and short stature. Since the first description of the disease, the clinical manifestations of STAT3 GOF mutations have expanded considerably. However, due to the complexity of immunological characteristics in patients carrying STAT3 GOF mutations, most of attention was focused on the immune alterations. This review summarizes current knowledge on STAT3 GOF mutations with special focus on the growth defects, since short stature is a predominant feature in this condition. Underlying mechanisms of STAT3 GOF disease are still poorly understood, and potential effects of STAT3 GOF mutations on the growth hormone signaling pathway are unclear. Functional studies of STAT3 GOF mutations and the broadening of clinical growth-related data in these patients are necessary to better delineate implications of STAT3 GOF mutations on growth.

Cytosolic delivery of peptidic STAT3 SH2 domain inhibitors.

The signal transducer and activator of transcription 3 (STAT3) protein is constitutively activated in several cancers. STAT3 activity can be blocked by inhibiting its Src Homology 2 (SH2) domain, but phosphotyrosine and its isosteres have poor bioavailability. In this work, we develop peptide-based inhibitors of STAT3-SH2 by combining chemical strategies that have proven effective for targeting other SH2 domains. These strategies include a STAT3-specific selectivity sequence, non-hydrolyzable phosphotyrosine isosteres, and a high-efficiency cell-penetrating peptide. Peptides that combined these three strategies had substantial biological stability and cytosolic delivery, as measured using highly quantitative cell-based assays. However, these peptides did not inhibit STAT3 activity in cells. By comparing in vitro binding affinity, cell penetration, and proteolytic stability, this work explores the delicate balance of factors that contribute to biological activity for peptidic inhibitors of STAT3.

Imperatorin alleviates cancer cachexia and prevents muscle wasting via directly inhibiting STAT3.

Skeletal muscle wasting is the most remarkable phenotypic feature of cancer cachexia that increases the risk of morbidity and mortality. Imperatorin (IMP), a main bioactive component of Angelica dahurica Radix, has been reported to possess several pharmacological effects including potential anti-colitis, anti-arthritis and anti-tumor activities. In this work, we demonstrated that IMP is a promising agent for the treatment of muscle wasting in cancer cachexia. IMP (5-20 µM) dose-dependently attenuated TCM-induced C2C12 myotube atrophy and prevented the induction of E3 ubiquitin ligases muscle RING-finger containing protein-1 (MuRF1) and muscle atrophy Fbox protein (Atrogin-1/MAFbx). Moreove, IMP administration significantly improved chief features of cancer cachexia in vivo, with significant prevention of the loss of body weight and deleterious wasting of multiple tissues, including skeletal muscle, fat and kidney and decreased expression of MuRF1 and Atrogin-1 in cachectic muscles. Cellular signaling pathway analysis showed that IMP selectively inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in vitro and in vivo, and surface plasmon resonance (SPR) affinity experiments further demonstrated IMP bound to STAT3 in a concentration-dependent resonance manner. Molecular docking results revealed that IMP binds to the SH2 domain of STAT3, forming a hydrogen bond interaction with Arg-609, and a Sigma-Pi interaction with Lys-591. Mechanism analysis demonstrated that STAT3 overexpression markedly weakens the improvements of IMP on myotube atrophy and muscle wasting of cancer cachexia, indicating that STAT3 mediated the therapeutic effect of IMP. All these favorable results indicated that IMP is a new potential therapeutic candidate for cancer cachexia.