技术资料

Protein citrullination has been shown to regulate numerous physiological pathways (e.g.,the innate immune response and gene transcription) and is,when dysregulated,known to be associated with numerous human diseases,including cancer,rheumatoid arthritis,and multiple sclerosis. This modification,also termed deimination,is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals,there are five PAD family members (i.e.,PADs 1,2,3,4,and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported,and these efforts guided the development of the two most potent PAD4 inhibitors (i.e.,F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors,we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3,thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated,the development of PAD-selective inhibitors is of paramount importance. To aid that goal,we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein,we report the results of these studies,which suggest that,like PAD4,PADs 1 and 3 employ a reverse protonation mechanism. Additionally,the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds,denoted F4- and Cl4-amidine,are the most potent PAD3 inhibitors ever described. View Publication

Introducing a reactive carbonyl to a scaffold that does not otherwise have an electrophilic functionality to create a reversible covalent inhibitor is a potentially useful strategy for enhancing compound potency. However,aldehydes are metabolically unstable,which precludes the use of this strategy for compounds to be tested in animal models or in human clinical studies. To overcome this limitation,we designed ketone-based functionalities capable of forming reversible covalent adducts,while displaying high metabolic stability,and imparting improved water solubility to their pendant scaffold. We tested this strategy on the ferroptosis inducer and experimental therapeutic erastin,and observed substantial increases in compound potency. In particular,a new carbonyl erastin analog,termed IKE,displayed improved potency,solubility and metabolic stability,thus representing an ideal candidate for future in vivo cancer therapeutic applications. View Publication

A low-molecular-weight receptor tyrosine kinase inhibitor,1-(6,7-dihydro-5H-benzo(6,7)cyclohepta(1,2-c)pyridazin-3-yl)-N3-((7-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo(7)annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine (R428) with high affinity and selectivity for the growth arrest-specific protein 6 (GAS6) receptor Axl was used to study a potential role of GAS6 signaling in adiposity. In vitro,R428 caused a concentration-dependent inhibition of preadipocyte differentiation into mature adipocytes,as evidenced by reduced lipid uptake. Inhibition of Axl-mediated signaling was confirmed by reduced levels of phospho-Akt activity. In vivo,oral administration of R428 for 5 weeks to mice kept on a high-fat diet resulted in significantly reduced weight gain and subcutaneous and gonadal fat mass. This was associated with marked adipocyte hypotrophy,enhanced macrophage infiltration,and apoptosis. Thus,affecting GAS6 signaling through receptor antagonism using a low-molecular-weight Axl antagonist impairs adipocyte differentiation and reduces adipose tissue development in a murine model of nutritionally induced obesity. View Publication

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth,survival,and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here,we report the characterization of Torin2,a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50),28 nmol/L),ATR (EC(50),35 nmol/L),and DNA-PK (EC(50),118 nmol/L; PRKDC),the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors,Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors,but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together,our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role. View Publication

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance,cerebrospinal fluid movement,and fertility. In the airways,hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria,viruses,and harmful particulates. During multiciliated cell differentiation,motile cilia are templated from basal bodies,each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here,we demonstrate that among the many motile cilia of a multiciliated cell,a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells,originates from parental centrioles,and its cellular position is biased and dependent on ciliary beating. Furthermore,we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment. View Publication

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach,intratumoral TSA-reactive CD4+,CD8+ T cells,and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs,TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype,TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle,FucoID should have the potential of accelerating the pace of personalized cancer treatment. View Publication

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox),a drug used in antitumor therapy,triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore,the p53 inhibitor pifithrin-alpha (PFT-alpha) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However,Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-alpha and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally,Dox triggered Bcl-2 down-regulation,cytochrome c release from mitochondria,and the activation of caspases 9 and 3,suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox,related to its ability to induce apoptosis in proliferating endothelial cells. View Publication

Protein arginine deiminase 4 (PAD4) is a transcriptional coregulator that catalyzes the calcium-dependent conversion of specific arginine residues in proteins to citrulline. Recently,we reported the synthesis and characterization of F-amidine,a potent and bioavailable irreversible inactivator of PAD4. Herein,we report our efforts to identify the steric and leaving group requirements for F-amidine-induced PAD4 inactivation,the structure of the PAD4-F-amidine x calcium complex,and in vivo studies with N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine),a PAD4 inactivator with enhanced potency. The PAD4 inactivators described herein will be useful pharmacological probes in characterizing the incompletely defined physiological role(s) of this enzyme. In addition,they represent potential lead compounds for the treatment of rheumatoid arthritis because a growing body of evidence supports a role for PAD4 in the onset and progression of this chronic autoimmune disorder. View Publication

OBJECTIVE To review the literature concerning the mechanism of action and pharmacodynamics of mifepristone (RU486),potential new uses of RU486,and its current use not only as an abortifacient but also as therapy for endometriosis,leiomyoma,breast cancer,and meningioma. DATA IDENTIFICATION AND SELECTION Studies that relate to RU486 were identified through a MEDLINE search. CONCLUSION(S) RU486 is an 11 beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. The receptor binding is not followed by transcription of P-dependent genes. Mifepristone effectively blocks P receptors in the placenta,resulting in the termination of pregnancy. In addition,it has been used in the treatment of leiomyomata,endometriosis,advanced breast cancer,and meningioma. It is a powerful tool to study the molecular action of P and in the future may be used as an estrogen-free contraceptive. Through an online search of MEDLINE,the authors reviewed the literature on the development of mifepristone (RU-486); RU-486's mechanism of action,pharmacodynamics,and distribution; the physiologic action of RU-486; potential new uses for RU-486; and its current use as both an abortifacient and therapy for endometriosis,leiomyoma,breast cancer,and meningioma. RU-486 is an 11beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. Receptor binding is not followed by the transcription of P-dependent genes. RU-486 effectively blocks P receptors in the placenta,resulting in the termination of pregnancy. It has also been used to treat leiomyomata,endometriosis,advanced breast cancer,and meningioma. The following therapeutic uses of RU-486 are discussed: the termination of early pregnancy,treatment with RU-486 in combination with prostaglandins,the termination of second-trimester pregnancy,cervical ripening,labor induction,postcoital contraception,uterine leiomyomata,endometriosis,breast cancer,and meningioma. View Publication

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN,in concert with cytotoxic drugs,efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study,we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO),an inhibitor of GSH biosynthesis,leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C,a representative example of MYCN-amplified cells,has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore,the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53,bcl-2,and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches. View Publication

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control,a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control,and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy,suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2,termed Ad26.COV2.S,is currently being evaluated in clinical trials. View Publication

This study quantifies uptake of a fluorescent glucose analog,(2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG),in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I),which phosphorylates 2-NBDG,were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 lM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies,and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG,as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents,lonidamine (LND),and a-cyano-hydroxycinnamate (a-Cinn),respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely,when cells were treated with a-Cinn,a drug used to increase glycolysis,2-NBDG uptake was increased. Furthermore,tamoxifen (tam),a common endocrine therapy,was administered to estrogen receptor positive and negative (ER?/-) breast cells and demonstrated a decreased 2-NBDG uptake in ER? cells,reflecting a decrease in glycolysis. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development. View Publication