技术资料

Existing antibiotics are inadequate to defeat tuberculosis (TB),a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used high-throughput CRISPR knockout and CRISPR interference (CRISPRi) screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Gu{\'{e}}rin),as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191,respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus,high-throughput functional genomic screens,by elucidating highly complex host-pathogen interactions,can serve to identify HDTs to potentially improve TB treatment. View Publication

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation,injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate,Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence,we hypothesized that CDDO-Me would reduce lung inflammation,fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis,mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation,while fibrosis and lung function were evaluated on day 21. On day 7,CDDO-Me reduced total BALF protein by 50{\%},alveolar macrophage infiltration by 40{\%},neutrophil infiltration by 90{\%} (p≤0.01),inhibited production of the inflammatory cytokines KC and IL-6 by over 90{\%} (p≤0.001),and excess production of the pro-fibrotic cytokine TGF$\beta$ by 50{\%}. CDDO-Me also inhibited $\alpha$-smooth muscle actin and fibronectin mRNA by 50{\%} (p≤0.05). On day 21,CDDO-Me treatment reduced histological fibrosis,collagen deposition and $\alpha$SMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me,as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases. View Publication

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines,with inhibition of CDC7 resulting in cell cycle arrest. In this paper,we disclose the discovery of a potent and selective CDC7 inhibitor,XL413 (14),which was advanced into Phase 1 clinical trials. Starting from advanced lead 3,described in a preceding communication,we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model. View Publication

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

CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However,circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression,and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically,TE cells treated with TGR undergo metabolic remodelling that,after glucose re-exposure,supports enhanced glucose uptake,increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state-all indicators of a more anabolic programme to support their enhanced functionality. Thus,metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy. View Publication

Perhexiline is a potent prophylactic anti-anginal agent that has been shown to inhibit myocardial utilization of long-chain fatty acids and to inhibit the mitochondrial enzyme carnitine palmitoyltransferase (CPT)-1. We compared the hemodynamic and biochemical effects of perhexiline (0.5 and 2.0 microM) and of another CPT-1 inhibitor,oxfenicine (0.5 mM),in Langendorff-perfused rat hearts subjected to 60 min of low-flow ischemia (95{\%} flow reduction) followed by 30 min of reperfusion. Both perhexiline (2 microM only) and oxfenicine attenuated (p {\textless} 0.003,p {\textless} 0.0002,respectively) increases in diastolic tension during ischemia,without significant effects on developed tension,or on cardiac function during reperfusion. Myocardial concentrations of long-chain acylcarnitines (LCAC),products of CPT-1 action,were decreased (p {\textless} 0.05) by oxfenicine,unaffected by 2 microM perhexiline,and increased slightly by 0.5 microM perhexiline. Perhexiline,but not the active metabolite of oxfenicine,also inhibited cardiac CPT-2 with similar IC50 and Emax,although lower Hill slope,compared with CPT-1. Oxfenicine,but not perhexiline,reduced concentrations of the endogenous CPT-1 inhibitor,malonyl-CoA. Perhexiline,but not oxfenicine,inhibited myocardial release of lactate during normal flow. We conclude that (a) perhexiline protects against diastolic dysfunction during ischemia in this model,independent of major changes in LCAC accumulation and (b) this may result from simultaneous effects of perhexiline on myocardial CPT-1 and CPT-2. View Publication

The mechanism of the anti-anginal effect of perhexiline is unclear but appears to involve a shift in cardiac metabolism from utilization of fatty acid to that of carbohydrate. We tested the hypothesis that perhexiline inhibits the enzyme carnitine palmitoyltransferase-1 (CPT-1),which controls access of long chain fatty acids to the mitochondrial site of beta-oxidation. Perhexiline produced a concentration-dependent inhibition of CPT-1 in rat cardiac and hepatic mitochondria in vitro,with half-maximal inhibition (IC50) at 77 and 148 mumol/L,respectively. Amiodarone,another drug with anti-anginal properties,also inhibited cardiac CPT-1 (IC50 = 228 mumol/L). The rank order of potency for inhibition was malonyl-CoA {\textgreater} 4-hydroxyphenylglyoxylate (HPG) = perhexiline {\textgreater} amiodarone = monohydroxy-perhexiline. Kinetic analysis revealed competitive inhibition of cardiac and hepatic CPT-1 by perhexiline with respect to palmitoyl-CoA but non-competitive inhibition with respect to carnitine. Curvilinear Dixon plots generated apparent inhibitory constant (Ki)" values for perhexiline which indicated a greater sensitivity of the cardiac than the hepatic enzyme to inhibition by perhexiline. Perhexiline inhibition of CPT-1 unlike that of malonyl-CoA and HPG was unaffected by pretreatment with the protease nagarse. These data establish for the first time that two agents with proven anti-anginal effects inhibit cardiac CPT-1. This action is likely to contribute to the anti-ischaemic effects of both perhexiline and amiodarone." View Publication

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix (ECM) changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however,whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production,we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) co-culture model. RSV infection of BEC/HLF co-cultures led to decreased hyaluronidase expression by HLFs,increased accumulation of HA,and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however,BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared to control mice which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared to SPC-Cre(-) RSV-infected littermates. Taken together,these data demonstrate that altered ECM accumulation of HA occurs following RSV infection and may contribute to airway inflammation. Additionally,loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment. View Publication

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density,but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model,enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common ($\Delta$F508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether,targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking,but also introduces a new tool for deconstructing the ubiquitin code in situ. View Publication

The endoplasmic reticular Ca(2+)-ATPase inhibitor,thapsigargin,was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition,confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator,bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus,we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis. View Publication

Perhexiline maleate,originally classified as a calcium antagonist,is in use as an antianginal agent. The mechanism of its protective effect is unknown,but there is speculation that it involves a modification of myocardial substrate utilization,in which glycolytic sources are used rather than fatty acids. This hypothesis was tested by employing [13C]NMR isotopomer analysis to measure substrate selection in the working rat heart. Substrate utilization was measured from a mixture of substrates present at their physiological concentration,as follows: acetoacetate,glucose,lactate and long-chain fatty acids. Control perfusions were compared with those perfused with perhexiline. It was found that perhexiline increased lactate utilization,which reduced the extent of fatty acid and endogenous substrate oxidation. There was also a significant increase in cardiac output for a small and insignificant increase in oxygen consumption,which suggested an improvement in myocardial efficiency. Thus,it was confirmed by direct measurement that this drug does modify substrate oxidation,which suggests that further investigations of the role that this agent can play in the management of ischemic heart disease would be beneficial. View Publication

Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here,we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+,a principal regulator of energy metabolism and cellular fate,in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression,increased expression of senescence markers (p16INK4a,p21Waf/Cip1,ApoJ,CTGF and $\beta$-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This,we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element. View Publication