Structure-guided combination therapy to potently improve the function of mutant CFTRs.

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50-100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

The nuclear receptor LXR is a glucose sensor.

The liver has a central role in glucose homeostasis, as it has the distinctive ability to produce and consume glucose. On feeding, glucose influx triggers gene expression changes in hepatocytes to suppress endogenous glucose production and convert excess glucose into glycogen or fatty acids to be stored in adipose tissue. This process is controlled by insulin, although debate exists as to whether insulin acts directly or indirectly on the liver. In addition to stimulating pancreatic insulin release, glucose also regulates the activity of ChREBP, a transcription factor that modulates lipogenesis. Here we describe another mechanism whereby glucose determines its own fate: we show that glucose binds and stimulates the transcriptional activity of the liver X receptor (LXR), a nuclear receptor that coordinates hepatic lipid metabolism. d-Glucose and d-glucose-6-phosphate are direct agonists of both LXR-alpha and LXR-beta. Glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands. Cholesterol homeostasis genes that require LXR for expression are upregulated in liver and intestine of fasted mice re-fed with a glucose diet, indicating that glucose is an endogenous LXR ligand. Our results identify LXR as a transcriptional switch that integrates hepatic glucose metabolism and fatty acid synthesis.

Identification of the tyrosine phosphatase PTP-MEG2 as an antagonist of hepatic insulin signaling.

Insulin resistance is a primary defect in type 2 diabetes characterized by impaired peripheral glucose uptake and insufficient suppression of hepatic glucose output. Insulin signaling inhibits liver glucose production by inducing nuclear exclusion of the gluconeogenic transcription factor FOXO1 in an Akt-dependent manner. Through the concomitant application of genome-scale functional screening and quantitative image analysis, we have identified PTP-MEG2 as a modulator of insulin-dependent FOXO1 subcellular localization. Ectopic expression of PTP-MEG2 in cells inhibited insulin-induced phosphorylation of the insulin receptor, while RNAi-mediated reduction of PTP-MEG2 transcript levels enhanced insulin action. Additionally, adenoviral-mediated depletion of PTP-MEG2 in livers of diabetic (db/db) mice resulted in insulin sensitization and normalization of hyperglycemia. These data implicate PTP-MEG2 as a mediator of blood glucose homeostasis through antagonism of insulin signaling, and suggest that modulation of PTP-MEG2 activity may be an effective strategy in the treatment of type 2 diabetes.

A role for the apoptosis inhibitory factor AIM/Spalpha/Api6 in atherosclerosis development.

Macrophages play a central role in the development of atherosclerosis through the accumulation of oxidized LDL (oxLDL). AIM (Spalpha/Api6) has previously been shown to promote macrophage survival; however, its function in atherogenesis is unknown. Here we identify AIM as a critical factor that protects macrophages from the apoptotic effects of oxidized lipids. AIM protein is induced in response to oxLDL loading and is highly expressed in foam cells within atherosclerotic lesions. Interestingly, both expression of AIM in lesions and its induction by oxidized lipids require the action of LXR/RXR heterodimers. AIM-/- macrophages are highly susceptible to oxLDL-induced apoptosis in vitro and undergo accelerated apoptosis in atherosclerotic lesions in vivo. Moreover, early atherosclerotic lesions in AIM-/-LDLR-/- double knockout mice are dramatically reduced when compared to AIM+/+LDLR-/- controls. We conclude that AIM production facilitates macrophage survival within atherosclerotic lesions and that loss of AIM decreases early lesion development by increasing macrophage apoptosis.

LXR-dependent gene expression is important for macrophage survival and the innate immune response.

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

Phospholipid transfer protein is present in human atherosclerotic lesions and is expressed by macrophages and foam cells.

Phospholipid transfer protein (PLTP) in plasma promotes phospholipid transfer from triglyceride-rich lipoproteins to HDL and plays a major role in HDL remodeling. Recent in vivo observations also support a key role for PLTP in cholesterol metabolism. Our immunohistochemical analysis of human carotid endarterectomy samples identified immunoreactive PLTP in areas that colocalized with CD68-positive macrophages, suggesting that PLTP could be produced locally by intimal macrophages. Using RT-PCR, Western blot analysis with a monoclonal anti-PLTP antibody, and a PLTP activity assay, we observed PLTP mRNA and protein expression in human macrophages. In adherent peripheral blood human macrophages, this PLTP expression was increased by culture with granulocyte macrophage colony-stimulating factor. Incubation of macrophages with acetylated-LDL induced an increase in PLTP mRNA and protein expression that paralleled cholesterol loading. PLTP expression was observed in elicited mouse peritoneal macrophages and in cultured Raw264.7 cells as well. Thus, this study demonstrates that PLTP is expressed by macrophages, is regulated by cholesterol loading, and is present in atherosclerotic lesions.

Identification of PLTP as an LXR target gene and apoE as an FXR target gene reveals overlapping targets for the two nuclear receptors.

Affymetrix microarray data and Northern blot assays demonstrated that phospholipid transfer protein (PLTP) was induced 6-fold when either murine or human macrophages were incubated in the presence of ligands for the liver X receptor (LXR) and the retinoid X receptor. Two functional LXR response elements (LXREs) were identified and characterized in the proximal promoter of the human PLTP gene. One LXRE corresponds to a traditional direct repeat separated by 4 bp. However, the second LXRE is novel in that it corresponds to an inverted repeat separated by 1 bp, and is identical to the farnesoid X receptor response element. These studies demonstrate that PLTP is a direct target for activated LXR and farnesoid X receptor (FXR). In addition, apolipoprotein E (apoE), a known LXR target gene in macrophages, was shown to be activated in liver cells by FXR ligands. Taken together, the current data suggest that a small number of genes that currently include PLTP, apoE, and apoC-II, are induced in macrophages by activated LXR and in liver by activated FXR.

Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta.

Lipid-loaded macrophage "foam cells" accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2-14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXRalpha/beta null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells.