New Targets for Antiviral Therapy: Inhibitory Receptors and Immune Checkpoints on Myeloid Cells.

Immune homeostasis is achieved by balancing the activating and inhibitory signal transduction pathways mediated via cell surface receptors. Activation allows the host to mount an immune response to endogenous and exogenous antigens; suppressive modulation via inhibitory signaling protects the host from excessive inflammatory damage. The checkpoint regulation of myeloid cells during immune homeostasis raised their profile as important cellular targets for treating allergy, cancer and infectious disease. This review focuses on the structure and signaling of inhibitory receptors on myeloid cells, with particular attention placed on how the interplay between viruses and these receptors regulates antiviral immunity. The status of targeting inhibitory receptors on myeloid cells as a new therapeutic approach for antiviral treatment will be analyzed.

Screening for Inhibitors of YAP Nuclear Localization Identifies Aurora Kinase A as a Modulator of Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFß (Transforming Growth Factor ß) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.

Targeting Cathepsin C in PR3-ANCA Vasculitis.

BACKGROUND: The ANCA autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO) are exclusively expressed by neutrophils and monocytes. ANCA-mediated activation of these cells is the key driver of the vascular injury process in ANCA-associated vasculitis (AAV), and neutrophil serine proteases (NSPs) are disease mediators. Cathepsin C (CatC) from zymogens activates the proteolytic function of NSPs, including PR3. Lack of NSP zymogen activation results in neutrophils with strongly reduced NSP proteins. METHODS: To explore AAV-relevant consequences of blocking NSP zymogen activation by CatC, we used myeloid cells from patients with Papillon-Lefèvre syndrome, a genetic deficiency of CatC, to assess NSPs and NSP-mediated endothelial cell injury. We also examined pharmacologic CatC inhibition in neutrophil-differentiated human hematopoietic stem cells, primary human umbilical vein cells, and primary glomerular microvascular endothelial cells. RESULTS: Patients with Papillon-Lefèvre syndrome showed strongly reduced NSPs in neutrophils and monocytes. Neutrophils from these patients produced a negative PR3-ANCA test, presented less PR3 on the surface of viable and apoptotic cells, and caused significantly less damage in human umbilical vein cells. These findings were recapitulated in human stem cells, in which a highly specific CatC inhibitor, but not prednisolone, reduced NSPs without affecting neutrophil differentiation, reduced membrane PR3, and diminished neutrophil activation upon PR3-ANCA but not MPO-ANCA stimulation. Compared with healthy controls, neutrophils from patients with Papillon-Lefèvre syndrome transferred less proteolytically active NSPs to glomerular microvascular endothelial cells, the cell type targeted in ANCA-induced necrotizing crescentic glomerulonephritis. Finally, both genetic CatC deficiency and pharmacologic inhibition, but not prednisolone, reduced neutrophil-induced glomerular microvascular endothelial cell damage. CONCLUSIONS: These findings may offer encouragement for clinical studies of adjunctive CatC inhibitor in patients with PR3-AAV.

Discovery and Toxicological Profiling of Aminopyridines as Orally Bioavailable Selective Inhibitors of PI3-Kinase γ.

Using a novel physiologically relevant in vitro human whole blood neutrophil shape change assay, an aminopyrazine series of selective PI3Kγ inhibitors was identified and prioritized for further optimization. Severe solubility limitations associated with the series leading to low oral bioavailability and poor exposures, especially at higher doses, were overcome by moving to an aminopyridine core. Compound 33, with the optimal balance of on-target activity, selectivity, and pharmacokinetic parameters, progressed into in vivo studies and demonstrated good efficacy (10 mg/kg) in a rat model of airway inflammation. Sufficient exposures were achieved at high doses to support toxicological studies, where unexpected inflammatory cell infiltrates in cardiovascular tissue prevented further compound development.

Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction.

Purpose: We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. Methods: We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. Results: AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. Conclusions: We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

Screening for YAP Inhibitors Identifies Statins as Modulators of Fibrosis.

Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in vivo in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.

Transcriptomic characterization of culture-associated changes in murine and human precision-cut tissue slices.

Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development.

Human lung fibroblast-to-myofibroblast transformation is not driven by an LDH5-dependent metabolic shift towards aerobic glycolysis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by aberrant fibroblast activation and progressive fibrotic remodelling of the lungs. Though the exact pathophysiological mechanisms of IPF remain unknown, TGF-ß1 is thought to act as a main driver of the disease by mediating fibroblast-to-myofibroblast transformation (FMT). Recent reports have indicated that a metabolic shift towards aerobic glycolysis takes place during FMT and that metabolic shifts can directly influence aberrant cell function. This has led to the hypothesis that inhibition of lactate dehydrogenase 5 (LDH5), an enzyme responsible for converting pyruvate into lactate, could constitute a therapeutic concept for IPF. METHODS: In this study, we investigated the potential link between aerobic glycolysis and FMT using a potent LDH5 inhibitor (Compound 408, Genentech). Seahorse analysis was performed to determine the effect of Compound 408 on TGF-ß1-driven glycolysis in WI-38 fibroblasts. TGF-ß1-mediated FMT was measured by quantifying α-smooth muscle actin (α-SMA) and fibronectin in primary human lung fibroblasts following treatment with Compound 408. Lactate and pyruvate levels in the cell culture supernatant were assessed by LC-MS/MS. In addition to pharmacological LDH5 inhibition, the effect of siRNA-mediated knockdown of LDHA and LDHB on FMT was examined. RESULTS: We show that treatment of lung fibroblasts with Compound 408 efficiently inhibits LDH5 and attenuates the TGF-ß1-mediated metabolic shift towards aerobic glycolysis. Additionally, we demonstrate that LDH5 inhibition has no significant effect on TGF-ß1-mediated FMT in primary human lung fibroblasts by analysing α-SMA fibre formation and fibronectin expression. CONCLUSIONS: Our data strongly suggest that while LDH5 inhibition can prevent metabolic shifts in fibroblasts, it has no influence on FMT and therefore glycolytic dysregulation is unlikely to be the sole driver of FMT.

FKBP51 modulates steroid sensitivity and NFκB signalling: A novel anti-inflammatory drug target.

Steroid refractory inflammation is an unmet medical need in the management of inflammatory diseases. Thus, mechanisms, improving steroid sensitivity and simultaneously decreasing inflammation have potential therapeutic utility. The FK506-binding protein 51 (FKBP51) is reported to influence steroid sensitivity in mental disorders. Moreover, biochemical data highlight a connection between FKBP51 and the IKK complex. The aim of this study was to elucidate whether FKBP51 inhibition had utility in modulating steroid resistant inflammation by increasing the sensitivity of the glucocorticoid receptor (GR) signalling and simultaneously inhibiting NFκB-driven inflammation. We have demonstrated that FKBP51 silencing in a bronchial epithelial cell line resulted in a 10-fold increased potency for dexamethasone towards IL1beta-induced IL6 and IL8, whilst FKBP51 over-expression of FKBP51 reduced significantly the prednisolone sensitivity in a murine HDM-driven pulmonary inflammation model. Immunoprecipitation experiments with anti-FKBP51 antibodies, confirmed the presence of FKBP51 in a complex comprising Hsp90, GR and members of the IKK family. FKBP51 silencing reduced NFκB (p50/p65) nucleus translocation, resulting in reduced ICAM expression, cytokine and chemokine secretion. In conclusion, we demonstrate that FKBP51 has the potential to control inflammation in steroid insensitive patients in a steroid-dependent and independent manner and thus may be worthy of further study as a drug target.

Discovery of BI-2545: A Novel Autotaxin Inhibitor That Significantly Reduces LPA Levels in Vivo.

In an effort to find new therapeutic interventions addressing the unmet medical need of patients with idiopathic pulmonary fibrosis, we initiated a program to identify new autotaxin (ATX) inhibitors. Starting from a recently published compound (PF-8380), we identified several highly potent ATX inhibitors with improved pharmacokinetic and safety profiles. Further optimization efforts resulted in the identification of a single-digit nanomolar lead compound (BI-2545) that shows substantial lowering of LPA in vivo and is therefore considered a valuable tool for further studies.

Ultrafast and Predictive Mass Spectrometry-Based Autotaxin Assays for Label-Free Potency Screening.

Autotaxin (ATX) is a promising drug target for the treatment of several diseases, such as cancer and fibrosis. ATX hydrolyzes lysophosphatidyl choline (LPC) into bioactive lysophosphatidic acid (LPA). The potency of ATX inhibitors can be readily determined by using fluorescence-based LPC derivatives. While such assays are ultra-high throughput, they are prone to false positives compared to assays based on natural LPC. Here we report the development of ultrafast mass spectrometry-based ATX assays enabling the measurement of data points within 13 s, which is 10 times faster than classic liquid chromatography-mass spectrometry. To this end, we set up a novel in vitro and whole-blood assay. We demonstrate that the potencies determined with these assays are in good agreement with the in vivo efficacy and that the whole-blood assay has the best predictive power. This high-throughput label-free approach paired with the translatable data quality is highly attractive for appropriate guidance of medicinal chemists for constructing strong structure-activity relationships.

Design and synthesis of a library of chemokine antagonists.

A library of chemokine antagonists has been synthesized using a combination of solid and solution-phase chemistry. Structures of known chemokine antagonists were used to produce a pharmacophore which served to guide monomer selection. Several combinations of monomers have resulted in providing novel chemokine antagonists which in some cases display dual chemokine receptor antagonism.

Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension.

BACKGROUND: Inflammation is a feature of pulmonary arterial hypertension (PAH), and increased circulating levels of cytokines are reported in patients with PAH. However, to date, no information exists on the significance of elevated cytokines or their potential as biomarkers. We sought to determine the levels of a range of cytokines in PAH and to examine their impact on survival and relationship to hemodynamic indexes. METHODS AND RESULTS: We measured levels of serum cytokines (tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta, -2, -4, -5, -6, -8, -10, -12p70, and -13) using ELISAs in idiopathic and heritable PAH patients (n=60). Concurrent clinical data included hemodynamics, 6-minute walk distance, and survival time from sampling to death or transplantation. Healthy volunteers served as control subjects (n=21). PAH patients had significantly higher levels of interleukin-1beta, -2, -4, -6, -8, -10, and -12p70 and tumor necrosis factor-alpha compared with healthy control subjects. Kaplan-Meier analysis showed that levels of interleukin-6, 8, 10, and 12p70 predicted survival in patients. For example, 5-year survival with interleukin-6 levels of >9 pg/mL was 30% compared with 63% for patients with levels < or = 9 pg/mL (P=0.008). In this PAH cohort, cytokine levels were superior to traditional markers of prognosis such as 6-minute walk distance and hemodynamics. CONCLUSIONS: This study illustrates dysregulation of a broad range of inflammatory mediators in idiopathic and familial PAH and demonstrates that cytokine levels have a previously unrecognized impact on patient survival. They may prove to be useful biomarkers and provide insight into the contribution of inflammation in PAH.

Bidirectional transport of amino acids regulates mTOR and autophagy.

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions.

The aim of this study was to explore the potential of recombinant cytochrome P450 (P450) enzymes for human metabolic clearance prediction. The relative abundance and relative activity approaches were compared as methods to bridge the gap between catalytic activities in recombinant P450 enzymes and human liver microsomes (HLMs). Relative activity factors were measured by determining the intrinsic clearance (CL(int)) of probe substrates (bufuralol-CYP2D6, diclofenac-CYP2C9, midazolam-CYP3A4, and phenacetin-CYP1A2) in recombinant P450s and 16 HLM donors. Simultaneous determination of drug depletion and metabolite formation profiles has enabled a direct comparison of these methods for CL(int) determination. Of the 110 drugs tested, 66% were metabolized by one or more P450 enzymes; of these 44% of were metabolized by CYP3A4 (0.3-21 microl/min/pmol of P450), 41% by CYP2D6 (0.6-60 microl/min/pmol of P450), 26% by CYP2C19 (0.4-8.1 microl/min/pmol of P450), 9% by CYP1A2 (0.4-2.5 microl/min/pmol of P450), and 4% by CYP2C9 (0.9-6.4 microl/min/pmol of P450). Recombinant enzymes demonstrated improved prediction reliability relative to HLMs and hepatocytes. The most reliable correlations in terms of lowest bias and highest precision were observed by comparing in vivo CL(int), calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CL(int) determined using relative activity factors and adjusted for nonspecific binding. Predictions were less reliable using the relative abundance approach. For these drugs, recombinant P450 enzymes offer improved assay sensitivity compared with HLMs and cryopreserved hepatocytes for CL(int) determination using the drug depletion method.

Potent and selective xanthine-based inhibitors of phosphodiesterase 5.

Inhibitors of PDE5 are useful therapeutic agents for treatment of erectile dysfunction. A series of novel xanthine derivatives has been identified as potent inhibitors of PDE5, with good levels of selectivity against other PDE isoforms, including PDE6. Studies in the dog indicate excellent oral bioavailability for compound 21.

Effects of cyclosporin A and a rapamycin derivative (SAR943) on chronic allergic inflammation in sensitized rats.

Immunomodulators such as cyclosporin A (CsA) and SAR943 (32-deoxorapamycin) inhibit single allergen-induced allergic inflammation such as eosinophilic and lymphocytic infiltration and mRNA expression for interleukin (IL)-4 and IL-5. We examined the effects of CsA and SAR943, administered orally, on asthmatic responses in a rat model of chronic allergic inflammation. Sensitized Brown-Norway (BN) rats were exposed to ovalbumin (OVA) aerosol every third day on six occasions. CsA (5 mg/kg/day), SAR943 (2.5 mg/kg/day) or vehicle (Neoral) was administered orally, once a day, from days 10 to 21 (a total of 12 doses). We measured eosinophilic and T-cell inflammation in the airways, proliferation of airway cells by incorporation of bromodeoxyuridine (BrdU) and bronchial responsiveness to acetylcholine. CsA had no effects, while SAR943 inhibited airway smooth muscle (ASM, P < 0.05) and epithelial cell (P < 0.01) BrdU incorporation, and the number of CD4+ T cells (P < 0.05), without effects on BHR. ASM thickness was not significantly increased following chronic allergen exposure. Therefore, CsA and SAR943 have no effect on chronic eosinophilic inflammation, while SAR943, but not CsA, had a small effect on the proliferation of ASM and epithelium.

8-Aryl xanthines potent inhibitors of phosphodiesterase 5.

In clinical studies, several inhibitors of phosphodiesterase 5 (PDE5) have demonstrated utility in the treatment of erectile dysfunction. We describe herein a series of 8-aryl xanthine derivatives which function as potent PDE5 inhibitors with, in many cases, high levels of selectivity versus other PDE isoforms.