Transcriptomic and Proteomic Changes Driving Pulmonary Fibrosis Resolution in Young and Old Mice.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFß antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.

Soluble Guanylyl Cyclase Activator BI 685509 Reduces Portal Hypertension and Portosystemic Shunting in a Rat Thioacetamide-Induced Cirrhosis Model.

Portal hypertension (PT) commonly occurs in cirrhosis. Nitric oxide (NO) imbalance contributes to PT via reduced soluble guanylyl cyclase (sGC) activation and cGMP production, resulting in vasoconstriction, endothelial cell dysfunction, and fibrosis. We assessed the effects of BI 685509, an NO-independent sGC activator, on fibrosis and extrahepatic complications in a thioacetamide (TAA)-induced cirrhosis and PT model. Male Sprague-Dawley rats received TAA twice-weekly for 15 weeks (300-150 mg/kg i.p.). BI 685509 was administered daily for the last 12 weeks (0.3, 1, and 3 mg/kg p.o.; n = 8-11 per group) or the final week only (Acute, 3 mg/kg p.o.; n = 6). Rats were anesthetized to measure portal venous pressure. Pharmacokinetics and hepatic cGMP (target engagement) were measured by mass spectrometry. Hepatic Sirius Red morphometry (SRM) and alpha-smooth muscle actin (αSMA) were measured by immunohistochemistry; portosystemic shunting was measured using colored microspheres. BI 685509 dose-dependently increased hepatic cGMP at 1 and 3 mg/kg (3.92 ± 0.34 and 5.14 ± 0.44 versus 2.50 ± 0.19 nM in TAA alone; P < 0.05). TAA increased hepatic SRM, αSMA, PT, and portosystemic shunting. Compared with TAA, 3 mg/kg BI 685509 reduced SRM by 38%, αSMA area by 55%, portal venous pressure by 26%, and portosystemic shunting by 10% (P < 0.05). Acute BI 685509 reduced SRM and PT by 45% and 21%, respectively (P < 0.05). BI 685509 improved hepatic and extrahepatic cirrhosis pathophysiology in TAA-induced cirrhosis. These data support the clinical investigation of BI 685509 for PT in patients with cirrhosis. SIGNIFICANCE STATEMENT: BI 685509 is an NO-independent sGC activator that was tested in a preclinical rat model of TAA-induced nodular, liver fibrosis, portal hypertension, and portal systemic shunting. BI 685509 reduced liver fibrosis, portal hypertension, and portal-systemic shunting in a dose-dependent manner, supporting its clinical assessment to treat portal hypertension in patients with cirrhosis.

Diminished Myoinositol in Ventromedial Prefrontal Cortex Modulates the Endophenotype of Impulsivity.

Maladaptive impulsivity manifests in a variety of disorders, including attention-deficit hyperactivity disorder (ADHD), depression, and substance use disorder. However, the etiological mechanisms of impulsivity remain poorly understood. In the present study, we used in-vivo proton magnetic resonance spectroscopy (1H-MRS) to investigate neurometabolite content in the prefrontal cortex (PFC) and striatum of rats exhibiting low- versus high-impulsive (LI, HI) behavior on a visual attentional task. We validated our 1H-MRS findings using regionally resolved ex-vivo mass spectroscopy, transcriptomics, and site-directed RNA interference in the ventromedial PFC. We report a significant reduction in myoinositol levels in the PFC but not the striatum of HI rats compared with LI rats. Reduced myoinositol content was localized to the infralimbic (IL) cortex, where significant reductions in transcript levels of key proteins involved in the synthesis and recycling of myoinositol (IMPase1) were also present. Knockdown of IMPase1in the IL cortex increased impulsivity in nonimpulsive rats when the demand on inhibitory response control was increased. We conclude that diminished myoinositol levels in ventromedial PFC causally mediate a specific form of impulsivity linked to vulnerability for stimulant addiction in rodents. Myoinositol and related signaling substrates may thus offer novel opportunities for treating neuropsychiatric disorders comorbid with impulsive symptomology.

A smart approach to enable preclinical studies in pharmaceutical industry: PLGA-based extended release formulation platform for subcutaneous applications.

Objective: Validation of a prospective new therapeutic concept in a proof of concept study is costly and time-consuming. In particular, pharmacologically active tool compounds often lack suitable pharmacokinetic (PK) properties for subsequent studies. The current work describes a PLGA-based formulation platform, encapsulating different preclinical research compounds into extended release microparticles, to optimize their PK properties after subcutaneous administration.Significance: Developing a PLGA-based formulation platform offers the advantage of enabling early proof of concept studies in pharmaceutical research for a variety of preclinical compounds by providing a tailor-made PK profile.Methods: Different model compounds were encapsulated into PLGA microparticles, utilizing emulsification solvent evaporation or spray drying techniques. Formulations aiming different release rates were manufactured and characterized. Optimized formulations were assessed in in vivo studies to determine their PK properties, with the mean residence time (MRT) as one key PK parameter.Results: Utilizing both manufacturing methods, tested tool compounds were encapsulated successfully, with a drug load between 5% and 40% w/w, and an extended release time up to 250 h. In the following PK studies, the MRT was extended by a factor of 90, resulting in prolonged coverage of the required target through level. This approach was confirmed to be equally successful for additional internal compounds, verifying a general applicability of the platform.Conclusion: For different active pharmaceutical ingredients (API), an optimized, tailor-made PK profile was obtained utilizing the described formulation platform. This approach is applicable for a variety of pharmacologically active tool compounds, reducing timelines and costs in preclinical research.

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.

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.

Vinylogous chain branching catalysed by a dedicated polyketide synthase module.

Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like ß-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching-acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis.

The global yield of bananas-one of the most important food crops-is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant-nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques (1)H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant's defense system, and derived strategies for improving banana resistance are highlighted.

Biomarker candidates for the detection of an infectious etiology of febrile neutropenia.

PURPOSE: Infections and subsequent septicemia are major complications in neutropenic patients with hematological malignancies. Here, we identify biomarker candidates for the early detection of an infectious origin, and monitoring of febrile neutropenia (FN). METHODS: Proteome, metabolome, and conventional biomarkers from 20 patients with febrile neutropenia without proven infection (FNPI) were compared to 28 patients with proven infection, including 17 patients with bacteremia. RESULTS: Three peptides (mass to charge ratio 1017.4-1057.3; p-values 0.011-0.024), six proteins (mass to charge ratio 6881-17,215; p-values 0.002-0.004), and six phosphatidylcholines (p-values 0.007-0.037) were identified that differed in FNPI patients compared to patients with infection or bacteremia. Seven of these marker candidates discriminated FNPI from infection at fever onset with higher sensitivity and specificity (ROC-AUC 0.688-0.824) than conventional biomarkers i.e., procalcitonin, C-reactive protein, or interleukin-6 (ROC-AUC 0.535-0.672). In a post hoc analysis, monitoring the time course of four lysophosphatidylcholines, threonine, and tryptophan allowed for discrimination of patients with or without resolution of FN (ROC-AUC 0.648-0.919) with higher accuracy compared to conventional markers (ROC-AUC 0.514-0.871). CONCLUSIONS: Twenty-one promising biomarker candidates for the early detection of an infectious origin or for monitoring the course of FN were found which might overcome known shortcomings of conventional markers.

Enzymatic polyketide chain branching to give substituted lactone, lactam, and glutarimide heterocycles.

Polyketides typically result from head-to-tail condensation of acyl thioesters to produce highly functionalized linear chains. The biosynthesis of the phytotoxin rhizoxin, however, involves a polyketide synthase (PKS) module that introduces a δ-lactone chain branch through Michael addition of a malonyl extender to an α,ß-unsaturated intermediate unit. To evaluate the scope of the branching module, polyketide mimics were synthesized and their biotransformation by the reconstituted PKS module from the Rhizopus symbiont Burkholderia rhizoxinica was monitored in vitro. The impact of the type and configuration of the δ-substituents was probed and it was found that amino-substituted surrogates yield the corresponding lactams. A carboxamide analogue was transformed into a glutarimide unit, which can be found in many natural products. Our findings illuminate the biosynthesis of glutarimide-bearing polyketides and also demonstrate the utility of this branching module for synthetic biology.

mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Synthetic remodeling of the chartreusin pathway to tune antiproliferative and antibacterial activities.

Natural products of the benzonaphthopyranone class, such as chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of chartreusin derivatives. Pathway engineering of the chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of vinyl- and ethynyl-substituted derivatives with blue light resulted in an improved antiproliferative potency against a colorectal cancer cell line. In contrast, the replacement of a methyl group by hydrogen caused a drastically decreased cytotoxicity but markedly enhanced antimycobacterial activity. Furthermore, mutasynthesis of bromochartreusin led to the first crystal structure of a chartreusin derivative that is not modified in the glycoside residue. Beyond showcasing the possibility of converting diverse, fully synthetic polyphenolic aglycones into the corresponding glycosides in a whole-cell approach, this work identified new chartreusins with fine-tuned properties as promising candidates for further development as therapeutics.

A ketosynthase homolog uses malonyl units to form esters in cervimycin biosynthesis.

Ketosynthases produce the carbon backbones of a vast number of biologically active polyketides by catalyzing Claisen condensations of activated acyl and malonyl building blocks. Here we report that a ketosynthase homolog from Streptomyces tendae, CerJ, unexpectedly forms malonyl esters during the biosynthesis of cervimycin, a glycoside antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). Deletion of cerJ yielded a substantially more active cervimycin variant lacking the malonyl side chain, and in vitro biotransformations revealed that CerJ is capable of transferring malonyl, methylmalonyl and dimethylmalonyl units onto the glycoside. According to phylogenetic analyses and elucidation of the crystal structure, CerJ is functionally and structurally positioned between the ketosynthase catalyzing Claisen condensations and acyl-ACP shuttles, and it features a noncanonical catalytic triad. Site-directed mutagenesis and structures of CerJ in complex with substrates not only allowed us to establish a model for the reaction mechanism but also provided insights into the evolution of this important subclass of the thiolase superfamily.

Discovery of BI 7446: A Potent Cyclic Dinucleotide STING Agonist with Broad-Spectrum Variant Activity for the Treatment of Cancer.

Activating the stimulator of interferon genes (STING) pathway with STING agonists is an attractive immune oncology concept to treat patients with tumors that are refractory to single-agent anti-PD-1 therapy. For best clinical translatability and broad application to cancer patients, STING agonists with potent cellular activation of all STING variants are desired. Novel cyclic dinucleotide (CDN)-based selective STING agonists were designed and synthesized comprising noncanonical nucleobase, ribose, and phosphorothioate moieties. This strategy led to the discovery of 2',3'-CDN 13 (BI 7446), which features unprecedented potency and activates all five STING variants in cellular assays. ADME profiling revealed that CDN 13 has attractive drug-like properties for development as an intratumoral agent. Injection of low doses of CDN 13 into tumors in mice induced long-lasting, tumor-specific immune-mediated tumor rejection. Based on its compelling preclinical profile, BI 7446 has been advanced to clinical trials (monotherapy and in combination with anti-PD-1 antibody).

Discovery of a Novel Potent and Selective HSD17B13 Inhibitor, BI-3231, a Well-Characterized Chemical Probe Available for Open Science.

Genome-wide association studies in patients revealed HSD17B13 as a potential new target for the treatment of nonalcoholic steatohepatitis (NASH) and other liver diseases. However, the physiological function and the disease-relevant substrate of HSD17B13 remain unknown. In addition, no suitable chemical probe for HSD17B13 has been published yet. Herein, we report the identification of the novel potent and selective HSD17B13 inhibitor BI-3231. Through high-throughput screening (HTS), using estradiol as substrate, compound 1 was identified and selected for subsequent optimization resulting in compound 45 (BI-3231). In addition to the characterization of compound 45 for its functional, physicochemical, and drug metabolism and pharmacokinetic (DMPK) properties, NAD+ dependency was investigated. To support Open Science, the chemical HSD17B13 probe BI-3231 will be available to the scientific community for free via the opnMe platform, and thus can help to elucidate the pharmacology of HSD17B13.

Imaging mass spectrometry and genome mining reveal highly antifungal virulence factor of mushroom soft rot pathogen.

Caught in the act: imaging mass spectrometry of a button mushroom infected with the soft rot pathogen Janthinobacterium agaricidamnosum in conjunction with genome mining revealed jagaricin as a highly antifungal virulence factor that is not produced under standard cultivation conditions. The structure of jagaricin was rigorously elucidated by a combination of physicochemical analyses, chemical derivatization, and bioinformatics.

Differential analyte derivatization enables unbiased MALDI-TOF-based high-throughput screening: A proof-of-concept study for the discovery of catechol-o-methyltransferase inhibitors.

Recent advances in label-free high-throughput screening via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) offer unprecedented opportunities for the identification of novel chemical starting points in target-based drug discovery. A clear advantage of the technology is the possibility for label-free, direct quantification of analytes with high precision and robustness. Here we have expanded the range of analytes and biology that can be addressed via MALDI-TOF HTS, by developing a method based on post-reaction pyrylium-based derivatization to detect 3-methoxytyramine, the physiological enzyme product of the catechol-O-methyltransferase (COMT) enzyme. The introduction of pyrylium-type reagents as universal derivatization strategy under aqueous conditions for molecules containing primary amines represents a valuable addition to the toolbox of MALDI-TOF assay development. Characterization of COMT's enzymatic activity and inhibition by reference inhibitors, and comparison of the results obtained in our assay with data from previous mechanistic studies validated the performance of this new method. To address the problem of isobaric interference, a source of false results in MALDI-TOF assays measuring low molecular weight analytes, we devised a differential derivatization workflow which can potentially replace other counter- or orthogonal assays in future screening campaigns. Finally, we report on the first label-free HTS campaign for the identification of COMT inhibitors performed in miniaturized 1536-well microtiter plate format via MALDI-TOF MS analysis.

Effects of GPR139 agonism on effort expenditure for food reward in rodent models: Evidence for pro-motivational actions.

Apathy, deficiency of motivation including willingness to exert effort for reward, is a common symptom in many psychiatric and neurological disorders, including depression and schizophrenia. Despite improved understanding of the neurocircuitry and neurochemistry underlying normal and deficient motivation, there is still no approved pharmacological treatment for such a deficiency. GPR139 is an orphan G protein-coupled receptor expressed in brain regions which contribute to the neural circuitry that controls motivation including effortful responding for reward, typically sweet gustatory reward. The GPR139 agonist TAK-041 is currently under development for treatment of negative symptoms in schizophrenia which include apathy. To date, however, there are no published preclinical data regarding its potential effect on reward motivation or deficiencies thereof. Here we report in vitro evidence confirming that TAK-041 increases intracellular Ca2+ mobilization and has high selectivity for GPR139. In vivo, TAK-041 was brain penetrant and showed a favorable pharmacokinetic profile. It was without effect on extracellular dopamine concentration in the nucleus accumbens. In addition, TAK-041 did not alter the effort exerted to obtain sweet gustatory reward in rats that were moderately food deprived. By contrast, TAK-041 increased the effort exerted to obtain sweet gustatory reward in mice that were only minimally food deprived; furthermore, this effect of TAK-041 occurred both in control mice and in mice in which deficient effortful responding was induced by chronic social stress. Overall, this study provides preclinical evidence in support of GPR139 agonism as a molecular target mechanism for treatment of apathy.

Mapping the metabolomic and lipidomic changes in the bleomycin model of pulmonary fibrosis in young and aged mice.

Alterations in metabolic pathways were recently recognized as potential underlying drivers of idiopathic pulmonary fibrosis (IPF), translating into novel therapeutic targets. However, knowledge of metabolic and lipid regulation in fibrotic lungs is limited. To comprehensively characterize metabolic perturbations in the bleomycin mouse model of IPF, we analyzed the metabolome and lipidome by mass spectrometry. We identified increased tissue turnover and repair, evident by enhanced breakdown of proteins, nucleic acids and lipids and extracellular matrix turnover. Energy production was upregulated, including glycolysis, the tricarboxylic acid cycle, glutaminolysis, lactate production and fatty acid oxidation. Higher eicosanoid synthesis indicated inflammatory processes. Because the risk of IPF increases with age, we investigated how age influences metabolomic and lipidomic changes in the bleomycin-induced pulmonary fibrosis model. Surprisingly, except for cytidine, we did not detect any significantly differential metabolites or lipids between old and young bleomycin-treated lungs. Together, we identified metabolomic and lipidomic changes in fibrosis that reflect higher energy demand, proliferation, tissue remodeling, collagen deposition and inflammation, which might serve to improve diagnostic and therapeutic options for fibrotic lung diseases in the future.

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein-Ligand Interactions at High Throughput.

Demonstration of in vitro target engagement for small-molecule ligands by measuring binding to a molecular target is an established approach in early drug discovery and a pivotal step in high-throughput screening (HTS)-based compound triaging. We describe the setup, evaluation, and application of a ligand binding assay platform combining automated affinity selection (AS)-based sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The platform enables mass spectrometry (MS)-based HTS for small-molecule target interactions from single-compound incubation mixtures and is embedded into a regular assay automation environment. Efficient separation of target-ligand complexes is achieved by in-plate size exclusion chromatography (SEC), and small-molecule ligands are subsequently identified by MALDI-TOF analysis. In contrast to alternative HTS-capable binding assay formats, MALDI-TOF AS-MS is capable of identifying orthosteric and allosteric ligands, as shown for the model system protein tyrosine phosphatase 1B (PTP1B), irrespective of protein function. Furthermore, determining relative binding affinities (RBAs) enabled ligand ranking in accordance with functional inhibition and reference data for PTP1B and a number of diverse protein targets. Finally, we present a validation screen of more than 23,000 compounds within 24 h, demonstrating the general applicability of the platform for the HTS-compatible assessment of protein-ligand interactions.