Hepatic steatosis, a reversible state of metabolic dysregulation, can promote the onset of nonalcoholic steatohepatitis (NASH), and its transition is thought to be critical in disease evolution. The ...association between endoplasmic reticulum (ER) stress response and hepatocyte metabolism disorders prompted us to characterize ER stress-induced hepatic metabolic dysfunction in human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep), to explore regulatory pathways and validate a phenotypic
model for progression of liver steatosis. We treated hiPSC-Hep with a ratio of unsaturated and saturated fatty acids in the presence of an inducer of ER stress to synergistically promote triglyceride accumulation and dysregulate lipid metabolism. We monitored lipid accumulation by high-content imaging and measured gene regulation by RNA sequencing and reverse transcription quantitative PCR analyses. Our results show that ER stress potentiated intracellular lipid accumulation by 5-fold in hiPSC-Hep in the absence of apoptosis. Transcriptome pathway analysis identified ER stress pathways as the most significantly dysregulated of all pathways affected. Obeticholic acid dose dependently inhibited lipid accumulation and modulated gene expression downstream of the farnesoid X receptor. We were able to identify modulation of hepatic markers and gene pathways known to be involved in steatosis and nonalcoholic fatty liver disease (NAFLD), in support of a hiPSC-Hep disease model that is relevant to clinical data for human NASH. Our results show that the model can serve as a translational discovery platform for the understanding of molecular pathways involved in NAFLD, and can facilitate the identification of novel therapeutic molecules based on high-throughput screening strategies.
OBJECTIVES/GOALS: Our team has developed a high-throughput 3D patient-derived muscle platform to study signaling pathways associated with skeletal muscle disease. This platform will be used to study ...pathologies of human muscle that arise from genetic mutations and processes of aging along with pharmacologic interventions to improve mass, function, and performance. METHODS/STUDY POPULATION: In the current study, 3D skeletal muscle is formed from young healthy male samples. Samples are treated with urocortin II (UCNII) or vehicle for ten days and evaluated for tissue performance. Functional assessments include real-time contraction magnitudes using digital image correlation (DIC) analysis of video collected during electrical pulse stimulation and end-point measures of initial and repeated tetanic force production. Functional measures provide indices of patient muscle synchronicity, strength, and endurance related to drug efficacy and toxicity which we will correlate to pro-growth protein signaling via Luminex. A subset of these samples will also be analyzed by histology and microscopy to assess muscle fiber density, type, and size, as well as myotube fusion index and sarcomere uniformity. RESULTS/ANTICIPATED RESULTS: We anticipate that healthy muscle treated with UCNII will have increased synchronicity and contraction magnitudes in DIC analysis throughout their seven-day electrical pulse stimulation protocol. We also expect to see sustained contraction magnitudes in DIC analysis at the end of electrical pulse stimulation indicating fatigue resistance in the drug treated group compared to no-drug control. Like our real-time DIC data, we anticipate increases to initial and sustained maximal force production in the drug treated group. We expect that drug treated muscle will present with an increased fiber density, fiber diameter, and fusion index with uniform sarcomeres. Finally, we expect heightened pro-growth signaling pathways in treated vs. controls. DISCUSSION/SIGNIFICANCE: The current study will serve as an initial investigation of the endogenous ligand UCNII for enhancing skeletal muscle mass and performance in human muscle laying the framework for future drug efficacy and toxicity studies. This platform will ultimately enhance the study of muscle diseases and translation of therapeutics to clinical settings.
The particulate guanylyl cyclase A receptor (GC-A), via activation by its endogenous ligands atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), possesses beneficial biological ...properties such as blood pressure regulation, natriuresis, suppression of adverse remodeling, inhibition of the renin-angiotensin-aldosterone system, and favorable metabolic actions through the generation of its second messenger cyclic guanosine monophosphate (cGMP). Thus, the GC-A represents an important molecular therapeutic target for cardiovascular disease and its associated risk factors. However, a small molecule that is orally bioavailable and directly targets the GC-A to potentiate cGMP has yet to be discovered. Here, we performed a cell-based high-throughput screening campaign of the NIH Molecular Libraries Small Molecule Repository, and we successfully identified small molecule GC-A positive allosteric modulator (PAM) scaffolds. Further medicinal chemistry structure-activity relationship efforts of the lead scaffold resulted in the development of a GC-A PAM, MCUF-651, which enhanced ANP-mediated cGMP generation in human cardiac, renal, and fat cells and inhibited cardiomyocyte hypertrophy in vitro. Further, binding analysis confirmed MCUF-651 binds to GC-A and selectively enhances the binding of ANP to GC-A. Moreover, MCUF-651 is orally bioavailable in mice and enhances the ability of endogenous ANP and BNP, found in the plasma of normal subjects and patients with hypertension or heart failure, to generate GC-A-mediated cGMP ex vivo. In this work, we report the discovery and development of an oral, small molecule GC-A PAM that holds great potential as a therapeutic for cardiovascular, renal, and metabolic diseases.
Diabetes produces a chronic inflammatory state that contributes to the development of vascular disease and impaired wound healing. Despite the known individual and societal impacts of diabetic ...ulcers, there are limited therapies effective at improving healing. Stromal cell-derived factor 1α (SDF-1α) is a CXC chemokine that functions via activation of the CXC chemokine receptor type 4 (CXCR4) receptor to recruit hematopoietic cells to locations of tissue injury and promote tissue repair. The expression of SDF-1α is reduced in diabetic wounds, suggesting a potential contribution to wound healing impairment and presenting the CXCR4 receptor as a target for therapeutic investigations. We developed a high-throughput β-arrestin recruitment assay and conducted structure-activity relationship (SAR) studies to screen compounds for utility as CXCR4 agonists. We identified CXCR4 agonist UCUF-728 from our studies and further validated its activity in vitro in diabetic fibroblasts. UCUF-728 reduced overexpression of miRNA-15b and miRNA-29a, negative regulators of angiogenesis and type I collagen production, respectively, in diabetic fibroblasts. In vivo, UCUF-728 reduced the wound closure time by 36% and increased the evidence of angiogenesis in diabetic mice. Together, this work demonstrates the clinical potential of small molecule CXCR4 agonists as novel therapies for pathologic wound healing in diabetes.
Non-alcoholic fatty liver disease (NAFLD) has a large impact on global health. At the onset of disease, NAFLD is characterized by hepatic steatosis defined by the accumulation of triglycerides stored ...as lipid droplets. Developing therapeutics against NAFLD and progression to non-alcoholic steatohepatitis (NASH) remains a high priority in the medical and scientific community. Drug discovery programs to identify potential therapeutic compounds have supported high throughput/high-content screening of in vitro human-relevant models of NAFLD to accelerate development of efficacious anti-steatotic medicines. Human induced pluripotent stem cell (hiPSC) technology is a powerful platform for disease modeling and therapeutic assessment for cell-based therapy and personalized medicine. In this study, we applied AstraZeneca's chemogenomic library, hiPSC technology and multiplexed high content screening to identify compounds that significantly reduced intracellular neutral lipid content. Among 13,000 compounds screened, we identified hits that protect against hiPSC-derived hepatic endoplasmic reticulum stress-induced steatosis by a mechanism of action including inhibition of the cyclin D3-cyclin-dependent kinase 2-4 (CDK2-4)/CCAAT-enhancer-binding proteins (C/EBPα)/diacylglycerol acyltransferase 2 (DGAT2) pathway, followed by alteration of the expression of downstream genes related to NAFLD. These findings demonstrate that our phenotypic platform provides a reliable approach in drug discovery, to identify novel drugs for treatment of fatty liver disease as well as to elucidate their underlying mechanisms.
High-throughput screening (HTS) is a powerful approach to drug discovery, but many lead compounds are found to be unsuitable for use in vivo after initial screening. Screening in small animals like ...C. elegans can help avoid these problems, but this system has been limited to screens with low-throughput or no specific molecular target. We report the first in vivo 1536-well plate assay for a specific genetic pathway in C. elegans. Our assay measures induction of a gene regulated by SKN-1, a master regulator of detoxification genes. SKN-1 inhibitors will be used to study and potentially reverse multidrug resistance in parasitic nematodes. Screens of two small commercial libraries and the full Molecular Libraries Small Molecule Repository (MLSMR) of ∼364,000 compounds validate our platform for ultra HTS. Our platform overcomes current limitations of many whole-animal screens and can be widely adopted for other inducible genetic pathways in nematodes and humans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by ...astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS.
Mutations in the merlin tumor suppressor gene cause Neurofibromatosis type 2 (NF2), which is a disease characterized by development of multiple benign tumors in the nervous system. The current ...standard of care for NF2 calls for surgical resection of the characteristic tumors, often with devastating neurological consequences. There are currently no approved non-surgical therapies for NF2. In an attempt to identify much needed targets and therapeutically active compounds for NF2 treatment, we employed a chemical biology approach using ultra-high-throughput screening. To support this goal, we created a merlin-null mouse Schwann cell (MSC) line to screen for compounds that selectively decrease their viability and proliferation. We optimized conditions for 384-well plate assays and executed a proof-of-concept screen of the Library of Pharmacologically Active Compounds. Further confirmatory and selectivity assays identified phosphatidylinositol 3-kinase (PI3K) as a potential NF2 drug target. Notably, loss of merlin function is associated with activation of the PI3K/Akt pathway in human schwannomas. We report that AS605240, a PI3K inhibitor, decreased merlin-null MSC viability in a dose-dependent manner without significantly decreasing viability of control Schwann cells. AS605240 exerted its action on merlin-null MSCs by promoting caspase-dependent apoptosis and inducing autophagy. Additional PI3K inhibitors tested also decreased viability of merlin-null MSCs in a dose-dependent manner. In summary, our chemical genomic screen and subsequent hit validation studies have identified PI3K as potential target for NF2 therapy.
Analogues of the known H1-antihistamine R-dimethindene were profiled as potential agents for the treatment of insomnia. Several highly selective compounds were efficacious in rodent sleep models. On ...the basis of overall profile, indene 1d and benzothiophene 2a had pharmacokinetic properties suitable for evaluation in night time dosing. Compound 2a did not show an in vivo cardiovascular effect from weak hERG channel inhibition.
Abstract only
Introduction:
Hypertension (HTN) represents one of the greatest burdens in human health and is associated with increased morbidity and mortality. Despite the availability of several ...antihypertensive drugs, most patients have suboptimal blood pressure (BP) control, thus underscoring the need for new therapeutic drugs. The natriuretic peptide receptor A (NPRA) possesses potent vasodilating, natriuretic and diuretic actions via activation by ANP or BNP and generation of its second messenger, cGMP. Recently, we reported the discovery of a small molecule positive allosteric modulator (PAM) for NPRA, MCUF-651, which enhances the ANP/BNP/NPRA/cGMP pathway (PNAS,2021). To date, the in vivo actions of MCUF-651 remains unknown. Here we tested the hypothesis that MCUF-651 would have BP lowering and renal enhancing properties in experimental HTN.
Methods:
A single IV bolus of MCUF-651 (10mg/kg; n=4) or vehicle (V; n=5) was injected into anesthetized spontaneous hypertensive rats (SHRs). Plasma and urinary cGMP (PcGMP and UcGMP), mean arterial pressure (MAP) and urinary volume (UV) and sodium (UNa) excretion were assessed at baseline (BL) and over 60-mins post-bolus.
Results:
In SHRs, PcGMP and UcGMP were significantly elevated with MCUF-651 compared to the vehicle group (ΔPcGMP MCUF-651: 16±5, ΔPcGMP V: -2±3, pmol/mL;
P
=0.003; ΔUcGMP MCUF-651: 78±29, ΔUcGMP V: 30±8, pmol/min,
P
=0.01), thus supporting NPRA target engagement. Notably, the elevations in cGMP with MCUF-651 were associated with a significant reduction in MAP at 15-mins post bolus compared to vehicle (ΔMAP MCUF-651: -29±14, ΔMAP V: -6±2 mmHg;
P
=0.04) and this MAP reduction was sustained over 60 mins. Further, UV and UNa excretion were significantly increased, from BL, with MCUF-651 (BL UV: 6 ± 2, 60 mins post bolus UV: 56± 18 μL/min,
P
<0.001; BL UNa: 1±0.2, 60 mins post bolus UNa: 8±2 μmol/min,
P
<0.001).
Conclusions:
We report for the first time that MCUF-651, a novel NPRA small molecule PAM, engages NPRA, increases cGMP and reduces MAP while enhancing sodium and water excretion in experimental HTN. These findings support the development of MCUF-651 as a potential new therapeutic modality to optimize BP lowering goals and reduce adverse outcomes in human HTN.
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