scholarly journals Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models

2020 ◽  
Author(s):  
Masato Sasaki ◽  
Mina Delawary ◽  
Hidetaka Sakurai ◽  
Hideki Kobayashi ◽  
Naoki Nakao ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
Affinity Purification ◽  
Atherosclerotic Lesion ◽  
Hdl Cholesterol ◽  
Lecithin:Cholesterol Acyltransferase ◽  
Lesion Area ◽  
Acyltransferase Activity ◽  
Hdl Function

Objective: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (ca. 2.1-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO × hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO × hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [ 3 H]-cholesterol and confirmed significant increases of [ 3 H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a–treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a–immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. Conclusions: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. This is also the first report describing that a small-molecule direct LCAT activator achieved HDL cholesterol elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

A novel small-molecule screening strategy identifies mitoxantrone as a RhoGTPase inhibitor

2013 ◽  
Vol 450 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Aurélien Bidaud-Meynard ◽  
Daniela Arma ◽  
Said Taouji ◽  
Michel Laguerre ◽  
Jean Dessolin ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
Large Scale ◽  
Topoisomerase Ii ◽  
Screening Strategy ◽  
Filamentous Actin ◽  
Library Screen ◽  
Rac1 Gtpase ◽  
Gtpase Activation

RhoGTPases are GDP/GTP molecular switches that control a wide variety of cellular processes, thereby contributing to many diseases, including cancer. As a consequence, there is great interest in the identification of small-molecule inhibitors of RhoGTPases. In the present paper, using the property of GTP-loaded RhoGTPases to bind to their effectors, we describe a miniaturized and robust assay to monitor Rac1 GTPase activation that is suitable for large-scale high-throughput screening. A pilot compound library screen revealed that the topoisomerase II poison MTX (mitoxantrone) is an inhibitor of Rac1, and also inhibits RhoA and Cdc42 in vitro. We show that MTX prevents GTP binding to RhoA/Rac1/Cdc42 in vitro. Furthermore, MTX strongly inhibits RhoGTPase-mediated F-actin (filamentous actin) reorganization and cell migration. Hence, we report a novel biochemical assay yielding the identification of RhoGTPase inhibitors and we present a proof-of-concept validation with the identification of MTX as a novel pan-RhoGTPase inhibitor.

scholarly journals A direct fluorometric activity assay for lipid kinases and phosphatases

2020 ◽  
Vol 61 (6) ◽  
pp. 945-952
Author(s):  
Jiachen Sun ◽  
Indira Singaram ◽  
Mona Hoseini Soflaee ◽  
Wonhwa Cho
Keyword(s):  
Real Time ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Modular Design ◽  
Acyl Chain ◽  
High Sensitivity ◽  
Activity Assay ◽  
Lipid Species ◽  
Lipid Kinases

Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.

scholarly journals Inhibitors of Helicobacter pylori ATPase Cagα block CagA transport and cag virulence

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2919-2930 ◽  
Author(s):  
Markus Hilleringmann ◽  
Werner Pansegrau ◽  
Michael Doyle ◽  
Susan Kaufman ◽  
Mary Lee MacKichan ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Host Cells ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Ags Cells ◽  
Antibacterial Compounds ◽  
Cellular Effects

With the steadily increasing occurrence of antibiotic resistance in bacteria, there is a great need for new antibacterial compounds. The approach described here involves targeting virulence-related bacterial type IV secretion systems (TFSSs) with small-molecule inhibitors. The cag TFSS of Helicobacter pylori was chosen as a model, and novel inhibitors directed against the cag VirB11-type ATPase Cagα were identified. The cag genes encode proteins that are components of a contact-dependent secretion system used by the bacterium to translocate the effector molecule CagA into host cells. Translocated CagA is associated with severe gastritis, and carcinoma. Furthermore, functional TFSSs and immunodominant CagA play a role in interleukin (IL)-8 induction, which is an important factor for chronic inflammation. Inhibitors of Cagα were identified by high-throughput screening of chemical libraries that comprised 524 400 small molecules. The ATPase activity of Cagα was inhibited by the selected compounds in an in vitro enzymic assay using the purified enzyme. The most active compound, CHIR-1, reduced TFSS function to an extent that cellular effects on AGS cells mediated by CagA were virtually undetectable, while reduced levels of IL-8 induction were observed. Gastric colonization by CHIR-1-pre-treated bacteria was found to be impaired in a dose-dependent manner using a mouse model of infection. Small-molecule Cagα inhibitors, the first described inhibitors of a TFSS, are potential candidates for the development of new antibacterial compounds that may lead to alternative medical treatments. The compounds are expected to impose weak selective pressure, since they target virulence functions. Moreover, the targeted virulence protein is conserved in a variety of bacterial pathogens. Additionally, TFSS inhibitors are potent tools to study the biology of TFSSs.

Identification of small-molecule inhibitors of the JIP–JNK interaction

2009 ◽  
Vol 420 (2) ◽  
pp. 283-296 ◽  
Author(s):  
Tracy Chen ◽  
Natasha Kablaoui ◽  
Jeremy Little ◽  
Sergei Timofeevski ◽  
William R. Tschantz ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Small Molecule ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Mitogen Activated Protein Kinase ◽  
Interacting Protein ◽  
Fluorescence Polarization Assay

JNK1 (c-Jun N-terminal kinase 1) plays a crucial role in the regulation of obesity-induced insulin resistance and is implicated in the pathology of Type 2 diabetes. Its partner, JIP1 (JNK-interacting protein 1), serves a scaffolding function that facilitates JNK1 activation by MKK4 [MAPK (mitogen-activated protein kinase) kinase 4] and MKK7 (MAPK kinase 7). For example, reduced insulin resistance and JNK activation are observed in JIP1-deficient mice. On the basis of the in vivo efficacy of a cell-permeable JIP peptide, the JIP–JNK interaction appears to be a potential target for JNK inhibition. The goal of the present study was to identify small-molecule inhibitors that disrupt the JIP–JNK interaction to provide an alternative approach for JNK inhibition to ATP-competitive inhibitors. High-throughput screening was performed by utilizing a fluorescence polarization assay that measured the binding of JNK1 to the JIP peptide. Multiple chemical series were identified, revealing two categories of JIP/JNK inhibitors: ‘dual inhibitors’ that are ATP competitive and probably inhibit JIP–JNK binding allosterically, and ‘JIP-site binders’ that block binding through interaction with the JIP site. A series of polychloropyrimidines from the second category was characterized by biochemical methods and explored through medicinal-chemistry efforts. As predicted, these inhibitors also inhibited full-length JIP–JNK binding and were selective against a panel of 34 representative kinases, including ones in the MAPK family. Overall, this work demonstrates that small molecules can inhibit protein–protein interactions in vitro in the MAPK family effectively and provides strategies for similar approaches within other target families.

scholarly journals The Capability of O-Acetyl-ADP-Ribose, an Epigenetic Metabolic Small Molecule, on Promoting the Further Spreading of Sir3 along the Telomeric Chromatin

Genes ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 577
Author(s):  
Tung ◽  
Wang ◽  
Lee ◽  
Tsai ◽  
Su ◽  
...  
Keyword(s):  
Small Molecule ◽  
Histone Deacetylases ◽  
Affinity Purification ◽  
Direct Interaction ◽  
Structural Rearrangement ◽  
Histone Deacetylation ◽  
Titration Calorimetry ◽  
On Chip

O-acetyl-ADP-ribose (AAR) is a metabolic small molecule relevant in epigenetics that is generated by NAD-dependent histone deacetylases, such as Sir2. The formation of silent heterochromatin in yeast requires histone deacetylation by Sir2, structural rearrangement of SIR complexes, spreading of SIR complexes along the chromatin, and additional maturation processing. AAR affects the interactions of the SIR-nucleosome in vitro and enhances the chromatin epigenetic silencing effect in vivo. In this study, using isothermal titration calorimetry (ITC) and dot blotting methods, we showed the direct interaction of AAR with Sir3. Furthermore, through chromatin immunoprecipitation (ChIP)-on-chip and chromatin affinity purification (ChAP)-on chip assays, we discovered that AAR is capable of increasing the extended spreading of Sir3 along telomeres, but not Sir2. In addition, the findings of a quantitative real-time polymerase chain reaction (qRT-PCR) and examinations of an in vitro assembly system of SIR-nucleosome heterochromatin filament were consistent with these results. This study provides evidence indicating another important effect of AAR in vivo. AAR may play a specific modulating role in the formation of silent SIR-nucleosome heterochromatin in yeast.

scholarly journals Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons

2018 ◽  
Vol 115 (41) ◽  
pp. 10481-10486 ◽  
Author(s):  
Jordi Pujols ◽  
Samuel Peña-Díaz ◽  
Diana F. Lázaro ◽  
Francesca Peccati ◽  
Francisca Pinheiro ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Small Molecule ◽  
Dopaminergic Neurons ◽  
High Throughput Screening ◽  
Protein Misfolding ◽  
Lewy Bodies ◽  
Molar Ratio ◽  
Lewy Neurites

Parkinson’s disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D–treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein–induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson’s disease.

scholarly journals Target Analysis of the Experimental Measles Therapeutic AS-136A

2009 ◽  
Vol 53 (9) ◽  
pp. 3860-3870 ◽  
Author(s):  
Jeong-Joong Yoon ◽  
Stefanie A. Krumm ◽  
J. Maina Ndungu ◽  
Vanessa Hoffman ◽  
Bettina Bankamp ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
Measles Virus ◽  
Pcr Analysis ◽  
Resistance Mutations ◽  
Protein Subunit ◽  
Rdrp Activity ◽  
Conserved Domains ◽  
L Protein

ABSTRACT No effective therapeutic is currently in place for improved case management of severe measles or the rapid control of outbreaks. Through high-throughput screening, we recently identified a novel small-molecule class that potently blocks activity of the measles virus (MeV) RNA-dependent RNA polymerase (RdRp) complex in transient replicon assays. However, the nature of the block in RdRp activity and the physical target of the compound remained elusive. Through real-time reverse transcription-PCR analysis, we demonstrate that the lead compound AS-136A blocks viral RNA synthesis in the context of an infection. Adaptation of different MeV strains to growth in the presence of the compound identified three candidate hot spots for resistance that are located in conserved domains of the viral polymerase (L protein) subunit of the RdRp complex. Rebuilding of individual mutations in RdRp-driven reporter assays and recombinant MeV traced the molecular basis for resistance to specific mutations in L. Mutations responsible for resistance cluster in the immediate vicinity of the proposed catalytic center for phosphodiester bond formation and neighboring conserved domains of L, providing support for effective inhibition of a paramyxovirus RdRp complex through interaction of a nonnucleoside small-molecule inhibitor with the L protein. Resistance mutations are located in regions of L that are fully conserved among viral isolates, and recombinant MeV harboring individual resistance mutations show some delay in the onset of viral growth in vitro. Taken together, these data support the hypothesis that acquiring mutations in these L domains may reduce virus fitness.

Discovery of small molecule guanylyl cyclase A receptor positive allosteric modulators

2021 ◽  
Vol 118 (52) ◽  
pp. e2109386118
Author(s):  
S. Jeson Sangaralingham ◽  
Kanupriya Whig ◽  
Satyamaheshwar Peddibhotla ◽  
R. Jason Kirby ◽  
Hampton E. Sessions ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Small Molecule ◽  
Guanylyl Cyclase ◽  
High Throughput Screening ◽  
Metabolic Diseases ◽  
Ex Vivo ◽  
Guanosine Monophosphate ◽  
Cardiomyocyte Hypertrophy ◽  
Orally Bioavailable

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.

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