scholarly journals Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D

2021 ◽  
Vol 22 (2) ◽  
pp. 594
Author(s):  
Yi-Hsun Ho ◽  
Lan Chen ◽  
Rong Huang
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
High Throughput Screening ◽  
Coenzyme A ◽  
Therapeutic Potential ◽  
Biological Functions ◽  
Histone H4 ◽  
Fluorescent Signal ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

scholarly journals Development of a continuous fluorescence-based assay for N-terminal acetyltransferase D

2020 ◽  
Author(s):  
Yi-Hsun Ho ◽  
Lan Chen ◽  
Rong Huang
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
High Throughput Screening ◽  
Coenzyme A ◽  
Therapeutic Potential ◽  
Biological Functions ◽  
Histone H4 ◽  
Fluorescent Signal ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

AbstractN-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z’-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

scholarly journals Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting

2021 ◽  
Author(s):  
Beatrice Parma ◽  
Vignesh Ramesh ◽  
Paradesi Naidu Gollavilli ◽  
Aarif Siddiqui ◽  
Luisa Pinna ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
Therapeutic Potential ◽  
Small Cell ◽  
Small Cell Lung ◽  
Lung Cancers ◽  
Metabolism Enzymes ◽  
Shock Proteins

ABSTRACTThe identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures. Here, we show HSPD1 (HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients’ prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on OXPHOS and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B. These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NCSLC.SignificanceHSPD1 elimination or disruption interferes with NSCLC metabolic activity causing a strong OXPHOS-dependent energetic breakdown, which the cancer cells fail to overcome, highlighting HSPD1 as a potential theranostic marker for improving lung cancer therapy.

scholarly journals FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay

2016 ◽  
Vol 21 (7) ◽  
pp. 701-712 ◽  
Author(s):  
Andrea Caporale ◽  
Fabiola Mascanzoni ◽  
Biancamaria Farina ◽  
Mattia Sturlese ◽  
Gianluigi Di Sorbo ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Cyclophilin A ◽  
Reaction Rates ◽  
Docking Studies ◽  
Isomerase Activity ◽  
Shape Complementarity ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds ◽  
Micromolar Range

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.

scholarly journals Pilot-Scale Compound Screening against RNA Editing Identifies Trypanocidal Agents

2014 ◽  
Vol 20 (1) ◽  
pp. 92-100 ◽  
Author(s):  
Houtan Moshiri ◽  
Vaibhav Mehta ◽  
Chun Wai Yip ◽  
Reza Salavati
Keyword(s):  
Rna Editing ◽  
High Throughput Screening ◽  
Pilot Scale ◽  
Messenger Rnas ◽  
Unique Type ◽  
Compound Screening ◽  
Parasite Viability ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Most mitochondrial messenger RNAs in trypanosomatid pathogens undergo a unique type of posttranscriptional modification involving insertion and/or deletion of uridylates. This process, RNA editing, is catalyzed by a multiprotein complex (~1.6 MDa), the editosome. Knockdown of core editosome proteins compromises mitochondrial function and, ultimately, parasite viability. Hence, because the editosome is restricted to trypanosomatids, it serves as a unique drug target in these pathogens. Currently, there is a lack of editosome inhibitors for antitrypanosomatid drug development or that could serve as unique tools for perturbing and characterizing editosome interactions or RNA editing reaction stages. Here, we screened a library of pharmacologically active compounds (LOPAC1280) using high-throughput screening to identify RNA editing inhibitors. We report that aurintricarboxylic acid, mitoxantrone, PPNDS, and NF449 are potent inhibitors of deletion RNA editing (IC50 range, 1–5 µM). However, none of these compounds could specifically inhibit the catalytic steps of RNA editing. Mitoxantrone blocked editing by inducing RNA-protein aggregates, whereas the other three compounds interfered with editosome-RNA interactions to varying extents. Furthermore, NF449, a suramin analogue, was effective at killing Trypanosoma brucei in vitro. Thus, new tools for editosome characterization and downstream RNA editing inhibitor have been identified.

scholarly journals NatD promotes lung cancer progression by preventing histone H4 serine phosphorylation to activate Slug expression

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Junyi Ju ◽  
Aiping Chen ◽  
Yexuan Deng ◽  
Ming Liu ◽  
Ying Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
Serine Phosphorylation ◽  
Histone H4 ◽  
Slug Expression

scholarly journals Biological Functions of Gasdermins in Cancer: From Molecular Mechanisms to Therapeutic Potential

2021 ◽  
Vol 9 ◽  
Author(s):  
Man Wang ◽  
Xinzhe Chen ◽  
Yuan Zhang
Keyword(s):  
Cell Death ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Cancer Biology ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Effector Proteins ◽  
Biological Functions ◽  
Cancer Cell Growth ◽  
Cancer Pathogenesis

Pyroptosis is a type of lytic programmed cell death triggered by various inflammasomes that sense danger signals. Pyroptosis has recently attracted great attention owing to its contributory role in cancer. Pyroptosis plays an important role in cancer progression by inducing cancer cell death or eliciting anticancer immunity. The participation of gasdermins (GSDMs) in pyroptosis is a noteworthy recent discovery. GSDMs have emerged as a group of pore-forming proteins that serve important roles in innate immunity and are composed of GSDMA-E and Pejvakin (PJVK) in human. The N-terminal domains of GSDMs, expect PJVK, can form pores on the cell membrane and function as effector proteins of pyroptosis. Remarkably, it has been found that GSDMs are abnormally expressed in several forms of cancers. Moreover, GSDMs are involved in cancer cell growth, invasion, metastasis and chemoresistance. Additionally, increasing evidence has indicated an association between GSDMs and clinicopathological features in cancer patients. These findings suggest the feasibility of using GSDMs as prospective biomarkers for cancer diagnosis, therapeutic intervention and prognosis. Here, we review the progress in unveiling the characteristics and biological functions of GSDMs. We also focus on the implication and molecular mechanisms of GSDMs in cancer pathogenesis. Investigating the relationship between GSDMs and cancer biology could assist us to explore new therapeutic avenues for cancer prevention and treatment.

scholarly journals A review on phytochemical and ethnopharmacological studies of Ajuga Bracteosa Wall. Ex Benth.

2019 ◽  
Vol 9 (2) ◽  
pp. 489-492
Author(s):  
Tabasum Ali ◽  
Zahida Shah ◽  
Rabiah Bashir
Keyword(s):  
Therapeutic Potential ◽  
Iridoid Glycosides ◽  
Herbal Medicines ◽  
Review Article ◽  
Pharmacological Activities ◽  
Cardiotonic Activity ◽  
Traditional System ◽  
The Family ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Herbal medicines as the major remedy in traditional system of medicine have been used in medical practices since antiquity. The plants of genus Ajuga are evergreen, clump-forming rhizomatous perennial or annual herbaceous flowering species, with Ajuga being one of the 266 genera of the family Lamiaceae. There are at least 301 species of the genus Ajuga with many variations. Ajugabracteosa Wall. ex Benth (A. bracteosa) is an important medicinal plant of Himalaya regions. Medicinal potential is due to presence of of various pharmacologically active compounds such as neo-clerodane diterpenoids, flavonol glycosides, iridoid glycosides, ergosterol-5,8- endoperoxide and phytoecdysones. The aim of this review article was to gather information about A. bracteosa which is currently scattered in form of various publications. This review article tried to attract the attention from people for therapeutic potential of A. bracteosa. The present review comprises upto date information of,traditional uses, botanical aspects, active ingredients and pharmacological activities such as antitumor, antimicrobial, antimalarial, anti-inflammatory, cardiotonic activity, antiarthritic activity, antioxidant activity . A large variety of compounds have so far been isolated from Ajuga bracteosa. Keywords:  Ajuga bracteosa, Herbal medicines, pharmacological activities.

scholarly journals Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology

2016 ◽  
Vol 22 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Jing Wang ◽  
Pengfei Fang ◽  
Peter Chase ◽  
Sagi Tshori ◽  
Ehud Razin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
High Throughput Screening ◽  
Treatment Resistance ◽  
Dna Interaction ◽  
Pigment Formation ◽  
Assay Performance ◽  
Microphthalmia Transcription Factor ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z′ factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

scholarly journals The Important Role of N6-methyladenosine RNA Modification in Non-Small Cell Lung Cancer

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 440
Author(s):  
Yue Cheng ◽  
Meiqi Wang ◽  
Junliang Zhou ◽  
Huanhuan Dong ◽  
Shuqing Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cancer Progression ◽  
Cell Lung Cancer ◽  
Vital Role ◽  
Small Cell ◽  
Rna Modification ◽  
Biological Functions ◽  
Small Cell Lung ◽  
Diagnosis And Prognosis

N6-methyladenosine (m6A) is one of the most prevalent epigenetic modifications of eukaryotic RNA. The m6A modification is a dynamic and reversible process, regulated by three kinds of regulator, including m6A methyltransferases, demethylases and m6A-binding proteins, and this modification plays a vital role in many diseases, especially in cancers. Accumulated evidence has proven that this modification has a significant effect on cellular biological functions and cancer progression; however, little is known about the effects of the m6A modification in non-small cell lung cancer (NSCLC). In this review, we summarized how various m6A regulators modulate m6A RNA metabolism and demonstrated the effect of m6A modification on the progression and cellular biological functions of NSCLC. We also discussed how m6A modification affects the treatment, drug resistance, diagnosis and prognosis of NSCLC patients.

scholarly journals Structure of Mpro from COVID-19 virus and discovery of its inhibitors

2020 ◽  
Author(s):  
Zhenming Jin ◽  
Xiaoyu Du ◽  
Yechun Xu ◽  
Yongqiang Deng ◽  
Meiqin Liu ◽  
...  
Keyword(s):  
Drug Design ◽  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Screening Strategy ◽  
Drug Candidates ◽  
Approved Drugs ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds ◽  
Drug Leads

SUMMARYA new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1–4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

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