scholarly journals Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies

2019 ◽  
Vol 476 (15) ◽  
pp. 2209-2219 ◽  
Author(s):  
Catherine E. Scull ◽  
Yinfeng Zhang ◽  
Nichole Tower ◽  
Lynn Rasmussen ◽  
Indira Padmalayam ◽  
...  
Keyword(s):  
Cell Growth ◽  
High Throughput ◽  
High Throughput Screening ◽  
Ribosome Biogenesis ◽  
Rrna Synthesis ◽  
Synthesis Rate ◽  
Screening Strategies ◽  
Secondary Screen ◽  
Cell Growth And Proliferation ◽  
High Throughput Screens

Abstract Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

scholarly journals The trials and tribulations of a robotic screening core

1995 ◽  
Vol 17 (2) ◽  
pp. 55-58 ◽  
Author(s):  
John Babiak ◽  
Brian Lucotch ◽  
Anthony Russo ◽  
Linda Heydt ◽  
Sharon Williams ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Customer Relations ◽  
Chemical Compounds ◽  
Screen Design ◽  
Therapeutic Benefits ◽  
Primary Means ◽  
Effective Use ◽  
High Throughput Screens ◽  
Time Frames

It is well recognized within the pharmaceutical industry that high throughput screening is a valuable and rapid tool to identify novel chemical compounds that may lead to tomorrow's drugs. High throughput screening involves testing as many chemical compounds as quickly as possible against a defined molecular or cellular ‘target’ (for example an enzyme) in the hope that interacting compounds may provide significant therapeutic benefits.At Wyeth-Ayerst Research, a Robotics and Automation Research Core Group has been established which serves as the in-house resource for high throughput screening. The robotics group has three missions: (1) develop and perform high throughput screens for customers in all therapeutic departments in the company; (2) educate customers in issues related to screen design; and (3) help customers to bring automated workstations into their laboratories. The mission, therefore, requires the effective use of automation, as well as building a strong collaboration with customers.The challenges that have been faced fall into two categories: technology limiting and customer relations. Technological challenges arise because it is necessary to develop and implement assays with very different formats and biochemical endpoints within extremely shortened time frames. The primary means to meet these challenges is with flexible robotics and flexible people. Challenges in the area of customer relations include setting realistic expectations, maintaining a sense of collaboration (and not merely service), educating investigators as to how to deal with the huge amount of data generated and seeking feedback. Effective and frequent communication, and an awareness of each individual's perspective, are essential to provide the most appropriate service.

scholarly journals A High Throughput Screen for Inhibitors of Fungal Cell Wall Synthesis

2002 ◽  
Vol 7 (4) ◽  
pp. 359-366 ◽  
Author(s):  
Jonathan M. Evans ◽  
Phillip G. Zaworski ◽  
Christian N. Parker
Keyword(s):  
Cell Wall ◽  
Cell Growth ◽  
High Throughput ◽  
High Throughput Screening ◽  
Osmotic Shock ◽  
Fungal Cell Wall ◽  
Cell Wall Synthesis ◽  
Fungal Cell ◽  
Unique Nature ◽  
Pharmacologically Active

Fungal cell wall synthesis is essential for viability, requiring the activity of genes involved in environmental sensing, precursor synthesis, transport, secretion, and assembly. This multitude of potential targets, the availability of known agents targeting this pathway, and the unique nature of fungal cell wall synthesis make this pathway an appealing target for drug discovery. Here we describe the adaptation of an assay monitoring cell wall synthesis for high-throughput screening. The assay requires fungal cell growth, in the presence of the test compound, for 3 h before the cells are subjected to osmotic shock in the presence of a dye that stains DNA. Miniaturization of the assay to a 384-well plate format and removing a mechanical transfer led to subtle changes in the assay characteristics. Validation of the assay with a library of known pharmacologically active agents has identified a number of different classes of compounds that are active in this assay, causing aberrant cell wall morphology and in many cases the inhibition of fungal cell growth.

scholarly journals High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

2020 ◽  
Author(s):  
Jicong Cao ◽  
Eva Maria Novoa ◽  
Zhizhuo Zhang ◽  
William C.W. Chen ◽  
Dianbo Liu ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Viral Gene ◽  
Cmv Promoter ◽  
Position Effects ◽  
Gene Therapies ◽  
Integration Strategy ◽  
Combinatorial Assembly ◽  
High Throughput Screens

ABSTRACTDespite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5’ untranslated regions (5’ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5’UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5’ UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5’ UTRs. A total of ∼12,000 5’ UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identified three synthetic 5’ UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5’ UTRs enabled even higher protein expression than obtained with each individual 5’ UTR. In summary, we demonstrate that high-throughput screening of 5’ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

scholarly journals A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc

Development ◽  
1998 ◽  
Vol 125 (18) ◽  
pp. 3571-3584 ◽  
Author(s):  
S. Zaffran ◽  
A. Chartier ◽  
P. Gallant ◽  
M. Astier ◽  
N. Arquier ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Ribosome Biogenesis ◽  
Protein Biosynthesis ◽  
Ectopic Expression ◽  
Rna Helicase ◽  
Potential Target ◽  
Dead Box ◽  
First Instar ◽  
Cell Growth And Proliferation

This article describes the characterization of a new Drosophila gene that we have called pitchoune (pit) (meaning small in Provence) because mutations in this gene produce larvae that cannot grow beyond the first instar larval stage although they can live as long as 7–10 days. All the tissues are equally affected and the perfectly shaped larvae are indistinguishable from first instar wild-type animals. Analysis of mutant somatic clones suggests a function in cell growth and proliferation, which is supported by the fact that cell proliferation is promoted by pit overexpression. Tagged-Pit, when transfected in S2 cells, localizes mainly to the nucleolus, pointing towards a possible role in ribosome biogenesis and, consequently, in protein biosynthesis. pit encodes a DEAD-box RNA helicase, a family of proteins involved in the control of RNA structure in many cellular processes and its closest homologue is a human DEAD-box RNA helicase, MrDb, whose corresponding gene transcription is directly activated by Myc-Max heterodimers (Grandori, C., Mac, J., Siebelt, F., Ayer, D. E. and Eisenman, R. N. (1996) EMBO J. 15, 4344–4357). The patterns of expression of d-myc and pit are superimposable. Ectopic expression of myc in the nervous system drives an ectopic expression of pit in this tissue indicating that in Drosophila as well, pit is a potential target of d-Myc. These results suggest that myc might promote cell proliferation by activating genes that are required in protein biosynthesis, thus linking cell growth and cell proliferation.

scholarly journals TGFβ/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands

Open Biology ◽  
2017 ◽  
Vol 7 (1) ◽  
pp. 160258 ◽  
Author(s):  
Torcato Martins ◽  
Nadia Eusebio ◽  
Andreia Correia ◽  
Joana Marinho ◽  
Fernando Casares ◽  
...  
Keyword(s):  
Cell Growth ◽  
Salivary Glands ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Growth Control ◽  
Tissue Growth ◽  
Cellular Growth ◽  
Nucleolar Structure ◽  
Cell Growth And Proliferation ◽  
Tgfβ Superfamily

Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila , the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis.

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