A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening

Caballero, Iván; Lafuente, María José; Gamo, Francisco‐Javier; Cid, Concepción

doi:10.1016/j.ab.2016.04.013

Cited by 8 publications

(5 citation statements)

References 30 publications

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“…Above this pH resorufin has higher fluorescence but enzyme is less active, while below this pH the enzyme is more active but due to protonation of resorufin fluorescence of that product is decreased. Similar results were obtained also during development of resazurin based assay for dihydroorotate dehydrogenase that also has an acidic pH optimum [38].…”

Section: Discussionsupporting

confidence: 80%

Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay

et al. 2019

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Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10 mM lactose and 0.08 mM cellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.

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Section: Discussionsupporting

confidence: 80%

Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay

et al. 2019

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“…Compounds 3 , 4a , and 5c were screened using the fluorescence-based assay to detect the fluorescent signal from the resorufin method by Reaction Biology Co. (, accessed on 27-12-2022). hDHODH catalyzes the oxidation of L-DHO to orotate, which is followed by resazurin catalysis to resorufin . Stop and detection step was employed for orotate addition to stop the reaction and detect the fluorescent signal from resorufin.…”

Section: Methodsmentioning

confidence: 99%

“…hDHODH catalyzes the oxidation of L-DHO to orotate, which is followed by resazurin catalysis to resorufin. 35 Stop and detection step was employed for orotate addition to stop the reaction and detect the fluorescent signal from resorufin. The assay involved two steps: (i) Enzymatic step for hDHODH catalyzes the oxidation of L-DHO to orotate and followed by resazurin catalyzed to resorufin.…”

Section: Methodsmentioning

confidence: 99%

Identification of Potent hDHODH Inhibitors for Lung Cancer via Virtual Screening of a Rationally Designed Small Combinatorial Library

et al. 2023

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Cancer is characterized by altered cellular metabolism, and metabolic enzymes are considered as a promising target for anticancer therapy. Pyrimidine metabolism dysregulation is associated with various types of cancer, particularly lung cancer, which is one of the leading causes of cancer-related mortality worldwide. Recent studies have shown that small-cell lung cancer cells are particularly reliant on the pyrimidine biosynthesis pathway and are sensitive to its disruption. DHODH, the rate-limiting enzyme of the de novo pyrimidine production pathway, is essential in the production of RNA and DNA and is overexpressed in malignancies such as AML, skin cancer, breast cancer, and lung cancer, thereby highlighting DHODH as a viable target for developing drugs to combat lung cancer. Herein, rational drug design and computational techniques were used to discover novel DHODH inhibitors. A small combinatorial library was generated, and the top hits were synthesized and tested for anticancer activity against three lung cancer cell lines. Among the tested compounds, compound 5c possessed a stronger cytotoxicity (TC 50 of 11 μM) compared to the standard FDA-approved drug (Regorafenib, TC 50 of 13 μM) on the A549 cell line. Furthermore, compound 5c demonstrated potent inhibitory activity against hDHODH at a nanomolar level of 421 nM. DFT, molecular docking, molecular dynamic simulations, and free energy calculations were also carried out to understand the inhibitory mechanisms of the synthesized scaffolds. These in silico studies identified key mechanisms and structural features that will be crucial for future studies.

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“…[53][54][55][56] A fluorescence intensity (FLINT) high-throughput assay to monitor the oxidation of L-DHO to orotate was also recently developed. 57 The assay was originally developed for…”

Section: General Introductionmentioning

confidence: 99%

“…Resazurin is a redox-sensitive fluorogenic dye that varies from a blue non-fluorescent state to a pink, highly fluorescent state upon reduction to resorufin, monitored at 590 nm ( Figure S1B, in appendices). 57 Considering this global introduction regarding DHODHs, we will describe Figure 4 illustrates the distribution of these species worldwide. 60 This disease affects over 206 million people (3.3 million DALYs) in more than 70 countries, including sub-Saharan Africa, the Middle East, Southwest Asia and parts of South America.…”

Section: General Introductionmentioning

confidence: 99%

Reposicionamento de drogas antimaláricas no tratamento da esquistossomose baseado na inibição seletiva da enzima diidroorotato desidrogenase

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A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening

Cited by 8 publications

References 30 publications

Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay

Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay

Identification of Potent hDHODH Inhibitors for Lung Cancer via Virtual Screening of a Rationally Designed Small Combinatorial Library

Reposicionamento de drogas antimaláricas no tratamento da esquistossomose baseado na inibição seletiva da enzima diidroorotato desidrogenase

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