Dataset Information

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology.

ABSTRACT: The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.

SUBMITTER: Ladds MJGW

PROVIDER: S-EPMC7939445 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology.

Ladds Marcus J G W MJGW Popova Gergana G Pastor-Fernández Andrés A Kannan Srinivasaraghavan S van Leeuwen Ingeborg M M IMM Håkansson Maria M Walse Björn B Tholander Fredrik F Bhatia Ravi R Verma Chandra S CS Lane David P DP Laín Sonia S

The Journal of biological chemistry 20200908 52

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to ...[more]

PMID: 32900849

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Project description:Neuroblastoma is the most common heterogeneous solid tumor in childhood, with limited treatments available for high-risk patients. Previous studies have shown that Dihydroorotate dehydrogenase (DHODH) plays an essential role in the de novo synthesis of pyrimidine and have identified it as a potential therapeutic target in cancer. Our previous studies have shown that the higher expression of DHODH is associated with the worst survival of neuroblastoma patients. Through knockdown experiments, we validated that reducing DHODH expression in neuroblastoma cells leads to a decrease in cell viability. Subsequently, we harnessed virtual screening software to repurpose drugs, targeting DHODH as potential neuroblastoma therapeutics. We conducted molecular docking simulations with a database of 2702 FDA-approved drugs on DHODH and identified a number of drugs that demonstrated high affinity for DHODH. Among these drugs, we found regorafenib, a multi-kinase inhibitor, effectively inhibited neuroblastoma cell proliferation and was more efficient than leflunomide, an FDA-approved DHODH inhibitor. To determine whether regorafenib is a novel DHODH inhibitor, we employed thermal shift and enzyme fluorescence assays. These assays revealed that regorafenib not only enhanced binding stability but also reduced the enzymatic activity of DHODH. Since regorafenib is a multi-kinase inhibitor, we confirm changes in downstream proteins by comparison with shDHODH to determine whether these alterations result from targeting DHODH. To further investigate the molecular mechanism of regorafenib in targeting DHODH, we conducted a proteome analysis using tandem mass tag (TMT) labeling. This involved comparing samples treated with regorafenib to those with DHODH knockdown. Using Liquid Chromatograph-mass spectrometry/ mass spectrometry (LC-MS/MS), a total of 4471 proteins and 31518 peptides were identified. Furthermore, we identified 343 differentially expressed proteins in the shDHODH sample and 225 in the regorafenib treatment sample. We performed gene ontology (GO) enrichment analysis on these two sets of samples. The results of the GO enrichment analysis, focusing on biological function, indicated that proteins were correlated with lipid metabolism. After a research search, the proteome data and validation through western blot revealed common differentially expressed proteins related to the mevalonate pathway and subsequently linked to ferroptosis. Additionally, previous studies suggest a connection between DHODH and ferroptosis, leading us to investigate whether regorafenib treatment influences ferroptosis as confirmation that our drug targets DHODH. Moreover, neuroblastoma cells could be rescued by ferroptosis inhibitor liproxstatin-1. In summary, our findings highlight the potential of using regorafenib to target DHODH, presenting a promising therapeutic strategy for high-risk neuroblastoma.

Project description:Neuroblastoma is the most common heterogeneous solid tumor in childhood, with limited treatments available for high-risk patients. Previous studies have shown that Dihydroorotate dehydrogenase (DHODH) plays an essential role in the de novo synthesis of pyrimidine and have identified it as a potential therapeutic target in cancer. Our previous studies have shown that the higher expression of DHODH is associated with the worst survival of neuroblastoma patients. Through knockdown experiments, we validated that reducing DHODH expression in neuroblastoma cells leads to a decrease in cell viability. Subsequently, we harnessed virtual screening software to repurpose drugs, targeting DHODH as potential neuroblastoma therapeutics. We conducted molecular docking simulations with a database of 2702 FDA-approved drugs on DHODH and identified a number of drugs that demonstrated high affinity for DHODH. Among these drugs, we found Regorafenib, a multi-kinase inhibitor, effectively inhibited neuroblastoma cell proliferation and was more efficient than leflunomide, an FDA-approved DHODH inhibitor. To determine whether Regorafenib is a novel DHODH inhibitor, we employed thermal shift and enzyme fluorescence assays. These assays revealed that Regorafenib not only enhanced binding stability but also reduced the enzymatic activity of DHODH. Since Regorafenib is a multi-kinase inhibitor, we confirm changes in downstream proteins by comparison with shDHODH to determine whether these alterations result from targeting DHODH. To further investigate the molecular mechanism of Regorafenib in targeting DHODH, we conducted a proteome analysis using tandem mass tag (TMT) labeling. This involved comparing samples treated with Regorafenib to those with DHODH knockdown. Using Liquid Chromatograph-mass spectrometry/ mass spectrometry (LC-MS/MS), a total of 4471 proteins and 31518 peptides were identified. Furthermore, we identified 377 differentially expressed proteins in the shDHODH sample and 225 in the Regorafenib treatment sample. We performed gene ontology (GO) enrichment analysis on these two sets of samples. The results of the GO enrichment analysis indicate that these proteins are associated with lipid metabolism in terms of biological functions. Through literature searches, we found that the results from proteomics analysis and Western blot validation both show that the commonly differentially expressed proteins are related to the mevalonate pathway. This pathway, when affected, influences ferroptosis. Additionally, we discovered a relationship between DHODH inhibition and lipid droplet production. In summary, previous studies have shown a connection between DHODH and ferroptosis. However, we identified a new mechanism where DHODH inhibition induces ferroptosis by affecting the mevalonate pathway. Therefore, our findings underscore the potential of using Regorafenib to target DHODH, proposing a novel and promising therapeutic strategy for high-risk neuroblastoma.

Dataset Information

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology.

Publications

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology.

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