Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Redox Biol ; 62: 102703, 2023 06.
Article in English | MEDLINE | ID: mdl-37087975

ABSTRACT

Ferroptosis is defined as cell death triggered by iron-dependent lipid peroxidation that is preventable by antioxidant compounds such as ferrostatin-1. Endogenous suppressors of ferroptosis include FSP-1 and the selenoprotein GPX4, the latter of which directly enzymatically reduces lipid hydroperoxides. Small molecules that trigger ferroptosis include RSL3, ML162, and ML210; these compounds are often used in studies of ferroptosis and are generally considered as GPX4 inhibitors. Here, we found that RSL3 and ML162 completely lack capacity of inhibiting the enzymatic activity of recombinant selenoprotein GPX4. Surprisingly, these compounds were instead found to be efficient inhibitors of another selenoprotein, TXNRD1. Other known inhibitors of TXNRD1, including auranofin, TRi-1 and TRi-2, are also efficient inducers of cell death but that cell death could not be suppressed with ferrostatin-1. Our results collectively suggest that prior studies using RSL3 and ML162 may need to be reevaluated in the context of ferroptosis with regards to additional enzyme targets and mechanisms of action that may be involved.


Subject(s)
Ferroptosis , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Cyclohexylamines/pharmacology , Lipid Peroxidation
2.
Science ; 379(6628): eabl3837, 2023 01 13.
Article in English | MEDLINE | ID: mdl-36634189

ABSTRACT

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.


Subject(s)
Arachnid Vectors , Host-Parasite Interactions , Ixodes , Janus Kinases , Receptors, Cytokine , STAT Transcription Factors , Animals , Interferon-gamma/metabolism , Ixodes/genetics , Ixodes/immunology , Janus Kinases/genetics , Janus Kinases/metabolism , Signal Transduction , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Host-Parasite Interactions/immunology , Receptors, Cytokine/metabolism , Arachnid Vectors/immunology
3.
Front Pharmacol ; 13: 1040039, 2022.
Article in English | MEDLINE | ID: mdl-36506591

ABSTRACT

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

4.
ACS Chem Biol ; 17(9): 2471-2482, 2022 09 16.
Article in English | MEDLINE | ID: mdl-36049119

ABSTRACT

Determining a molecule's mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein's unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered to withstand unfolding at temperatures greater than 90 °C and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.


Subject(s)
Biological Assay , Drug Discovery , Biological Assay/methods , Drug Discovery/methods , Lactate Dehydrogenases , Ligands
SELECTION OF CITATIONS
SEARCH DETAIL
...