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1.
Proc Natl Acad Sci U S A ; 121(18): e2400752121, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38648484

ABSTRACT

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.


Subject(s)
Atherosclerosis , Endothelial Cells , Lamin Type A , Muscle, Smooth, Vascular , Progeria , Animals , Mice , Atherosclerosis/genetics , Atherosclerosis/metabolism , Atherosclerosis/pathology , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Lamin Type A/metabolism , Lamin Type A/genetics , Mice, Transgenic , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Progeria/metabolism , Progeria/genetics , Progeria/pathology , Proprotein Convertase 9/metabolism , Proprotein Convertase 9/genetics
2.
J Med Chem ; 64(8): 4623-4661, 2021 04 22.
Article in English | MEDLINE | ID: mdl-33818106

ABSTRACT

Targeting the protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1-Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound 8 with a 220-380-fold stronger affinity (Ki = 16 µM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with Ki values of 0.04-0.5 µM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand-protein interactions, and identify new potent inhibitors of the Keap1-Nrf2 PPI.


Subject(s)
Kelch-Like ECH-Associated Protein 1/antagonists & inhibitors , Small Molecule Libraries/chemistry , Binding Sites , Crystallography, X-Ray , Drug Stability , Humans , Kelch-Like ECH-Associated Protein 1/metabolism , Ligands , Magnetic Resonance Spectroscopy , Microsomes/metabolism , Molecular Dynamics Simulation , NF-E2-Related Factor 2/chemistry , NF-E2-Related Factor 2/metabolism , Protein Binding , Protein Interaction Maps/drug effects , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Surface Plasmon Resonance
3.
J Med Chem ; 62(17): 8028-8052, 2019 09 12.
Article in English | MEDLINE | ID: mdl-31411465

ABSTRACT

Inhibiting the protein-protein interaction (PPI) between the transcription factor Nrf2 and its repressor protein Keap1 has emerged as a promising strategy to target oxidative stress in diseases, including central nervous system (CNS) disorders. Numerous non-covalent small-molecule Keap1-Nrf2 PPI inhibitors have been reported to date, but many feature suboptimal physicochemical properties for permeating the blood-brain barrier, while others contain problematic structural moieties. Here, we present the first side-by-side assessment of all reported Keap1-Nrf2 PPI inhibitor classes using fluorescence polarization, thermal shift assay, and surface plasmon resonance-and further evaluate the compounds in an NQO1 induction cell assay and in counter tests for nonspecific activities. Surprisingly, half of the compounds were inactive or deviated substantially from reported activities, while we confirm the cross-assay activities for others. Through this study, we have identified the most promising Keap1-Nrf2 inhibitors that can serve as pharmacological probes or starting points for developing CNS-active Keap1 inhibitors.


Subject(s)
Kelch-Like ECH-Associated Protein 1/antagonists & inhibitors , NF-E2-Related Factor 2/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Kelch-Like ECH-Associated Protein 1/chemistry , Kelch-Like ECH-Associated Protein 1/metabolism , Models, Molecular , Molecular Structure , NF-E2-Related Factor 2/chemistry , NF-E2-Related Factor 2/metabolism , Protein Binding/drug effects , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Surface Plasmon Resonance
4.
DNA Repair (Amst) ; 54: 40-45, 2017 06.
Article in English | MEDLINE | ID: mdl-28460268

ABSTRACT

Non-homologous end joining (NHEJ) is the main mechanism for double strand break (DSB) DNA repair. The error-prone DNA polymerase mu (Polµ) is involved in immunoglobulin variable region rearrangement and in general, NHEJ in non-lymphoid cells. Deletion of NHEJ factors in P53-/- mice, which are highly prone to development of T cell lymphoma, generally increases cancer incidence and shifts the tumor spectrum towards aggressive pro-B lymphoma. In contrast, Polµ deletion increased sarcoma incidence, proportionally reducing pro-B lymphoma development on the P53-deficient background. Array comparative genomic hybridization (aCGH) analyses showed DNA copy number alterations in both P53-/- and Polµ-/-P53-/- lymphomas. Our results also indicate that the increase in sarcoma incidence in Polµ-/-P53-/- mice could be associated with Cdk4 and Kub3 amplification and overexpression. These results identify a role for Polµ in the prevention of sarcomagenesis on a murine P53-deficient background, in contrast to most other NHEJ factors.


Subject(s)
Carcinogenesis , DNA End-Joining Repair , DNA-Directed DNA Polymerase/genetics , Sarcoma/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Carrier Proteins/genetics , Cyclin-Dependent Kinase 4/genetics , DNA/metabolism , DNA Copy Number Variations , Gene Amplification , Gene Deletion , Genomic Instability , Lymphoma/genetics , Lymphoma/metabolism , Lymphoma/pathology , Mice , Mice, Knockout , Sarcoma/genetics , Sarcoma/pathology , Up-Regulation
5.
PLoS One ; 9(4): e93074, 2014.
Article in English | MEDLINE | ID: mdl-24691161

ABSTRACT

Polµ is an error-prone PolX polymerase that contributes to classical NHEJ DNA repair. Mice lacking Polµ (Polµ(-/-)) show altered hematopoiesis homeostasis and DSB repair and a more pronounced nucleolytic resection of some V(D)J junctions. We previously showed that Polµ(-/-) mice have increased learning capacity at old ages, suggesting delayed brain aging. Here we investigated the effect of Polµ(-/-) deficiency on liver aging. We found that old Polµ(-/-) mice (>20 month) have greater liver regenerative capacity compared with wt animals. Old Polµ(-/-) liver showed reduced genomic instability and increased apoptosis resistance. However, Polµ(-/-) mice did not show an extended life span and other organs (e.g., heart) aged normally. Our results suggest that Polµ deficiency activates transcriptional networks that reduce constitutive apoptosis, leading to enhanced liver repair at old age.


Subject(s)
Aging/pathology , DNA-Directed DNA Polymerase/deficiency , Liver/pathology , Oxidative Stress , Animals , Genomic Instability , Liver/physiopathology , Liver Function Tests , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Biological , Myocardium/pathology , Phenotype , Sister Chromatid Exchange
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