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1.
Nat Commun ; 14(1): 4812, 2023 08 09.
Article in English | MEDLINE | ID: mdl-37558654

ABSTRACT

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.


Subject(s)
Amino Acids, Branched-Chain , Thiophenes , Mice , Animals , Amino Acids, Branched-Chain/metabolism , Phosphorylation , Thiophenes/pharmacology , Oxidoreductases/metabolism
2.
J Biol Chem ; 299(3): 102959, 2023 03.
Article in English | MEDLINE | ID: mdl-36717078

ABSTRACT

The mammalian mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched-chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched-chain amino acids and branched-chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker class antihypertension drugs and angiotensin receptor blocker-like compounds were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and antihypertension.


Subject(s)
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) , Angiotensin Receptor Antagonists , Humans , 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/metabolism , Amino Acids, Branched-Chain/metabolism , Angiotensin Receptor Antagonists/pharmacology , Multienzyme Complexes/metabolism , Heart Failure , Hypertension
3.
Mol Metab ; 66: 101611, 2022 12.
Article in English | MEDLINE | ID: mdl-36220546

ABSTRACT

OBJECTIVE: Branched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements. METHODS: The rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density lipoprotein receptor null mouse model (Ldlr-/-). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice. RESULTS: A rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr-/- mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-mediated gene expression in mice. CONCLUSIONS: These data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a "fed signal" to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a "fasting signal" to prolong the catabolic state.


Subject(s)
Fatty Liver , PPAR alpha , Animals , Mice , 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/metabolism , Amino Acids, Branched-Chain/metabolism , Fasting , Mice, Knockout , RNA, Small Interfering
4.
PLoS One ; 13(7): e0200358, 2018.
Article in English | MEDLINE | ID: mdl-30044847

ABSTRACT

Neuroblastoma is the second most common childhood tumor. Survival is poor even with intensive therapy. In a search for therapies to neuroblastoma, we assessed the oncolytic potential of Zika virus. Zika virus is an emerging mosquito-borne pathogen unique among flaviviruses because of its association with congenital defects. Recent studies have shown that neuronal progenitor cells are likely the human target of Zika virus. Neuroblastoma has been shown to be responsive to infection. In this study, we show that neuroblastoma cells are widely permissive to Zika infection, revealing extensive cytopathic effects (CPE) and producing high titers of virus. However, a single cell line appeared poorly responsive to infection, producing undetectable levels of non-structural protein 1 (NS1), limited CPE, and low virus titers. A comparison of these poorly permissive cells to highly permissive neuroblastoma cells revealed a dramatic loss in the expression of the cell surface glycoprotein CD24 in poorly permissive cells. Complementation of CD24 expression in these cells led to the production of detectable levels of NS1 expression after infection with Zika, as well as dramatic increases in viral titers and CPE. Complementary studies using the Zika virus index strain and a north African isolate confirmed these phenotypes. These results suggest a possible role for CD24 in host cell specificity by Zika virus and offer a potential therapeutic target for its treatment. In addition, Zika viral therapy can serve as an adjunctive treatment for neuroblastoma by targeting tumor cells that can lead to recurrent disease and treatment failure.


Subject(s)
CD24 Antigen/metabolism , Neuroblastoma/metabolism , Neuroblastoma/therapy , Oncolytic Virotherapy , Zika Virus , Cell Line, Tumor , Cell Survival , Cytopathogenic Effect, Viral , Humans , Neuroblastoma/pathology , Zika Virus/isolation & purification
5.
Nanoscale ; 9(29): 10375-10387, 2017 Jul 27.
Article in English | MEDLINE | ID: mdl-28702620

ABSTRACT

In this study, several formulations of nanoceria and dextran-nanoceria with curcumin, each demonstrated to have anti-cancer properties, were synthesized and applied as treatment for human childhood neuroblastoma. The anti-cancer activities of these formulations were explored in neuroblastoma models of both MYCN-amplified and non-amplified cell lines. Ceria nanoparticles, coated with dextran and loaded with curcumin, were found to induce substantial cell death in neuroblastoma cells (up to a 2-fold and a 1.6-fold decrease in cell viability for MYCN-upregulated and normal expressing cell lines, respectively; *p < 0.05) while producing no or only minor toxicity in healthy cells (no toxicity at 100 µM; **p < 0.01). This formulation evokes prolonged oxidative stress, stabilizing HIF-1α, and inducing caspase-dependent apoptosis (up to a 2.4-fold increase over control; *p < 0.05). Overall, nano-therapeutic treatments showed a more pronounced effect in MYCN-amplified cells, which are traditionally more resistant to drug therapies. These results represent a very promising alternative to small molecule drug therapies for robust cancers.


Subject(s)
Apoptosis , Curcumin/administration & dosage , Drug Delivery Systems , Nanoparticles , Neuroblastoma/pathology , Cell Hypoxia , Cell Line, Tumor , Humans , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , bcl-2-Associated X Protein/metabolism
6.
Oncotarget ; 8(4): 6589-6607, 2017 Jan 24.
Article in English | MEDLINE | ID: mdl-28035057

ABSTRACT

The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death.


Subject(s)
Alternative Splicing , Apoptosis , BRCA1 Protein/genetics , Biomarkers, Tumor/genetics , Cell Cycle Checkpoints , Cell Proliferation , Neuroblastoma/genetics , RNA, Long Noncoding/genetics , Tumor Suppressor Protein p53/genetics , BRCA1 Protein/metabolism , Biomarkers, Tumor/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/metabolism , Neuroblastoma/pathology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , RNA, Long Noncoding/metabolism , Signal Transduction , Time Factors , Transfection , Tumor Suppressor Protein p53/metabolism
7.
ACS Chem Neurosci ; 5(12): 1253-65, 2014 Dec 17.
Article in English | MEDLINE | ID: mdl-25299732

ABSTRACT

Casein kinase 1 delta (CK1δ) and casein kinase 1 epsilon (CK1ε) inhibitors are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing a CNS kinase inhibitor has been limited by an inability to identify safe brain-penetrant compounds with high kinome selectivity. Guided by structure-based drug design, potent and selective CK1δ/ε inhibitors have now been identified that address this gap, through the design and synthesis of novel 4-[4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl]pyrimidin-2-amine derivatives. PF-5006739 (6) possesses a desirable profile, with low nanomolar in vitro potency for CK1δ/ε (IC50 = 3.9 and 17.0 nM, respectively) and high kinome selectivity. In vivo, 6 demonstrated robust centrally mediated circadian rhythm phase-delaying effects in both nocturnal and diurnal animal models. Further, 6 dose-dependently attenuated opioid drug-seeking behavior in a rodent operant reinstatement model in animals trained to self-administer fentanyl. Collectively, our data supports further development of 6 as a promising candidate to test the hypothesis of CK1δ/ε inhibition in treating multiple indications in the clinic.


Subject(s)
Analgesics, Opioid/administration & dosage , Casein Kinase I/antagonists & inhibitors , Drug-Seeking Behavior/drug effects , Enzyme Inhibitors/pharmacology , Fentanyl/administration & dosage , Analysis of Variance , Animals , Circadian Rhythm/drug effects , Conditioning, Operant/drug effects , Cross-Over Studies , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Humans , Isoxazoles/pharmacology , Macaca fascicularis , Mice , Mice, Inbred C57BL , Protein Transport/drug effects , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Reinforcement, Psychology , Self Administration , Time Factors
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