Targeting WEE1 Kinase in Cancer Matheson, Christopher J; Backos, Donald S; Reigan, Philip
Trends in pharmacological sciences (Regular ed.),
10/2016, Letnik:
37, Številka:
10
Journal Article
Recenzirano
WEE1 kinase plays a crucial role in the G2–M cell-cycle checkpoint arrest for DNA repair before mitotic entry. Normal cells repair damaged DNA during G1 arrest; however, cancer cells often have a ...deficient G1–S checkpoint and depend on a functional G2–M checkpoint for DNA repair. WEE1 is expressed at high levels in various cancer types including breast cancers, leukemia, melanoma, and adult and pediatric brain tumors. Many of these cancers are treated with DNA-damaging agents; therefore, targeting WEE1 for inhibition and compromising the G2–M checkpoint presents an opportunity to potentiate therapy. In this review we summarize the current WEE1 inhibitors, the potential for further inhibitor development, and the challenges in the clinic for the WEE1 inhibitor strategy.
When nanoparticles are intravenously injected into the body, complement proteins deposit on the surface of nanoparticles in a process called opsonization. These proteins prime the particle for ...removal by immune cells and may contribute toward infusion-related adverse effects such as allergic responses. The ways complement proteins assemble on nanoparticles have remained unclear. Here, we show that dextran-coated superparamagnetic iron oxide core-shell nanoworms incubated in human serum and plasma are rapidly opsonized with the third complement component (C3) via the alternative pathway. Serum and plasma proteins bound to the nanoworms are mostly intercalated into the nanoworm shell. We show that C3 covalently binds to these absorbed proteins rather than the dextran shell and the protein-bound C3 undergoes dynamic exchange in vitro. Surface-bound proteins accelerate the assembly of the complement components of the alternative pathway on the nanoworm surface. When nanoworms pre-coated with human plasma were injected into mice, C3 and other adsorbed proteins undergo rapid loss. Our results provide important insight into dynamics of protein adsorption and complement opsonization of nanomedicines.
We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by ...immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.
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•Lysine glutarylation is a protein posttranslational modification•SIRT5 can catalyze the enzymatic removal of lysine glutarylation•Proteomic analyses identify a link between lysine glutarylation and metabolism•Glutarylation suppresses CPS1 activity, which is targeted by SIRT5 for removal
Tan et al. report a new type of evolutionarily conserved posttranslational modification, lysine glutarylation, targeted by SIRT5 which impacts metabolic processes. CPS1, the rate-limiting enzyme in urea cycle, is suppressed by glutarylation in glutaric academia type I disease and is targeted by SIRT5 for deglutarylation.
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates the expression of genes related to cell cycle, cell survival, and immune response associated with ...cancer progression and malignancy in a number of cancer types. Once activated, STAT3 forms a homodimer and translocates to the nucleus where it binds DNA promoting the translation of target genes associated with antiapoptosis, angiogenesis, and invasion/migration. In normal cells, levels of activated STAT3 remain transient; however, STAT3 remains constitutively active in approximately 70% of human solid tumors. The pivotal role of STAT3 in tumor progression has promoted a campaign in drug discovery to identify small molecules that disrupt the function of STAT3. A range of approaches have been used to identify novel small molecule inhibitors of STAT3, including high-throughput screening of chemical libraries, computational-based virtual screening, and fragment-based design strategies. The most common approaches in targeting STAT3 activity are either via the inhibition of tyrosine kinases capable of phosphorylating and thereby activating STAT3 or by preventing the formation of functional STAT3 dimers through disruption of the SH2 domains. However, the targeting of the STAT3 DNA-binding domain and disruption of binding of STAT3 to its DNA promoter have not been thoroughly examined, mainly due to the lack of adequate assay systems. This review summarizes the development of STAT3 inhibitors organized by the approach used to inhibit STAT3, the current inhibitors of each class, and the assay systems used to evaluate STAT3 inhibition and offers an insight into future approaches for small molecule STAT3 inhibitor development.
The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that ...undergo intramolecular catalysis to form reactive intermediates that non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the "acylomes" of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins and define a new regulatory paradigm in metabolism.
The glutathione (GSH) biosynthetic pathway in brain tumors. (A) Most brain tumors arise from 3 main cell types or their progenitors: neurons (green), oligodendrocytes (cyan), and astrocytes ...(magenta). (B) GSH biosynthesis consists of two ATP-dependent reactions: (1) glutamate cysteine ligase (GCL)-mediated formation of γ-glutamyl-cysteine (γGluCys) from glutamate (Glu) and cysteine (Cys) and (2) GSH synthetase (GS)-catalyzed formation of GSH from γGluCys and glycine (Gly). Buthionine sulfoximine (BSO) is an irreversible inhibitor of GCL. Detoxification of chemotherapeutic agents proceeds via the GSH-S-transferase (GST)-mediated formation of GSH-drug conjugates (GSH-X) followed by efflux by multidrug resistance protein (Mrp) transporters. γ-Glutamyl transpeptidase (γGT) can break GSH down into Glu and cysteinylglycine (CysGly). CysGly is further hydrolyzed by cellular dipeptidases (DP) followed by the transporter-mediated uptake of the constituent amino acids that can serve as substrates for either cellular GSH production or protein synthesis. Additional Cys may also be obtained via the glutamate-cystine antiporter (Xc-)-mediated uptake of extracellular cystine (CysCys).
Chemotherapy is central to the current treatment modality for primary human brain tumors, but despite high-dose and intensive treatment regimens there has been little improvement in patient outcome. The development of tumor chemoresistance has been proposed as a major contributor to this lack of response. While there have been some improvements in our understanding of the molecular mechanisms underlying brain tumor drug resistance over the past decade, the contribution of glutathione (GSH) and the GSH-related enzymes to drug resistance in brain tumors have been largely overlooked. GSH constitutes a major antioxidant defense system in the brain and together with the GSH-related enzymes plays an important role in protecting cells against free radical damage and dictating tumor cell response to adjuvant cancer therapies, including irradiation and chemotherapy. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferases (GST), and GSH complex export transporters (GS-X pumps) are major components of the GSH-dependent enzyme system that function in a dynamic cascade to maintain redox homeostasis. In many tumors, the GSH system is often dysregulated, resulting in a more drug resistant phenotype. This is commonly associated with GST-mediated GSH conjugation of various anticancer agents leading to the formation of less toxic GSH–drug complexes, which can be readily exported from the cell. Advances in our understanding of the mechanisms of drug resistance and patient selection based on biomarker profiles will be crucial to adapt therapeutic strategies and improve outcomes for patients with primary malignant brain tumors.
Redox modulation of NQO1 Siegel, David; Dehn, Donna D; Bokatzian, Samantha S ...
PloS one,
01/2018, Letnik:
13, Številka:
1
Journal Article
Recenzirano
Odprti dostop
NQO1 is a FAD containing NAD(P)H-dependent oxidoreductase that catalyzes the reduction of quinones and related substrates. In cells, NQO1 participates in a number of binding interactions with other ...proteins and mRNA and these interactions may be influenced by the concentrations of reduced pyridine nucleotides. NAD(P)H can protect NQO1 from proteolytic digestion suggesting that binding of reduced pyridine nucleotides results in a change in NQO1 structure. We have used purified NQO1 to demonstrate the addition of NAD(P)H induces a change in the structure of NQO1; this results in the loss of immunoreactivity to antibodies that bind to the C-terminal domain and to helix 7 of the catalytic core domain. Under normal cellular conditions NQO1 is not immunoprecipitated by these antibodies, however, following treatment with β-lapachone which caused rapid oxidation of NAD(P)H NQO1 could be readily pulled-down. Similarly, immunostaining for NQO1 was significantly increased in cells following treatment with β-lapachone demonstrating that under non-denaturing conditions the immunoreactivity of NQO1 is reflective of the NAD(P)+/NAD(P)H ratio. In untreated human cells, regions with high intensity immunostaining for NQO1 co-localize with acetyl α-tubulin and the NAD+-dependent deacetylase Sirt2 on the centrosome(s), the mitotic spindle and midbody during cell division. These data provide evidence that during the centriole duplication cycle NQO1 may provide NAD+ for Sirt2-mediated deacetylation of microtubules. Overall, NQO1 may act as a redox-dependent switch where the protein responds to the NAD(P)+/NAD(P)H redox environment by altering its structure promoting the binding or dissociation of NQO1 with target macromolecules.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondrial oxidative stress and damage have been implicated in the etiology of temporal lobe epilepsy, but whether or not they have a functional impact on mitochondrial processes during epilepsy ...development (epileptogenesis) is unknown. One consequence of increased steady-state mitochondrial reactive oxygen species levels is protein post-translational modification (PTM). We hypothesize that complex I (CI), a protein complex of the mitochondrial electron transport chain, is a target for oxidant-induced PTMs, such as carbonylation, leading to impaired function during epileptogenesis. The goal of this study was to determine whether oxidative modifications occur and what impact they have on CI enzymatic activity in the rat hippocampus in response to kainate (KA)-induced epileptogenesis. Rats were injected with a single high dose of KA or vehicle and evidence for CI modifications was measured during the acute, latent, and chronic stages of epilepsy. Mitochondrial-specific carbonylation was increased acutely (48 h) and chronically (6 week), coincident with decreased CI activity. Mass spectrometry analysis of immunocaptured CI identified specific metal catalyzed carbonylation to Arg76 within the 75 kDa subunit concomitant with inhibition of CI activity during epileptogenesis. Computational-based molecular modeling studies revealed that Arg76 is in close proximity to the active site of CI and carbonylation of the residue is predicted to induce substantial structural alterations to the protein complex. These data provide evidence for the occurrence of a specific and irreversible oxidative modification of an important mitochondrial enzyme complex critical for cellular bioenergetics during the process of epileptogenesis.
Isoxazolo3,4-d pyridazinones (3,4-ds) were previously shown to have selective positive modulation at the metabotropic glutamate receptor (mGluR) Subtypes 2 and 4, with no functional cross-reactivity ...at mGluR1a, mGluR5, or mGluR8. Additional analogs were prepared to access more of the allosteric pocket and achieve higher binding affinity, as suggested by homology modeling. Two different sets of analogs were generated. One uses the fully formed 3,4-d with an N6-aryl with and without halogens. These underwent successful selective lateral metalation and electrophilic quenching (LM&EQ) at the C3 of the isoxazole. In a second set of analogs, a phenyl group was introduced at the C4 position of the 3,4-d ring via a condensation of 4-phenylacetyl-3-ethoxcarbonyl-5-methyl isoxazole with the corresponding hydrazine to generate the 3,4-ds 2b and 2j to 2n.
Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone ...deacylases that use NAD+ as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD+/NADH ratio, but it has not been formally tested for all the mammalian sirtuins in vitro. To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C1) to palmitoyl (C16) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, “long chain” acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1–3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD+-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD+/NADH ratio.
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