Covalent Inhibition by a Natural Product-Inspired Latent Electrophile.

Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.

Potent Inhibition of Thioredoxin Reductase by the Rh Derivatives of Anticancer M(arene/Cp*)(NHC)Cl2 Complexes.

Metal complexes provide a versatile platform to develop novel anticancer pharmacophores, and they form stable compounds with N-heterocyclic carbene (NHC) ligands, some of which have been shown to inhibit the cancer-related selenoenzyme thioredoxin reductase (TrxR). To expand a library of isostructural NHC complexes, we report here the preparation of RhIII- and IrIII(Cp*)(NHC)Cl2 (Cp* = η5-pentamethylcyclopentadienyl) compounds and comparison of their properties to the RuII- and OsII(cym) analogues (cym = η6-p-cymene). Like the RuII- and OsII(cym) complexes, the RhIII- and IrIII(Cp*) derivatives exhibit cytotoxic activity with half maximal inhibitory concentration (IC50) values in the low micromolar range against a set of four human cancer cell lines. In studies on the uptake and localization of the compounds in cancer cells by X-ray fluorescence microscopy, the Ru and Os derivatives were shown to accumulate in the cytoplasmic region of treated cells. In an attempt to tie the localization of the compounds to the inhibition of the tentative target TrxR, it was surprisingly found that only the Rh complexes showed significant inhibitory activity at IC50 values of ∼1 µM, independent of the substituents on the NHC ligand. This indicates that, although TrxR may be a potential target for anticancer metal complexes, it is unlikely the main target or the sole target for the Ru, Os, and Ir compounds described here, and other targets should be considered. In contrast, Rh(Cp*)(NHC)Cl2 complexes may be a scaffold for the development of TrxR inhibitors.

Correction to: Selenite-mediated production of superoxide radical anions in A549 cancer cells is accompanied by a selective increase in SOD1 concentration, enhanced apoptosis and Se-Cu bonding.

In the original article there were errors in the methods section. Thus, within Table 1: (i) the primer sequence pair for SOD-2 was incorrectly cited; (ii) the primer sequence pair used for SOD 1 was incorrect and did not target the gene of interest. Additional experiments were performed with correctly designed SOD1 primer pair and the outcomes documented here.

Inhibition of Copper Transport Induces Apoptosis in Triple-Negative Breast Cancer Cells and Suppresses Tumor Angiogenesis.

Treatment of advanced breast cancer remains challenging. Copper and some of the copper-dependent proteins are emerging therapeutic targets because they are essential for cell proliferation and survival, and have been shown to stimulate angiogenesis and metastasis. Here, we show that DCAC50, a recently developed small-molecule inhibitor of the intracellular copper chaperones, ATOX1 and CCS, reduces cell proliferation and elevates oxidative stress, triggering apoptosis in a panel of triple-negative breast cancer (TNBC) cells. Inhibition of ATOX1 activity with DCAC50 disrupts copper homeostasis, leading to increased copper levels, altered spatial copper redistribution, and accumulation of ATP7B to the cellular perinuclear region. The extent and impact of this disruption to copper homeostasis vary across cell lines and correlate with cellular baseline copper and glutathione levels. Ultimately, treatment with DCAC50 attenuates tumor growth and suppresses angiogenesis in a xenograft mouse model, and prevents endothelial cell network formation in vitro Co-treatment with paclitaxel and DCAC50 enhances cytotoxicity in TNBC and results in favorable dose reduction of both drugs. These data demonstrate that inhibition of intracellular copper transport targets tumor cells and the tumor microenvironment, and is a promising approach to treat breast cancer.

Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide Dismutase (SOD) Mimetics: Fluorescently Labeled MnSOD Mimetics, X-ray Absorption Spectroscopy, and X-ray Fluorescence Microscopy Studies.

Manganese(II) pentaazamacrocyclic complexes (MnPAMs) can act as small-molecule mimics of manganese superoxide dismutase (MnSOD) with potential therapeutic application in conditions linked to oxidative stress. Previously, the in vitro mechanism of action has been determined, their activity has been demonstrated in cells, and some representatives of this class of MnSOD mimetics have entered clinical trials. However, MnPAM uptake, distribution, and metabolism in cells are largely unknown. Therefore, we have used X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS) to study the cellular fate of a number of MnPAMs. We have also synthesized and characterized fluorescently labeled (pyrene and rhodamine) manganese(II) pyane [manganese(II) trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene] derivatives and investigated their utility for cellular imaging of MnPAMs. Their SOD activity was determined via a direct stopped-flow technique. XFM experiments show that treatment with amine-based manganese(II) pyane type pentaazamacrocycles leads to a 10-100-fold increase in the overall cellular manganese levels compared to the physiological levels of manganese in control cells. In treated cells in general, manganese was distributed throughout the cell body, with a couple of notable exceptions. The lipophilicity of the MnPAMs, examined by partitioning in octanol-buffer system, was a good predictor of the relative cellular manganese levels. Analysis of the XAS data of treated cells revealed that some fraction of amine-based MnPAMs taken up by the cells remained intact, with the rest transformed into SOD-active manganese(II) phosphate. Higher phosphate binding constants, determined from the effect of the phosphate concentration on in vitro SOD activity, were associated with more extensive metabolism of the amine-based MnPAMs to manganese(II) phosphate. In contrast, the imine-based manganese(II) pydiene complex that is prone to hydrolysis was entirely decomposed after uptake and free manganese(II) was oxidized to a manganese(III) oxide type species, in cytosolic compartments, possibly mitochondria. Complex stability constants (determined for some of the MnPAMs) are less indicative of the cellular fate of the complexes than the corresponding phosphate binding constants.

Developing drugs targeting transition metal homeostasis.

Metal dyshomeostasis is involved in the pathogenesis and progression of diseases including cancer and neurodegenerative diseases. Metal chelators and ionophores are well known modulators of transition metal homeostasis, and a number of these molecules are in clinical trials. Metal-binding compounds are not the only drugs capable of targeting transition metal homeostasis. This review presents recent highlights in the development of chelators and ionophores for the treatment of cancer and neurodegenerative disease. Moreover, we discuss the development of small molecules that alter copper and iron homeostasis by inhibiting metal transport proteins. Finally, we consider the emergence of metal regulatory factor 1 as a drug target in diseases where it mediates zinc-induced signalling cascades leading to pathogenesis.

XAS studies of Se speciation in selenite-fed rats.

The biological activity of selenium is dependent on its chemical form. Therefore, knowledge of Se chemistry in vivo is required for efficacious use of selenium compounds in disease prevention and treatment. Using X-ray absorption spectroscopy, Se speciation in the kidney, liver, heart, spleen, testis and red blood cells of rats fed control (∼0.3 ppm Se) or selenite-supplemented (1 ppm or 5 ppm Se) diets for 3 or 6 weeks, was investigated. X-ray absorption spectroscopy revealed the presence of Se-Se and Se-C species in the kidney and liver, and Se-S species in the kidney, but not the liver. X-ray absorption near edge structure (XANES) spectra showed that there was variation in speciation in the liver and kidneys, but Se speciation was much more uniform in the remaining organs. Using principal component analysis (PCA) to interpret the Se K-edge X-ray absorption spectra, we were able to directly compare the speciation of Se in two different models of selenite metabolism--human lung cancer cells and rat tissues. The effects of Se dose, tissue type and duration of diet on selenium speciation in rat tissues were investigated, and a relationship between the duration of the diet (3 weeks versus 6 weeks) and selenium speciation was observed.

XAS and XFM studies of selenium and copper speciation and distribution in the kidneys of selenite-supplemented rats.

Dietary selenium has been implicated in the prevention of cancer and other diseases, but its safety and efficacy is dependent on the supplemented form and its metabolites. In this study, X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM) have been used to investigate the speciation and distribution of Se and Cu in vivo. In kidneys isolated from rats fed a diet containing 5 ppm Se as selenite for 3 weeks, Se levels increased 5-fold. XFM revealed a strong correlation between the distribution of Se and the distribution of Cu in the kidney, a phenomenon that has previously been observed in cell culture (Weekley et al., JBIC, J. Biol. Inorg. Chem., 2014, DOI: 10.1007/s00775-014-1113-x). However, X-ray absorption spectra suggest that most of the Se in the kidney is found as Se-Se species, rather than Cu-bound, and that most of the Cu is bound to S and N, presumably to amino acid residues in proteins. Furthermore, SOD1 expression did not change in response to the high Se diet. We cannot rule out the possibility of some Cu-Se bonding in the tissues, but our results suggest mechanisms other than the formation of Cu-Se species and SOD1 upregulation are responsible for the highly correlated distributions of Se and Cu in the kidneys of rats fed high selenite diets.

Selenite-mediated production of superoxide radical anions in A549 cancer cells is accompanied by a selective increase in SOD1 concentration, enhanced apoptosis and Se-Cu bonding.

Selenite may exert its cytotoxic effects against cancer cells via the generation of reactive oxygen species (ROS). We investigated sources of, and the cellular response to, superoxide radical anion (O2 (·-)) generated in human A549 lung cancer cells after treatment with selenite. A temporal delay was observed between selenite treatment and increases in O2 (·-) production and biomarkers of apoptosis/necrosis, indicating that the reduction of selenite by the glutathione reductase/NADPH system (yielding O2 (·-)) is a minor contributor to ROS production under these conditions. By contrast, mitochondrial and NADPH oxidase O2 (·-) generation were the major contributors. Treatment with a ROS scavenger [poly(ethylene glycol)-conjugated superoxide dismutase (SOD) or sodium 4,5-dihydroxybenzene-1,3-disulfonate] 20 h after the initial selenite treatment inhibited both ROS generation and apoptosis determined at 24 h. In addition, SOD1 was selectively upregulated and its perinuclear cytoplasmic distribution was colocalised with the cellular distribution of selenium. Interestingly, messenger RNA for manganese superoxide dismutase, catalase, inducible haem oxygenase 1 and glutathione peroxidase either remained unchanged or showed a delayed response to selenite treatment. Colocalisation of Cu and Se in these cells (Weekley et al. in J. Am. Chem. Soc. 133:18272-18279, 2011) potentially results from the formation of a Cu-Se species, as indicated by Cu K-edge extended X-ray absorption fine structure spectra. Overall, SOD1 is upregulated in response to selenite-mediated ROS generation, and this likely leads to an accumulation of toxic hydrogen peroxide that is temporally related to decreased cancer cell viability. Increased expression of SOD1 gene/protein coupled with formation of a Cu-Se species may explain the colocalisation of Cu and Se observed in these cells.

Which form is that? The importance of selenium speciation and metabolism in the prevention and treatment of disease.

The biological activity of selenium is dependent upon its speciation. We aim to integrate selenium speciation and metabolism into a discussion of the mechanisms by which selenium exerts its biological activity. First, we present the current status of selenium in the prevention of cancer, cardiovascular and neurodegenerative diseases with particular attention paid to the results of major chemoprevention trials involving selenium supplementation. A comprehensive review of the current understanding of the metabolism of common dietary selenium compounds - selenite, selenomethionine, methylselenocysteine and selenocystine - is presented, with discussion of the evidence for the various metabolic pathways and their products. The antioxidant, prooxidant and other mechanisms of the dietary selenium compounds have been linked to their disease prevention and treatment properties. The evidence for these various mechanisms -in vitro, in cells and in vivo- is evaluated with emphasis on the selenium metabolites involved. We conclude that dietary selenium compounds should be considered prodrugs, whose biological activity will depend on the activity of the various metabolic pathways in, and the redox status of, cells and tissues. These factors should be considered in future laboratory research and in selecting selenium compounds for trials of disease prevention and treatment by selenium supplementation.

Selenium metabolism in cancer cells: the combined application of XAS and XFM techniques to the problem of selenium speciation in biological systems.

Determining the speciation of selenium in vivo is crucial to understanding the biological activity of this essential element, which is a popular dietary supplement due to its anti-cancer properties. Hyphenated techniques that combine separation and detection methods are traditionally and effectively used in selenium speciation analysis, but require extensive sample preparation that may affect speciation. Synchrotron-based X-ray absorption and fluorescence techniques offer an alternative approach to selenium speciation analysis that requires minimal sample preparation. We present a brief summary of some key HPLC-ICP-MS and ESI-MS/MS studies of the speciation of selenium in cells and rat tissues. We review the results of a top-down approach to selenium speciation in human lung cancer cells that aims to link the speciation and distribution of selenium to its biological activity using a combination of X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM). The results of this approach highlight the distinct fates of selenomethionine, methylselenocysteine and selenite in terms of their speciation and distribution within cells: organic selenium metabolites were widely distributed throughout the cells, whereas inorganic selenium metabolites were compartmentalized and associated with copper. New data from the XFM mapping of electrophoretically-separated cell lysates show the distribution of selenium in the proteins of selenomethionine-treated cells. Future applications of this top-down approach are discussed.

Selenium inhibits renal oxidation and inflammation but not acute kidney injury in an animal model of rhabdomyolysis.

UNLABELLED: Acute kidney injury (AKI) is a manifestation of rhabdomyolysis (RM). Extracellular myoglobin accumulating in the kidney after RM promotes oxidative damage, which is implicated in AKI. AIM: To test whether selenium (Se) supplementation diminishes AKI and improves renal function. RESULTS: Dietary selenite increased Se in the renal cortex, as demonstrated by X-ray fluorescence microscopy. Experimental RM-stimulated AKI as judged by increased urinary protein/creatinine, clusterin, and kidney injury molecule-1 (KIM-1), decreased creatinine clearance (CCr), increased plasma urea, and damage to renal tubules. Concentrations of cholesterylester (hydro)peroxides and F2-isoprostanes increased in plasma and renal tissues after RM, while aortic and renal cyclic guanidine monophosphate (cGMP; marker of nitric oxide (NO) bioavailability) decreased. Renal superoxide dismutase-1, phospho-P65, TNFα gene, MCP-1 protein, and the 3-chloro-tyrosine/tyrosine ratio (Cl-Tyr/Tyr; marker of neutrophil activation) all increased after RM. Dietary Se significantly decreased renal lipid oxidation, phospho-P65, TNFα gene expression, MCP-1 and Cl-Tyr/Tyr, improved NO bioavailability in aorta but not in the renal microvasculature, and inhibited proteinuria. However, CCr, plasma urea and creatinine, urinary clusterin, and histopathological assessment of AKI remained unchanged. Except for the Se++ group, renal angiotensin-receptor-1/2 gene/protein expression increased after RM with parallel increases in MEK1/2 inhibitor-sensitive MAPkinase (ERK) activity. INNOVATION: We employed synchrotron radiation to identify Se distribution in kidneys, in addition to assessing reno-protection after RM. CONCLUSION: Se treatment has some potential as a therapeutic for AKI as it inhibits oxidative damage and inflammation and decreases proteinuria, albeit histopathological changes to the kidney and some plasma and urinary markers of AKI remain unaffected after RM.

Distinct cellular fates for KP1019 and NAMI-A determined by X-ray fluorescence imaging of single cells.

Small molecule ruthenium complexes show great promise as anticancer pharmaceuticals, but further rational development of these as drugs is stymied by an incomplete understanding of the mechanisms that give rise to markedly different biological behaviour for structurally similar species. X-ray fluorescence imaging at two incident energies was used to reveal the intracellular distribution of Ru in single human cells treated with KP1019, showing Ru localised in both cytosol and in the nuclear region. In addition the imaging showed that treatment with KP1019 modulated Fe distribution to resemble the Ru distribution, without affecting cellular Fe content. In stark contrast, Ru could not be visualised in cells treated with NAMI-A, indicating that it was not internalised and supporting the proposition that its activity is exerted through a membrane-binding mechanism.

Methylselenocysteine treatment leads to diselenide formation in human cancer cells: evidence from X-ray absorption spectroscopy studies.

The selenoamino acids methylselenocysteine (MeSeCys) and selenomethionine (SeMet) have disparate efficacies as anticancer agents. Herein, we use X-ray absorption spectroscopy to determine the chemical form of selenium in human neuroblastoma cells. Cells treated with MeSeCys contain a significant diselenide component, which is absent from SeMet-treated cells and suggests that metabolites of MeSeCys are capable of altering the redox status of the cells. The differences in the speciation of Se in the selenoamino acid-treated cells may provide insight into the differing anticancer activities of MeSeCys and SeMet.

Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies.

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (µ-XANES) showed that the highly localized Se species was mostly elemental Se.

Uptake, distribution, and speciation of selenoamino acids by human cancer cells: X-ray absorption and fluorescence methods.

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.