Butein Derivatives Prevent Obesity and Improve Insulin Resistance through the Induction of Energy Expenditure in High-Fat Diet-Fed Obese Mice.

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3s at 5 mg/kg prevented weight gain by 13.6% in high-fat diet-fed mice without any toxicological observation. In addition, compound 3s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.

The FGFR Family Inhibitor AZD4547 Exerts an Antitumor Effect in Ovarian Cancer Cells.

The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.

Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 µM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α.

Protein Kinase C Isoforms Differentially Regulate Hypoxia-Inducible Factor-1α Accumulation in Cancer Cells.

Hypoxia-inducible factor-1α (HIF-1α) is one of the key transcription factors that mediate adaptation to hypoxia. Despite increasing evidence implicating the PKC family as potential modulators of HIF-1α, the molecular mechanisms of PKC isoform-dependent HIF-1α activity under hypoxic conditions have not been systematically elucidated in cancer cell lines. Here, we collectively investigated how each isoform of the PKC family contributes to HIF-1α accumulation in the human cervical cancer cell line HeLa. Among the abundant PKC isoforms, blockade of either PKCα or PKCδ was found to substantially reduce HIF-1α accumulation and transcriptional activity in hypoxic cells. Knockdown of PKCδ resulted in a reduction of HIF-1α mRNA levels, whereas the HIF-1α mRNA level was unchanged regardless of PKCα knockdown. Upon searching for the downstream effectors of these kinases, we found that PKCα controls HIF-1α translation via AKT-mTOR under hypoxic conditions. On the other hand, one of the well-known transcriptional regulation pathways of HIF-1α, nuclear factor-κB (NF-κB) is identified as a downstream effector of PKCδ. Taken together, our findings provide insights into the roles of PKC isoforms as additional, discrete modulators of hypoxia-stimulated HIF-1α accumulation through different signaling pathways.

Selective inhibition of the hypoxia-inducible factor prolyl hydroxylase PHD3 by Zn(II).

We report herein that Zn(II) selectively inhibits the hypoxia-inducible factor prolyl hydroxylase PHD3 over PHD2, and does not compete with Fe(II). Independent of the oligomer formation induced by Zn(II), inhibition of the activity of PHD3 by Zn(II) involves Cys42 and Cys52 residues distantly located from the active site.

Visualization of hypoxia-inducible factor 1α-p300 interactions in live cells by fluorescence resonance energy transfer.

Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1α and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1α containing plasmid. A mutant HIF-1α incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1α-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications.

Menadione and ethacrynic acid inhibit the hypoxia-inducible factor (HIF) pathway by disrupting HIF-1α interaction with p300.

Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation.

Probing enzymatic activity inside living cells using a nanowire-cell "sandwich" assay.

Developing a detailed understanding of enzyme function in the context of an intracellular signal transduction pathway requires minimally invasive methods for probing enzyme activity in situ. Here, we describe a new method for monitoring enzyme activity in living cells by sandwiching live cells between two vertical silicon nanowire (NW) arrays. Specifically, we use the first NW array to immobilize the cells and then present enzymatic substrates intracellularly via the second NW array by utilizing the NWs' ability to penetrate cellular membranes without affecting cells' viability or function. This strategy, when coupled with fluorescence microscopy and mass spectrometry, enables intracellular examination of protease, phosphatase, and protein kinase activities, demonstrating the assay's potential in uncovering the physiological roles of various enzymes.

Effects of clioquinol analogues on the hypoxia-inducible factor pathway and intracelullar mobilization of metal ions.

We previously found that clioquinol (CQ) increases functional hypoxia-inducible factor-1α (HIF-1α) with enhanced transcription of its target genes. Here we report that compounds derived from 8-hydroxyquinoline including CQ, broxyquinoline (BQ), iodoquinol (IQ) and chloroacetoxyquinoline (CAQ) promote neovascularization effectively based on chick chorioallantoic membrane assays. The CQ analogues induce stabilization of HIF-1α as well as enhance HIF-1-mediated vascular endothelial growth factor transcription. These analogues also exert inhibitory effects on the activity of prolyl and asparaginyl hydroxylations of HIF-1α in vitro. Despite metal ion-dependent restoration of the inhibited HIF-1α hydroxylase activity, the cellular HIF-1α-inducing effects of the CQ analogues are reversed to varying degrees by Zn(2+) and Fe(2+). While CQ and BQ are completely reversed by Zn(2+), co-administration of Zn(2+) and IQ has only a partial reversing effect. On the other hand, CAQ-mediated stabilization of HIF-1α is reversed by Fe(2+) but not by Zn(2+). These phenomena are found to coincide with elevation of the intracellular Zn(2+) and Fe(2+) levels by the CQ analogues, suggesting that metal ion effects on HIF-1α in cells likely reflect the differential transporting capability of the analogues.

Investigating protein unfolding kinetics by pulse proteolysis.

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed "pulse proteolysis" to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP*maltose complex, we have determined the dissociation equilibrium constant (K(d)) of the complex from unfolding kinetic constants, which is also in good agreement with known K(d) values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.

Specific interactions of serpins in their native forms attenuate their conformational transitions.

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein superfamily. Serpins are unique in that their native forms are not the most thermodynamically stable conformation; instead, a more stable, latent conformation exists. During the transition to the latent form, the first strand of beta-sheet C (s1C) in the serpin is peeled away from the beta-sheet, and the reactive center loop (RCL) is inserted into beta-sheet A, rendering the serpin inactive. To elucidate the contribution of specific interactions in the metastable native form to the latency transition, we examined the effect of mutations at the s1C of PAI-1, specifically in positions P4' through P10'. Several mutations strengthened the interactions between these residues and the core protein, and slowed the transition of the protein from the metastable native form to the latent form. In particular, anchoring of the strand to the protein's hydrophobic core at the beginning (P4' site) and center of the strand (P8' site) greatly retarded the latency transition. Mutations that weakened the interactions at the s1C region facilitated the conformational conversion of the protein to the latent form. PAI-1's overall structural stability was largely unchanged by the mutations, as evaluated by urea-induced equilibrium unfolding monitored via fluorescence emission. Therefore, the mutations likely exerted their effects by modulating the height of the energy barrier from the native to the latent form. Our results show that interactions found only in the metastable native form of serpins are important structural features that attenuate folding of the proteins into their latent forms.

The native metastability and misfolding of serine protease inhibitors.

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.

The length of the reactive center loop modulates the latency transition of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1.

Retarded protein folding of deficient human alpha 1-antitrypsin D256V and L41P variants.

alpha(1)-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human alpha(1)-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant alpha(1)-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient alpha(1)-antitrypsin variants. Unlike most alpha(1)-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing alpha(1)-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional.