Kyeong Lee

Recent advances in hypoxia-inducible factor (HIF)-1 inhibitors.

Tumor hypoxia has been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor-1 (HIF-1), a molecular determinant of responses to hypoxia in mammalian cells, has renewed enthusiasm for discovery and development of targeted therapies exploiting the hypoxic tumor microenvironment. HIF-1 activity in tumors depends on availability of the HIF-1α subunit, the levels of which increase under hypoxic conditions and through activation of oncogenes and/or inactivation of tumor suppressor genes. Increased HIF-1 has been correlated with increased angiogenesis, aggressive tumor growth, and poor patient prognosis, leading to current interest in HIF-1 as promising anticancer drug target. In spite of an ever increasing number of putative small molecule inhibitors of HIF-1, only a few are progressing through preclinical and early clinical development. In this review, we will discuss recent advances in discovery and development of small molecule inhibitors that target the HIF-1 pathway as potential anticancer agents.

Bioactive benzofuran derivatives: moracins A-Z in medicinal chemistry.

Benzofuran heterocycles are fundamental structural units in a variety of biologically active natural products as well as synthetic materials. Over the time, benzofuran derivatives have attracted many researchers due to the broad scope of their biological activity, which include anticancer, antimicrobial, immunomodulatory, antioxidant and anti-inflammatory properties. Egonol, homoegonol and moracin families are biologically active natural products containing benzofuran heterocycle as basic structural units. This paper focuses on the moracin family (moracin A to Z). Morus, a genus of flowering plants in the family Moraceae, comprises 10-16 species of deciduous trees commonly known as mulberries. The root bark, stem bark and leaves of Morus alba, M. lhou, Morus macroura are the main sources for arylbenzofuran derivatives including the moracins. A large volume of research has been carried out on moracins and their derivatives, which has shown the pharmacological importance of this benzofuran heterocyclic nucleus. In this mini-review, we attempt to highlight the importance of moracins, as they have been a major source for drug development. Herein, we also summarize the current state of the art concerning the synthesis and medicinal use of moracins A-Z.

LW6, a novel HIF-1 inhibitor, promotes proteasomal degradation of HIF-1α via upregulation of VHL in a colon cancer cell line

Hypoxia-inducible factor HIF-1 is responsible for radiation resistance and poor prognosis in cancer therapy. As part of our drug discovery program, a novel HIF inhibitor, LW6, was identified as a small compound that inhibits the accumulation of HIF-1alpha. We found that LW6 decreased HIF-1alpha protein expression without affecting HIF-1beta expression. MG132, a proteasome inhibitor, protected HIF-1alpha from LW6-induced proteasomal degradation, indicating that LW6 affects the stability of the HIF-1alpha protein. We found that LW6 promoted the degradation of wild type HIF-1alpha, but not of a DM-HIF-1alpha with modifications of P402A and P564A, at hydroxylation sites in the oxygen-dependent degradation domain (ODDD). LW6 did not affect the activity of prolyl hydroxylase (PHD), but induced the expression of von Hippel-Lindau (VHL), which interacts with prolyl-hydroxylated HIF-1alpha for proteasomal degradation. In the presence of LW6, knockdown of VHL did not abolish HIF-1alpha protein accumulation, indicating that LW6 degraded HIF-1alpha via regulation of VHL expression. In mice carrying xenografts of human colon cancer HCT116 cells, LW6 demonstrated strong anti-tumor efficacy in vivo and caused a decrease in HIF-1alpha expression in frozen-tissue immunohistochemical staining. These data suggest that LW6 may be valuable in the development of a HIF-1alpha inhibitor for cancer treatment.

Identification of malate dehydrogenase 2 as a target protein of the HIF-1 inhibitor LW6 using chemical probes

Hypoxia-inducible factor (HIF) regulates tumor angiogenesis and metastasis in response to low oxygen tension. In the presence of oxygen, HIF-1a is rapidly degraded through the ubiquitin–proteasome pathway. In hypoxic conditions, stabilized HIF-1a dimerizes with HIF-1b. The HIF-1a/b heterodimer binds to hypoxia response elements (HRE) in gene promoters and induces the expression of target genes involved in angiogenesis, metastasis, glycolysis, cell proliferation, and resistance to apoptosis. Increased expression of HIF-1a in many solid tumors correlates with aggressive tumor growth, therapeutic resistance, and a poor clinical outcome. HIF-1a shifts the metabolism from oxidative phosphorylation to anaerobic glycolysis. Therefore, HIF-1a is an important therapeutic target for cancer. We previously synthesized and evaluated aryloxyacetylamino benzoic acid analogues. LW6 (1 in Figure 1A) potently inhibited HIF-1a accumulation by degrading HIF1a without affecting the HIF-1a mRNA levels during hypoxia. LW6, which is commercially available, has been used in various studies as an HIF-1a inhibitor. However, the molecular target of LW6 remains unknown. To identify a drug target, chemical biological methods such as activity-based probes (ABPs), photoaffinity labeling, biotinylation, and click conjugation have been used. Herein, we identify the molecular target of 1 using chemical probes. Cellular images and direct protein interactions of 1 were examined in living cells with a series of chemical probes (2–6), which were designed using the structure– activity relationship (SAR) of 1. Synthesis and characterization data for these probes are available in the Supporting Information. The distribution of drug molecules within subcellular compartments can provide information about the mechanism of drug action. The intracellular localization of LW6 was visualized through click chemistry with probe 2, containing an acetylene group, in colon cancer HCT116 cells (Figure 1A). Both 1 and 2 suppressed HIF-1a accumulation (Figure 1B) and HRE-luciferase activity (Figure 1A; Supporting Information, Figure S12). Subsequently, the cellular localization of probe 2 was determined by a click reaction with an azidelinked Alexa Fluor 488 molecule. Notably, copper-catalyzed azide–alkyne cycloadditions (click reactions) are highly specific and efficient bio-orthogonal reactions to visualize intracellular probe distribution. We found that compound 2 was localized primarily in the cytoplasm (Figure 1C). The colocalization of compound 2 (3 mm) with the mitochondriaselective probe, MitoTracker (500 nm), indicated that 2 is specifically localized in the mitochondria, whereas the localization of an adamantyl-free probe 4 was not observed (Figure S13). The mitochondrial localization of probe 2 was Figure 1. Biological activities and cellular localization of a chemical probe for LW6. A) Formula of 1 and its clickable probe 2. B) Inhibitory effects of 1 and probe 2 on HIF-1a accumulation were determined by immunoblot analysis. C) Localization of probe 2 (3 mm, green) was detected through a click reaction using azide-linked Alexa Fluor 488 in HCT116 cells. Mitochondria were selectively stained with the MitoTracker probe (red). Nuclei (blue) were stained with 4,6-diamidino-2phenylindole (DAPI). D) Competitive binding of probe 2 (3 mm) to its target molecules in the presence or absence of 1 (10 mm). Scale bars = 20 mm.

Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21Cip1. In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21Cip1 was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21Cip1 have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21Cip1 facilitated shikonin-induced apoptosis, implying that p21Cip1 delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21Cip1 to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21Cip1 actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21Cip1, leading to p38 MAPK activation, and finally, promoting apoptosis.

A Novel Approach to Cancer Therapy using PX-478 as a HIF-1α Inhibitor

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor produced by tumor cells under hypoxic conditions, and a key regulator of a number of genes important in cancer biology. Over-expression of HIF-1α in human tumors is associated with poor prognosis and poor therapeutic outcomes and HIF-1α has been suggested as a novel target for cancer therapy. This article provides a review of PX-478 as the first novel HIF-1α inhibitor in clinical stage for the treatment of solid tumors.

A Novel Small Molecule Facilitates the Reprogramming of Human Somatic Cells into a Pluripotent State and Supports the Maintenance of an Undifferentiated State of Human Pluripotent Stem Cells

Booster of pluripotency: RSC133, a new synthetic derivative of indoleacrylic acid/indolepropionic acid, exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells (hPSCs).

HIF-1α inhibitors: Synthesis and biological evaluation of novel moracin O and P analogues

The natural products moracins O and P exhibited potent in vitro inhibitory activity against hypoxia-inducible factor (HIF-1), which is a key mediator during adaptation of cancer cells to tumour hypoxia. Systematic variations of the structures of benzofuran type moracins were made and structure-activity relationship analysis showed the importance of the 2-arylbenzofuran ring and the (R)-configuration of the core scaffold. Further evaluation of the representative compound 5 showed its inhibitory effect on HIF-1α protein accumulation and target gene expression under hypoxia.

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Therapeutic Strategies for Metabolic Diseases: Small‐Molecule Diacylglycerol Acyltransferase (DGAT) Inhibitors

Metabolic diseases such as atherogenic dyslipidemia, hepatic steatosis, obesity, and type II diabetes are emerging as major global health problems. Acyl‐CoA:diacylglycerol acyltransferase (DGAT) is responsible for catalyzing the final reaction in the glycerol phosphate pathway of triglycerol synthesis. It has two isoforms, DGAT‐1 and DGAT‐2, which are widely expressed and present in white adipose tissue. DGAT‐1 is most highly expressed in the small intestine, whereas DGAT‐2 is primarily expressed in the liver. Therefore, the selective inhibition of DGAT‐1 has become an attractive target with growing potential for the treatment of obesity and type II diabetes. Furthermore, DGAT‐2 has been suggested as a new target for the treatment of DGAT‐2‐related liver diseases including hepatic steatosis, hepatic injury, and fibrosis. In view the discovery of drugs that target DGAT, herein we attempt to provide insight into the scope and further reasons for optimization of DGAT inhibitors.