Hyun Seung Ban

Identification of HSP60 as a Primary Target of o-Carboranylphenoxyacetanilide, an HIF-1α Inhibitor

We succeeded in the design and synthesis of multifunctional chemical probes of the HIF-1alpha inhibitor carboranylphenoxyacetanilide (1) that combine photoaffinity labeling and click reaction to identify the target protein. HSP60 was identified as a primary target protein of 1 using the chemical probes 2 and 3. Furthermore, HSP60 inhibitor 4 suppressed hypoxia-induced HIF activation, indicating that HSP60 affects HIF-1alpha accumulation directly or indirectly.

Development of High Boron Content Liposomes and Their Promising Antitumor Effect for Neutron Capture Therapy of Cancers

Mercaptoundecahydrododecaborate (BSH)-encapsulating 10% distearoyl boron lipid (DSBL) liposomes were developed as a boron delivery vehicle for neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in addition to its encapsulated agents. BSH-encapsulating 10% DSBL liposomes have high boron content (B/P ratio: 2.6) that enables us to prepare liposome solution with 5000 ppm boron concentration. BSH-encapsulating 10% DSBL liposomes displayed excellent boron delivery efficacy to tumor: boron concentrations reached 174, 93, and 32 ppm at doses of 50, 30, and 15 mg B/kg, respectively. Magnescope was also encapsulated in the 10% DSBL liposomes and the real-time biodistribution of the Magnescope-encapsulating DSBL liposomes was measured in a living body using MRI. Significant antitumor effect was observed in mice injected with BSH-encapsulating 10% DSBL liposomes even at the dose of 15 mg B/kg; the tumor completely disappeared three weeks after thermal neutron irradiation ((1.5-1.8) × 10(12) neutrons/cm(2)). The current results enabled us to reduce the total dose of liposomes to less than one-fifth compared with that of the BSH-encapsulating liposomes without reducing the efficacy of boron neutron capture therapy (BNCT).

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.

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy.

Closo-dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 x 10(12)neutrons/cm(2)).

Boron-containing phenoxyacetanilide derivatives as hypoxia-inducible factor (HIF)-1α inhibitors

A series of boron-containing phenoxyacetanilide derivatives 8a-f, 9a-f, 15, and 16 were synthesized as hypoxia-inducible factor (HIF)-1alpha inhibitors. Among the compounds synthesized, carboranylphenoxyacetanilide 16 (GN26361) was found to be a potent inhibitor against HIF-1alpha accumulation under hypoxic conditions and inhibited the hypoxia-induced HIF-1 transcriptional activity in HeLa cells (IC50=0.74 microM). Compound 16 suppressed hypoxia-induced HIF-1alpha accumulation and vascular endothelial growth factor mRNA expression in a concentration-dependent manner without affecting the expression of HIF-1alpha mRNA.

Boron-Based Drug Design.

The use of the element boron, which is not generally observed in a living body, possesses a high potential for the discovery of new biological activity in pharmaceutical drug design. In this account, we describe our recent developments in boron-based drug design, including boronic acid containing protein tyrosine kinase inhibitors, proteasome inhibitors, and tubulin polymerization inhibitors, and ortho-carborane-containing proteasome activators, hypoxia-inducible factor 1 inhibitors, and topoisomerase inhibitors. Furthermore, we applied a closo-dodecaborate as a water-soluble moiety as well as a boron-10 source for the design of boron carriers in boron neutron capture therapy, such as boronated porphyrins and boron lipids for a liposomal boron delivery system.

Hypoxia-inducible factor inhibitors: a survey of recent patented compounds (2004 -- 2010)

Introduction: Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor consisting of α and β subunits that regulates the expression of angiogenic factors, including VEGF, which are involved in angiogenesis, invasion/metastasis, glucose uptake and cell survival during cancer development. Areas covered: This review summarizes the information about patented HIF inhibitors over the last 7 years (2004 – 2010). The reader will gain an outline of the structure and biological activity of recently developed HIF inhibitors. Expert opinion: Inhibition of HIF is an attractive therapeutic target for tumor angiogenesis and, until now, various HIF inhibitors have been discovered and evaluated. It is expected that development of more potent and selective HIF inhibitors will provide an effective treatment of cancer and other HIF-related diseases, including inflammation and cardiovascular disorder. As VEGF plays an important role in angiogenesis during tumor growth and ischemic diseases, the inhibition of VEGF-induced HIF is an attractive approach for the suppression of hypoxia-mediated pathological angiogenesis. HIF inhibitors may not only have cytostatic antitumor effects with fewer side effects, but also synergetic effects combined with radiotherapy.

Boron‐Containing Protoporphyrin IX Derivatives and Their Modification for Boron Neutron Capture Therapy: Synthesis, Characterization, and Comparative In Vitro Toxicity Evaluation

A novel series of boronated porphyrins for potential use in boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) for tumor suppression is described. Protoporphyrin IX {i.e., bis(alpha-methyl-beta-pentylethylether)protoporphyrin IX, and bis(alpha-methyl-beta-dodecanylethylether)protoporphyrin IX} bearing polyhedral borane anions (B(12)H(11)SH(2-), B(12)H(11)NH(3) (-), or B(12)H(11)OH(2-)) were synthesized with reasonable yields. Modification of the protoporphyrin IX structure was achieved by variation of the lengths of the alkyl chains (pentyl and dodecanyl) attached through ether linkages to the former vinyl groups. The goal of this modification was to develop boronated porphyrins with chemical and physical properties that differed from those of protoporphyrin IX. Performance of an MTT assay with each derivative revealed that the synthesized boronated porphyrins showed low cytotoxicities in a variety of cancer cells. Of these compounds, B(12)H(11)NH(2) (2-)-conjugated porphyrin induced a significant increase in the level of boron accumulation and PDT efficacy against HeLa cells.

Discovery of boron-conjugated 4-anilinoquinazoline as a prolonged inhibitor of EGFR tyrosine kinase.

Boron-conjugated 4-anilinoquinazolines were designed and synthesized as inhibitors of EGFR tyrosine kinase with possible covalent bond interactions between the boron atom and the nucleophilic groups of the EGFR kinase domain. Among the compounds synthesized, compounds 6c, 7b, and 7d reduced the EGF-mediated phosphorylation of EGFR tyrosine kinase and its downstream kinases including ERK and Akt in A431 cells. The cell growth was inhibited by these compounds through arrest of G1 cell cycle, which induced apoptosis. A time-dependent in vitro preincubation assay demonstrated the irreversible inhibition of compound 7d against EGFR tyrosine kinase. Quantum mechanical docking simulation revealed that the boronic acid moiety of compound 7d formed a covalent B-O bond with Asp800 in addition to hydrogen bonds with Asp800 and Cys797, which may cause the prolonged inhibition of compound 7d toward EGFR tyrosine kinase.

1-[4-(N-Benzylamino)phenyl]-3-phenylurea derivatives as a new class of hypoxia-inducible factor-1α inhibitors

A series of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives 2a-r were synthesized as HIF-1alpha inhibitors. Among the compounds synthesized, compound 2k was found to be a potent inhibitor against HIF-1alpha accumulation under hypoxic condition and inhibited the hypoxia-induced HIF-1 transcriptional activity in HEK293 cells (IC(50)=7.2 microM). Furthermore, compound 2k suppressed the hypoxia-induced secretion of VEGF in HeLa cells (IC(50)=15 microM).