A Guanidine-Based Synthetic Compound Suppresses Angiogenesis

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A guanidine-based synthetic compound suppresses angiogenesis via inhibition of acid ceramidase Sung Min Cho, Hyung Keun Lee, Qing Liu, Ming-Wei Wang, and Ho Jeong Kwon ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00558 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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ACS Chemical Biology

A guanidine-based synthetic compound suppresses angiogenesis in human umbilical vein endothelial cells via inhibition of acid ceramidase 160x79mm (96 x 96 DPI)

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ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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A guanidine-based synthetic compound suppresses angiogenesis via

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inhibition of acid ceramidase

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Sung Min Cho1,+, Hyung Keun Lee1,+, Qing Liu2, Ming-Wei Wang2,3,*, and

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Ho Jeong Kwon1,*

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1Chemical

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Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.

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2The

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Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

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Genomics Global Research Laboratory, Department of Biotechnology, College of

National Center for Drug Screening and CAS Key Laboratory of Receptor Research,

3School

of Pharmacy, Fudan University, Shanghai 201203, China

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+ These

authors contributed equally to this work

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*Corresponding

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authors

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Correspondence should be addressed to:

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Ho Jeong Kwon, Ph.D., Professor, Chemical Genomics Global Research Laboratory,

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Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea, Tel: 82-

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2-2123-5883; E-mail: [email protected]

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Ming-Wei Wang, M.D., Ph.D., The National Center for Drug Screening, 189 Guo Shou Jing

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Road, Shanghai 201203, China, Tel: 86-21-50800598; E-mail: [email protected]

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ABSTRACT

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Angiogenesis generates new blood vessels from pre-existing vessels. Tumours induce the

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formation of new blood vessels to ensure sufficient oxygen and nutrients for their growth.

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Normally, angiogenesis is induced by various pro-angiogenesis factors, including vascular

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endothelial growth factor (VEGF). Inhibition of VEGF is a promising approach to cancer

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treatment. A guanidine-based synthetic compound, E2, was identified as a potent hit from 68

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guanidine-based derivatives by screening for angiogenesis inhibitors showing anti-proliferative

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activity in human umbilical vein endothelial cells (HUVECs). To explore the mode of action

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of E2, target proteins were investigated using phage display biopanning and acid ceramidase 1

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(ASAH1) was identified as an E2-binding protein. Drug affinity responsive target stability

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(DARTS) and ASAH1 activity assays revealed the direct binding of E2 to ASAH1. Moreover,

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siRNA knockdown of ASAH1 demonstrated its role as an angiogenesis factor. Consequently,

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E2 inhibited chemoinvasion and tube formation of HUVECs in a dose-dependent manner. E2

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also potently suppressed neo-vascularization of chorioallantoic membranes in vivo.

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Collectively, these data suggest that E2 is a novel angiogenesis inhibitor and ASAH1 is

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proposed to be a new anti-angiogenesis target.

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INTRODUCTION

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Angiogenesis, the growth of new capillary blood vessels from pre-existing vessels, is an

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important physiological process for wound healing and reproduction.1 This process is

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modulated by a feedback balance of growth and inhibitory factors such as vascular endothelial

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growth factor (VEGF) family members and their receptors.2 When the levels of these proteins

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are unbalanced, abnormal angiogenesis contributes to various diseases, including cancer,

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diabetic retinopathy, and rheumatoid arthritis.3

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In order to identify new angiogenesis inhibitors, 68 guanidine-based derivatives were

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screened due to their previously reported antiviral,4 antifungal,4 antibacterial5 and antitumor

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activities.6 As a result, [1,1'-biphenyl]-4-yl 2-(4-guanidinophenyl)acetate (E2) was identified

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as the most potent anti-angiogenesis hit among the derivatives tested. To address the impact of

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these compounds on phenotypic activity of interest, target protein identification is crucial as it

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could aid in elucidating the mode of action underlying biological effects. Phage display is one

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of the reverse chemical proteomics technologies that has been used as a powerful tool for direct

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identification of target proteins of a compound.7 This method requires engineered

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bacteriophages, which have the ability to express peptides encoded by human cDNA sequences

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and display them on the surface of a virion. Target proteins could be found by an affinity

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selection approach called biopanning, where a phage library is incubated with an immobilized

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biotinylated compound. To discover target proteins, human coding cDNA is amplified by PCR

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and analysed by DNA sequencing. This method is not only efficient and robust to identify

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target proteins, but also economically useful.8-10

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To validate the direct interaction of E2 with a target protein identified by phage display

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method, drug affinity responsive target stability (DARTS) was used in the present study.11, 12 3


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This approach employs a label-free compound to keep its original properties and utilises whole-

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cell lysate to maintain the tertiary structure of the protein. In DARTS, proteolytic enzymes are

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used to discover the stability of a specific protein, because small molecule-bound protein

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undergoes a conformational change that alters its susceptibility to cleavage by protease.10, 13

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By combinations of these technologies, we were able to identify and validate acid

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ceramidase 1 (ASAH1) as the target protein of E2 for its anti-angiogenesis activity. ASAH1 is

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an enzyme which cleaves ceramide into sphingosine and fatty acid and is known for high

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expression in several cancer cell lines.14 Inhibition of ASAH1 induces ceramide accumulation

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that can generate mitochondrial stress resulting in caspase-dependent apoptosis in cancer

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cells.15 On the other hand, the level of sphingosine and sphingosine-1-phosphate (S1P) is

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reduced by inhibition of ASAH1. S1P normally amplifies autocrine and/or paracrine signals

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through S1P receptors present on the cell membrane. S1P is implicated in multiple biological

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processes, including cell migration, proliferation, allergic responses and angiogenesis.16

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Although ASAH1 was proposed to be a target for cancer therapy due to its in vivo functions,

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its link with angiogenesis in HUVECs has not been revealed. Here, we report that ASAH1 is

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involved in angiogenesis and is the target protein of E2, a new anti-angiogenic synthetic

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compound.

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RESULTS AND DISCUSSION

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Screening of guanidine derivatives for anti-angiogenic activity

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In our screening program for discovery of anti-angiogenic inhibitors, we first examined the

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effects of compounds in question on endothelial cell proliferation. A total of 68 guanidine-

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bearing compounds were treated with HUVECs for 72 h and 13 of them inhibited the

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proliferation of HUVECs at a concentration of 10 µM (Figures 1a and b; Supplementary

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Figures 1a–c). Subsequent cell viability assay showed that 7 compounds were not cytotoxic. In

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an adipocyte-derived leucine aminopeptidase (A-LAP) assay, the 13 compounds were

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examined to distinguish anti-angiogenic action from that of antibacterial because their parent

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compound NE-2001 (Supplementary Figure 2) displayed specific regulation on A-LAP.17

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Three compounds with non- cytotoxicity to HUVECs and non-inhibitory effect on A-LAP

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activity were subjected to chemoinvasion assay for evaluation of their anti-angiogenic activity

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(Figure 1c). VEGF is known to enhance cell invasion by increasing vascular permeability.18, 19

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As shown in Figure 1d, VEGF efficiently induced HUVEC invasion, which was blocked by

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the three compounds at a concentration of 5 µM. Among them, E2 exhibited the best efficacy

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(Figure 1d).

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Identification of E2 binding protein using phage display

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To explore the mode of action of E2 on angiogenesis, its target needs to be identified. Phage

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display, a target identification method, requires a biotinylated compound. For this reason,

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biotinyl-E2 was synthesized (Figure 2a, Supplementary Methods). In an MTT assay10, 20, E2

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inhibited the proliferation of HUVECs in a dose-dependent manner with an IC50 of 17 µM

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(Figure 2b). Cell viability after E2 treatment was not affected at the concentrations up to 40 5


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µM (Figure 2c). This was followed by the phage display biopanning assay21 with biotinyl-E2

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whose bioactivity was shown to be between 30 and 40 µM (Supplementary Figure 3). Four

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different T7 phage encoding human cDNA libraries from lung, breast and colon cancers as well

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as normal heart tissue were utilized as proteome sources. Biotinyl-E2 was immobilized onto

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streptavidin-coated wells and affinity selection of E2 was in proportion to each round of

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biopanning, implying that specific E2-binding phage was enriched during the process (Figure

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2d and Supplementary Figure 4a). After 5 consecutive rounds, standard PCR was performed

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with isolated phage particles to determine expressed DNA (Supplementary Figure 4b). The

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amplified PCR products were sequenced to identify possible targets.8,

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Decoded DNA

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sequences were searched with BLAST for homology alignment (Table 1), which identified

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ASAH1 as one of the enriched binding proteins of E2 (Figure 2e). Phage binding assay was in

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agreement with this finding as ASAH1 specifically bound to E2, but not biotin (Figure 2f).

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Drug affinity responsive target stability assay for target validation

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DARTS has the advantages of using a label-free compound and whole-cell lysate11, 12 to

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investigate the interaction between a target protein and the compound in question.10, 22

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Proteolysis of ASAH1, ASAH2 and β-actin in HUVECs lysate was monitored in the presence

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of increasing amounts of E2 (0 to 400 μM) and pronase (0 to 10 μg mL-1) (Figure 3a). For β-

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actin and ASAH2, hydrolysis proceeded in an E2-independent manner. In contrast, ASAH1

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was more stable in an E2 dose-dependent manner (Figure 3b; P=0.0054), suggesting that E2

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selectively binds to ASAH1, but not other proteins (Figures 3c and 3d).

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E2 is a competitive inhibitor of ASAH1 revealed by biochemical and docking analyses

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ASAH1 (N-acylsphingosine amidohydrolase; EC 3.5.1.23) is a lysosomal enzyme which

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hydrolyses the N-acyl linkage in ceramide to produce sphingosine and free fatty acids.23, 24 To

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determine the inhibitory effect of E2 on acid ceramidase activity in vitro, a fluorescence-based

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enzymatic activity assay was performed with HUVECs lysate as the source of acid ceramidase.

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BODIPY C5-ceramide was applied as the substrate in the acidic condition (pH 4.5).25, 26, 27 It

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was shown that E2 specifically and competitively inhibited ASAH1 activity since the latter was

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recovered by increasing the substrate concentration. When the substrate concentration was 20

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μM, ASAH1 activity decreased by 61% (P 80%). d)

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Three hits were confirmed at 5 µM by the phenotypic chemoinvasion assay (NT, DMSO

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control without VEGF. CUR, curcumin, a positive control). Data presented are means ± SEM

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from three independent experiments. *P < 0.05 and ** P < 0.005.

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Figure 2. Identification of E2 binding protein using a phage display biopanning method. a)

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Chemical structures of E2 and biotinyl-E2. b) Effect of E2 on HUVEC proliferation. HUVECs

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were treated with various concentrations of E2 (0–80 µM) for 72 h in triplicate and their

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proliferation measured by MTT assay. c) Viability of E2-treated HUVECs at concentrations

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between 0 and 40 µM in trypan blue assay. d)–f) Identification of E2 binding protein using

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phage display biopanning technique. d) Specific E2-binding T7 phage was enriched after five

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rounds of biopanning. ‘Wash’ indicates nonspecific phage bound to E2 and ‘elute’ indicates

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specific phage bound to E2. e) The partial domain of ASAH1 (H154–S203 domain) was

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identified as a possible binding protein for E2. (SP, signal peptide; red arrows, R159 and D162

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are enzyme active site; blue grid, phage coding domain) f) Specific binding of ASAH1 (H154–

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S203 domain) expressing phage to biotinyl-E2. Data presented are means ± SD from three

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independent experiments. *** P < 0.001. 21


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Figure 3. Validation of E2 binding protein (ASAH1) using a label-free DARTS assay. a)

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Western blotting of ASAH1, ASAH2 and β-actin (loading control) with variable E2 and

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pronase concentrations. b)–d) Graphical representation of b) for ASAH1 (p = 0.0054), ASAH2

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c) and β-actin d), respectively. **P < 0.01.

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Figure 4. Competitive binding of E2 to the hydrophobic pocket of ASAH1. a) Michaelis-

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Menten equation of active compound E2 and inactive compound A11 (Fluorescein intensity).

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b) Lineweaver-Burk plot of E2 and A11. Y-intercept means 1/Vmax and X-intercept means -

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1/Km. c) In silico docking of ASAH1 with E2. Brown colour represents high hydrophobicity

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and blue colour represents high hydrophilicity. d) E2-ASAH1 interaction on a 2-D diagram.

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Figure 5. The efficacy of E2 depends on the expression level of ASAH1. a) Expression levels

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of ASAH1 in various cell lines. b) Relative quantification of ASAH1 in various cell lines. c)

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Pearson correlation graph between IC50 of E2 and expression level of ASAH1. d) ASAH1 was

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knocked down in live HUVECs with various concentrations of siRNAs (lanes 2–4 and 6–8)

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relative to scramble siRNA (lanes 1 and 5). e) Proliferation of HUVECs depleted of ASAH1.

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f) The efficacy of E2 is enhanced in HUVECs depleted of ASAH1.

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Figure 6. E2 suppresses VEGF expression through Akt signalling pathway by inhibiting

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ASAH1. a) Akt signalling pathway was affected in the same pattern in HUVECs by E2 or

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siASAH1 treatment for 24 h. DMSO and scramble siRNA were used as controls. b) Protein

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and mRNA levels of HIF-1α. Cells were pre-treated with E2 for 1 h and incubated under 22



1

hypoxic conditions (Nor, Normoxia; Hyp, Hypoxia; IB, Immunoblotting for protein; RT-PCR,

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reverse transcription-PCR for mRNA). c) Effect of S1P on HUVEC proliferation. HUVECs

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were treated with various concentrations of S1P (0–1 µM) for 72 h in triplicate and their

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proliferation measured by MTT assay. d) The efficacy of E2 is reduced in HUVECs treated

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with S1P. HUVECs were treated with various concentrations of E2 (0–80 µM) and S1P (1 µM)

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for 72 h in triplicate and their proliferation measured by MTT assay. e) Suppression of Akt

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signalling pathway by E2 was rescued by S1P treatment in HUVECs. HUVECs were treated

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with E2 (10 µM) and S1P (1 µM) for 24 h. Data presented are means ± SEM from three

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independent experiments. **P < 0.01 and *** P < 0.001.

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Figure 7. E2 inhibits angiogenesis in vitro (phenotypic assay) and in vivo (chorioallantoic

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membrane assay). a) Microscopic observation of invaded cells. b) Arrows indicate narrow or

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broken tubes formed by VEGF-stimulated HUVECs after inhibitor treatment (Con, DMSO

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control without VEGF). Data presented are means ± SD from three independent experiments.

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**P < 0.01 and *** P < 0.001. c) E2 was applied to the CAM and neo-vessel formation was

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observed (NT, MeOH control; RA, 1 μg of retinoic acid; E2, 1 μg). d) Proposed mechanism of

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E2-mediated angiogenesis inhibition. E2 binds to ASAH1 and inhibits its activity leading to

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the suppression of HIF-1α protein translation due to Akt/S6K pathway blockade. VEGF down

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-regulation results in angiogenesis inhibition (blue arrows).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 6

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Figure 7

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1

Table 1. BLAST of PCR products from isolated phage cDNA in round 5 of phage display

2

biopanning. Identities Plaques

Account (%)

1,2,3,4,5,6,

Gene Identified from DNA Sequence

Strand Match

Total

Pct (%)

152

152

100

Homo sapiens N-acylsphingosine 19

7,12,14,16,17,18,19,

Plus amidohydrolase (acid ceramidase) 1

(60%) 20,21,22,23,24,27

/ Plus (ASAH1)

1

Homo sapiens bestrophin 1 (BEST1) on

13

Plus 173

(3%)

173

100

chromosome 11

/ Minus

Human DNA sequence from clone RP5971N18 on chromosome 20p12 Contains the 2

Plus PHKBP1 pseudogene (phosphorylase kinase,

25, 28

256

256

100

(7%)

/ Minus beta pseudogene 1), two novel genes and a CpG island, complete sequence

1

Homo sapiens chromosome 17, clone RP11-

26

Plus 469

(3%)

469

100

89H15, complete sequence

/ Plus

Homo sapiens chromosome 2 open reading 1 29

Plus / frame 3, mRNA (cDNA clone MGC:75039

464

464

100

(3%)

Plus IMAGE:5548897), complete cds

1

Homo sapiens PAC clone RP5-905J8 from

30

Plus 395

(3%)

396

99

7p11.2-p21, complete sequence

/ Minus

5 8,9,10,11,15

2 band (16%)

3 4 5 6 7 8 31


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“Table of Contents (TOC)”

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A Guanidine-Based Synthetic Compound Suppresses Angiogenesis

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