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Drug Repurposing of Histone Deacetylase Inhibitors that Alleviate Neutrophilic Inflammation in Acute Lung Injury and Idiopathic Pulmonary Fibrosis via Inhibiting Leukotriene A4 Hydrolase and Blocking LTB4 Biosynthesis Weiqiang Lu, Xue Yao, Ping Ouyang, Ningning Dong, Dang Wu, Xingwu Jiang, Zengrui Wu, Chen Zhang, Zhongyu Xu, Yun Tang, Shien Zou, Mingyao Liu, Jian Li, Ming-Hua Zeng, Ping Lin, Feixiong Cheng, and Jin Huang J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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Journal of Medicinal Chemistry

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Drug Repurposing of Histone Deacetylase Inhibitors that Alleviate Neutrophilic Inflammation in Acute Lung Injury and Idiopathic Pulmonary Fibrosis via Inhibiting Leukotriene A4 Hydrolase and Blocking LTB4 Biosynthesis

Weiqiang Lu1,2,†, Xue Yao1,†, Ping Ouyang1, Ningning Dong1, Dang Wu1, Xingwu Jiang2, Zengrui Wu1, Chen Zhang1, Zhongyu Xu1, Yun Tang1, Shien Zou3, Mingyao Liu2, Jian Li1, Minghua Zeng4, Ping Lin5, Feixiong Cheng5,6,7,*, Jin Huang1,* 1

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, Shanghai 200237, China; 2

Shanghai Key Laboratory of Regulatory Biology, The Institute of Biomedical Sciences

and School of Life Sciences, East China Normal University, Shanghai 200241, China; 3

Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University,

Shanghai 200011, China; 4

Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources

(Ministry of Education), School of Chemistry & Chemical Engineering, Guangxi Normal University, Guilin 541004, China; 5

State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and

Collaborative Innovation Center for Biotherapy, Chengdu 610041, Sichuan, China; 6

Center for Complex Networks Research, Northeastern University, Boston, MA 02115,

USA 7

Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber

Cancer Institute, Harvard Medical School, Boston, MA 02215, USA


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ABSTRACT Acute lung injury (ALI) and idiopathic pulmonary fibrosis (IPF) are both serious public health problems with high incidence and mortality rate in adults, and with few drugs available for the efficient treatment in clinic. In this study, we identified that two known histone deacetylase (HDAC) inhibitors, suberanilohydroxamic acid (SAHA, 1) and its analog 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide (2), are effective inhibitors of Leukotriene A4 hydrolase (LTA4H), a key enzyme in the biosynthesis of leukotriene B4 (LTB4), across a panel of 18 HDAC inhibitors, using enzymatic assay, thermofluor assay, and X-ray Crystallographic investigation. Importantly, both 1 and 2 markedly diminish early neutrophilic inflammation in mouse models of ALI and IPF under a clinical safety dose. Detailed mechanisms of downregulation of proinflammatory cytokines by 1 or 2 were determined in vivo. Collectively, 1 and 2 would provide promising agents with well-known clinical safety for potential treatment in patients with ALI and IPF via pharmacologically inhibiting LAT4H and blocking LTB4 biosynthesis.



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INTRODUCTION Acute lung injury (ALI), a syndrome consisting of acute hypoxemic respiratory failure with bilateral pulmonary infiltrates, is commonly characterized by alveolar injury, cytokine induction, and neutrophil recruitment in the lung tissue with a high lung injury incidence of approximately 190,000 adult patients per year in the United States (U.S.).1, 2 Idiopathic pulmonary fibrosis (IPF) is the most common and most lethal diffuse fibrosing lung disease.3, 4 Furthermore, a mortality rate of IPF exceeds that of many cancers.3 Although ALI and IPF are both serious public health problems with high incidence and mortality rate in adults, there are few drugs available for the efficient treatment of ALI and IPF in clinic.4 There have been many clinical trials of novel therapies investigated for IPF in the past decade, while the results have mostly been disappointing.4 Several main issues include the disease heterogeneity of IPF and ALI, lack of predictive biomarkers, and drug safety profiles.1, 4 With faster development time, well-known safety, good pharmacokinetic profiles, and a low commercial risk, the prospect of drug repurposing5-8 provides a promising strategy for the development of novel pharmacological therapies for various emerging diseases, such as ALI and IPF. Neutrophils, the most abundant leukocytes in circulation, can be recruited to local inflammatory sites, and are capable of ‘cleaning up’ pathogenic microorganisms by various mechanisms.9 Recent studies have suggested that neutrophilic inflammation is frequently observed in several lung diseases, such as ALI or IPF.10-12 Furthermore, blockage of neutrophilic inflammation has suggested to provide an effective strategy for treatment of ALI or IPF.13, 14 A previous study has reported that the processes of


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neutrophil migration and infiltration in neutrophilic inflammation were regulated by complex networks of chemokine and chemotactic agents.14 Among various chemotactic agents, leukotriene is a group of key lipid mediators of the arachidonic acid (AA) metabolism products that are mainly biosynthesized by leukocytes.15 Leukotriene B4 (LTB4), a predominant member of the leukotriene family, is bio-synthesized by the sequential action of 5-lipoxygenase (5-LOX) and leukotriene A4 hydrolase (LTA4H),16 and is well known to regulate chemotactic activity of human neutrophils.17 LTB4 is secreted by neutrophils at inflammation sites in response to formyl peptides, playing an important role in neutrophil activation and migration to formyl peptides.17 LTB4 levels were persistently elevated in bronchoalveolar lavage fluid (BALF) of lipopolysaccharide (LPS)-induced ALI,18 and the leukotriene levels in pulmonary edema fluid were significantly higher in ALI patients than that in the control patients with hydrostatic pulmonary edema.19 In addition, LTB4 level was increased in lung homogenates and BALF of patients with IPF and the level of LTB4 correlate with the extent of fibrosis in histological sections.20 Put together, inhibiting LTB4 biosynthesis and subsequently neutrophilic inflammation may provide a potential strategy for the treatment of ALI or IPF. Suberanilohydroxamic acid (SAHA), 1, is the first U.S. FDA-approved histone deacetylases (HDAC) inhibitor in 2006 for the treatment of cutaneous T-cell lymphoma.21 Recent studies have suggested that 1 showed potential anti-inflammatory activity, while the underlying mechanisms of the favorable anti-inflammatory effect by 1 remained unclear. In this study, we identified that 1 and its analog 4-(dimethylamino)-N[7-(hydroxyamino)-7-oxoheptyl]benzamide (2)22 are potent inhibitors of LTA4H, a key



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enzyme in the biosynthesis of LTB4, among a panel of 18 HDAC inhibitors by integrating enzymatic assays, thermofluor assays, and X-ray crystallographic observations. Furthermore, we demonstrated that 1 potently blocked LTB4 biosynthesis and repressed neutrophils infiltration in mouse models of both ALI and IPF under a clinical safety dose. As a more potent inhibitor of LTA4H, 2 showed a higher potential than 1 in mouse models of ALI and IPF. Specifically, we demonstrated that 1 and 2 (HDAC and LTA4H dual inhibitors) are more effective than a specific HDAC inhibitor (N-(6-(2-aminophenylamino)-6-oxyhexyl)-4-methylbenzamide, 17)23 without a LTA4H inhibitory activity in LPS-induced ALI. 1 and 2 protect against bleomycin (BLM)induced pulmonary fibrosis, which is comparable to nintedanib4, a triple-tyrosine-kinase inhibitor approved by FDA for the treatment of IPF. Finally, the crystal structure analyses of apo-LTA4H, 1-LTA4H, and 2-LTA4H and molecular modeling revealed novel structural information for further lead optimization by decreasing the inhibitory activity of aminopeptidase in order to reduce the side effects of 1 and 2 resulting from their aminopeptidase’s inhibition in clinic. Collectively, this study provides a novel molecular mechanism and therapeutic strategy to 1 and 2 for patients with ALI and IPF via pharmacologically inhibiting LAT4H and blocking LTB4 biosynthesis.

RESULTS AND DISCUSSION Discovery of known HDAC inhibitors as potent inhibitors of LTA4H We collected a panel of 18 available HDAC inhibitors to evaluate their inhibitory activities against LTA4H. Among 18 HDAC inhibitors, only 1 and its analog 2 revealed high inhibitory activities against LTA4H, a final enzyme in the biosynthesis of LTB4


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(Table 1). LTB4 is an AA metabolism product, bio-synthesized by the sequential action of cytosolic phospholipases A2 (cPLA2), LOX, and LTA4H. To explore the mechanism of 1 as an inhibitor of LTB4 production, we conducted a systematic evaluation of 1 against cPLA2, 5-LOX, 12-LOX, 15-LOX, and LTA4H. Interestingly, we found that 1 selectively inhibited the activity of LTA4H (Fig. 1A). In contrast, 1 exhibited considerably less inhibitory activity on other AA metabolizing enzymes. The epoxide hydrolase activity of human LTA4H was inhibited by 1 with an IC50 value of 7.65 µM. Similarly, aminopeptidase activity of human LTA4H was inhibited by 1 in a dosedependent manner, with an IC50 value of 1.67 µM. These data revealed that LTA4H was a new target of 1. 2, a 1’s analog, was identified as a more potent inhibitor of LTA4H than 1, and further exhibited a dose-dependent inhibitory activity on LTA4H aminopeptidase and hydrolase with IC50 values of 0.3 µM and 0.68 µM, respectively. Similar as 1, 2 also exhibited less inhibitory activity on other AA metabolizing enzymes (Fig. 1B). LTA4H is a direct target of the known HDAC inhibitors by thermofluor assays To further examine the interactions between LTA4H and the HDAC inhibitors, we tested 18 HDAC inhibitors (Fig. 1C) on LTA4H in vitro via a thermofluor assay that is used to study thermal stabilization of proteins upon ligand binding.24 The purified recombinant human LTA4H protein was subjected to thermal scanning in the absence and presence of the experimental compounds, and melting temperature (Tm) was calculated from the melt curve. As shown in Fig. 1C, a known LTA4H inhibitor, 3-[methyl[3-[4(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl (19, 10 µM)25, significantly increases the Tm value about 2.95 °C. Among 18 HDAC inhibitors, only 1 and 2



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stabilized the LTA4H protein as a reflection of the increasing Tm with 1.47 °C and 2.35 °C, further suggesting the direct binding of 1 and 2 to LTA4H. Crystallization and complex structures of LTA4H-1 and LTA4H-2 We next solved the crystal structures of LTA4H and complex structures with 1 and 2 to examine the details of binging modes of LTA4H with 1 and 2. The structures of LTA4H (PDB entry 4RVB), LTA4H-1 (PDB entry 4R7L) and LTA4H-2 (PDB entry 4RSY) were determined at 1.93 Å, 1.66 Å, and 1.93 Å, respectively. Statistics of data collection and structural refinement were summarized in Supplementary Table 1. We found that LTA4H folded into three domains and created a deep cleft harboring the catalytic Zn2+ site, forming the active site with an L-shaped hydrophobic pocket deep into the protein, consistent with previous studies.26, 27 The crystal structure reveals that 1 bound at the end of the L-shaped hydrophobic cavity, which accommodates the ω-end of LTA4H and blocks substrate access (Fig. 1D and 1E). The carbonyl oxygen of the hydroxamic acid group of 1 is coordinated with Zn2+ through its carbonyl (2.0 Å) and hydroxyl groups (2.2 Å). Moreover, 1 is tightly packed in LTA4H through several hydrogen bonds with the general bases in the active site and a π-π interaction with Phe314. Specifically, the hydroxamic acid group of 1 is anchored to the main chain of LTA4H through hydrogen bonds to the carbonyl oxygens of Gly269 (2.8 Å), Try383 (2.6 Å) and Glu296 (2.5 Å and 3.1 Å). The cap group of 1 consists of an amide carbonyl group. In the crystal structure, the amide group near the cap forms a hydrogen bond with Gln136 (3.2 Å) and interactions with Asp375 and Tyr267 through a water bridge. An additional van der Waals contact of 1 with Phe314, Pro374, Val 367 and Tyr 357, fixes the six carbon-long aliphatic chain of 1 packing in the L-shaped hydrophobic cavity of LTA4H. Fig. 1F and


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1G revealed that LTA4H-2 had a very similar binding model with LTA4H-1, while 2 showed more contact with the L-shaped pocket of the LTA4H since it had an additional dimethylamino group. Compared with the two complex crystal structures, we found that ligands with a higher binding affinity to LTA4H should satisfy all of three main interactions: (i) ligands bind to active site Zn2+ with two coordinate bonds, (ii) three residues (Gly269, Glu296, and Tyr383) around Zn2+ interact with the ligand by hydrogen bonds, and (iii) hydrophobic part of the ligand locates into the L-shape hydrophobic pocket and interacts with Phe314 by a π-π contact (Fig. 2). 1 and 2 inhibit LTB4 biosynthesis in neutrophils and repress neutrophil migration by targeting LTA4H We next tested the effect of 1 and 2 on endogenous LTB4 biosynthesis in neutrophils. We found that both 1 and 2 significantly decreased LTB4 levels in neutrophil with IC50 values of 6.15 µM and 0.79 µM (Fig. 3A), respectively. In contrast, 17, a specific inhibitor of HDAC,23 has no inhibitory activity on LTB4 biosynthesis in neutrophils (data not shown). In response to formyl peptide N-formylmethionyl-leucyl-phenyl-alanine (fMLP), neutrophils will quickly secrete LTB4 and induce neutrophil chemotaxis. 1 or 2 treatment completely reduces fMLP-induced neutrophil chemotaxis in a dose-dependent manner from 1 to 10 µM (Fig. 3B-E). In contrast, 17 exhibits a limited effect on fMLPinduced neutrophil migration (Fig. 3D). These results suggest that the additional inhibition of LTA4H may be responsible for the better anti-migration capacities of 1 and 2. Furthermore, we then investigated a synergetic anti-migration effect of dual inhibitions on HDAC and LTA4H by using two control compounds: 17 and a known LTA4H



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inhibitor (S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanoic acid (20). 20 is a well-characterized, oral, small-molecule inhibitor of LTA4H without a HDAC inhibitory activity.28 Notably, 1 and 2 showed more potent anti-migration effects than 17. In addition, co-treatment of 17 and 20 synergistically represses the migration of neutrophil and LTB4-induce neutrophil migration was not affected by these treatments. Taken together, these data show that the LTA4H is a functional and pharmacologically relevant target of 1 and 2 for their anti-neutrophilic inflammation activities. 1 attenuates LPS-induced lung neutrophilic inflammation in vivo To investigate the anti-inflammatory activity of 1, a murine experimental model of ALI was induced by LPS inhalation. Histopathological analysis of lung tissue using hematoxylin-eosin (H&E) staining revealed that LPS treatment caused serious pulmonary damage, including intra-alveolar hemorrhage, interstitial edema, and alveolar collapse. In the 1-treated group, LPS-induced pathological changes were significantly attenuated. Furthermore, no significant lesions were observed in the lung parenchyma and the structure of alveolar remained relatively intact, with only a mild hyperemia of pulmonary alveolar septa (Fig. 4A). Alveolar vascular permeability was then examined by intravenous injection (i.v.) of Evans blue dye in the experimental mice. LPS indeed increased lung vascular permeability and leakage, while this symptom was relieved after treatment with 1 (Fig. 4B). LPS-induced lung inflammatory responses were then examined by measuring the total number of cells and the protein concentration in BALF at 24 h after inhalation of LPS. The increased total number of leucocytes and protein concentrations in LPS-induced mice were attenuated by 1 (Fig. 4C and 4D). We further determined myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, in


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sonicated whole lung homogenates to monitor the neutrophil accumulation. Fig. 4E shows that 1 suppresses the increased MPO activity in LPS-induced mice model. Meanwhile, 1 effectively attenuates pulmonary edema as indicated by lung Wet/Dry ratio in mouse (Fig. 4F). These results suggest that 1 efficiently represses LPS-induced inflammatory responses. 1 inhibits LPS-induced inflammatory mediators’ production We next examined mRNA and protein levels of pro-inflammatory cytokines in lung tissue and BALF using quantitative Real-time fluorescence PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) to further investigate the underlying mechanisms of anti-inflammatory activities by 1. We found that inhalation of LPS significantly increased the mRNA and protein expression of several pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 (Fig. S1). While, the elevations of both mRNA and protein levels were dramatically suppressed after dose-dependent pretreatment with 1. For example, TNF-α levels in LPS-injected mice (203.04 pg/mL) were 10 times higher than those in the control group (19.16 pg/mL). Treatment with 25 mg/kg of 1 reduced this level in LPS-injected mice to 80.54 pg/mL, representing an approximately 70% reduction compared to the TNF-α level in mice receiving LPS alone. LTB4 is a potent activator and chemoattractant for neutrophils, and numerous neutrophils were accumulated in BALF following LPS treatment, inspiring us to test the concentration of LTB4 in BALF. Upon LPS administration, LTB4 levels remarkably increased in vehicle-treated mice. Interestingly, 1 significantly inhibited LTB4 biosynthesis at 10, 25, and 50 mg/kg in a dose dependent manner (Fig. 4G).



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HDAC and LTA4H dual inhibitors are more effective than a specific HDAC inhibitor in LPS-induced ALI We next examined the in vivo potency of a specific HDAC inhibitor (17), the HDAC and LTA4H dual inhibitors (1 and 2), and a combination therapy of 17 with a specific LTA4H inhibitor (20) under the same dosage on LPS-induced ALI using the same experimental setup (Fig. 5A and 5B). 1 (25 mg/kg) and 2 (25 mg/kg) show similar effects with single dose of 20 or 20 and 17 combination group, while they are more effective than single dose of 17 on LPS-induced pathological changes. Similarly, less leucocytes, protein concentrations, and MPO activity, were observed in 1 and 2 treated group (Fig. 5C-E). Meanwhile, 1 and 2 more effectively attenuate pulmonary edema condition than 17 in mice as indicated by lung Wet/Dry ratio (Fig. 5F). Specifically, 25 mg/kg of 1, 2 and 20 possessed the potent inhibitory activities against the LTB4 biosynthesis (Fig. 5G). However, 17 displayed a minor suppression on production of LTB4 in vivo. Similar with the results of the LTA4H inhibition, neutrophil migration repression and the protection of ALI, 1 and 2 showed a higher effect than 17 on both mRNA and protein levels of proinflammatory cytokines in lung tissues and BALF (Fig. S2). Taken together, we suggested that: 1) The high inhibition of LTA4H by 1 or 2 is responsible for its favorable anti-neutrophilic inflammation; 2) Comparing to 17, 1 or 2 exhibits a better protective effect on neutrophilic inflammation when administrated at the same dosage may due to their dual inhibition of HDAC and LTA4H. The plasma concentration of 1 ranges from 0.01 to 6 µM in clinical trials and is approximately 1 to 10 µM in mouse model,29, 30 which is enough to block HDAC or LTA4H hydrolase in vivo. In addition, computational evaluations via the admetSAR tool31 show that both 1 and 2 have great pharmacokinetics


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properties, including non-Cytochrome P450 enzyme inhibition, non-P-glycoprotein inhibition, non-carcinogens, and low risk of rat acute oral toxicity (Supplementary Table 2). Collectively, both 1 and 2 with a good preclinical safety profiles provide potential therapies in clinic for ALI or IPF. Further clinical study will be needed, which we hope will be prompted by the findings herein. 1 and 2 relieve BLM-induced lung neutrophilic inflammation We further investigated the effects of 1 and 2 on LTB4 production and neutrophil infiltration in vivo using a murine experimental model of BLM-induced lung neutrophilic inflammation. Nintedanib is used as a positive control.4 Histopathological evaluation of mice lung sections using H&E staining showed that BLM administration induced severe pulmonary damage, including thickening of alveolar walls and accumulation of substantial inflammatory cells (such as neutrophils) at day 7 (Fig. 6A). After treatment with 1 and 2, the inflammatory response in lung tissues induced by BLM was significantly attenuated. Administration of BLM significantly up-regulates total inflammatory cell populations in BALF on day 7 (Fig. 6B), including neutrophils, macrophages and lymphocytes. Specifically, 1 and 2 at low doses (25 mg/kg and 10 mg/kg, respectively) significantly reduce the number of neutrophils, while nintedanib (30 mg/kg) mainly reduces lymphocytes. We further measured the MPO activity in sonicated whole lung homogenates on day 7. Administration of 1 (50 mg/kg) or 2 (25 mg/kg) significantly suppressed the MPO activity (decreased from 0.98 U/g to 0.67 U/g and 0.56 U/g, respectively) compared to nintedanib (Fig. 6C), consistent with the neutrophils counts in BALF. In line with these results, the level of LTB4 in BALF was also reduced from 139.23 pg/ml to 43.19 pg/ml and 37.23 pg/ml following 1 (50 mg/kg) and 2 (25



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mg/kg) treatment (Fig. 6D), respectively. With the extensive infiltration of inflammatory cells, BLM instillation significantly increased the protein content of BALF on day 7 compared with control (increased from 0.22 mg/ml to 2.39 mg/ml), which was markedly suppressed by 1 (1.79 mg/ml at 50 mg/kg) and 2 (1.92 mg/ml at 25 mg/kg) (Fig. 6E). The mRNA and protein levels of pro-inflammatory cytokines in lung tissue and BALF were examined using qRT-PCR and ELISA (Fig. S3). BLM instillation significantly increases the level of IL-1β and IL-6 in lung tissue compared to the untreated group on day 7, and the elevated level was diminished by 1 and 2 treatments in a dose-dependent manner. In addition to the decreased mRNA level of IL-1β and IL-6 in lung tissue, the protein level of IL-1β and IL-6 were notably reduced after administration of 1 and 2 compared to that of the BLM-treated group. Collectively, administration of 1 and 2 reduced BLM-induced lung inflammatory responses. 1 and 2 protect the BLM-induced pulmonary fibrosis Pulmonary fibrosis appears following the early inflammation process in BLM-induced mouse model of IPF.32 The deposition of collagen fibers was greatly reduced after 1 and 2 treatment, as illustrated by Masson’s trichrome staining (Fig. 7A). To explore the mechanism of 1 and 2 on BLM-induced fibrogenic changes, we first examined the biologically active TGF-β1 levels in BALF, since TGF-β1 was recognized as a profibrogenic master cytokine (Fig. 7B). TGF-β1 was increased both on day 7 and day 14 in the BLM treated group, but dramatically inhibited by 1 (50 mg/kg) and 2 (25 mg/kg). Specifically, the TGF-β level of the 1 and 2 treated groups decreased from 75.39 pg/ml to 34.09 pg/ml and 31.74 pg/ml on day 7, respectively. On day 14, TGF-β levels decreased from 99.30 pg/ml to 49.74 pg/ml and 38.22 pg/ml, respectively. Similar decreased


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mRNA levels of TGF-β in lung tissues were observed (Fig. 7C). We then measured the hydroxyproline in lung tissues on day 14, as hydroxyproline was a major constituent of collagen (Fig. 7D). Compared with the BLM group, the hydroxyproline level was reduced by approximately 37% and 34% following treatment with 1 (50 mg/kg) and 2 (25 mg/kg), respectively, suggesting a significant protective effect of 1 and 2 in vivo by counteracting extracellular matrix (ECM) accumulation. Molecular Modeling of HDAC inhibitors against LTA4H hydrolase and aminopeptidase Human LTA4H is a bifunctional enzyme with the hydrolase and aminopeptidase functions utilizing the same or overlapping binding sites.33 The aminopeptidase functions mediate side effects for the hydrolase functions. Previous studies showed that the aminopeptidase binding site shares a similar structure to LTA4H at its ligand binding sites.34, 35 We performed molecular docking studies of 18 HDAC inhibitors (Table 1) into two isoforms of aminopeptidases: ERAP1 and ERAP2. We found that aminopeptidase and LTA4H showed similar binding modes with only slight differences in the Zn2+ binding region and the L-shaped hydrophobic pocket (Fig. S4). Hence, enhancing the hydrolase binding mode of ligands by increasing hydrogen bonds with Gly269, Glu296, Tyr283 and π-π contacts with Phe314 may reduce the undesirable effects caused by aminopeptidase in hydrolase functional studies of LTA4H (Fig. 2 and Fig. S4). In addition, aminopeptidase binding effects may be also decreased through reducing hydrogen bonds with Glu and Tyr around Zn2+ sites and hydrophobic interactions for 1 and 2 on the aminopeptidase protein (Fig. S4). Taken together, molecular modeling would provide potential structural information for further lead optimization by avoiding



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the inhibitory activity of aminopeptidase in order to reduce the side effects of 1 and 2 in clinic.

CONCLUSION In this study, we demonstrated that an FDA approved HDAC inhibitor, 1, significantly reduced the production of LTB4 in a dose dependent manner in mouse models of both ALI and IPF. Furthermore, we identified that 1 and its analog 2 were potent inhibitors of LTA4H, a critical enzyme in the biosynthesis of LTB4. The crystal structures of LTA4H and complex structures of LTA4H with 1 or 2 provide novel structure-activity relationship of HDAC inhibitors on LTA4H inhibitory activities. Many studies have suggested that HDAC inhibitor such as 1 exhibited potential anti-inflammatory activity through suppressing the expression of inflammatory cytokines via epigenetic regulation. To the best knowledge of the authors, this is the first time to identify the novel mechanism underlying the favorable anti-inflammatory effects for 1 and 2 by inhibiting LTA4H and further blocking LTB4 biosynthesis. Furthermore, we demonstrated that both 1 and 2 markedly diminished early neutrophilic inflammation in an IPF mouse model. Via repurposing FDA approved drugs with a well-known safety profile, this study would speed up development of dual HDAC and LTA4H inhibitors as potential therapies in clinic for ALI or IPF with the novel molecular mechanisms.

EXPERIMENTAL SECTION Chemistry


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All HDAC inhibitors were commercially available (Selleck Chemicals LLC, Huston, TX, USA), compound 20 was from MedChemExpress (Shanghai, China) and the purities of compounds were ≥ 95% as determined by HPLC. X-ray crystallography, data collection and structure validation Human LTA4H cDNA (NM_000895) was purchased from Bioworld, China. The PCR product of human LTA4H cDNA were digested with NdeI and BamHI and cloned into pET28a for expression in BL21 (DE3) pLysS. Detailed expression and purification procedures were provided in the Supporting Information. Purified human LTA4H proteins (10~15 mg/mL in 10 mM Tris-HCl buffer pH 8.0) was incubated with compounds (2 mM) for 24 h on ice.36 A mixture of 1 µl protein solution and 1 µl reservoir solution was equilibrated against 500 µl reservoir solution. Reservoir solution composed of 10~15% (wt/vol) PEG 8000, 100 mM NaAc, 100 mM Imidazole buffer (pH 6.0~6.8), and 5 mM YbCl3. Plateshaped crystals grew to maximum dimensions of 0.5×0.3×0.05 mm within 7 days at 293 K. For data collection, crystals were then transferred in the cryobuffer (13% PEG 8000, 100 mM NaAc, 100 mM imidazole/HCl pH 6.2, 30% Glycerol and 5 mM YbCl3) for a few seconds and then flash-frozen by insertion in liquid nitrogen using a fibre loop. X-ray diffraction data was collected on beamline BL17U at Shanghai Synchrotron Radiation Facility (SSRF). For LTA4H in complex with 1 and 2, a single dataset (resolution of 33.5~1.66 Å and 28.6~1.93 Å respectively) of 180 images was collected with an oscillation angle of 1° per image and a crystal-to-detector distance of 280 mm. The data indexing, integration, and scaling for both proteins were done with the HKL2000. The data collection statistics for datasets of LTA4H in complex with 1 and 2 are summarized in Supplementary Table 1. Crystal



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structures were solved by the molecular replacement using MOLREP. All structures were refined in REFMAC5, and manual inspection and model building were performed in COOT. The structure refinement was done using PHENIX and Refmac 5. The figures were prepared using PyMOL. Statistics for data processing and refinement are listed in Supplementary Table 1. The coordinates and structure factors of the final models of LTA4H were deposited in the PDB (www.rcsb.org, PDB IDs: 4R7L, 4RSY, and 4RVB). Animals Female, 6 weeks C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences and maintained in a 12 h light/dark cycle. All experimental methods were carried out accordance with the guidelines of the IACUC of Shanghai and the National Research Council Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the Institutional Animal Ethics Committee at East China Normal University Isolation of mouse neutrophils from bone marrow The female C57BL/6 mice (6-8 weeks) neutrophils were prepared as described previously.17, 37 Mice were sacrificed and, the femurs and tibias were removed from both hind legs and freed of soft tissue attachments before being soaked in the HBSS (without calcium and magnesium) supplemented with 100 U/mL penicillin, 100 U/mL streptomycin. After washing with 75% ethanol once and PBS twice, HBSS with 2 mM EDTA was forced through the bones with a syringe, and the solution was filtered through a cell strainer. Cells were centrifuged at 500 g for 10 min, and the remaining red cells in the neutrophil pellet were eliminated by hypotonic lysis and spun down (500 g, 10 min, and 4 °C). Cells were washed once with phosphate buffered saline (PBS), resuspended in


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PBS, and layered on top of a discontinuous Histopaque gradient containing 3 mL of Histopaque (Sigma-Aldrich, St. Louis, MO, USA) at 1.119 g/mL at the bottom and 3 mL of Histopaque at 1.077 g/mL on top. Cells were centrifuged for 45 min at 834 g without braking. Cells at the interface of the two layers were collected and washed twice with PBS. We obtained approximately 1×107 living cells per mouse, and verified that 85 ± 1% of them were neutrophils using trypan blue staining and Wright’s staining. Measurement for the release of LTB4 in neutrophils The neutrophils were suspended in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (GIBCO, Carlsbad, NY, USA) at 2×106 cells/ mL and incubated with compounds at indicated concentrations for 30 min at 37 °C. Cells were stimulated with 5 µM ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3benzoxazole-4-carboxylic acid (21)38 for another 30 min and supernatants were collected and frozen at -80 °C for the measurement of LTB4 release.17 LTB4 was measured using an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Transwell migration assay Neutrophils (1× 105) from mouse bone marrow were resuspended in 50 µL HBSS (without calcium and magnesium) containing 1% bovine serum albumin (BSA) and incubated with compounds at indicated concentrations for 30 min at 37 °C. Cells were placed on the top of a 96-well chemotaxis plate using polycarbonate filters with 5 µm pore diameter (MAMIC 5S10, Millipore, Billerica, MA, USA) and indicated concentrations of fMLP or LTB4 (Sigma-Aldrich, St. Louis, MO, USA) were added to



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stimulate the migration of neutrophil at 37 °C for 1 h.37, 39 Cells that had migrated through the pores to the lower chamber were collected and counted using a Cell Counter (Nexcelom, Lawrence, MA, USA). LPS-induced ALI in mice The female C57BL/6 mice (6-8 weeks) were randomly divided into six groups: control group, LPS group, 1-treated group (10, 25, 50 mg/kg) and Dexamethasone (Dex)-treated group (10 mg/kg). Each group was composed of ten mice. After being anesthetized, LPS (O55:B5; 3.75 mg/kg) in 40 µL PBS was given intratracheally. Control mice were intratracheally injected with an equal volume of PBS. 1 (10, 25, 50 mg/kg) and Dex (10 mg/kg) was intraperitoneal injected for 1 h prior to administration of LPS. Mice from the control and LPS groups received an equal volume of 0.5% CMCNa. 24 h after LPS administration, animals were euthanized to collect BALF and lung tissue samples.40 BLM induced IPF in mice BLM was known to induce acute pulmonary inflammation response during the first week, followed by fibrogenic changes leading to ECM accumulation.32 The female C57BL/6 mice (6-8 weeks) were randomly divided into eight groups: control group, BLM group, 1-treated group (25, 50 mg/kg), 2-treated group (10, 25 mg/kg) and nintedanib-treated group (30 mg/kg). Each group was composed of ten mice. The animals received a single dose of BLM at 2 mg/kg by intratracheal instillation as described previously.41, 42 1, 2 and nintedanib were intraperitoneal injected daily for 7 or 14 days after administration of BLM. BALF and lung tissue samples were collected at day 7 and 14.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The Supplemental Figures 1-4, Supplemental Tables 1-3, a separate CSV file of molecular formula strings and the associated biochemical and biological data, and supplementary biological methods are provided in supporting information.

Accession Codes The coordinates and structure factors for the LTA4H and its complexes with compound 1 and compound 2 have been deposited in the Protein Data Bank, www.rcsb.org, under accession codes 4RVB, 4R7L, and 5TUO, respectively. Authors will release the atomic coordinates an experimental data upon article publication.

AUTHOR INFORMATION Corresponding Authors *For J.H.: phone, +86-021-64253681; E-mail, [email protected]. *For F.C.: phone, +1-615-8924046; E-mail, [email protected]. Author Contributions †

These authors contributed equally to this manuscript. All authors read and approved the

finalized manuscript. Notes



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The authors declare no competing financial interest

ACKNOWLEDGMENTS We thank Michaela S. Fooksa and Prof. Donald H. Rubin at Vanderbilt University School of Medicine for improving the English of the manuscript. We thank the staff at SSRF beamline BL17U for assistances with data collection. We thank Prof. Colin D Funk (Department of Biochemistry, Queen’s University, Canada) for pCDNA3-5-LOX, pCDNA3-p-12-LOX, and pCDNA3-15-LOX-1 and Prof. Marcia E. Newcomer (Department of Biological Sciences, Louisiana State University) for stable 5-LOX. Supported by grants from the National Natural Science Foundation of China (grants 81402482, 21222211, 91313303, 81573020), the Shanghai Committee of Science and Technology (grants 14ZR1411100, 15431902000), China Postdoctoral Science Foundation grant (2014M551361, 2015T80415), BAGUI scholar program (2014A001), Guangxi Committee of Science and Technology (2014GXNSFFA118003) and the Project of Talents Highland of Guangxi Province.

ABBREVIATIONS USED 5-LOX, 5-Lipoxygenase; 12-LOX, 12-Lipoxygenase; 15-LOX, 15-Lipoxygenase; ALI, Acute lung injury; AA, Arachidonic acid; BALF, Bronchoalveolar lavage fluid; BLM, bleomycin; cPLA2, Cytosolic phospholipases A2; i.v., Dex, Dexamethasone; ECM, Extracellular matrix; Intravenous injection; ELISA, Enzyme-linked immunosorbent assay;


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fMLP, N-formylmethionyl-leucyl-phenyl-alanine; IPF, Idiopathic pulmonary fibrosis; LPS, Lipopolysaccharide; LTA4H, Leukotriene A4 hydrolase; LTB4, Leukotriene B4; qRT-PCR, quantitative Real-time fluorescence PCR; SSRF, Shanghai Synchrotron Radiation Facility

REFERENCES (1) Rubenfeld, G. D.; Caldwell, E.; Peabody, E.; Weaver, J.; Martin, D. P.; Neff, M.; Stern, E. J.; Hudson, L. D. Incidence and outcomes of acute lung injury. N. Engl. J. Med. 2005, 353, 1685-1693. (2) Matthay, M. A.; Ware, L. B.; Zimmerman, G. A. The acute respiratory distress syndrome. J. Clin. Invest. 2012, 122, 2731-2740. (3) du Bois, R. M. Strategies for treating idiopathic pulmonary fibrosis. Nat. Rev. Drug Discovery 2010, 9, 129-140. (4) Richeldi, L.; du Bois, R. M.; Raghu, G.; Azuma, A.; Brown, K. K.; Costabel, U.; Cottin, V.; Flaherty, K. R.; Hansell, D. M.; Inoue, Y.; Kim, D. S.; Kolb, M.; Nicholson, A. G.; Noble, P. W.; Selman, M.; Taniguchi, H.; Brun, M.; Le Maulf, F.; Girard, M.; Stowasser, S.; Schlenker-Herceg, R.; Disse, B.; Collard, H. R.; INPULSIS Trial Investigators. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N. Engl. J. Med. 2014, 370, 2071-2082. (5) Cheng, F.; Liu, C.; Jiang, J.; Lu, W.; Li, W.; Liu, G.; Zhou, W.; Huang, J.; Tang, Y. Prediction of drug-target interactions and drug repositioning via network-based inference. PLoS Comput. Biol. 2012, 8, e1002503.



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

Page 24 of 41

(6) Wu, Z.; Lu, W.; Wu, D.; Luo, A.; Bian, H.; Li, J.; Li, W.; Liu, G.; Huang, J.; Cheng, F.; Tang, Y. In silico prediction of chemical mechanism of action via an improved network-based inference method. Br. J. Pharmacol. 2016, 173, 3372-3385. (7) Cheng, F.; Murray, J. L.; Zhao, J.; Sheng, J.; Zhao, Z.; Rubin, D. H. Systems biologybased investigation of cellular antiviral drug targets identified by gene-trap insertional mutagenesis. PLoS Comput. Biol. 2016, 12, e1005074. (8) Cheng, F.; Hong, H.; Yang, S.; Wei, Q. Individualized network-based drug repositioning infrastructure for precision oncology in the panomics era. Briefings Bioinf. DOI:

10.1093/bib/bbw051.

Publication

online:

June

12,

2016.

https://academic.oup.com/bib/article-lookup/doi/10.1093/bib/bbw051 (accessed October 30, 2016). (9) Mantovani, A.; Cassatella, M. A.; Costantini, C.; Jaillon, S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat. Rev. Immunol. 2011, 11, 519-531. (10) Grommes, J.; Soehnlein, O. Contribution of neutrophils to acute lung injury. Mol. Med. 2011, 17, 293-307. (11) Hunninghake, G. W.; Gadek, J. E.; Lawley, T. J.; Crystal, R. Mechanisms of neutrophil accumulation in the lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Invest. 1981, 68, 259-269. (12) Bringardner, B. D.; Baran, C. P.; Eubank, T. D.; Marsh, C. B. The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis. Antioxid. Redox Signaling 2008, 10, 287-302.


Page 25 of 41

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


(13) Kinder, B. W.; Brown, K. K.; Schwarz, M. I.; Ix, J. H.; Kervitsky, A.; King, T. E. Baseline BAL neutrophilia predicts early mortality in idiopathic pulmonary fibrosis. Chest 2008, 133, 226-232. (14) Kolaczkowska, E.; Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 2013, 13, 159-175. (15) Ford-Hutchinson, A.; Bray, M.; Doig, M. V.; Shipley, M.; Smith, M. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 1980, 286, 264-265. (16) Tager, A. M.; Luster, A. D. BLT1 and BLT2: the leukotriene B 4 receptors. Prostaglandins, Leukotrienes Essent. Fatty Acids 2003, 69, 123-134. (17) Afonso, P. V.; Janka-Junttila, M.; Lee, Y. J.; McCann, C. P.; Oliver, C. M.; Aamer, K. A.; Losert, W.; Cicerone, M. T.; Parent, C. A. LTB 4 is a signal-relay molecule during neutrophil chemotaxis. Dev. Cell 2012, 22, 1079-1091. (18) Wang, L.; Zhao, L.; Lv, J.; Yin, Q.; Liang, X.; Chu, Y.; He, R. BLT1-dependent alveolar recruitment of CD4+ CD25+ Foxp3+ regulatory T cells is important for resolution of acute lung injury. Am. J. Respir. Crit. Care Med. 2012, 186, 989-998. (19) Frank, J. A.; Matthay, M. A. Leukotrienes in acute lung injury: a potential therapeutic target? Am. J. Respir. Crit. Care Med. 2005, 172, 261-262. (20) Wilborn, J.; Bailie, M.; Coffey, M.; Burdick, M.; Strieter, R.; Peters-Golden, M. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Invest. 1996, 97, 1827-1836. (21) Marks, P. A.; Breslow, R. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat. Biotechnol. 2007, 25, 84-90.



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

Page 26 of 41

(22) Jung, M.; Brosch, G.; Kölle, D.; Scherf, H.; Gerhäuser, C.; Loidl, P. Amide analogues of trichostatin A as inhibitors of histone deacetylase and inducers of terminal cell differentiation. J. Med. Chem. 1999, 42, 4669-4679. (23) Rai, M.; Soragni, E.; Chou, C. J.; Barnes, G.; Jones, S.; Rusche, J. R.; Gottesfeld, J. M.; Pandolfo, M. Two new pimelic diphenylamide HDAC inhibitors induce sustained frataxin upregulation in cells from Friedreich's ataxia patients and in a mouse model. PLoS One 2010, 5, e8825. (24) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2007, 2, 22122221. (25) Askonas, L. J.; Kachur, J. F.; Villani-Price, D.; Liang, C. D.; Russell, M. A.; Smith, W.

G.

Pharmacological

characterization

of

SC-57461A

(3-[methyl[3-[4-

(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl), a potent and selective inhibitor of leukotriene A(4) hydrolase I: in vitro studies. J. Pharmacol. Exp. Ther. 2002, 300, 577-582. (26) Thunnissen, M. M.; Nordlund, P.; Haeggström, J. Z. Crystal structure of human leukotriene A4 hydrolase, a bifunctional enzyme in inflammation. Nat. Struct. Mol. Biol. 2001, 8, 131-135. (27) Thunnissen, M. M.; Andersson, B.; Samuelsson, B.; Wong, C.-H.; Haeggström, J. Z. Crystal structures of leukotriene A4 hydrolase in complex with captopril and two competitive tight-binding inhibitors. FASEB J. 2002, 16, 1648-1650.


Page 27 of 41

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


(28) Orning, L.; Krivi, G.; Fitzpatrick, F. A. Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes. J. Biol. Chem. 1991, 266, 1375-1378. (29) Ramalingam, S. S.; Parise, R. A.; Ramanathan, R. K.; Lagattuta, T. F.; Musguire, L. A.; Stoller, R. G.; Potter, D. M.; Argiris, A. E.; Zwiebel, J. A.; Egorin, M. J.; Belani, C. P. Phase I and pharmacokinetic study of vorinostat, a histone deacetylase inhibitor, in combination with carboplatin and paclitaxel for advanced solid malignancies. Clin. Cancer Res. 2007, 13, 3605-3610. (30) Novotny-Diermayr, V.; Sangthongpitag, K.; Hu, C. Y.; Wu, X.; Sausgruber, N.; Yeo, P.; Greicius, G.; Pettersson, S.; Liang, A. L.; Loh, Y. K.; Bonday, Z.; Goh, K. C.; Hentze, H.; Hart, S.; Wang, H.; Ethirajulu, K.; Wood, J. M. SB939, a novel potent and orally active histone deacetylase inhibitor with high tumor exposure and efficacy in mouse models of colorectal cancer. Mol. Cancer Ther. 2010, 9, 642-652. (31) Cheng, F.; Li, W.; Zhou, Y.; Shen, J.; Wu, Z.; Liu, G.; Lee, P. W.; Tang, Y. admetSAR: a comprehensive source and free tool for assessment of chemical ADMET properties. J. Chem. Inf. Model. 2012, 52, 3099-3105. (32) Moeller, A.; Ask, K.; Warburton, D.; Gauldie, J.; Kolb, M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int. J. Biochem. Cell Biol. 2008, 40, 362-382. (33) Thangapandian, S.; John, S.; Arooj, M.; Lee, K. W. Molecular dynamics simulation study and hybrid pharmacophore model development in human LTA4H inhibitor design. PLoS One 2012, 7, e34593.



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

(34) Kochan, G.; Krojer, T.; Harvey, D.; Fischer, R.; Chen, L.; Vollmar, M.; von Delft, F.; Kavanagh, K. L.; Brown, M. A.; Bowness, P.; Wordsworth, P.; Kessler, B. M.; Oppermann, U. Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 7745-7750. (35) Birtley, J. R.; Saridakis, E.; Stratikos, E.; Mavridis, I. M. The crystal structure of human endoplasmic reticulum aminopeptidase 2 reveals the atomic basis for distinct roles in antigen processing. Biochemistry (Mosc) 2012, 51, 286-295. (36) Thunnissen, M. M.; Andersson, B.; Samuelsson, B.; Wong, C.-H.; Haeggström, J. Z. Crystal structures of leukotriene A4 hydrolase in complex with captopril and two competitive tight-binding inhibitors. FASEB J. 2002, 16, 1648-1650. (37) Boxio, R.; Bossenmeyer-Pourié, C.; Steinckwich, N.; Dournon, C.; Nüsse, O. Mouse bone marrow contains large numbers of functionally competent neutrophils. J. Leukocyte Biol. 2004, 75, 604-611. (38) Wu, Q.; Liang, J.; Lin, S.; Zhou, X.; Bai, L.; Deng, Z.; Wang, Z. Characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic calcimycin (A23187) in Streptomyces chartreusis NRRL 3882. Antimicrob. Agents Chemother. 2011, 55, 974982. (39) Helgadottir, A.; Manolescu, A.; Thorleifsson, G.; Gretarsdottir, S.; Jonsdottir, H.; Thorsteinsdottir, U.; Samani, N. J.; Gudmundsson, G.; Grant, S. F.; Thorgeirsson, G. The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nat. Genet. 2004, 36, 233-239.


Page 28 of 41

Page 29 of 41

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(40) Singh, R. K.; Furze, R. C.; Birrell, M. A.; Rankin, S. M.; Hume, A. N.; Seabra, M. C. A role for Rab27 in neutrophil chemotaxis and lung recruitment. BMC Cell Biol. 2014, 15, 39. (41) Wollin, L.; Maillet, I.; Quesniaux, V.; Holweg, A.; Ryffel, B. Antifibrotic and antiinflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis. J. Pharmacol. Exp. Ther. 2014, 349, 209-220. (42) Helms, M. N.; Torres-Gonzalez, E.; Goodson, P.; Rojas, M. Direct tracheal instillation of solutes into mouse lung. J. Visualized Exp. 2010, 42, e1941. (43) Cai, X.; Zhai, H. X.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai, C. J.; Bao, R.; Qian, C. Discovery of 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-Nhydroxyheptanamide (CUDc-101) as a potent multi-acting HDAC, EGFR, and HER2 inhibitor for the treatment of cancer. J. Med. Chem. 2010, 53, 2000-2009. (44) Arts, J.; King, P.; Marien, A.; Floren, W.; Belien, A.; Janssen, L.; Pilatte, I.; Roux, B.; Decrane, L.; Gilissen, R.; Hickson, I.; Vreys, V.; Cox, E.; Bol, K.; Talloen, W.; Goris, I.; Andries, L.; Du Jardin, M.; Janicot, M.; Page, M.; van Emelen, K.; Angibaud, P. JNJ26481585, a novel "second-generation" oral histone deacetylase inhibitor, shows broadspectrum preclinical antitumoral activity. Clin. Cancer Res. 2009, 15, 6841-6851. (45) Johnston, T. H.; Huot, P.; Damude, S.; Fox, S. H.; Jones, S. W.; Rusche, J. R.; Brotchie, J. M. RGFP109, a histone deacetylase inhibitor attenuates L-DOPA-induced dyskinesia in the MPTP-lesioned marmoset: a proof-of-concept study. Parkinsonism Relat. Disord. 2013, 19, 260-264.



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Table 1. Inhibitory activities of the 18 known HDAC inhibitors against LTA4H in enzyme activity assays.

Compounds

Structure

Aminopeptidase Hydrolase IC50 (µM) IC50 (µM)

∆Tm (°C)

SAHA (1)

1.67

7.65

1.47

M344 (2)

0.30a

0.68

2.35

Scriptaid (3)

4.40

>10

-0.68

Trichostatin A (4)

>10

>10

0.17

Panobinostat (5)

>10

>10

0.12

Rocilinostat (6)

>10

>10

0.55

Belinostat (7)

>10

>10

0.11

Resminostat (8)

>10

>10

0.44

CUDC-10143 (9)

>10

>10

0.30

Pracinostat (10)

>10

>10

0.31


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Tubacin (11)

>10

>10

0.34

Givinostat (12)

>10

>10

0.19

>10

>10

0.24

JNJ-2648158544 (14)

>10

>10

0.52

Sodium valproate (15)

>10

>10

0.36

Entinostat (16)

>10

>10

0.39

RG283345 (17)

>10

>10

0.39

Mocetinostat (18)

>10

>10

-0.33

O H N

O

Abexinostat (13)

O

N H O

OH

N

a

IC50 values were determined from the results of at least three independent tests, and attempts to

determine IC50 values were made if the inhibition rate at 10 µM was larger than 50%.



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FIGURE LEGENDS Figure 1. The interaction of 1 and 2 with LTA4H. Inhibitory activities of 1 (A) and 2 (B) against cPLA2, LOXs and LTA4H. (C) Melt curves of LTA4H alone (black) or with 50 µM compounds. The detailed data for 18 drugs labeled in Figure 1 are provided in Table 1. Co-crystal complexes of 1 and 2 with LTA4H (D-G). (D) The LTA4H binding pocket with 1 (PDB ID 4R7L). (E)The detailed binding model of 1-LTA4H in co-crystal complex. (F) The LTA4H binding pocket with 2 (PDB ID 4RSY). (G) The detailed binding model of 2-LTA4H in co-crystal complex. 4R7L and 4RSY were shown in cartoon with color of sky blue and orange, respectively. 1, 2 and conserved binding residues discussed in this study were shown in sticks. The atom Zn2+ was shown with sphere and light blue. The hydrogen bond is represented by black dash line.

Figure 2. Two-dimensional interaction schemes of co-crystal structures of 1 (A, PDB ID 4R7L) or 2 (B, PDB ID 4RSY) with LTA4H. The ligands are shows as sticks and the non-carbon atoms are colored by atom types. Critical residues of the binding pocket are shown.

Figure 3. 1 and 2 inhibits LTB4 biosynthesis and represses neutrophil migration by targeting LTA4H. (A) Neutrophils isolated from the bone marrow of mouse were pretreated with various concentrations of 1 and 2 for 30 min and then stimulated with 21 (5 µM) for 30 min, the LTB4 content in the supernatant was measured by ELISA as described in methods. (B-E) Isolated mouse neutrophils were incubated with various compounds at indicated concentrations for 30 min at 37°C. Cells migration was determined in the present of 10 µM fMLP (B, D) or 10 nM LTB4 (C, E) at 37°C for 1 h using a 96-well chemotaxis plate with 5 µm pore diameter. Migrated cells were collected and counted. Data are representative of three independent experiments. Bar groups represent the mean ± SD. *P

Drug Repurposing of Histone Deacetylase ... - ACS Publications

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