ToP-DNJ, a selective inhibitor of endoplasmic reticulum Î±-glucosidase

Subscriber access provided by READING UNIV

Letter

ToP-DNJ, a selective inhibitor of endoplasmic reticulum #-glucosidase II exhibiting anti-flaviviral activity J.L. Kiappes, Michelle L. Hill, Dominic S Alonzi, Joanna L. Miller, Ren Iwaki, Andrew C. Sayce, Alessandro T. Caputo, Atsushi Kato, and Nicole Zitzmann ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00870 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

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

ACS Chemical Biology

Letter

ToP-DNJ, a selective inhibitor of endoplasmic reticulum α-glucosidase II exhibiting anti-flaviviral activity

J. L. Kiappes1, Michelle L. Hill1, Dominic S. Alonzi1, Joanna L. Miller1, Ren Iwaki2, Andrew C. Sayce1, Alessandro T. Caputo1, Atsushi Kato2 and Nicole Zitzmann1*

Author Affiliations 1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU (UK) 2. Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 9300194 (Japan)

Corresponding Author Professor Nicole Zitzmann: [email protected]

1 ACS Paragon Plus Environment


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

1

Abstract

2

Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as

3

glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in

4

making a compound that is selective for the glycosidase associated with a given disease. We

5

report the synthesis of ToP-DNJ, an antiviral iminosugar–tocopherol conjugate. Tocopherol

6

was incorporated into the design of the iminosugar in order to direct the drug to the liver and

7

immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER

8

α−glucosidase II at low micromolar concentrations and selectively accumulated in the liver

9

in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the

10

myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite

11

into an iminosugar provides selectivity with respect to target enzyme, target cell and target

12

tissue.

13

Document Text

14

Iminosugars are characterized by their nitrogen-containing heterocycles and ability to mimic

15

monosaccharides. They have been investigated for their therapeutic potential against a variety

16

of diseases including diabetes, genetic sphingolipidoses, and viral infections, with several

17

iminosugar drugs in clinical use and clinical trials.1-11 A major challenge for the development

18

of these molecules is that the ubiquity of sugars and sugar processing enzymes throughout the

19

body, which allows iminosugars to be therapeutic with respect to so many diseases, also leads

20

to a variety of side effects.2 We perceived two ways of improving selectivity: (i) developing a

21

drug that has activity against only the target enzyme and (ii) directing the drug preferentially

22

to target cells and tissues.


Page 2 of 21

Page 3 of 21

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


23

Our interests lie in the application of iminosugars as antivirals, particularly for the

24

treatment of hepatitis C virus (HCV) and dengue virus (DENV). The therapeutic effect is

25

theorized to occur via inhibition of the endoplasmic reticulum α-glucosidases I and II (GluI

26

and GluII).4,6,10,11 These two enzymes are antiviral targets due to their key role in regulating

27

entry into the endoplasmic reticulum quality control (ERQC) pathway. As N-linked

28

glycoproteins are being translocated into the ER, a 14-sugar oligosaccharide is transferred en

29

bloc onto the nascent polypeptide. The first two steps of glycan processing are carried out by

30

GluI and GluII, sequentially removing the two terminal glucose residues of the

31

oligosaccharide (Figure 1A). The resulting monoglucosylated glycan serves as “tag” for

32

recognition by calnexin and calreticulin, which mediate interactions with host chaperones that

33

make up ERQC to allow proper glycoprotein folding. GluII acts a second time to remove the

34

final glucose residue, which means the protein can no longer interact with calnexin and

35

calreticulin. Enveloped viruses that contain N-glycosylated envelope and nonstructural

36

proteins rely on this host glycoprotein folding process. Inhibition of GluI and GluII by

37

iminosugars

38

monoglucosylated stage, preventing these proteins from interacting with ERQC which in turn

39

can lead to misfolding. Without properly folded viral glycoproteins, morphogenesis and/or

40

infectivity of the virus are compromised. Iminosugars are effective against a host of viruses

41

including HCV, DENV, HIV, hepatitis B virus, influenza and others.6,12

keeps

the

protein-linked

N-glycans

from

being

processed

to

the

42

Because limited high-resolution structural information was available for these

43

enzymes when the project began, a structure-based inhibitor design approach was intractable.

44

Instead, we conceived that cell/tissue targeting could be achieved by conjugation of an

45

iminosugar to a metabolite, thus directing the attached iminosugar according to the

46

metabolite’s own distribution. Previously, iminosugars have been successfully targeted to the

47

ER via encapsulation into liposomes;13 however, degradation of liposomes “on the shelf” 3 ACS Paragon Plus Environment


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

48

made this methodology undesirable for long term storage or treatment. In contrast, by uniting

49

the pharmacophore and targeting moieties into a single molecule, the stability is defined by

50

that of the molecule itself rather than a vehicle.

51

We selected D-1-deoxynojirimycin (DNJ) 1 (Figure 1B) as the iminosugar component

52

due to the broad-spectrum antiviral activity of 1 and its N-alkyl derivatives (e.g. N-butylDNJ

53

2).6 D-α-tocopherol 3, a form of vitamin E (Figure 1C), was selected as the metabolite to be

54

conjugated to the iminosugar as it is non-toxic.14 As 3 accumulates in two places desirable for

55

antiviral therapy, we hoped it could serve as a tissue-targeting moiety. After being absorbed

56

in the gut, 3 is packaged in the liver, the target organ of HCV and a potential reservoir for

57

DENV.15 Coupling 3 to siRNA directs the conjugate to the liver in vivo,16 though this has

58

never been demonstrated for a small molecule conjugate, nor via oral administration. 3 also

59

accumulates in the membranes of immune cells,17 one of the target cell types of DENV. So

60

that both the DNJ and tocopherol moieties could be recognized independently by biological

61

partners, a 5-carbon chain was employed as a flexible linker, resulting in the target structure,

62

5’-tocopheroxypentyl-DNJ (ToP-DNJ, 4).

63

A convergent route (Scheme 1) was designed with eventual exploration of structure

64

activity relationships (SAR) in mind, with straightforward replacement of any of the 3

65

subunits (linker, iminosugar, or metabolite) possible to allow optimization of each

66

component. Desymmetrization of 1,5-pentanediol provided linker compound 5 (Scheme

67

S1).18-20 Deprotonation of tocopherol 3 (NaH, DMF, 0 °C) yielded a nucleophilic phenolate

68

anion that attacked alkyl tosylate 5 to give tocopheryl ether 6 (86% yield). With the

69

metabolite attached, the other end of the linker could be unmasked (p-TsOH, H2O) to reveal

70

aldehyde 7 (99% yield). Due to the lipophilicity of the tocopherol moiety, 7 was coupled to

71

tetra-O-acetyl DNJ 821 rather than free DNJ 1, so that the two reactants could be dissolved in


Page 4 of 21

Page 5 of 21

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


72

a single phase. The reductive amination (Na(AcO)3BH, cat. HOAc, Na2SO4) proceeded

73

smoothly to give per-acetylated ToP-DNJ 9 (53% yield), and subsequent removal (NaOMe,

74

MeOH) of the acetyl protecting groups provided 4 (71% yield). Attempts were made to

75

couple 7 directly with DNJ 1, but the lack of mutual solubility resulted in lower yields of 4.

76

With 4 in hand, we examined the biological activities of the lead compound as a proof

77

of concept. To begin characterization of 4, we addressed whether the addition of the large

78

tocopherol moiety abolished the ability of the iminosugar to inhibit glucosidases. In vitro

79

inhibition studies of isolated glucosidases22 were carried out (Supplemental Table 1 and

80

Supplemental Figure 1). In addition to the targeted enzymes GluI and GluII, the effects of 4

81

on α-glucosidases (intestinal maltase, intestinal isomaltase, intestinal sucrase, and lysosomal

82

glucosidase) and on a β-glucosidase (intestinal cellobiase) were analysed, as off-target

83

inhibition of these can cause undesirable gastrointestinal side effects.2 The activity of 4 was

84

compared to that of the parent compound 1 and the clinically approved drug 2,10 both of

85

which inhibit all of the tested α-glucosidases. Suprisingly, 4 showed a remarkable selectivity

86

for GluII. It has a comparable IC50 (concentration which gives 50% inhibition) to 1 and 2

87

with regard to GluII (IC50s 9.0, 13, and 16 µM for 4, 2, and 1, respectively), but shows less

88

than 50% inhibition of the other tested enzymes at the maximum tested concentration of 50

89

µM. This selectivity for GluII has not been reported for any other DNJ compound and

90

represents a huge step towards developing an antiviral of this class of iminosugars (which

91

requires ER α-glucosidase inhibition) without associated gastrointestinal side effects (due to

92

inhibition of the intestinal glucosidases). GluII and the intestinal α-glucosidases are all

93

members of glycoside hydrolase family 31; it is therefore difficult to suggest a molecular

94

explanation for the selectivity of 4 towards the ER-resident enzyme. Preliminary comparison



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

95

of the active site of recently reported crystal structures of GluII23,24 and that of intestinal

96

maltase/glucoamylase25,26 does not reveal the molecular origin of selectivity.

97

After demonstrating inhibition of GluII in vitro, 4 was next evaluated for its ability to

98

disrupt activity of the same enzyme in the host cells in which we routinely evaluate

99

compounds for inhibition of HCV and DENV: primary human monocyte-derived

100

macrophages (MDMΦ, for DENV) and the human hepatoma Huh7.5 cell line (for HCV and

101

DENV). Inhibition of GluI and GluII in cell culture is measured by the free oligosaccharide

102

(FOS) assay.27 In the absence of ER α-glucosidase inhibition, the only glucosylated FOS

103

observed are low levels of Glc1Man5GlcNAc1. In general, when cells are treated with 2,

104

mono-, di- and triglucosylated species can be observed indicating that both GluI and GluII

105

are inhibited.27 Accumulation of mono- and diglucosylated species (Glc1Man4GlcNAc1 and

106

Glc2Man4GlcNAc1, respectively) result from inhibition of GluII, while triglucosylated

107

glycans (Glc3Man4GlcNAc1) serve as a biomarker for inhibition of GluI. In contrast to 2, 4

108

demonstrated selectivity both in terms of enzyme and cell type. Glucosylated FOS were

109

observed in naïve MDMΦ (Figure 2A) but not in naïve Huh7.5 cells. Only monoglucosylated

110

FOS accumulated in 4-treated MDMΦ, while mono- and triglucosylated FOS were observed

111

in cells similarly treated with 2. This further confirms the results of in vitro enzyme assays

112

that 4 inhibits only GluII, while 2 inhibits both ER-resident glucosidases. In the Huh7.5 cells,

113

no glucosylated FOS were observed, indicating that 4 inhibited neither GluI nor GluII in

114

these cells, while 2 inhibits both enzymes in the same cells (Supplemental Figure 2A).

115

To examine the cell-type selectivity more thoroughly, additional human cell lines

116

were treated with 4 and analysed for FOS. Glu1Man4GlcNAc1 was detected in HL60

117

(promyelocytic) cells (Supplemtnal Figure 2B), but not in Jurkat (T lymphocyte,

118

Supplemental Figure 2C) nor Raji (B lymphocyte, Supplemental Figure 2D) cells. The fact


Page 6 of 21

Page 7 of 21

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


119

that FOS were observed only in the MDMΦ and HL60 cells indicates that 4 affects only

120

myeloid lineage immune cells. The GluII enzyme is the same in all human cells, suggesting

121

that 4 is more effectively absorbed by myeloid lineage cell types than others. This is

122

consistent with our initial hypothesis that the biological uptake of 4 would be influenced by

123

the patterns of the constituent 3, as immune cells are known to have increased amounts of 3

124

in their membranes, suggesting they likely have mechanisms for enhanced uptake of this

125

moiety. This opens up an exciting new strategy for targeting specific host cells, thereby

126

reducing off-target effects typical of iminosugars.

127

The FOS produced under treatment with 4 in both the primary MDMΦ and HL60

128

cells included only monoglucosylated species, indicating inhibition of the second reaction

129

catalyzed by GluII. However, no diglucosylated species were detected, raising the question

130

whether 4 inhibits only one of the reactions catalyzed by GluII. In an effort to address this

131

question, we measured the inhibition of GluII in vitro using a fluorescently labelled analogue

132

of a native glycan substrate (Glc2Man7GlcNAc1), rather than α-p-nitrophenylglucoside. 4

133

inhibited both reactions of GluII, with a more potent influence on the first reaction catalyzed

134

by the enzyme (IC50 of 14 µM for the conversion of diglucosylated to monoglucosylated vs.

135

56 µM for the conversion of monoglucosylated to non-glucosylated glycan). This suggests

136

that the lack of diglucosylated FOS in the primary MDMΦ and HL60 experiments is due to

137

the kinetics of cellular glycan processing, rather than a property of the inhibitor 4.

138

Given the more selective inhibition profile of 4, it remained to be determined whether

139

it retained the antiviral activity observed for other members of the iminosugar class. 4 was

140

antiviral with respect to DENV in primary MDMΦ cells (Figure 2B), in which GluII is

141

inhibited as shown by FOS assay, but in Huh7.5 cells, in which GluII is not inhibited, neither

142

an anti-DENV nor anti-HCV effect was observed (Supplemental Figure 3). This is consistent



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

143

with the theory that inhibition of GluII is key to the antiviral effect. The IC50 of 4 (12.7 µM)

144

for DENV inhibition in MDMΦ cells (7 donors) is similar to that determined for 2 (6.00

145

µM)28 in the same system. Notably, 4 has an antiviral effect in MDMΦ cells in the absence of

146

any GluI inhibition activity, suggesting that GluII inhibition alone is sufficient for an anti-

147

DENV effect.

148

The in vitro and whole cell assays characterized the targeted effects of the conjugated

149

tocopherol on selectivity for specific glucosidases and cell types. However, to see whether it

150

influenced the distribution of the iminosugar in different tissues, biodistribution studies were

151

carried out in 4-treated mice, with investigations of oral and intravenous administration

152

routes. In both cases, 4 was detected in the highest amounts in the liver (Figure 3A,

153

intravenous (IV) data; orally administered (per os, PO), Supplemental Figure 4), whereas 2 is

154

found in the highest amounts in the kidneys and bladder as it is excreted.29,30 The amount of 4

155

present in the liver did not decrease between the 4 h and 24 h time points. In contrast, simple

156

alkylated iminosugars are eliminated from the liver by more than 80% during the same time

157

period.29 In addition, the plasma terminal half-life of 4 was 8.79 h (Figure 3B), compared to 5

158

h for 2.30 The increase in half-life both in plasma and in the liver means that a single dose will

159

have longer term therapeutic effects than previously investigated iminosugars. 4 has poor oral

160

bioavailability, achieving a maximum blood plasma concentration of 46 nM after 8 h (Figure

161

3B). However, the absorption of 3 depends on co-consumed nutrients. By consuming 3 with a

162

lipid-rich meal, ten-fold higher plasma levels are observed.31 4 might similarly require other

163

hydrophobic molecules for efficient absorption after oral administration. Despite this

164

limitation with regard to oral absorption, the high liver levels of 4 demonstrate that tissue-

165

targeting of small molecules can be accomplished by the incorporation of the tocopherol

166

moiety, regardless of administration route.


Page 8 of 21

Page 9 of 21

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


167

In conclusion, we prepared DNJ-tocopherol conjugate 4, the first reported DNJ

168

derivative to be a selective inhibitor of GluII. This selectivity was demonstrated both for

169

isolated enzymes and in a whole cell system. By eliminating inhibition of the intestinal

170

glucosidases, 4 could overcome the side effects both in antiviral iminosugar clinical

171

candidates and FDA-approved 2. With a selective GluII inhibitor in hand, we discovered that

172

GluI inhibition is not obligate for antiviral activity. Given the cell type specificity of 4 and

173

the correlation of FOS and antiviral effect, it was substantiated that ER α-glucosidase

174

inhibition, and not an alternative effect of DNJ compounds, is the mechanism of action for

175

anti-DENV activity. Furthermore, incorporation of vitamin E into the iminosugar bestowed

176

improved plasma and liver half-lives, as well as cell type dependent activity, which could be

177

exploited for tailor-making drugs for host cells of specific viruses, leaving other cell types

178

less affected. While we continue to study the properties of 4, investigations are also

179

proceeding to establish SAR for each of these novel iminosugar capabilities, as well as work

180

on further iminosugar-metabolite conjugates.

181

Methods

182

Chemistry. Methods for the synthesis of ToP-DNJ 4, compound characterization and spectra

183

are described in detail in the Supporting Information.

184

Biological Assays. Methods for in vitro inhibition of isolated enzymes can be found in the

185

Supporting Information. Methods used for evaluating the cellular inhibition of the ER

186

glucosidase, as well as inhibition of HCV and DENV, are detailed in the Supporting

187

Information, which also describes the procedures used in the biodistribution studies.

188

Acknowledgements



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

189

Financial support for this work was provided by the Oxford Glycobiology

190

Endowment, Unither Virology LLC, a Lerner–Fink Fellowship in Medicinal

191

Chemistry (to J.L.K.) and Wellcome Trust predoctoral fellowship (to A.T.C.,

192

097300/Z/11/Z). The authors thank R. Dwek and K. Scott for useful discussions and

193

M. Wormald and A. Kumar for NMR spectroscopy and mass spectrometry assistance,

194

respectively.

195

Supporting Information

196

Scheme 1 with reagents and conditions, Supplemental Scheme 1, Supplemental Table 1,

197

Supplemental Figures 1–4, NMR spectra and in-depth experimental procedures are available

198

free of charge via the Internet from http://pubs.acs.org.

199

References

200

(1) Mehta, A., Zitzmann, N., Rudd, P. M., Block, T. M., and Dwek, R. A. (1998) Alpha-

201

glucosidase inhibitors as potential broad based anti-viral agents. FEBS Lett. 430, 17–22.

202

(2) Butters, T. D., Dwek, R. A., and Platt, F. M. (2005) Imino sugar inhibitors for treating the

203

lysosomal glycosphingolipidoses. Glycobiology 15, 43R–52R.

204

(3) Martin, O. (2007) Les Iminosucres : applications thérapeutiques actuelles et futures. Ann.

205

Pharm. Fr. 65, 5–13.

206

(4) Sayce, A. C., Miller, J. L., and Zitzmann, N. (2010) Targeting a host process as an

207

antiviral approach against dengue virus. Trends Microbiol. 18, 323–330.

208

(5) Nash, R. J., Kato, A., Yu, C.-Y., and Fleet, G. W. (2011) Iminosugars as therapeutic

209

agents: recent advances and promising trends. Future Med. Chem. 3, 1513–1521.

210

(6) Chang, J., Block, T. M., and Guo, J.-T. (2013) Antiviral therapies targeting host ER

211

alpha-glucosidases: Current status and future directions. Antiviral Res. 99, 251–260.

212

(7) Dalziel, M., Crispin, M., Scanlan, C. N., Zitzmann, N., and Dwek, R. A. (2014) Emerging 10 ACS Paragon Plus Environment

Page 10 of 21

Page 11 of 21

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


213

principles for the therapeutic exploitation of glycosylation. Science (Washington, DC, U. S.)

214

343, 1235681.

215

(8) Low, J. G., Sung, C., Wijaya, L., Wei, Y., Rathore, A. P. S., Watanabe, S., Tan, B. H.,

216

Toh, L., Chua, L. T., Hou, Y., Chow, A., Howe, S., Chan, W. K., Tan, K. H., Chung, J. S.,

217

Cherng, B. P., Lye, D. C., Tambayah, P. A., Ng, L. C., Connolly, J., Hibberd, M. L., Leo, Y.

218

S., Cheung, Y. B., Ooi, E. E., and Vasudevan, S. G. (2014) Efficacy and safety of celgosivir

219

in patients with dengue fever (CELADEN): a phase 1b, randomised, double-blind, placebo-

220

controlled, proof-of-concept trial. Lancet Infect. Dis. 14, 706–715.

221

(9) Watanabe, S., Chan, K. W.-K., Dow, G., Ooi, E. E., Low, J. G., and Vasudevan, S. G.

222

(2016) Optimizing celgosivir therapy in mouse models of dengue virus infection of serotypes

223

1 and 2: The search for a window for potential therapeutic efficacy. Antiviral Res. 127, 10–

224

19.

225

(10) Sayce, A. C., Alonzi, D. S., Killingbeck, S. S., Tyrrell, B. E., Hill, M. L., Caputo, A. T.,

226

Iwaki, R., Kinami, K., Ide, D., Kiappes, J. L., Beatty, P. R., Kato, A., Harris, E., Dwek, R. A.,

227

Miller, J. L., and Zitzmann, N. (2016) Iminosugars Inhibit Dengue Virus Production via

228

Inhibition of ER Alpha-Glucosidases-Not Glycolipid Processing Enzymes. PLoS Neglected

229

Trop. Dis. (Beasley, D. W. C., Ed.) 10, e0004524.

230

(11) Warfield, K. L., Plummer, E. M., Sayce, A. C., Alonzi, D. S., Tang, W., Tyrrell, B. E.,

231

Hill, M. L., Caputo, A. T., Killingbeck, S. S., Beatty, P. R., Harris, E., Iwaki, R., Kinami, K.,

232

Ide, D., Kiappes, J. L., Kato, A., Buck, M. D., King, K., Eddy, W., Khaliq, M., Sampath, A.,

233

Treston, A. M., Dwek, R. A., Enterlein, S. G., Miller, J. L., Zitzmann, N., Ramstedt, U., and

234

Shresta, S. (2016) Inhibition of endoplasmic reticulum glucosidases is required for in vitro

235

and in vivo dengue antiviral activity by the iminosugar UV-4. Antiviral Res. 129, 93–98.

236

(12) Alonzi, D. S., Scott, K. A., Dwek, R. A., and Zitzmann, N. (2017) Iminosugar antivirals:

237

the therapeutic sweet spot. Biochem. Soc. Trans. 45, 571–582.



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

238

(13) Pollock, S., Antrobus, R., Newton, L., Kampa, B., Rossa, J., Latham, S., Nichita, N. B.,

239

Dwek, R. A., and Zitzmann, N. (2010) Uptake and trafficking of liposomes to the

240

endoplasmic reticulum. FASEB J. 24, 1866–1878.

241

(14) Kappus, H., and Diplock, A. T. (1992) Tolerance and Safety of Vitamin-E - a

242

Toxicological Position Report. Free Radical Biol. Med. 13, 55–74.

243

(15) Balsitis, S. J., Coloma, J., Castro, G., Alava, A., Flores, D., McKerrow, J. H., Beatty, P.

244

R., and Harris, E. (2009) Tropism of dengue virus in mice and humans defined by viral

245

nonstructural protein 3-specific immunostaining. Am. J. Trop. Med. Hyg. 80, 416–424.

246

(16) Nishina, K., Unno, T., Uno, Y., Kubodera, T., Kanouchi, T., Mizusawa, H., and Yokota,

247

T. (2008) Efficient In Vivo Delivery of siRNA to the Liver by Conjugation of α-Tocopherol.

248

Mol. Ther. 16, 734–740.

249

(17) Wang, X., and Quinn, P. J. (2000) The location and function of vitamin E in membranes

250

(Review). Mol. Membr. Biol. 17, 143–156.

251

(18) Bouzide, A., and Sauvé, G. (1997) Highly selective silver (I) oxide mediated

252

monoprotection of symmetrical diols. Tetrahedron Lett. 38, 5945–5948.

253

(19) Davison, E. C., Fox, M. E., Holmes, A. B., Roughley, S. D., Smith, C. J., Williams, G.

254

M., Davies, J. E., Raithby, P. R., Adams, J. P., Forbes, I. T., Press, N. J., and Thompson, M.

255

J. (2002) Nitrone dipolar cycloaddition routes to piperidines and indolizidines. Part 9. Formal

256

synthesis of (–)-pinidine and total synthesis of (–)-histrionicotoxin, (+)-histrionicotoxin and

257

(–)-histrionicotoxin 235A. J. Chem. Soc., Perkin Trans. 1 1494–1514.

258

(20) Wennekes, T., van den Berg, R. J. B. H. N., Donker, W., van der Marel, G. A., Strijland,

259

A., Aerts, J. M. F. G., and Overkleeft, H. S. (2007) Development of adamantan-1-yl-

260

methoxy-functionalized 1-deoxynojirimycin derivatives as selective inhibitors of

261

glucosylceramide metabolism in man. J. Org. Chem. 72, 1088–1097.

262

(21) Heuckendorff, M., Pedersen, C. M., and Bols, M. (2010) Quantifying Electronic Effects


Page 12 of 21

Page 13 of 21

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


263

of Common Carbohydrate Protecting Groups in a Piperidine Model System. Chem. - Eur. J.

264

16, 13982–13994.

265

(22) Asano, N., Nishida, M., Kato, A., Kizu, H., Matsui, K., Shimada, Y., Itoh, T., Baba, M.,

266

Watson, A. A., Nash, R. J., Lilley, P. M., Watkin, D. J., and Fleet, G. W. J. (1998)

267

Homonojirimycin isomers and N-alkylated homonojirimycins: structural and conformational

268

basis of inhibition of glycosidases. J. Med. Chem. 41, 2565–2571.

269

(23) Satoh, T., Toshimori, T., Yan, G., Yamaguchi, T., and Kato, K. (2016) Structural basis

270

for two-step glucose trimming by glucosidase II involved in ER glycoprotein quality control.

271

Sci. Rep. 6, 20575.

272

(24) Caputo, A. T., Alonzi, D. S., Marti, L., Reca, I. B., Kiappes, J. L., Struwe, W. B., Cross,

273

A., Basu, S., Lowe, E. D., Darlot, B., Santino, A., Roversi, P., and Zitzmann, N. (2016)

274

Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum

275

iminosugar antivirals. Proc. Natl. Acad. Sci. U. S. A. 113, E4630–8.

276

(25) Sim, L., Willemsma, C., Mohan, S., Naim, H. Y., Pinto, B. M., and Rose, D. R. (2010)

277

Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-

278

isomaltase N-terminal domains. J. Biol. Chem. 285, 17763–17770.

279

(26) Ren, L., Qin, X., Cao, X., Wang, L., Bai, F., Bai, G., and Shen, Y. (2011) Structural

280

insight into substrate specificity of human intestinal maltase-glucoamylase. Protein Cell 2,

281

827–836.

282

(27) Alonzi, D. S., Neville, D. C. A., Lachmann, R. H., Dwek, R. A., and Butters, T. D.

283

(2008) Glucosylated free oligosaccharides are biomarkers of endoplasmic- reticulum alpha-

284

glucosidase inhibition. Biochem. J. 409, 571–580.

285

(28) Miller, J. L., Lachica, R., Sayce, A. C., Williams, J. P., Bapat, M., Dwek, R., Beatty, P.

286

R., Harris, E., and Zitzmann, N. (2012) Liposome-mediated delivery of iminosugars enhances

287

efficacy against dengue virus in vivo. Antimicrob. Agents Chemother. 56, 6379–6386.



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

288

(29) Mellor, H. R., Nolan, J., Pickering, L., Wormald, M. R., Platt, F. M., Dwek, R. A., Fleet,

289

G. W. J., and Butters, T. D. (2002) Preparation, biochemical characterization and biological

290

properties of radiolabelled N-alkylated deoxynojirimycins. Biochem. J. 366, 225–233.

291

(30) Treiber, A., Morand, O., and Clozel, M. (2007) The pharmacokinetics and tissue

292

distribution of the glucosylceramide synthase inhibitor miglustat in the rat. Xenobiotica 37,

293

298–314.

294

(31) Jeanes, Y. M., Hall, W. L., Ellard, S., Lee, E., and Lodge, J. K. (2004) The absorption of

295

vitamin E is influenced by the amount of fat in a meal and the food matrix. Br. J. Nutr. 92,

296

575–579.

297 298

Figure Legends

299

Figure 1. Enzyme targets and structures of iminosugars. (A) Trimming of the N-glycan by

300

ER α-glucosidases I and II (GluI and GluII). (B) Deoxynojirimycin 1 and N-

301

butyldeoxynojirimycin 2 inhibit the ER-resident enzymes GluI (PDB ID 4J5T), GluII (PDB

302

ID 5F0E), and intestinal glucosidases (e.g. maltase/glucoamylase, PDB ID 3TOP), which,

303

like GluII, are members of glycoside hydrolase family 31. (C) D-(+)-α-tocopherol 3 and ToP-

304

DNJ 4.

305 306

Scheme 1. Synthesis of ToP-DNJ 4.

307 308

Figure 2. Effects of ToP-DNJ 4 treatment in monocyte-derived macrophages (MDMΦ). (A)

309

Protein-normalized free oligosaccharide levels of naïve MDMΦ (1 representative donor). The

310

bar represents the average; error bars show one standard deviation. (B) Infectious virus titer

311

produced by dengue-infected MDMΦ (7 donors) under ToP-DNJ 4 or α-tocopherol 3 14 ACS Paragon Plus Environment

Page 14 of 21

Page 15 of 21

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


312

treatment. 4 has an IC50 of 12.7 µM, while 3 showed no antiviral effect. The data points

313

represent the mean; error bars show standard error of the mean.

314



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

315

Figure 3. In vivo studies of ToP-DNJ 4 in BALB/c mice. (A) The concentration of 4 in all

316

organs after intravenous (IV) administration. (B) Serum concentration of 4 over time (IV and

317

per os, PO). On both graphs, the mean of 3 animals is shown; error bars show one standard

318

deviation.


Page 16 of 21

Page 17 of 21

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


Figure 1. Enzyme targets and structures of iminosugars. (A) Trimming of the N-glycan by ER αglucosidases I and II (GluI and GluII). (B) Deoxynojirimycin 1 and N-butyldeoxynojirimycin 2 inhibit the ERresident enzymes GluI (PDB ID 4J5T), GluII (PDB ID 5F0E), and intestinal glucosidases (e.g. maltase/glucoamylase, PDB ID 3TOP), which, like GluII, are members of glycoside hydrolase family 31. (C) D-(+)-α-tocopherol 3 and ToP-DNJ 4. 196x593mm (300 x 300 DPI)

ACS Paragon Plus Environment


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

Scheme 1. Synthesis of ToP-DNJ 4. 84x51mm (300 x 300 DPI)


Page 18 of 21

Page 19 of 21

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


Figure 2. Effects of ToP-DNJ 4 treatment in monocyte-derived macrophages (MDMΦ). (A) Proteinnormalized free oligosaccharide levels of naïve MDMΦ (1 representative donor). The bar represents the average; error bars show one standard deviation. (B) Infectious virus titer produced by dengue-infected MDMΦ (7 donors) under ToP-DNJ 4 or α-tocopherol 3 treatment. 4 has an IC50 of 12.7 µM, while 3 showed no antiviral effect. The data points represent the mean; error bars show standard error of the mean. 91x131mm (300 x 300 DPI)



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

Figure 3. In vivo studies of ToP-DNJ 4 in BALB/c mice. (A) The concentration of 4 in all organs after intravenous (IV) administration. (B) Serum concentration of 4 over time (IV and per os, PO). On both graphs, the mean of 3 animals is shown; error bars show one standard deviation. 91x134mm (300 x 300 DPI)


Page 20 of 21

Page 21 of 21

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


Table of Contents graphic 43x24mm (300 x 300 DPI)


ToP-DNJ, a selective inhibitor of endoplasmic reticulum Î±-glucosidase

Recommend Documents