2-Chloropropionamide As a Low-Reactivity Electrophile for

Jun 14, 2017 - ... Case Western Reserve University, Cleveland, Ohio 44106, United States ... W. Guddat , G. Paul Savage , Ahmed Chadli , Craig M. Will...
0 downloads 0 Views 1MB Size
Subscriber access provided by The University of New Mexico

Article

2-Chloropropionamide as a low-reactivity electrophile for irreversible small-molecule probe identification Dharmaraja Allimuthu, and Drew J. Adams ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00424 • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 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 28

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

1

2-chloropropionamide as a low-reactivity

2

electrophile for irreversible small-molecule

3

probe identification

4

5

Dharmaraja Allimuthu1, Drew J. Adams1*

6 7

Affiliations:

8

1

9

Medicine, Case Western Reserve University, Cleveland, OH 44106

Department of Genetics and Genome Sciences and Comprehensive Cancer Center, School of

10 11

* Corresponding author email: [email protected]

12 13

ACS Paragon Plus Environment

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

Page 2 of 28

Adams et al.

14

Abstract

15

Resurgent interest in covalent target engagement in drug discovery has demonstrated

16

that small molecules containing weakly reactive electrophiles can be safe and effective

17

therapies. Several recently FDA-approved drugs feature an acrylamide functionality to

18

selectively engage cysteine side chains of kinases (Ibrutinib, Afatinib, and Neratinib).

19

Additional electrophilic functionalities whose reactivity is compatible with highly selective

20

target engagement and in vivo application could open new avenues in covalent small

21

molecule discovery. Here we report the synthesis and evaluation of a library of small

22

molecules containing the 2-chloropropionamide functionality, which we demonstrate is

23

less reactive than typical acrylamide electrophiles. Although many library members do

24

not appear to label proteins in cells, we identified S-CW3554 as selectively labeling

25

protein disulfide isomerase and inhibiting its enzymatic activity. Subsequent profiling of

26

the library against five diverse cancer cell lines showed unique cytotoxicity for S-

27

CW3554 in cells derived from multiple myeloma, a cancer recently reported to be

28

sensitive to PDI inhibition. Our novel PDI inhibitor highlights the potential of 2-

29

chloropropionamides as weak and stereochemically-tunable electrophiles for covalent

30

drug discovery.

31

2 ACS Paragon Plus Environment

Page 3 of 28

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

Adams et al.

32

Introduction

33

Multiple safe and effective drugs target cellular proteins covalently, and

34

irreversible target binding can offer benefits over reversible binding, including potent

35

and durable target engagement.1-3 However, drug discovery efforts in recent decades

36

have generally selected against molecules with covalent mechanisms of action, owing

37

largely to concerns regarding the presumed lower selectivity of target engagement and

38

idiosyncratic toxicity of covalent binders in vivo.4-6 Some electrophilic functionalities

39

observed to recur in small-molecule probes identified by high-throughput screening

40

have recently been labeled Pan-Assay Interference (PAINS) motifs and are now

41

recognized as unlikely candidates for advancement to in vivo studies due to their high

42

chemical reactivity and apparent lack of selectivity across biological assays.7

43

A new generation of targeted covalent inhibitors has begun to leverage the

44

benefits of irreversible target engagement while lowering concerns regarding

45

unselective covalent labeling.3 As typified by three recently FDA-approved drugs—

46

Ibrutinib, Afatinib, and Neratinib—these molecules use a moderately electrophilic

47

acrylamide functionality to engage specific cysteine residues adjacent to the active site

48

of their kinase targets.8-10

49

structure-based design guided by X-ray co-crystal structures,8, 11-13 recent efforts have

50

also sought to use covalent attachment as a design element in the synthesis of focused

51

screening libraries, either of ‘electrophilic fragments’14,

52

molecular weight.16-18

Although these molecules were largely the product of

15

or molecules of more typical

53

We reasoned that additional electrophilic functionalities that react with biological

54

nucleophiles at rates similar to or less than typical acrylamide functionalities could be

55

productive starting points for the development of in vivo-compatible covalent small3 ACS Paragon Plus Environment

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

Page 4 of 28

Adams et al.

56

molecule probes.15,

19

57

sterically hindered version of the commonly-used chloroacetamide, as showing lower

58

reactivity than analogous acrylamides.

59

chloropropionamides highlighted the low proteome reactivity of this functionality but also

60

identified one molecule, S-CW3554, that selectively targeted a 57 kDa protein and also

61

uniquely among library members showed cytotoxicity to multiple myeloma cell lines.

62

The target of S-CW3554 was confirmed using multiple approaches as protein disulfide

63

isomerase (PDIA1), an ER-localized protein currently under investigation as a

64

therapeutic target in neurodegenerative disease and cancer.20-24

65

disulfide bond formation and disulfide exchange reactions required for proper folding of

66

a wide range of proteins.24, 25 Although irreversible16, 18, 20, 22, 23 and reversible21, 26 PDI

67

inhibitors have been reported, S-CW3554’s 2-chloropropionamide electrophile is the

68

least reactive among known probes, making S-CW3554 a candidate for further

69

optimization to a metabolically stable in vivo probe. Together our studies validate the 2-

70

chloropropionamide functionality as an electrophile with lower reactivity than

71

acrylamides and suggest its utility in various covalent drug discovery approaches.

We have validated the 2-chloropropionamide functionality, a

A library of 26 structurally-diverse 2-

72 73 74

4 ACS Paragon Plus Environment

PDIA1 catalyzes

Page 5 of 28

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

Adams et al.

75

Results and Discussion

76

In considering potentially useful weak electrophilic functionalities, we focused on

77

2-chloropropionamides for a variety of reasons. Although the related chloroacetamide

78

functionality has been a commonly-used electrophile for the identification of a wide

79

range of useful chemical probes,15, 16, 20, 27, 28 such molecules have in many cases not

80

progressed to in vivo application, presumably due to the relatively low stability of the

81

chloroacetamide. We expected the addition of an alkyl group would sterically hinder

82

alkylation by cysteine and other nucleophiles, potentially allowing more selective

83

engagement with cellular protein targets in analogy to recently FDA-approved

84

acrylamide-containing kinase inhibitors.

85

substituent makes the electrophilic carbon a stereogenic center, meaning that the two

86

stereoisomeric 2-chloropropionamide products could have distinct affinity for cellular

87

nucleophiles.

Moreover, the introduction of an alkyl

88

To explore the utility of 2-chloropropionamides, we first compared the chemical

89

reactivity of 3 to the structurally-analogous chloroacetamide 2 and acrylamide 1 (Figure

90

1a-e). We noted no significant reactivity of the 2-chloroproprionamide with a cysteine

91

derivative (N-(4-nitrobenzoyl)-cysteine, *Cys) in 1:1 acetonitrile:PBS (pH 7.4) medium at

92

37 ºC for 60 min (Figure 1c). By contrast, under identical conditions, we observed that

93

62% of our chloroacetamide substrate 2 and 28% of our acrylamide substrate 1 were

94

converted to the expected addition products (Figure 1a, b). When we monitored the

95

reactivity of electrophiles 1-3 over 6 h using high-performance liquid chromatography

96

(HPLC), 54% of 1, 95% of 2 and 27% of 3 were converted into their corresponding

97

addition products (Figure 1d, e, Supporting Figure 1-3), which were each confirmed by

98

liquid chromatography-mass spectrometry analysis (LC-MS) (Supporting Figure 4). We 5 ACS Paragon Plus Environment

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

Page 6 of 28

Adams et al.

99

next synthesized and assessed alkyne-containing probes 4, 5, and 6 in a Click-ABPP

100

assay29 to assess the relative abilities of generic acrylamide, chloroacetamide, and 2-

101

chloropropionamide electrophiles to covalently label cellular nucleophiles. The extent of

102

proteome labeling followed a similar trend, with chloroacetamide 5 showing the most

103

extensive proteome labeling and 2-chloropropionamide 6 showing less labeling than the

104

analogous acrylamide (Figure 1e). Based on the greatly reduced reactivity of 2-

105

chloropropionamides relative to chloroacetamide and even acrylamide electrophiles in

106

in vitro and cellular assays, we concluded that chemical probes containing this

107

functionality could be of sufficiently low inherent reactivity to be compatible with safe in

108

vivo application.

109

We synthesized a collection of 26 structurally-diverse 2-chloropropionamides

110

with the hypothesis that, in contrast to simplified fragment 6, one or more of these

111

molecules (ranging from 392 to 495 Da) may have sufficient noncovalent affinity for one

112

or more cellular proteins to enable alkylation of the otherwise poorly reactive

113

electrophile (Figure 2a). Our library includes two diversity elements connected

114

combinatorially: the first diversity element (R1) contains a terminal alkyne functionality to

115

enable Click chemistry-mediated derivatizations, while the second diversity element (R2)

116

comprises structurally diverse diamines that provide distinct bond paths to bridge the R1

117

and 2-chloropropionamide fragments (Figure 2a). We used a simple synthesis strategy

118

in which four R1 building blocks were coupled to each of seven R2 building blocks by

119

peptide bond formation. Protecting group removal then enabled a second amide

120

coupling using S-2-chloropropionyl chloride, performed under solution-phase parallel

121

synthesis conditions,30 to provide the final library members in yields and purities

6 ACS Paragon Plus Environment

Page 7 of 28

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

Adams et al.

122

adequate for screening (Supporting Table 3). Two planned library members failed

123

synthesis and were not pursued further.

124

Although this small library could be screened in any standard target-based or

125

phenotypic assay, the inclusion of a terminal alkyne functionality also enables Click-

126

ABPP to identify cellular proteins labeled by each library member. We first used this

127

approach in HEK293 cells to monitor cellular binding partners for all 26 molecules within

128

our collection at two concentrations, 10 µM and 2 µM (Supporting Figure 6-9). One

129

molecule, S-CW3554, gave rise to a strong band (ca. 60 kDa) at both doses, and two

130

additional library members, CW3555 and CW3684 weakly labeled a band of similar

131

apparent molecular weight (Supporting Figure 6, 7). No other compound in the

132

collection gave rise to a strong band, confirming the generally low reactivity of 2-

133

chloropropionamides within the soluble proteome. Notably, S-CW3554 and CW3555

134

share the same tryptophan-derived alkyne R1 building block, while S-CW3554 and

135

CW3684 shared the same m-xylenediamine R2 building block, suggesting that both the

136

R1 and R2 portions of S-CW3554 contribute to its labeling efficiency.

137

We resynthesized S-CW3554 and confirmed its labeling interaction in HEK293

138

cells (Figure 3b).

We also synthesized R-CW3554 and derivatives that varied the

139

electrophile (CW2334, chloroacetamide; CW2294, acrylamide) (Figure 2b, 2c). As

140

expected, CW2334 showed strong labeling of a wide range of proteins, again

141

highlighting the substantially higher reactivity of the chloroacetamide electrophile

142

relative to its 2-chloropropionamide analog. R-CW3554 showed clear labeling of a band

143

that appeared to migrate just below the band observed for its enantiomer (Figure 3b).

144

Use of a biotin-containing azide during Click chemistry enabled purification and mass

7 ACS Paragon Plus Environment

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

Page 8 of 28

Adams et al.

145

spectrometric identification of the protein targets of both R- and S-CW3554 (Supporting

146

Figure 12). As expected given the slightly different mobilities of the proteins during gel

147

electrophoresis, the enantiomeric molecules target different proteins, with S-CW3554

148

targeting protein disulfide isomerase (PDIA1, 57kDa) and R-CW3554 targeting an

149

aldehyde dehydrogenase (ALDH2, 56 kDa). Notably, no spectral counts for ALDH2

150

were obtained in the S-CW3554-treated sample and no spectral counts for PDIA1 were

151

obtained in the R-CW3554-treated sample (Supporting Figure 12). This finding confirms

152

the potential for the stereoconfiguration at the reactive center to play a dominant role in

153

determining cellular target engagement, a unique feature of 2-choropropionamides

154

relative to other electrophilic warheads.

155

As protein disulfide isomerase has been implicated in a wide range of disease

156

states,20,

21, 24, 31, 32

157

analysis suggested an EC50 for labeling PDI after 6 h in HEK293 cells of 2 µM (Figure

158

3c, d). Three further approaches were undertaken to confirm the assignment of S-

159

CW3554 as a PDI inhibitor. First, S-CW3554 labeled recombinant PDI as assessed

160

using Click chemistry labeling and in-gel fluorescence (Figure 3e). In close analogy to

161

our cell-based labeling experiments (Figure 3b), CW3555 also showed labeling of rPDI,

162

while CW3557 did not label recombinant or cellular PDI (Figure 3b, e and Supporting

163

Figure 6). Elevated doses of S-CW3554 enhanced labeling of rPDI (Figure 3e).

164

Chloroacetamide CW2334 also labeled rPDI in vitro (Supporting Figure 11), suggesting

165

that PDI is likely one of the many targets observed during Click-ABPP experiments

166

using this probe (Figure 3b). However, across a wide concentration range, CW2334

167

labeled several proteins of varying molecular weight, suggesting that in this case the

we focused attention on S-CW3554. A concentration-response

8 ACS Paragon Plus Environment

Page 9 of 28

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

Adams et al.

168

sterically more hindered 2-chloropropionamide electrophile enables more selective

169

targeting of PDI (Figure 3c, f).

170

Second, S-CW3554 and chloroacetamide CW2334 inhibited the enzymatic

171

activity of rPDI. Using an insulin refolding-based assay, we established that 16F16, a

172

known PDI inhibitor, inhibits PDI with IC50 94 µM, in line with prior findings of 75-125 µM

173

range (Figure 3g).18, 20 This potency is substantially lower than that seen in cell-based

174

assays using 16F16,18,

175

observed. Chloroacetamide CW2334 showed somewhat greater potency for rPDI

176

inhibition than 16F16 (IC50 12 µM), while S-CW3554 showed inhibition of rPDI at doses

177

higher than 16F16 (IC50 574 µM) (Figure 3g). Notably, S-CW3544 was a substantially

178

better inhibitor of rPDI enzymatic activity than R-CW3544, mirroring the relative ability of

179

these molecules to label PDI in cell-based Click-ABPP experiments (Figure 3g).

20

where EC50 values in the range of 5 - 10 µM have been

180

As a third approach, we were able to abrogate labeling of PDI by S-CW3554 in

181

HEK293 cells by pretreatment of cells with PDI inhibitor 16F16, which targets catalytic

182

cysteine residues C53 and C56 (Figure 3h). These residues reversibly form a disulfide

183

bridge during the catalytic cycle of PDI and are known to be among the most reactive

184

cysteine nucleophiles across the proteome.21

185

targets catalytic residues of PDI, buffer conditions that omit DTT and favor formation of

186

a disulfide bond between these two catalytic cysteines abrogate S-CW3544’s ability to

187

label rPDI (Figure 3i). In addition, pretreatment of rPDI with cystine, which would oxidize

188

the catalytic cysteines to their disulfide form, suppressed the labeling of rPDI by S-

189

CW3554, further strengthening the claim of S-CW3554 targeting the catalytic cysteines

As further evidence that S-CW3544

9 ACS Paragon Plus Environment

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

Page 10 of 28

Adams et al.

190

in PDI (Figure 3i). Together these complementary approaches confirm that S-CW3554

191

targets PDI in cells and in vitro, likely by labeling of catalytic cysteines.

192

In addition to Click-ABPP labeling to assess cellular target engagement with our

193

library, we also performed a series of phenotypic screens assessing cytotoxicity across

194

a range of cancer cell lines. Across three cell lines derived from solid tumors of diverse

195

sites of origin (cervix, lung, and breast) little cell killing was observed, with none of our

196

26 2-chloropropionamides showing EC50 values less than 50 µM (Figure 4a and

197

Supporting Figure 14). In contrast, in a cell line derived from the hematopoietic cancer

198

multiple myeloma (MM1.S), one molecule showed clear cytotoxicity and three others

199

showed weaker cell killing (Figure 4b and Supporting Figure 13, 15-17). Remarkably,

200

cytotoxicity to MM1.S closely mirrored cellular affinity for PDIA1 seen by Click-ABPP,

201

with S-CW3554 most potent (MM1.S cytotoxicity EC50, 10 µM), CW3555, CW3684, and

202

CW3694 less potent (EC50 52-58 µM), and none of the remaining 22 2-

203

cloropropionamides within our library showing cytotoxicity up to 80 µM (Figure 4b and

204

Supporting Figure 13, 15-17). Similar results were obtained in a second MM cell line

205

(KMS11), although potency was lower (Supporting Figure 13, 15-17). A recent report

206

also demonstrated efficacy of protein disulfide isomerase inhibitors in multiple myeloma,

207

a hematological cancer of plasma cells characterized by sensitivity to proteasome

208

inhibitors and other inducers of proteotoxic stress.22 As expected, the bona fide PDI

209

inhibitor 16F16 also induced significant cytotoxicity in each MM cell line, with MM1.S

210

again more sensitive than KMS11 (EC50 600 nM MM1.S vs. 2.5 µM KMS11) (Figure 4c).

211

The close correlation between PDI labeling and cell killing within this library

212

suggest that inhibition of PDI may be the relevant target responsible for inducing cell 10 ACS Paragon Plus Environment

Page 11 of 28

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

Adams et al.

213

death. As further evidence for this possibility, we directly compared S-CW3554 and R-

214

CW3554 (which targets ALDH2) and noted in two MM cell lines that the S-CW3554

215

isomer showed enhanced cytotoxicity relative to its R-configured isomer (Figure 4d and

216

Supporting Figure 15). Additionally, as with the bona fide PDI inhibitor 16F16, S-

217

CW3554 showed greater cell killing of MM1.S than KMS11 cells (Figure 4c), while R-

218

CW3554 showed similarly low potency for killing both cell lines (Supporting Figure 15).

219

Chloroacetamide analog CW2334 showed greater cell killing than S-CW3554 as well as

220

preferential killing of MM1.S, with EC50 values similar to the bona fide PDI inhibitor

221

16F16 across our panel of cancer cell lines (Figure 4e). CW2334’s enhanced potency

222

for cell killing relative to S-CW3554 is consistent with its enhanced potency for inhibition

223

of rPDI (Figure 3g). Click-ABPP labeling experiments using 2-chloropropionamides in

224

KMS11 cells mirrored results in HEK293, with clear labeling of a ca. 60 kDa band by S-

225

CW3554, diminished labeling by CW3555, and no labeling from R-CW3554 and inactive

226

library member CW3557 (Figure 4f). These results together demonstrate that PDI target

227

engagement, PDI inhibition, and myeloma cell death correlate for the known PDI

228

inhibitor 16F16 and our novel 2-chloropropionamide-containing S-CW3554, suggesting

229

that PDI inhibition may be a key mechanism underlying the observed cell killing.

230

These studies have established 2-chloropropionamides as a class of useful

231

electrophile whose reactivity is less than typical acrylamides and whose stereogenic

232

reactive center can strongly influence proteome labeling.

233

targets of S- and R-CW3554 (PDIA1 and ALDH2) are both known to contain unusually

234

reactive catalytic thiol residues, further suggesting that 2-chloropropionamides are

235

generally unreactive with protein thiol residues and that noncovalent target affinity

11 ACS Paragon Plus Environment

We note that the protein

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

Page 12 of 28

Adams et al.

236

and/or perturbed thiol reactivity are required for covalent attachment.24,

237

more structurally diverse libraries will likely access a broader range of protein targets

238

than those obtained for our library or one other recently reported 2-chloropropionamide-

239

containing fragment.15 Our identification of a 2-chloropropionamide inhibitor of PDI

240

marks the least reactive irreversible inhibitor of PDI reported to date.

241

molecules have used chloroacetamide,16,

242

PAINS-type electrophiles,16 which show substantially greater reactivity with thiol

243

nucleophiles than 2-chloropropionamides.15,

244

reactivity difference between chloroacetamide and 2-chloropropionamide electrophiles

245

(Figure 1b, c and d), and a recent report noted that vinyl sulfonate and propynoic amide

246

electrophiles reacted 6 to 8-fold faster with glutathione than an analogous

247

chloroacetamide.19 Although further optimization of S-CW3554’s potency is required, its

248

cellular selectivity and sub-acrylamide reactivity make it a strong starting point for the

249

optimization of selective in vivo probes of PDI with potential applications in multiple

250

myeloma and neurodegenerative disease.

251

their sterically-tuned derivatives may find future use in the optimization of highly

252

selective targeted covalent inhibitors.

253

chloropropionamide-containing screening libraries, we imagine application of this

254

electrophile in the re-engineering of existing chloroacetamide probes or acrylamide-

255

containing FDA-approved drugs to generate derivatives whose diminished chemical

256

reactivity may enhance selectivity and facilitate in vivo evaluation.

20

Larger and

Previous

vinyl sulfonate,18 propynoic amide23 and

19

We have demonstrated the large

More broadly, 2-chloropropionamides or

In addition to the synthesis of expanded 2-

257 258

33

Methods

259 12 ACS Paragon Plus Environment

Page 13 of 28

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

Adams et al.

260

Reactivity of Electrophilic molecules with N-(4-nitrobenzoyl)-cysteine: HPLC

261

study. Compounds 1, 2 and 3 (100 µL, 10 mM) were independently combined with N-

262

(4-nitrobenzoyl)-cysteine (100 µL, 10 mM) in 1:1 acetonitrile: pH 7.4 PBS at 37 °C for 6

263

h. After the incubation, at each time point the mixture was injected into an Agilent high

264

performance liquid chromatograph attached with a Phenomenex C-18 reverse phase

265

column (250 × 4.6 mm, 5µm) and a diode array detector (detection wavelength was 254

266

nm). A mobile phase of 60% acetonitrile in water was used with a run time of 10 min.

267

The reaction mixtures were separately analyzed by Liquid Chromatography-Mass

268

Spectrometry (LC-MS) for product identification.

269 270

In-gel fluorescence assay:

271

HEK293 cells were seeded in a 6-well plate at a density of 0.5 M cells/well and allowed

272

to attach overnight. After treatment with desired concentration of compounds for 6 h,

273

cells were washed with cold PBS and placed at -80 ºC for 90 min. Then, 50 µL of PBS

274

containing a phosphatase and protease inhibitor cocktail (Halt, Life Technologies) was

275

added to the cells, which were then collected with a cell scraper and lysed using a

276

probe sonicator on ice. The lysed cells were centrifuged for 45 min at 4 ºC, and the

277

supernatant containing the soluble proteome was collected for a Cu(I)-catalyzed click

278

reaction with TAMRA-N3. A Click reagent cocktail was freshly prepared (4.25 µL

279

containing 1.5 µL 20% SDS, 0.5 µL 50 mM CuSO4, 0.5 µL 50 mM TCEP, 1.25 µL 1 mM

280

TBTA and 0.5 µL 5 mM TAMRA-N3), added to 25 µL of cell lysate and incubated at RT

281

for 60 min. After the incubation, 10 µL of SDS-loading buffer containing 50 mM DTT was

282

added and the proteins were resolved by SDS-PAGE (Bolt™ 4-12% Bis-Tris Plus Gels, 13 ACS Paragon Plus Environment

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

Page 14 of 28

Adams et al.

283

Invitrogen). The labeled protein bands were visualized by in-gel fluorescence imaging

284

using a Typhoon gel scanner. Coomassie blue stained gel images are available with

285

the supporting figures.

286 287

Affinity pull-down experiment:

288

HEK293 cells were cultured in a T175 flask in Dulbecco’s Modified Eagle Media

289

(DMEM) supplemented with 10% fetal bovine serum to nearly 90% confluency and

290

exposed to 30 µM of probe (S-CW3554 or R-CW3554) for 6 h. Following the incubation,

291

the media was aspirated, cells were washed with cold PBS and placed at -80 ºC for 90

292

min. Then, 1 mL of PBS containing a phosphatase and protease inhibitor cocktail (Halt,

293

Life Technologies) was added to the cells, which were then collected with a cell scraper

294

and lysed using a probe sonicator on ice. The lysed cells were centrifuged for 45 min at

295

4 ºC, and the supernatant was collected for a Cu(I)-catalyzed click reaction with biotin-

296

N3. A freshly prepared Click reagent cocktail (40 µL of 20% SDS, 40 µL of 50 mM

297

CuSO4, 40 µL of 50 mM TCEP, 120 µL of 1 mM TBTA and 30 µL of 5 mM biotin-N3)

298

was added to cell lysate and incubated at RT for 60 min. After the incubation, the

299

mixture was poured into 10 mL of cold acetone and stored at -20 ºC overnight. The

300

bluish-white fluffy precipitate was pelleted (4000 x g, 4 ºC, 20 min), collected in a 2mL

301

vial and repeatedly washed by vortexing with cold acetone (3 times) and centrifuged

302

(4000 x g, 4 ºC, 20 min). Then the protein pellet was air dried for an hour and re-

303

dissolved in 1 ml Tris buffer (pH 8.0) containing 0.1% SDS. This solution was desalted

304

using a pre-equilibrated Zeba™ Spin desalting column (as recommended by Thermo

305

scientific). Eluted protein solution was collected in a clean tube and mixed with

14 ACS Paragon Plus Environment

Page 15 of 28

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

Adams et al.

306

prewashed streptavidin-magnetic beads (75 µL, Pierce™ Streptavidin magnetic beads).

307

This was allowed to rotate overnight at 4 ºC, then the supernatant was carefully

308

removed by placing each tube in a magnetic holder. The beads were washed twice with

309

PBS containing 1% NP-40 and 1% SDS and once with PBS. Then, 0.5% SDS in PBS

310

(0.5 mL) was added and rotated at 4 ºC for 5 min. Then, the supernatant was removed

311

and the beads were rinsed with PBS and DI-water. Any residual liquid was carefully

312

removed by placing the tube in a magnetic holder. A mixture of 40 µL containing 30 µL

313

of 90% formamide and 50 mM EDTA in PBS and 10 µL of SDS-loading buffer

314

containing 50 mM DTT were added. This heated to 90 ºC for 5 min and the supernatant

315

was collected in a fresh tube. The eluted protein was cooled to RT and loaded into a 4-

316

12% SDS-Page gel to resolve the proteins. The gel was stained using Sypro Ruby

317

protein gel stain (Invitrogen) and the fluorescent bands were sliced under UV- light

318

visualization.

319

(LC/MS/MS) analysis of proteins were carried out as described in the supporting

320

methods.

Tryptic

digestion

and

liquid

chromatography-mass

spectrometric

321 322

In vitro labeling of recombinant bovine PDI by probes:

323

A mixture of Bovine PDI (40 nM) and 2 µM dithiothreitol (DTT) in phosphate buffer (100

324

mM pH 7.0 potassium phosphate buffer, 0.5mM EDTA) was incubated with 10 µM of

325

probes (S-CW3554, CW3555, CW3557 and CW2334) for 30 min at 37 °C. Freshly

326

prepared Click reagents stock (4.25 µL containing 1.5 µL 20% SDS, 0.5 µL 50 mM

327

CuSO4, 0.5 µL 50 mM TCEP, 1.25 µL 1 mM TBTA and 0.5 µL 5 mM TAMRA-N3) was

328

added to the protein solution and incubated for 1 h at RT. Then, SDS-loading buffer was 15 ACS Paragon Plus Environment

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

Page 16 of 28

Adams et al.

329

added to the protein reaction mixture and resolved using 4-12% SDS-PAGE gel. In-gel

330

fluorescence scanning was performed using a Typhoon gel imager. Similarly, various

331

concentrations of S-CW3554 were added to bovine PDI (40 nM) and 8 µM dithiothreitol

332

(DTT) in phosphate buffer, and the labeling was visualized as described above. For the

333

studies of effect of DTT and cystine on labeling of PDI by S-CW3554, rPDI (1 µM) was

334

pre-incubated for 3 h at 37 °C with DTT (10 µM) and cysteine (1 mM) separately in

335

phosphate buffer, then treated with S-CW3554 (10 µM) for overnight at 37 °C, and the

336

labeling was visualized as described above.

337 338 339

In vitro rPDI inhibition studies:

340

The assay protocol was adopted from literature and used with slight modifications23.

341

Bovine PDI (200 nM in 100 mM pH 7.0 potassium phosphate buffer, 0.5mM EDTA, 8

342

µM DTT, 30 µL) was treated with probes at varying concentration (2% final DMSO

343

concentration) and incubated for 30 min at 37 °C. After incubation, a final concentration

344

of 0.5 mM DTT (in pH 7.0 phosphate buffer) and 0.16 mM insulin (in 0.1N HCl) were

345

added and incubated at 37 °C for 90 min before recording the optical density at 650 nm

346

using an Enspire microplate reader.

347 348

Cell viability assays:

349

MM1.S, KMS11, HeLa, A549, MDA-MB-231 were acquired from ATCC and cultured in

350

DMEM+10% fetal bovine serum (HeLa and A549) or RPMI+10% fetal bovine serum

351

(MM1.S, KMS11 and MDA-MB-231). Cells were plated at 500 per well in white 384-well 16 ACS Paragon Plus Environment

Page 17 of 28

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

Adams et al.

352

plates and allowed to attach overnight. After addition of compounds by pin transfer,

353

plates were incubated for 72 h. At that time, media was removed and replaced with a

354

solution of CellTiter-Glo reagent in PBS. Luminescence was read using an Enspire

355

microplate reader, and signal intensity was calculated relative to in-plate DMSO control

356

wells.

357 358

Acknowledgments

359

We thank K. Lundberg and D. Schlatzer of the CWRU Proteomics core for experimental

360

assistance and the CWRU School of Medicine and Comprehensive Cancer Center for

361

unrestricted support.

362

Supporting Information Available: Fourteen supporting figures, three supporting table, supporting

363

methods and NMR spectra. This information is available free of charge via the Internet.

17 ACS Paragon Plus Environment

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

Page 18 of 28

Adams et al.

364

References

365 366 367 368 369 370 371

[1] Baillie, T. A. (2016) Targeted Covalent Inhibitors for Drug Design, Angew. Chem. Int. Ed. 55, 13408-13421. [2] Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The resurgence of covalent drugs, Nat. Rev. Drug Discovery 10, 307-317. [3] Potashman, M. H., and Duggan, M. E. (2009) Covalent Modifiers: An Orthogonal Approach to Drug Design, J. Med. Chem. 52, 1231-1246.

372

[4] Nakayama, S., Atsumi, R., Takakusa, H., Kobayashi, Y., Kurihara, A., Nagai, Y.,

373

Nakai, D., and Okazaki, O. (2009) A Zone Classification System for Risk

374

Assessment of Idiosyncratic Drug Toxicity Using Daily Dose and Covalent

375

Binding, Drug Metab. Dispos. 37, 1970-1977.

376 377

[5] Uetrecht, J. (2009) Immune-Mediated Adverse Drug Reactions, Chem. Res. Toxicol. 22, 24-34.

378

[6] Zhang, X., Liu, F., Chen, X., Zhu, X., and Uetrecht, J. (2011) Involvement of the

379

Immune System in Idiosyncratic Drug Reactions, Drug Metab. Pharmacokinet.

380

26, 47-59.

381

[7] Baell, J. B., and Holloway, G. A. (2010) New Substructure Filters for Removal of Pan

382

Assay Interference Compounds (PAINS) from Screening Libraries and for Their

383

Exclusion in Bioassays, J. Med. Chem. 53, 2719-2740.

384

[8] Li, D., Ambrogio, L., Shimamura, T., Kubo, S., Takahashi, M., Chirieac, L. R.,

385

Padera, R. F., Shapiro, G. I., Baum, A., Himmelsbach, F., Rettig, W. J.,

386

Meyerson, M., Solca, F., Greulich, H., Wong, and K, K. (2008) BIBW2992, an

387

irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer

388

models, Oncogene 27, 4702-4711.

389

[9] Fry, D. W., Bridges, A. J., Denny, W. A., Doherty, A., Greis, K. D., Hicks, J. L., Hook,

390

K. E., Keller, P. R., Leopold, W. R., Loo, J. A., McNamara, D. J., Nelson, J. M.,

391

Sherwood, V., Smaill, J. B., Trumpp-Kallmeyer, S., and Dobrusin, E. M. (1998)

392

Specific, irreversible inactivation of the epidermal growth factor receptor and

393

erbB2, by a new class of tyrosine kinase inhibitor, Proc. Natl. Acad. Sci. U. S. A

394

95, 12022-12027. 18 ACS Paragon Plus Environment

Page 19 of 28

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

Adams et al.

395

[10] Pan, Z., Scheerens, H., Li, S.-J., Schultz, B. E., Sprengeler, P. A., Burrill, L. C.,

396

Mendonca, R. V., Sweeney, M. D., Scott, K. C. K., Grothaus, P. G., Jeffery, D.

397

A., Spoerke, J. M., Honigberg, L. A., Young, P. R., Dalrymple, S. A., and Palmer,

398

J. T. (2007) Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine

399

Kinase, ChemMedChem 2, 58-61.

400

[11] Yun, C.-H., Mengwasser, K. E., Toms, A. V., Woo, M. S., Greulich, H., Wong, K.-K.,

401

Meyerson, M., and Eck, M. J. (2008) The T790M mutation in EGFR kinase

402

causes drug resistance by increasing the affinity for ATP, Proc. Natl. Acad. Sci.

403

U. S. A 105, 2070-2075.

404

[12] Solca, F., Dahl, G., Zoephel, A., Bader, G., Sanderson, M., Klein, C., Kraemer, O.,

405

Himmelsbach, F., Haaksma, E., and Adolf, G. R. (2012) Target Binding

406

Properties and Cellular Activity of Afatinib (BIBW 2992), an Irreversible ErbB

407

Family Blocker, J. Pharmacol. Exp. Ther. 343, 342-350.

408

[13] Hossam, M., Lasheen, D. S., and Abouzid, K. A. M. (2016) Covalent EGFR

409

Inhibitors: Binding Mechanisms, Synthetic Approaches, and Clinical Profiles,

410

Arch. Pharm. 349, 573-593.

411

[14] Miller, R. M., Paavilainen, V. O., Krishnan, S., Serafimova, I. M., and Taunton, J.

412

(2013) Electrophilic Fragment-Based Design of Reversible Covalent Kinase

413

Inhibitors, J. Am. Chem. Soc. 135, 5298-5301.

414

[15] Backus, K. M., Correia, B. E., Lum, K. M., Forli, S., Horning, B. D., González-Páez,

415

G. E., Chatterjee, S., Lanning, B. R., Teijaro, J. R., Olson, A. J., Wolan, D. W.,

416

and Cravatt, B. F. (2016) Proteome-wide covalent ligand discovery in native

417

biological systems, Nature 534, 570-574.

418

[16] Banerjee, R., Pace, N. J., Brown, D. R., and Weerapana, E. (2013) 1,3,5-Triazine

419

as a Modular Scaffold for Covalent Inhibitors with Streamlined Target

420

Identification, J. Am. Chem. Soc. 135, 2497-2500.

421

[17] Evans, M. J., Saghatelian, A., Sorensen, E. J., and Cravatt, B. F. (2005) Target

422

discovery in small-molecule cell-based screens by in situ proteome reactivity

423

profiling, Nat. Biotechnol. 23, 1303-1307.

19 ACS Paragon Plus Environment

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

Page 20 of 28

Adams et al.

424

[18] Ge, J., Zhang, C.-J., Li, L., Chong, L. M., Wu, X., Hao, P., Sze, S. K., and Yao, S.

425

Q. (2013) Small Molecule Probe Suitable for In Situ Profiling and Inhibition of

426

Protein Disulfide Isomerase, ACS Chem. Biol. 8, 2577-2585.

427

[19] Flanagan, M. E., Abramite, J. A., Anderson, D. P., Aulabaugh, A., Dahal, U. P.,

428

Gilbert, A. M., Li, C., Montgomery, J., Oppenheimer, S. R., Ryder, T., Schuff, B.

429

P., Uccello, D. P., Walker, G. S., Wu, Y., Brown, M. F., Chen, J. M., Hayward, M.

430

M., Noe, M. C., Obach, R. S., Philippe, L., Shanmugasundaram, V., Shapiro, M.

431

J., Starr, J., Stroh, J., and Che, Y. (2014) Chemical and Computational Methods

432

for the Characterization of Covalent Reactive Groups for the Prospective Design

433

of Irreversible Inhibitors, J. Med. Chem. 57, 10072-10079.

434

[20] Hoffstrom, B. G., Kaplan, A., Letso, R., Schmid, R. S., Turmel, G. J., Lo, D. C., and

435

Stockwell, B. R. (2010) Inhibitors of protein disulfide isomerase suppress

436

apoptosis induced by misfolded proteins, Nat. Chem. Biol. 6, 900-906.

437

[21] Kaplan, A., Gaschler, M. M., Dunn, D. E., Colligan, R., Brown, L. M., Palmer, A. G.,

438

Lo, D. C., and Stockwell, B. R. (2015) Small molecule-induced oxidation of

439

protein disulfide isomerase is neuroprotective, Proc. Natl. Acad. Sci. U. S. A 112,

440

E2245-E2252.

441

[22] Vatolin, S., Phillips, J. G., Jha, B. K., Govindgari, S., Hu, J., Grabowski, D., Parker,

442

Y., Lindner, D. J., Zhong, F., Distelhorst, C. W., Smith, M. R., Cotta, C., Xu, Y.,

443

Chilakala, S., Kuang, R. R., Tall, S., and Reu, F. J. (2016) Novel Protein Disulfide

444

Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma, Cancer Res. 76,

445

3340-3350.

446

[23] Xu, S., Butkevich, A. N., Yamada, R., Zhou, Y., Debnath, B., Duncan, R., Zandi, E.,

447

Petasis, N. A., and Neamati, N. (2012) Discovery of an orally active small-

448

molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer

449

treatment, Proc. Natl. Acad. Sci. U. S. A 109, 16348-16353.

450 451 452 453

[24] Xu, S., Sankar, S., and Neamati, N. (2014) Protein disulfide isomerase: a promising target for cancer therapy, Drug Discovery Today 19, 222-240. [25] Wilkinson, B., and Gilbert, H. F. (2004) Protein disulfide isomerase, Biochim. Biophys. Acta 1699, 35-44.

20 ACS Paragon Plus Environment

Page 21 of 28

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

Adams et al.

454

[26] Eirich, J., Braig, S., Schyschka, L., Servatius, P., Hoffmann, J., Hecht, S., Fulda, S.,

455

Zahler, S., Antes, I., Kazmaier, U., Sieber, S. A., and Vollmar, A. M. (2014) A

456

Small Molecule Inhibits Protein Disulfide Isomerase and Triggers the

457

Chemosensitization of Cancer Cells, Angew. Chem. Int. Ed. 53, 12960-12965.

458 459

[27] Weerapana, E., Simon, G. M., and Cravatt, B. F. (2008) Disparate proteome reactivity profiles of carbon electrophiles, Nat. chem. biol. 4, 405-407.

460

[28] Dachert, J., Schoeneberger, H., Rohde, K., and Fulda, S. (2016) RSL3 and Erastin

461

differentially regulate redox signaling to promote Smac mimetic-induced cell

462

death, Oncotarget.

463 464 465 466

[29] Nomura, D. K., Dix, M. M., and Cravatt, B. F. (2010) Activity-based protein profiling for biochemical pathway discovery in cancer, Nat. Rev. Cancer 10, 630-638. [30] Suto, M. J., Gayo-Fung, L. M., Palanki, M. S. S., and Sullivan, R. (1998) Solutionphase parallel synthesis using ion-exchange resins, Tetrahedron 54, 4141-4150.

467

[31] Khan, M. M. G., Simizu, S., Lai, N. S., Kawatani, M., Shimizu, T., and Osada, H.

468

(2011) Discovery of a Small Molecule PDI Inhibitor That Inhibits Reduction of

469

HIV-1 Envelope Glycoprotein gp120, ACS Chem. Biol. 6, 245-251.

470 471

[32] Parakh, S., and Atkin, J. D. (2015) Novel roles for protein disulphide isomerase in disease states: a double edged sword?, Front. Cell Dev. Biol. 3.

472

[33] Steinmetz, C. G., Xie, P., Weiner, H., and Hurley, T. D. (1997) Structure of

473

mitochondrial aldehyde dehydrogenase: the genetic component of ethanol

474

aversion, Structure 5, 701-711.

475

21 ACS Paragon Plus Environment

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

Page 22 of 28

Adams et al.

476 477

Figure 1. Evaluation of the reactivity of 2-chloropropionamides with nucleophiles in in

478

vitro and in cells. (a-c) Electrophiles 1-3 were allowed to react with N-(4-nitrobenzoyl)-

479

cysteine (*Cys) and reaction progress after 60 minutes was monitored using HPLC. (d,

480

e) Quantification of remaining *Cys and electrophilic molecules (1, 2 and 3) over 6 h

481

under the conditions described above. Data are presented as the mean of 2

482

independent experiments +/- standard deviation (e) Click-ABPP in HEK293 cells treated

483

with alkyne-tagged model electrophilic molecules 4-6 at 20, 10 and 5 µM for 6 h.

484

22 ACS Paragon Plus Environment

Page 23 of 28

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

Adams et al.

485 486 487

Figure 2. Design and synthesis. (a) (top) Schematic depicting the modular structure of

488

2-chloropropionamide library members. (bottom) Building blocks R1 and R2 used to

489

synthesize a combinatorial library of 26 small molecules. Each box represents one

490

library member featuring the illustrated R1, R2, and R3 fragments. White blocks refer to

491

molecules not synthesized. Molecules that showed a clear band during Click-ABPP are

492

highlighted in the panel. For performance of all library members by Click-ABPP, see

493

Supporting Figure 2-5. Later, the target of S-CW3554 was identified as PDIA1

494

(Supporting Figure 8). (b) Synthesis of R-CW3554 and S-CW3554. Conditions: (a)

495

Propargyl chloroformate, aq. NaHCO3, 0ºC-RT, 1h; (b) 1-(N-Boc-aminomethyl)-3-

496

(aminomethyl)benzene, HCTU, DIPEA, DMF, RT, 5h; (c) 10% TFA/CH2Cl2, 0 ºC-RT,

497

2h; (d) (R)-(+)-2-chloropropionic acid (or) (S)-(-)-2-chloropropionic acid, HCTU, DIPEA,

498

DMF, RT, 5h. (c) Structures of electrophilic molecules evaluated.

499

23 ACS Paragon Plus Environment

ACS Chemical Biology

Adams et al.

b

d S-CW3554/∝M

CH 3

H N

25 12.5

Cl

6.3 3.2 1.6 0.8

0.4 0.2

O O O

HN

PDIA1 57kDa

O

ALDH2 56kDa

100

50

0 25 .0 12 .5 6. 3 3. 2 1. 6 0. 8 0. 4 0. 2

HN

HN

c

Relative % PDI labeling in HEK 293

a

Cis-CW3555

S-CW3554 (µM)

CH3 O

e

f CW2334/∝M

O O

HN

g

NH

HN

HN

Cl

10

2.5

% Recombinant PDI Activity

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 28

0.6 0.16 0.04 0.01

O CW3557

Cl

N N H

O O

O

16F16

100

50 16F16 CW2334 S-CW3554 R-CW3554

0 -1

h

i

0

1

2

3

Log [Inhibitor], µM

1

2

3

4

5

+ -

+ -

+ + -

+ +

+ + +

rPDI DTT (S)-CW3554 Cystine

500 501

Figure 3. Validation of S-CW3554 as a novel PDI inhibitor. (a) Structures of additional

502

2-chloropropionamides evaluated. (b, c, f) Click-ABPP in HEK 293 cells treated 6 h with

503

the indicated concentrations of probes. Mass spectrometric analysis of proteins

504

obtained by streptavidin-biotin affinity purification identified PDIA1 as the target of S-

505

CW3554 and ALDH2 as the target of R-CW3554 (Supporting Figure 8). (d)

506

Quantification of PDIA1 band intensities from Figure 2c. (e) Evaluation of the labeling of

507

recombinant PDI (rPDI) by the indicated concentrations of 2-chloropropionamide probes

508

for 30 min at 37 ºC. (g) Effects on PDIA1 enzymatic activity for the indicated

509

concentrations of probes. Data are presented as the mean of 3 independent

510

experiments +/- standard deviation. (h) HEK293 cells pretreated with 16F16 (5 µM) or

511

DMSO for 14h were subsequently exposed to S-CW3554 (10 µM) for 6 h and analyzed

24 ACS Paragon Plus Environment

Page 25 of 28

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

Adams et al.

512

using in-gel fluorescence. (i) Effect of DTT, cystine and iodoacetamide on labeling of

513

recombinant PDI (1 µM) by S-CW3554 (10 µM) at 37 ºC.

514

25 ACS Paragon Plus Environment

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

Page 26 of 28

Adams et al.

515 516

Figure 4. Effect of S-CW3554 on cancer cell line viability, as assessed by cellular ATP

517

levels (CellTiter-Glo). (a) Cell viability EC50 values of 2-chloropropionamides (S-

518

CW3554 and R-CW3554) and chloroacetamides (CW2334 and 16F16) against a panel

519

of five cancer cell lines. (b) Profiling of cell viability for 26 2-chloropropionamide library

520

members in MM1.S cells. Library members with a measurable EC50 are highlighted;

521

gray wells show EC50 > 80 µM. (c, e) Cell viability in two multiple myeloma cell lines.

522

(d) ATP levels in MM1.S cells after 72 h treatment with the indicated concentrations of

523

molecules. Data are represented as the mean of two independent experiments +/-

524

standard deviation. (f) Small molecules containing various electrophilic functionalities

525

were evaluated for their reactivity with proteome in KMS11 cells using Click-ABPP.

526

26 ACS Paragon Plus Environment

Page 27 of 28

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

Adams et al.

527

TOC Graphic

528

27 ACS Paragon Plus Environment

ACS Chemical Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14

ACS Paragon Plus Environment

Page 28 of 28