2-Chloropropionamide As a Low-Reactivity Electrophile for

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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

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ACS Chemical Biology

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2-chloropropionamide as a low-reactivity

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electrophile for irreversible small-molecule

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probe identification

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Dharmaraja Allimuthu1, Drew J. Adams1*

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Affiliations:

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1

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Medicine, Case Western Reserve University, Cleveland, OH 44106

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

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* Corresponding author email: [email protected]

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Abstract

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Resurgent interest in covalent target engagement in drug discovery has demonstrated

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

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

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

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Additional electrophilic functionalities whose reactivity is compatible with highly selective

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

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

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

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

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

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

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

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

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

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chloropropionamides as weak and stereochemically-tunable electrophiles for covalent

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drug discovery.

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Introduction

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Multiple safe and effective drugs target cellular proteins covalently, and

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

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

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

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

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

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

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

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

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

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

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benefits of irreversible target engagement while lowering concerns regarding

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

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

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

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of their kinase targets.8-10

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

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

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screening libraries, either of ‘electrophilic fragments’14,

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molecular weight.16-18

Although these molecules were largely the product of

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or molecules of more typical

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We reasoned that additional electrophilic functionalities that react with biological

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

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


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molecule probes.15,

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

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reactivity than analogous acrylamides.

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chloropropionamides highlighted the low proteome reactivity of this functionality but also

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

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

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

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

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therapeutic target in neurodegenerative disease and cancer.20-24

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

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

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

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

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

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chloropropionamide functionality as an electrophile with lower reactivity than

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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-

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PDIA1 catalyzes

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Results and Discussion

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In considering potentially useful weak electrophilic functionalities, we focused on

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

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

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

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

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

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alkylation by cysteine and other nucleophiles, potentially allowing more selective

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

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acrylamide-containing kinase inhibitors.

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

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stereoisomeric 2-chloropropionamide products could have distinct affinity for cellular

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nucleophiles.

Moreover, the introduction of an alkyl

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

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

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

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

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

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

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

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

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

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

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


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

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

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chloropropionamide electrophiles to covalently label cellular nucleophiles. The extent of

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

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

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

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chloropropionamides relative to chloroacetamide and even acrylamide electrophiles in

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

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

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vivo application.

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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synthesis and were not pursued further.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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cells (Figure 3b).

We also synthesized R-CW3554 and derivatives that varied the

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

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

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highlighting the substantially higher reactivity of the chloroacetamide electrophile

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

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

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



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

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

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

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

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

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

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

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

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

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relative to other electrophilic warheads.

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

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states,20,

21, 24, 31, 32

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

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

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

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

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

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

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

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

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

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

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

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


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sterically more hindered 2-chloropropionamide electrophile enables more selective

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targeting of PDI (Figure 3c, f).

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Second, S-CW3554 and chloroacetamide CW2334 inhibited the enzymatic

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

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

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

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assays using 16F16,18,

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observed. Chloroacetamide CW2334 showed somewhat greater potency for rPDI

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

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

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

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

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

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

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

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

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

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cysteine nucleophiles across the proteome.21

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

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

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

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

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

As further evidence that S-CW3544



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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a hematological cancer of plasma cells characterized by sensitivity to proteasome

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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electrophile whose reactivity is less than typical acrylamides and whose stereogenic

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reactive center can strongly influence proteome labeling.

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

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

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generally unreactive with protein thiol residues and that noncovalent target affinity


We note that the protein


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

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

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

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

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

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molecules have used chloroacetamide,16,

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

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nucleophiles than 2-chloropropionamides.15,

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reactivity difference between chloroacetamide and 2-chloropropionamide electrophiles

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

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

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

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

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

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myeloma and neurodegenerative disease.

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

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selective targeted covalent inhibitors.

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chloropropionamide-containing screening libraries, we imagine application of this

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

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containing FDA-approved drugs to generate derivatives whose diminished chemical

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reactivity may enhance selectivity and facilitate in vivo evaluation.

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Larger and

Previous

vinyl sulfonate,18 propynoic amide23 and

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We have demonstrated the large

More broadly, 2-chloropropionamides or

In addition to the synthesis of expanded 2-

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Methods

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

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

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

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

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

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

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

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The reaction mixtures were separately analyzed by Liquid Chromatography-Mass

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Spectrometry (LC-MS) for product identification.

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In-gel fluorescence assay:

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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the supporting figures.

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Affinity pull-down experiment:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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(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


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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


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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

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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.



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

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

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

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

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

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

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

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biological systems, Nature 534, 570-574.

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

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as a Modular Scaffold for Covalent Inhibitors with Streamlined Target

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

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

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

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

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

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

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

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

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

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for the Characterization of Covalent Reactive Groups for the Prospective Design

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

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

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

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

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

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

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

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E2245-E2252.

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

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

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

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

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3340-3350.

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

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

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molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer

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

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[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.


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

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Small Molecule Inhibits Protein Disulfide Isomerase and Triggers the

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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

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differentially regulate redox signaling to promote Smac mimetic-induced cell

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[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.

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

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

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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

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474

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475



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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


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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



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


Page 25 of 28

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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



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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


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TOC Graphic

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2-Chloropropionamide As a Low-Reactivity Electrophile for

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