Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11

Article

Epidithiodiketopiperazines Inhibit Protein

Degradation by Targeting Proteasome

Deubiquitinase Rpn11

Graphical Abstract

Authors

Jing Li, Yaru Zhang,

Bruno Da Silva Sil Dos Santos, ...,

Tsui-Fen Chou, Stephen T. Hilton,

Raymond J. Deshaies

Correspondence

jing.li@caltech.edu (J.L.),

deshaies@caltech.edu (R.J.D.)

In Brief

The deubiquitinase Rpn11 is a

component of 26S proteasome, is

required for proteasome activity, and is a

new target for proteasome inhibition in

cancer. We develop an assay that

enables measurement of the

proteasome’s protein breakdown activity

in cell lysate and identiﬁes ETPs as a new

class of Rpn11 inhibitors.

Highlights

d

An HTS assay is developed to measure protein breakdown

activity of 26S proteasome

It allows measurement of 26S proteasome activity in cell or

tissue lysate

ETPs inhibit JAMM proteases including the proteasomal

deubiquitinase Rpn11

ETPs block proliferation of cancer cells including those

resistant to bortezomib

Li et al., 2018, Cell Chemical Biology 25, 1–9

November 15, 2018 ª 2018 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2018.07.012

Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

Cell Chemical Biology

Article

Epidithiodiketopiperazines Inhibit Protein

Degradation by Targeting

Proteasome Deubiquitinase Rpn11

Jing Li,^1,8,Yaru Zhang,^1,8Bruno Da Silva Sil Dos Santos,²Feng Wang,³Yuyong Ma,⁴Christian Perez,⁴Yanling Yang,⁵

*

Junmin Peng,⁵Seth M. Cohen,⁴Tsui-Fen Chou,³Stephen T. Hilton,⁶and Raymond J. Deshaies^1,7,8,9,

*

¹Division of Biology and Biological Engineering, California Institute of Technology, Box 114-96, Pasadena, CA 91125, USA

²London Metropolitan University, 166–220 Holloway Road, London N7 8DB, UK

³Division of Medical Genetics, Department of Pediatrics, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute,

Torrance, CA 90502, USA

⁴Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA

⁵Departments of Structural Biology and Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA

⁶UCL School of Pharmacy, 29–39 Brunswick Square, London WC1N 1AX, UK

⁷Howard Hughes Medical Institute, Chevy Chase, MD 26335, USA

⁸Amgen Discovery Research, One Amgen Center Drive MS 29-M-B, Thousand Oaks, CA 91320, USA

⁹Lead Contact

*Correspondence: jing.li@caltech.edu (J.L.), deshaies@caltech.edu (R.J.D.)

https://doi.org/10.1016/j.chembiol.2018.07.012

SUMMARY

derived from a pair of sulfur atoms that form an unusual, intramo-

lecular disulﬁde bridge (Dolan et al., 2015; Scharf et al., 2016).

The 26S proteasome is the major proteolytic machine Gliotoxin poisons animal cells by inactivating multiple key en-

zymes through conjugation of their thiol groups. Known targets

of gliotoxin include nuclear factor kB (NF-kB), NADPH oxidase,

for breaking down cytosolic and nuclear proteins in

eukaryotes. Due to the lack of a suitable assay, it is

difﬁcult to measure routinely and quantitatively the

breakdown of proteins by the 26S proteasome

in vitro. In the present study, we developed an assay

to monitor proteasome-mediated protein degrada-

tion. Using this assay, we discovered that epidithiodi-

and glutaredoxin (Pahl et al., 1996; Srinivasan et al., 2006; Tsu-

nawaki et al., 2004). Glutathione (GSH) is important for gliotoxin

uptake and mediates its cytotoxicity in animal cells, as it reduces

gliotoxin to convert it into the toxic dithiol form (Dolan et al.,

2015). Further studies have revealed a mechanism for its inhibi-

tion involving zinc chelation: gliotoxin and other ETPs can inhibit

HIF1a interaction with p300 by ejecting the Zn²⁺from p300

through the formation of a Zn²⁺-ETP complex (Cook et al.,

2009). This disrupts HIF1a activation and provides a plausible

ketopiperazine (ETPs) blocked the degradation of our

model substrate in vitro. Further characterization re-

vealed that ETPs inhibited proteasome function by

targeting the essential proteasomal deubiquitinase molecular basis for the anti-angiogenesis effects of ETPs.

The proteasome, the major cellular machine for protein

degradation, is also reported as a target of gliotoxin (Kroll

Rpn11 (POH1/PSMD14). ETPs also inhibited other

JAMM (JAB1/MPN/Mov34 metalloenzyme) proteases

such as Csn5 and AMSH. An improved ETP with fewer

non-speciﬁc effects, SOP11, stabilized a subset of

proteasome substrates in cells, induced the unfolded

protein response, and led to cell death. SOP11 repre-

sents a class of Rpn11 inhibitor and provides an alter-

native route to develop proteasome inhibitors.

et al., 1999). Proteasomes are essential for various cellular pro-

cesses including protein quality control, regulation of gene

expression, and cell-cycle progression. Structurally, the protea-

some is composed of a 20S core particle (CP) and 19S regulatory

particles (RPs), which cap the ends of the CP. The RP recognizes

polyubiquitinated substrates and processes them for insertion

into the CP, which contains the proteolytic active sites (Finley,

2009). There are three distinct catalytic peptidase activities iden-

tiﬁed in the CP: chymotrypsin-like, trypsin-like, and caspase-like

INTRODUCTION

(Heinemeyer et al., 1997). Drugs such as bortezomib (BTZ) and

carﬁlzomib (CFZ), which inhibit the active sites in the CP, are

The fungus Aspergillus fumigatus is one of the most common important therapeutic agents for the treatment of multiple

species to cause disease in immunocompromised individuals, myeloma (Dimopoulos et al., 2015). However, patients ultimately

such as AIDS patients and organ transplant recipients (Dolan suffer relapse despite the clinical beneﬁt conferred by these

et al., 2015). A. fumigatus infection causes aspergillosis and drugs. Therefore, novel drugs working through different mecha-

immunosuppression (Scharf et al., 2016). The secondary metab- nisms are needed. Recently, we and others discovered small-

olites produced by Aspergillus are considered to be important molecule inhibitors targeting Rpn11, a JAMM protease that re-

virulence factors. Among them, gliotoxin, an epidithiodiketopi- moves polyubiquitin chains from substrate proteins (Lauinger

perazine (ETP), is the major and the most potent toxin (Scharf et al., 2017; Li et al., 2017; Perez et al., 2017). Inhibition of

et al., 2016). Most of the biological activities of gliotoxin are Rpn11 function results in proteasome malfunction and leads to

Cell Chemical Biology 25, 1–9, November 15, 2018 ª 2018 Elsevier Ltd.

1

Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

cell death (Li et al., 2017). Previous research suggests that glio- by SDS-PAGE. The ﬂuorescence-scanned gel showed that

toxin is a noncompetitive inhibitor of the chymotrypsin-like activ- both proteins were degraded, consistent with the prior report

ity of the 20S proteasome (Kroll et al., 1999). However, the (Kim and Huibregtse, 2009). The degradation was speciﬁc and

detailed mechanism of this inhibition remains unknown.

was blocked by a Rpn11 inhibitor (capzimin [CZM]), a 20S inhib-

Here, we developed an assay to measure the protein break- itor (CFZ), or a non-hydrolyzable ATP analog, AMP-PNP (Figures

down activity of the proteasome in puriﬁed systems and cell 1B and S1B).

extracts and identiﬁed ETPs as a scaffold for inhibiting JAMM

To simplify and accelerate data acquisition, we adapted the

proteases. ETPs inhibit proteasome function by targeting the degradation assay to a format compatible with high-throughput

essential proteasomal deubiquitinase Rpn11. Identiﬁcation of screening by monitoring the decrease in ﬂuorescence polariza-

ETPs provides an alternative route to inhibit proteasome function tion (FP) in a multi-well plate reader. The decreased FP indicated

and opens the door to the development of new Rpn11 inhibitors. that the ﬂuorescent UbⁿGST-Wbp2 substrate was cleaved into

smaller species. Substrate degradation monitored in this fashion

RESULTS

was speciﬁc in that it was inhibited by addition of the 20S and

Rpn11 inhibitors (Figures 1C, S1C, and S1D), or the slowly hydro-

lyzable ATP analog ATPgS (Figure S1C), and was completed by a

large excess of K48-linked Ub⁴chains (Figure S1E). Note that in-

Development of a Proteasome Substrate to Monitor

Protein Degradation In Vitro

There is no quantitative method for the simple and rapid assess- hibition of the decrease in FP by the 20S inhibitor was not as com-

ment of 26S proteasome protein degradation activity in vitro to plete as with the Rpn11 inhibitor (Figure 1C), because there re-

date. The Suc-LLVY-amc substrate widely used for the evalua- mained some decrease in FP due to substrate deubiquitination,

tion of 20S proteasome activity does not accurately reﬂect pro- which decreased its molecular weight (Figure 1B).

tein breakdown because it only measures the chymotrypsin-like

To test whether the FP assay could be used to measure 26S

active site of the b5 subunit. Meanwhile, it has been shown that proteasome activity directly in the cell lysate, we lysed

to block protein degradation it is necessary to inhibit both the HEK293T cells treated with different concentrations of CFZ

chymotryptic site, which is intrinsically the most sensitive to and performed the assay directly in the lysate by adding

the commonly used 20S inhibitors, as well as either the tryptic ^K48UbⁿGST-Wbp2 substrate and ATP. Ubiquitin aldehyde was

or caspase site, which are about an order of magnitude less sen- included in the assay buffer to eliminate the interference of

sitive (Demo et al., 2007; Kisselev and Goldberg, 2005). In addi- cysteine-based deubiquitinases (Hershko and Rose, 1987).

tion, Suc-LLVY-amc is not only cleaved by the 20S proteasome The IC₅₀for inhibition of ^K48UbⁿGST-Wbp2 degradation by

but also by other chymotrypsin-like proteases and by calpains CFZ was 110 nM, which was ꢀ20 fold higher than the IC₅₀ob-

(Giguere and Schnellmann, 2008).

tained in the Suc-LLVY-amc assay (Figure 1D). This is consistent

To measure protein breakdown by the proteasome, we modi- with the observation that inhibition of protein breakdown re-

ﬁed an existing method (Kim and Huibregtse, 2009) to generate a quires blockade of at least two of the three active sites (Kisselev

polyubiquitinated protein substrate, termed UbⁿGST-Wbp2 (WW and Goldberg, 2005). This assay is also compatible with lysate

domain-binding protein 2, n > 30) using enzymatic approaches prepared directly from tissue. In a recent study, we demon-

(Figures 1A and S1A). Wbp2 was originally isolated from a mouse strated that overexpression of the 11S proteasome subunit

embryo library. It contains an N-terminal pleckstrin homology- PA28a in the mouse retina does not alter ubiquitin-dependent

glucosyltransferase (GRAM) domain and three C-terminal protein degradation (Lobanova et al., 2018). This example illus-

PPxY motifs, which interact with multiple WW domain-contain- trates how our assay can be used to monitor 26S proteasome

ing proteins (Chen and Sudol, 1995). A previous study showed activity in various disease states. It is worth noting that the

that Wbp2 functions as a coactivator for estrogen receptor and ^K63UbⁿGST-Wbp2 substrate was not suitable for use in the cell

is closely linked to the development of breast cancer (Chen lysate, possibly due to the presence of a high level of K63 link-

et al., 2017). The original method described by Huibregtse and age-speciﬁc deubiquitinases (Cooper et al., 2009) (Figure S1F).

colleagues relies on Rsp5-mediated ubiquitination of a truncated

Wbp2 (Kim and Huibregtse, 2009). We modiﬁed their method Epidithiodiketopiperazines Block Protein Degradation

by inserting a C-terminal hexahistidine tag into the GST-Wbp2 Gliotoxin is the most well-known member of the family of ETP

construct and chemically labeling the puriﬁed protein with a compounds. Previous reports indicated that it functions as a

cysteine-reactive ﬂuorophore. Rsp5 assembles K63-linked ubiq- 20S proteasome inhibitor targeting chymotrypsin-like activity,

uitin chains and contains a WW domain that recognizes the PPxY and the inhibitory effect depends on an intact disulﬁde bond in

motifs in Wbp2. Swapping the HECT domain of Rsp5 with that gliotoxin (Kroll et al., 1999). However, gliotoxin exists almost

from E6AP yields a chimeric enzyme that preferentially synthe- exclusively in the dithiol form after uptake into cells due to the

sizes K48-linked ubiquitin chains (Kim and Huibregtse, 2009). reducing power of cellular glutathione (Bernardo et al., 2003).

The ubiquitin linkages formed on our substrate treated with the These contradictory observations motivated us to revisit the

wild-type (WT) and chimeric Rsp5 were quantiﬁed by mass interaction between ETPs and the proteasome.

spectrometry. 87% of the linkages formed by Rsp5 were via

Taking advantage of our FP assay, we investigated the effects

K63 (^K63UbⁿGST-Wbp2), whereas 94% of those formed by the of gliotoxin and its core scaffold compound, SOP6, on the

Rsp5–E6AP chimera were via K48 (^K48UbⁿGST-Wbp2; Table S1). proteasome-mediated degradation of ^K48UbⁿGST-Wbp2 (Fig-

To test whether ^K63UbⁿGST-Wbp2 and ^K48UbⁿGST-Wbp2 ure 2A). Both SOP6 and gliotoxin slowed down the decline

were proteasome substrates, we incubated them with puriﬁed in FP, suggesting that ETP compounds inhibited proteasome

human 26S proteasomes and ATP and analyzed the reactions function. To test this hypothesis, we treated HCT116 cells with

2

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Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

A

D

B

C

Figure 1. Development of an Assay to Monitor Protein Degradation

(A) Design of protein degradation assay.

(B) ^K48UbⁿGST-Wbp2 (20 nM) was incubated with 26S proteasome (5 nM) at 37^ꢁC for 2 hr in the absence and presence of different inhibitors (20 mM capzimin

[CZM] or 10 mM carﬁlzomib [CFZ]). Reactions were fractionated by SDS-PAGE and analyzed using a Typhoon ﬂuorescence scanner. Boxes mark the input high-

molecular-weight polyubiquitinated substrate and a deubiquitinated species that accumulated in reactions inhibited by CFZ (Verma et al., 2002).

(C) Measurement of proteasome activity using the ﬂuorescence polarization assay. ^K48UbⁿGST-Wbp2 (2.5 nM) was incubated with 26S proteasome (1 nM) at

37^ꢁC in the absence or presence of different inhibitors (10 mM CZM or 1 mM bortezomib[BTZ]).

(D) Measurement of proteasome protein breakdown activity in cell lysate in response to increasing concentrations of CFZ. Shown are the reaction kinetics (upper

panel) and dose-response (red curve in the bottom panel) of proteasome activity measured at 37^ꢁC using ^K48UbⁿGST-Wbp2 as substrate and lysate from cells

treated with different concentrations of CFZ. For comparison, a dose-response curve measured using Suc-LLVY-amc is plotted in black (bottom panel). Error

bars represent SD, n = 3 wells, from 1 representative of 3 independent experiments.

See also Table S2.

B

panel of related molecules that share the

core ETP scaffold and identiﬁed SOP11

as a candidate for future studies. Unlike

gliotoxin, SOP11 did not cause the cell-

rounding phenotype. Like CZM, SOP11

triggered a UPR and induced accumula-

Figure 5A), indicating that ETP-based and ‘‘omib’’ inhibitors tion of polyubiquitin conjugates and the speciﬁc proteasome sub-

worked through distinct mechanisms. In addition to inhibiting strates NFE2L1 and NFE2L2. SOP11 also mimicked CZM in that it

cell growth, ETPs induced cell apoptosis, as conﬁrmed by immu- did not inhibit zinc-dependent enzymes unrelated to Rpn11, such

noblotting for caspase-cleaved poly(ADP-ribose) polymerase as human carbonic anhydrase and matrix metalloproteinase 2.

(PARP1) (Figure 5B). DNA damage is a common event during However, SOP11 does inhibit other members of the JAMM family

apoptosis, which is reﬂected by the increased phosphorylation in vitro in addition to Rpn11, including Csn5 and AMSH. However,

of H2AX (gH2AX). ETPs strongly induced H2AX phosphorylation we did not observe effects on the Csn5 substrate Cul1 at concen-

after 24 hr of treatment, indicating the occurrence of the DNA trations of SOP11 that inhibited the proteasome in cells. Thus,

damage response (Figure 5B).

SOP11 is a promising starting point to develop Rpn11 inhibitors

based on the ETP scaffold. However, considerable medicinal

chemistry optimization will be required to generate ETPs that

have sufﬁcient potency and speciﬁcity to enable a rigorous test

DISCUSSION

Gliotoxin is the most well-studied ETP. It targets multiple pro- of the therapeutic potential of Rpn11 inhibition.

teins and enzymes with important cellular functions. The protea-

The proteasome is the central conduit through which all sub-

some, the central cellular machine for protein degradation, has strates of the ubiquitin-proteasome system (UPS) are degraded.

been reported as a target of gliotoxin (Kroll et al., 1999). It inhibits Genomic mutations, aneuploidy, and other alterations in cancer

the chymotrypsin-like activity of 20S proteasome at high con- cells can result in an imbalance between protein expression

centrations under oxidative conditions. However, gliotoxin is and the degradative capacity of the UPS (Cenci et al., 2008; De-

rapidly reduced upon its uptake by the cell (Bernardo et al., shaies, 2014). This has the potential to render cancer cells more

2003). We report here that multiple ETPs including gliotoxin dependent on proteasome activity than normal cells. Inhibitors

inhibit the proteasome in vitro and in cells by targeting its essen- targeting the proteolytic center of the 20S proteasome, such

tial deubiquitinase subunit Rpn11. Inhibition of Rpn11 function as BTZ, CFZ, and ixazomib, have been approved for the treat-

by gliotoxin and other ETPs led to proteasome malfunction and ment of multiple myeloma and mantle cell lymphoma, thereby

subsequent cell death. Our ﬁndings identify ETPs as an inhibitor validating the hypothesis that at least some cancer cells have

scaffold for targeting Rpn11 and open the door to further devel- heightened dependence on proteasome activity. However, pro-

opment of more speciﬁc and potent Rpn11 inhibitors.

teasome inhibitor therapy has not been successfully expanded

A key consideration in the characterization of inhibitors is their to the treatment of solid tumors, potentially due to reduced

target speciﬁcity. Whereas gliotoxin is one of the most potent sensitivity of solid tumor cells to proteasome inhibitors coupled

ETPs for inhibiting the proteasome, it is also relatively non-selec- with the pharmacological properties of the existing drugs (De-

tive and induces morphological changes in cells that are not shaies, 2014). Our study provides an alternative to achieve pro-

observed with the selective Rpn11 inhibitor CZM (Jordan and teasome inhibition, which might translate into a therapeutic

Pedersen, 1986). To address this non-selectivity, we surveyed a approach in the future.

Cell Chemical Biology 25, 1–9, November 15, 2018

7

Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

SUPPLEMENTAL INFORMATION

Changes in proteasome activity have been suggested to occur

during aging and in neurodegenerative diseases (Saez and Vil-

Supplemental Information includes four ﬁgures and two tables and can

chez, 2014). Proteasome dysfunction and decreased proteasome

be found with this article online at https://doi.org/10.1016/j.chembiol.2018.

activity might contribute to aging-related diseases. For example, it

has been proposed that toxic protein aggregates that accumulate

07.012.

in polyglutamine repeat diseases such as Huntington’s disease

may inhibit proteasome function (Diaz-Hernandez et al., 2006),

although this has been controversial (Hipp et al., 2012; Ortega

e t a l., 2007).Oneprobleminaddressingtheroleofproteasomeac-

tivity in aging and disease has been a lack of suitable assays to

directlymeasuretheproteinbreakdown activityoftheproteasome

in cell lysates. Consequently, these studies rely on measuring the

chymotryptic peptidase activity of the b5 subunit, which shows

poor correlation with protein breakdown activity (Bence et al.,

2001). The proteasome activity assay we developed in this study

may help to shed light on these simple yet important issues.

ACKNOWLEDGMENTS

We thank J.M. Huibregtse (The University of Texas at Austin) for the vector ex-

pressing GST-Wbp2-C-K222, Rsp5, and Rsp5-E6AP, A. Martin (University of

California, Berkeley) for the pETDuet-1 vector expressing the Rpn11-Rpn8

dimer, T.M. Kapoor (The Rockefeller University) for providing the RPE1 WT

and BTZ-resistant cell lines, H. Park for testing the cell lines for mycoplasma

contamination, A. Bowers (University of North Carolina) for the stimulating dis-

cussion on gliotoxin, and D. Sherman (Amgen Inc.) for critical reading of our

manuscript. This work was supported by grants from the Caltech Gates Grub-

stake Fund and Amgen to R.J.D. and from the NIH (CA164803) to R.J.D. and

S.M.C. S.T.H. and B.D.S.S.D.S. were supported by the funding from FCT

(grant no. SFRH/BD/65630/2009).

SIGNIFICANCE

AUTHOR CONTRIBUTIONS

J.L. designed, executed, and interpreted the experiments using Wbp2 as pro-

teasome activity substrate, in vitro Rpn11 assay and AMSH assay, and west-

ern blot. Y.Z. performed and analyzed the in vitro Csn5 assay, real-time qPCR

assay, and western blot. B.D.S.S.D.S. and S.T.H. synthesized ETP com-

pounds SOP1-11. Y.Y. and J.P. quantiﬁed the linkage type of ubiquitin chain

on Wbp2. F.W. and T.-F.C performed and analyzed the Ub^G76V-GFP degrada-

tion assay. Y.M., C.P. and S.M.C. performed and analyzed the hCAII and

MMP-2 assay. R.J.D. designed, interpreted, and oversaw the experiments

for the entire study. The manuscript was drafted by J.L. and R.J.D with input

from all authors.

We develop an assay to measure the protein breakdown ac-

tivity of the proteasome, and use this assay to demonstrate

that gliotoxin and other epidithiodiketopiperazines (ETPs)

inhibit proteasome activity by targeting the essential deubi-

quitinase Rpn11. These molecules quench protease activity

by chelating the active site Zn²⁺ion in Rpn11. An improved

ETP, SOP11, stabilizes proteasome substrates, triggers

the unfolded protein response, and blocks proliferation of

cancer cells. Importantly it does not cause cells to round

up, an off-target effect observed with gliotoxin and other

ETPs. In addition to characterizing proteasome inhibitors

such as ETPs, our assay can be used to monitor the protein

degradation activity of the proteasome in cell lysates, which

may facilitate studies on modulation of proteasome activity

in response to disease, aging, and metabolic state.

DECLARATION OF INTERESTS

R.J.D. is a founder, shareholder, and member of the Scientiﬁc Advisory Board

of Cleave Biosciences, which is engaged in discovery and development of

drugs that target enzymes involved in ubiquitin-dependent protein degrada-

tion. R.J.D. is currently Senior Vice President of discovery research at Amgen

Inc.. S.M.C. is a co-founder and has an equity interest, and receives income as

a member of the Scientiﬁc Advisory Board for Cleave Biosciences and is a co-

founder, has an equity interest, and a member of the Scientiﬁc Advisory Board

for Forge Therapeutics. Both companies may potentially beneﬁt from the

research results of certain projects in the laboratory of S.M.C. The terms of

this arrangement have been reviewed and approved by the University of Cal-

ifornia, San Diego in accordance with its conﬂict of interest policies.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

Received: February 15, 2018

Revised: May 23, 2018

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

d METHOD DETAILS

Accepted: July 25, 2018

Published: August 23, 2018

B Plasmid Construction and Protein Puriﬁcation

B Protein Labeling

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Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

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Cell Chemical Biology 25, 1–9, November 15, 2018

9

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Ubiquitin

Enzo Life Sciences

Cell Signaling

Santa Cruz

Bethyl

Cat# ADI-SPA-200-F; RRID: AB_10618379

Cat# 8052S; RRID: AB_11178947

Cat# sc-365949; RRID: AB_10917561

Cat# A303-373A; RRID: AB_10950130

Cat# sc-25778; RRID: AB_10167668

Cat# 2118S; RRID: AB_561053

NFE2L1

NFE2L2

Cul1

GAPDH antibody (FL-335) HRP

Santa Cruz

Cell Signaling

BioLegend

GAPDH

XBP1

Cat# 647502; RRID: AB_2241743

Cat# 2895P; RRID: AB_2089254

Cat# 5683P; RRID: AB_10841299

Cat# 3177P; RRID: AB_2119845

Cat# 05-636; RRID: AB_309864

Cat# 5625S; RRID: AB_10699459

Cat# ab32518; RRID: AB_733068

Cat# ab32072; RRID: AB_731658

Cat# 700555; RRID: AB_2532328

Cat# ab1101; RRID: AB_297667

CHOP

Cell Signaling

Millipore

PERK

Bip

gH2AX

PARP1

Cell Signaling

Abcam

IkBa

c-Myc

Abcam

MDM2

Thermo Fischer Scientiﬁc

Abcam

P53

Chemicals, Peptides, and Recombinant Proteins

Alexa Fluorꢀ 488 C5 Maleimide

gliotoxin

Thermo Fisher Scientiﬁc

Sigma-Aldrich

Cat# A10254

Cat# G9893

Cat# H8916

TNF-a

Sigma-Aldrich

Critical Commercial Assays

CellTiter-Gloꢁ Luminescent Cell Viability Assay

Experimental Models: Cell Lines

HCT116

Promega

Cat# G7572

ATCC

Cat# CCL-247ꢀ

Ub^G76V GFP Hela cell line

RPE1 WT and BTZ-resistant cell lines

Software and Algorithms

Graphpad

T.F. Chou, Harbor-UCLA and LA BioMed

T.M. Kapoor, The Rockefeller University

Prism

N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and request for resources and reagents should be directed and will be fulﬁlled by Raymond J. Deshaies

(deshaies@caltech.edu)

EXPERIMENTAL MODEL AND SUBJECT DETAILS

HCT116 cells and RPE1 cells were grown at 37^ꢁC with 5% CO2 in Dulbecco’s Modiﬁed Eagle Medium (DMEM) containing 10% fetal

bovine serum, penicillin-streptomycin, and 2 mM L-glutamine.

METHOD DETAILS

Plasmid Construction and Protein Puriﬁcation

Puriﬁcation of GST–Ube1 (E1), Rsp5 (E3 for generating K63-linked Ub chains) and Rsp5-E6AP (E3 for generating K48-linked Ub

chains) was carried out as described previously (Kim and Huibregtse, 2009). UbcH5a (E2) was purchased from Boston Biochem

e1 Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018

Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

Inc. The plasmid encoding Wbp2-C-K222 was a kind gift from Jon M. Huibregtse. A sequence encoding ‘‘LPETGHHHHHH’’ was

inserted at the 3’ end of the Wbp2-C-K222 coding sequence through PCR reaction and the resulting construct was cloned into

the pGEX-4T1 vector resulting in the addition of an N-terminal GST tag. To purify GST–Wbp2-C-K222-His6, Rosetta cells were trans-

formed with the plasmid (RDB3386) and grown at 37^ꢁC in Luria–Bertani broth containing 100 mg/mL ampicillin to an optical density of

about 0.6 at 600 nm. Then, the cells were induced with IPTG (ﬁnal concentration 0.5 mM) for three hours at 37^ꢁC. After lysis of the cells

by sonication, the fusion protein was puriﬁed from the soluble franction by GSTrapꢀ High Performance column (GE healthcare life

sciences), followed by size-exclusion chromatography on a Superdex 200 prep grade column (GE healthcare life sciences).

Protein Labeling

GST–Wbp2-C-K222-His6 was covalently labeled with the ﬂuorescent dye Alexa Fluor 488 C5 Maleimide (A10254, Thermo Fisher

Scientiﬁc) on cysteine residues by incubation with a ﬁve-fold molar excess of the dye under the conditions recommended by the

manufacturer. The reaction was quenched by the addition of 10 mM DTT. We then applied the reaction mixture to a Superdex

75HR column (GE Healthcare) to separate the free dye from the labeled protein. The concentration of the labeled protein and the

degree of labeling were determined according to the manufacturer’s instructions.

In Vitro Ubiquitination and Puriﬁcation

To ubiquitinate GST–Wbp2-C-K222-His6, two separate mixtures (E3 + substrate and GST–Ube1+UbcH5a+ubiquitin+ATP) were

preincubated for two minutes and then combined to initiate the reaction. Ubiquitination was carried out at room temperature for

4 hours. The reaction mixture was puriﬁed by Ni-NTA afﬁnity chromatography and desalting chromatography to remove E1, E2

and E3 and exchange the buffer.

Chemical Syntheses

SOP1 to SOP9 was synthesized as previously reported (Sil and Hilton, 2013). Synthetic protocols for SOP10 and SOP11 were

described below.

SOP-10

Ethyl-2-(allylamino)-2-(4-methoxybenzylthio)acetate (2).

MeO

1) Ethyl glyoxalate

2) para-Methoxybenzyl mercaptan

O

NH₂

S

O

Me

Toluene, 2 hours

RT

NH

1

2

Chemical Formula: C₃H₇N

Molecular Weight: 57.09

Chemical Formula: C₁₅H₂₁NO₃S

Molecular Weight: 295.40

Allylamine (1.90 mL, 25.2 mmol) (1) was added to a solution of ethyl glyoxalate (5.00 mL of a 50% solution in toluene, 25.2 mmol) in

toluene (30.0 mL) at room temperature (RT) and the solution stirred for 2 minutes, whereupon para-methoxybenzyl mercaptan

(3.51 mL, 25.22 mmol) was added and the mixture stirred for 2 hours. Solvent was removed under reduced pressure and the residue

puriﬁed via Biotageꢀ Horizon (3:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 50 g) to give ethyl-2-(allylamino)-2-(4-methoxyben-

zylthio)acetate (6.00 g, 81%) (2) as a colourless oil; n_max/cm^-13344 (N-H), 1732 (C=O); d H (400 MHz; CDCl₃) 1.27 (3H, t, J 7.1,

CH₂CH₃), 1.89 (1H, br s, NH), 3.11 (1H, dd, J 13.9 & 6.4, NCH₂), 3.28 (1H, dd, J 13.9 & 6.6, NCH₂), 3.69 (2H, s, SCH₂), 3.74 (3H, s,

OCH₃), 4.18 (2H, q, J 7.1, CH₂CH₃), 4.32 (1H, s, CHS), 5.06 – 5.10 (2H, m, CH=CH₂), 5.72 – 5.77 (1H, m, CH=CH₂), 6.80 (2H, d, J

8.6, Ar-H), 7.23 (2H, d, J 8.6, Ar-H); d C (101 MHz; CDCl₃) 13.89 (CH₂CH₃), 32.85 (CH₂), 47.59 (CH₂), 54.99 (OCH₃), 61.09 (CH₂),

63.36 (CHS), 113.66 (Ar-C-H), 116.64 (CH=CH₂), 129.72 (quaternary C), 129.78 (Ar-C-H), 135.07 (CH=CH₂), 158.46

(quaternary C), 169.68 (CO); m/z 296 (100%, [M+H]⁺): Found [M+H]⁺296.1322, C₁₅H₂₂NO₃S requires 296.1320.

2-(Allyl(2-ethoxy-1-(4-methoxybenzylthio)-2-oxoethyl)amino)-2-oxoethane-1,1-diyl diacetate (3).

MeO

O

MeO

O

S

Diacetoxyacetyl chloride

O

Me

S

O

Me

N

CH₂Cl₂/ H₂O

NaHCO₃

OAc

NH

2

3

Chemical Formula: C₁₅H₂₁NO₃S

Molecular Weight: 295.40

Chemical Formula: C₂₁H₂₇NO₈S

Molecular Weight: 453.51

A solution of diacetoxyacetyl chloride (4.74 g, 24.4 mmol) in dichloromethane (15.0 mL) was added dropwise to a rapidly stirred

biphasic mixture of ethyl-2-(allylamino)-2-(4-methoxybenzylthio)acetate (6.00 g, 20.3 mmol) (2) and sodium hydrogen carbonate

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Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

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(11.9 g, 142 mmol) in dichloromethane (200 mL) and water (100 mL) at RT and the resulting mixture stirred rapidly for 12 hours. The

layers were separated and the aqueous phase extracted with dichloromethane (2 x 100 mL). The combined organic extracts were

dried over MgSO₄, ﬁltered and solvent removed under reduced pressure to give 2-(allyl(2-ethoxy-1-((4-methoxybenzyl)thio)-2-ox-

oethyl)amino)-2-oxoethane-1,1-diyl diacetate (8.93 g, 97%) (3) as a colourless oil that did not require further puriﬁcation; n_max/cm^-1

1770 (C=O), 1743 (C=O), 1681 (C=O); d H (400 MHz; CDCl₃) 1.20 (3H, t, J 7.1, CH₂CH₃), 1.95 (3H, s, OC(O)CH₃), 2.11 (3H, s, OC(O)

CH₃), 3.67 (2H, s, SCH₂), 3.73 (3H, s, OCH₃), 3.99 (1H, dd, J 17.7 & 5.4, NCH₂), 4.09 (2H, d, J 7.1, CH₂CH₃), 4.25 (1H, dd, J 17.7 & 5.5,

NCH₂), 5.19 – 5.23 (2H, m, CH=CH₂), 5.84 – 5.89 (1H, m, CH=CH₂), 6.08 (1H, s, CHS), 6.80 (2H, d, J 8.6, Ar-H), 7.03 (1H, s, CH(OAc)₂),

7.19 (2H, d, J 8.6, Ar-H); d C (101 MHz; CDCl₃) 13.70 (CH₂CH₃), 20.29 (C(O)CH₃), 34.90 (CH₂), 47.12 (CH₂), 54.99 (OCH₃), 60.77 (CHS),

62.08 (CH₂), 83.86 (CH(OAc)₂), 113.80 (Ar-C-H), 117.56 (CH=CH₂), 128.48 (quaternary C), 130.02 (Ar-C-H), 133.22 (CH=CH₂), 158.73

(quaternary C), 165.41 (CO), 167.14 (CO), 168.37 (CO), 168.69 (CO); m/z 454 (100%, [M+H]⁺): Found [M+H]⁺454.1539, C₂₁H₂₈NO₈S

requires 454.1536.

( )-(3S,6S)-1,4-Diallyl-3,6-bis((4-methoxybenzyl)thio)piperazine-2,5-dione (4).

MeO

O

1) Allylamine

S

2) para-Methoxybenzyl mercaptan

S

N

O

Me

N

MeCN

S

OAc

DMAP (MW)

O

OMe

3

4

Chemical Formula: C₂₁H₂₇NO₈S

Molecular Weight: 453.51

Chemical Formula: C₂₆H₃₀N₂O₄S₂

Molecular Weight: 498.66

Allylamine (0.62 mL, 8.31 mmol) was added to a solution of 2-(allyl(2-ethoxy-1-((4-methoxybenzyl)thio)-2-oxoethyl)amino)-2-oxo-

ethane-1,1-diyl diacetate (2.09 g, 4.62 mmol) (3) in acetonitrile (15.0 mL) followed by addition of para-methoxybenzyl mercaptan

(1.29 mL, 9.23 mmol) and the resulting mixture stirred for 2 minutes. DMAP (0.28 g, 2.31 mmol) was added and the mixture heated

in the microwave at 140^ꢁC for 30 minutes, allowed to cool to room temperature and solvent removed under reduced pressure. The

residue was puriﬁed via Biotageꢀ Horizon (3:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 25 g) and further puriﬁed by recrystal-

lisation (petroleum spirit 40-60^ꢁC: ethyl acetate) to give ( )-(3S,6S)-1,4-diallyl-3,6-bis((4-methoxybenzyl)thio)piperazine-2,5-dione

(1.67 g, 72%) (4) as a colourless solid; m.p. 70-71^ꢁC; n_max/cm^-11675 (C=O), 1609 (C=C), 1465 (CH₂), 832 (C-H); d H (400 MHz,

CDCl₃) 3.82 – 3.91 (12H, m, NCH₂& OCH₃& SCH₂), 3.98 – 4.02 (2H, m, NCH₂), 4.45 (2H, s, CHS), 4.62 – 4.68 (2H, m, CH=CH₂),

4.99 – 5.05 (2H, m, CH=CH₂), 5.48 – 5.54 (2H, m, CH=CH₂), 6.88 – 6.90 (4H, m, Ar-H), 7.36 – 7.38 (4H, m, Ar-H); d C (101 MHz,

CDCl₃) 36.74 (CH₂), 45.85 (CH₂), 55.56 (OCH₃), 58.20 (CHS), 114.28 (CH=CH₂), 119.78 (CH₂), 129.04 (quaternary C), 130.91

(Ar-C-H), 130.95 (Ar-C-H), 159.36 (quaternary C), 165.09 (CO); m/z 499 (100%, [M+H]⁺); Found [M+H]⁺499.1723, C₂₆H₃₁N₂O₄S₂

requires 499.1725.

( )-(3S,6S)-1,3,4,6-Tetraallyl-3,6-bis((4-methoxybenzyl)thio)piperazine-2,5-dione (5).

MeO

O

S

Allyl bromide

N

LiHMDS

S

THF, -78 °C

O

OMe

4

5

Chemical Formula: C₂₆H₃₀N₂O₄S₂

Molecular Weight: 498.66

Chemical Formula: C₃₂H₃₈N₂O₄S₂

Molecular Weight: 578.79

LiHMDS (4.14 mL of a 1M solution in tetrahydrofuran, 4.14 mmol) was added dropwise to a solution of (3S,6S)-1,4-diallyl-3,6-

bis((4-methoxybenzyl)thio)piperazine-2,5-dione (0.83 g, 1.65 mmol) (4) and allyl bromide (0.43 mL, 4.96 mmol) in tetrahydrofuran

(20.0 mL) at -78^ꢁC and the resulting mixture stirred at this temperature for 1 hour and 1.5 hours at 0^ꢁC. Saturated aqueous sodium

hydrogen carbonate (10.0 mL) was added and solvent removed under reduced pressure. The residue was partitioned between water

(10.0 mL) and dichloromethane (20.0 mL). The aqueous phase was extracted with dichloromethane (2 x 10.0 mL) and the combined

extracts were dried (MgSO₄), ﬁltered and solvent removed under reduced pressure. The residue was puriﬁed via Biotageꢀ Horizon

(4:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 10 g) to give ( )-(3S,6S)-1,3,4,6-tetraallyl-3,6-bis((4-methoxybenzyl)thio)pipera-

zine-2,5-dione (0.53 g, 55%) (5) as a pale yellow oil; n_max/cm^-11656 (C=O), 1610 (C=C), 1001 (C-O-C), 835 (C-H); d H (400 MHz,

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CDCl₃) 2.69 (2H, d, J 14.2, CH₂CH=CH₂), 3.21 (2H, d, J 14.1, CH₂CH=CH₂), 3.79 (6H, s, OCH₃), 3.94 – 3.99 (2H, m, SCH₂), 4.05 – 4.09

(2H, m, SCH₂), 4.23 – 4.27 (4H, m, NCH₂), 5.13 – 5.22 (2H, m, CH=CH₂), 5.25 (4H, dd, J 10.2 & 1.2, CH=CH₂), 5.40 (2H, dd, J 10.3 & 1.4,

CH=CH₂), 5.52 – 5.63 (2H, m, CH=CH₂), 6.02 – 6.07 (2H, m, CH=CH₂), 6.81 – 6.85 (4H, m, Ar-H), 7.20 – 7.24 (4H, m, Ar-H); d C (101

MHz, CDCl₃) 35.66 (CH₂), 41.66 (CH₂), 47.69 (CH₂), 55.42 (OCH₃), 73.15 (quaternary C), 114.20 (CH=CH₂), 118.63 (CH₂), 121.15

(CH₂), 128.06 (quaternary C), 130.53 (CH=CH₂), 131.28 (Ar-C-H), 133.62 (Ar-C-H), 159.05 (quaternary C), 164.94 (CO); m/z 601

(100%, [M+Na]⁺); Found [M+Na]⁺601.2176, C₃₂H₃₈N₂O₄S₂Na requires 601.2170.

( )-(1S,4S)-1,2,4,5-Tetraallyl-7-thia-2,5-diazabicyclo[2.2.1]heptane-3,6-dione 7-sulﬁde (6).

MeO

O

S

BBr₃, CH₂Cl₂

N

S₂

O

N

-78 °C to RT,

I₂

S

O

OMe

5

6

Chemical Formula: C₃₂H₃₈N₂O₄S₂

Molecular Weight: 578.79

Chemical Formula: C₁₆H₂₀N₂O₂S₂

Molecular Weight: 336.47

Boron tribromide (0.14 mL, 1.50 mmol) was added dropwise to a solution of 1,3,4,6-tetraallyl-3,6-bis((4-methoxybenzyl)thio)piper-

azine-2,5-dione (0.44 g, 0.75 mmol) (5) in dichloromethane (20.0 mL) at -78^ꢁC. The resulting mixture was stirred for 15 minutes where-

upon Rochelle’s salt (20.0 mL of a saturated aqueous solution) was added and the biphasic mixture stirred for 15 minutes until the

yellow colour had dissipated. The resulting mixture was stirred at room temperature for 10 minutes, whereupon iodine was added

portionwise until the colour due to iodine just persisted and stirring was maintained for 2 minutes. Sodium thiosulfate was added

and the mixture stirred for 10 minutes, diluted with dichloromethane (10.0 mL) and water (20.0 mL). The layers were separated

and the aqueous phase extracted with dichloromethane (2 x 20.0 mL). The combined organic extracts were dried over MgSO₄,

ﬁltered and solvent removed under reduced pressure. The residue was puriﬁed via Biotageꢀ Horizon (4:1, petroleum spirit

40-60^ꢁC: ethyl acetate; snap 10 g) and further puriﬁed by recrystallisation (petroleum spirit 40-60^ꢁC: ethyl acetate) to give

( )-(1S,4S)-1,2,4,5-tetraallyl-7-thia-2,5-diazabicyclo[2.2.1]heptane-3,6-dione 7-sulﬁde (0.10 g, 41%) (6) as a colourless solid;

m.p. 180-182^ꢁC; n_max/cm^-11639 (C=O, C=C), 1462 (CH₂), 749 (CH₂); d H (400 MHz, CDCl₃) 3.20 – 3.27 (2H, m, CH₂CH=CH),

3.28 – 3.36 (2H, m, CH₂CH=CH), 4.04 (2H, dd, J 16.4 & 6.8, NCH₂), 4.62 (2H, dd, J 16.2 & 4.8, NCH₂), 5.21 – 5.41 (8H, m,

CH=CH₂), 5.89 – 5.94 (2H, m, CH=CH₂), 6.12 – 6.17 (2H, m, CH=CH₂); d C (101 MHz, CDCl₃) 36.44 (CH₂), 45.46 (CH₂), 74.50

(quaternary C), 118.31 (CH=CH₂), 120.60 (CH=CH₂), 131.74 (CH=CH₂), 132.01 (CH=CH₂), 165.02 (CO); m/z 337 (100%, [M+H]⁺);

Found [M+H]⁺337.1456, C₁₆H₂₁N₂O₂S₂requires 337.1448.

( )-(6aS,12aS)-7,10-Dihydro-6a,12a-epithiodipyrido[1,2-a:1’,2’-d] pyrazine-6,12(1H,4H)-dione 13-sulﬁde (SOP-10).

Me

N

Me

Cl

Me

Ru

O

Cl

O

Me

N

S₂

N

CH₂Cl₂

(MW)

O

6

SOP-10

Chemical Formula: C₁₆H₂₀N₂O₂S₂

Molecular Weight: 336.47

Chemical Formula: C₁₂H₁₂N₂O₂S₂

Molecular Weight: 280.37

Method A

A solution of ( )-(1S,4S)-1,2,4,5-tetraallyl-7-thia-2,5-diazabicyclo[2.2.1]heptane-3,6-dione 7-sulﬁde (0.04 g, 0.12 mmol) (6) in di-

chloromethane (2.00 mL) was degassed and taken under nitrogen atmosphere. To this (1,3-bis-(2,4,6-trimethylphenyl)-2-imidazoli-

dinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium (Grubbs-Hoveyda second-generation catalyst) (0.01 g, 0.01 mmol)

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was added and the resulting mixture heated at 80^ꢁC for 30 minutes in the microwave, cooled to RT and solvent removed under

reduced pressure. The residue was puriﬁed via Biotageꢀ Horizon (4:1, petroleum spirit 40-60^ꢁC: ethyl acetate; pipette column)

and further puriﬁed by recrystallisation (petroleum spirit 40-60^ꢁC: ethyl acetate) to give ( )-(6aS,12aS)-7,10-dihydro-6a,12a-epithio-

dipyrido[1,2-a:1’,2’-d]pyrazine-6,12(1H,4H)-dione 13-sulﬁde (0.01 g, 39%) (SOP-10) as a colourless solid; m.p. 135-137^ꢁC; n_max/cm^-

¹1683 (C=O, C=C), 1351 (C-H), 1321 (C-H); d H (400 MHz, CDCl₃) 2.60 – 2.72 (2H, m, CH₂CH=CH), 3.29 – 3.40 (2H, m, CH₂CH=CH),

3.87 – 4.08 (4H, m, NCH₂), 5.86 – 5.97 (4H, m, CH=CH); d C (101 MHz, CDCl₃) 28.60 (CH₂), 42.30 (CH₂), 70.29 (quaternary C), 121.14

(CH=CH), 122.32 (CH=CH), 165.15 (CO); m/z 303 (100%, [M+Na]⁺); Found [M+Na]⁺303.0253, C₁₂H₁₂N₂O₂S₂Na requires 303.0238.

Method B

A solution of ( )-(1S,4S)-1,2,4,5-tetraallyl-7-thia-2,5-diazabicyclo[2.2.1]heptane-3,6-dione 7-sulﬁde (0.04 g, 0.13 mmol) (6) in di-

chloromethane (2.00 mL) was degassed and taken under nitrogen atmosphere. To this (1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidi-

nylidene)dichloro (phenylmethylene)(tricyclohexylphosphine)ruthenium (Grubbs second-generation catalyst) (0.01 g, 0.01 mmol) was

added and the resulting mixture heated at 80^ꢁC for 30 minutes in the microwave, cooled to RT and solvent removed under reduced

pressure. The residue was puriﬁed via Biotageꢀ Horizon (4:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 5 g) and further puriﬁed

by recrystallisation (petroleum spirit 40-60^ꢁC: ethyl acetate) to give ( )-(6aS,12aS)-7,10-dihydro-6a,12a-epithiodipyrido[1,2-a:1’,2’-

d]pyrazine-6,12(1H,4H)-dione 13-sulﬁde (0.01 g, 7%) (SOP-10) as a colourless solid; identical spectroscopic data to that obtained

previously for (SOP-10).

SOP-11

Ethyl-2-(benzylamino)-2-((4-methoxybenzyl)thio)acetate (8).

MeO

1) Ethyl glyoxalate

2) para-Methoxybenzyl mercaptan

O

S

O

Me

NH₂

Toluene, 2 hours

RT

Ph

NH

7

8

Chemical Formula: C₇H₉N

Molecular Weight: 107.15

Chemical Formula: C₁₉H₂₃NO₃S

Molecular Weight: 345.46

Benzylamine (2.76 mL, 25.2 mmol) (7) was added to a solution of ethyl glyoxalate (5.00 mL of a 50% solution in toluene, 25.2 mmol)

in toluene (20.0 mL) at room temperature (RT) and stirred for 2 minutes, whereupon para-methoxybenzyl mercaptan (3.54 mL,

25.2 mmol) was added and the mixture stirred for 2 hours. Solvent was removed under reduced pressure and the residue puriﬁed

via Biotageꢀ Horizon (3:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 50 g) to give ethyl-2-(benzylamino)-2-((4-methoxybenzyl)

thio)acetate (7.56 g, 87%) (8) as a colourless oil; n_max/cm^-13347 (N-H), 1732 (C=O); d H (400 MHz; CDCl₃) 1.31 (3H, t, J 7.2,

CO₂CH₂CH₃), 1.88 (1H, br s, NH), 3.72 (1H, d, J 13.1, SCH₂), 3.75 – 3.77 (2H, m, NHCH₂), 3.79 (3H, s, OCH₃), 3.87 (1H, d, J 13.1,

SCH₂), 4.22 (2H, q, J 7.2, CO₂CH₂CH₃), 4.35 (1H, s, CH), 6.84 (2H, d, J 8.7, Ar-H), 7.23 – 7.29 (7H, m, Ar-H); d C (101 MHz;

CDCl₃) 14.15 (CO₂CH₂CH₃), 33.23 (SCH₂), 49.22 (NHCH₂), 55.28 (OCH₃), 61.41 (CO₂CH₂CH₃), 63.71 (CH), 114.00 (Ar-C-H),

127.19 (Ar-C-H), 128.41 (Ar-C-H), 128.48 (Ar-C-H), 130.07 (Ar-C-H), 138.79 (quaternary C), 158.76 (quaternary C), 169.97

(CO₂CH₂CH₃); m/z 346 (100%, [M+H]⁺): Found [M+H]⁺346.1477, C₁₉H₂₄NO₃S requires 346.1482.

2-(Benzyl(2-ethoxy-1-(4-methoxybenzylthio)-2-oxoethyl)amino)-2-oxoethane-1,1-diyl diacetate (9).

MeO

O

MeO

O

S

O

Me

Diacetoxyacetyl chloride

S

O

Me

Ph

N

OAc

Ph

NH

CH₂Cl₂/ H₂O

NaHCO₃

8

9

Chemical Formula: C₁₉H₂₃NO₃S

Molecular Weight: 345.46

Chemical Formula: C₂₅H₂₉NO₈S

Molecular Weight: 503.56

A solution of diacetoxyacetyl chloride (5.11 g, 26.3 mmol) in dichloromethane (20.0 mL) was added dropwise to a rapidly stirred

biphasic mixture of ethyl-2-(benzylamino)-2-((4-methoxybenzyl)thio)acetate (7.56 g, 21.9 mmol) (8) and sodium hydrogen carbonate

(12.9 g, 153 mmol) in dichloromethane (150 mL) and water (100 mL) at RT and the resulting mixture stirred rapidly for 12 hours. The

layers were separated and the aqueous phase extracted with dichloromethane (2 x 100 mL). The combined extracts were dried over

MgSO₄, ﬁltered and solvent removed under reduced pressure to give 2-(benzyl(2-ethoxy-1-((4-methoxybenzyl)thio)-2-oxoethyl)

amino)-2-oxoethane-1,1-diyl diacetate (10.5 g, 95%) (9) as a colourless oil that did not require further puriﬁcation; n_max/cm^-11770

(C=O), 1739 (C=O), 1685 (C=O); d H (400 MHz; CDCl₃) 1.15 (3H, t, J 7.1, CH₂CH₃), 1.95 (3H, s, OC(O)CH₃), 1.98 (3H, s, OC(O)

e5 Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018

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CH₃), 3.72 (2H, s, SCH₂), 3.75 (3H, s, OCH₃), 3.98 – 4.02 (2H, m, CH₂CH₃), 4.59 (1H, d, J 17.4, NCH₂), 4.86 (1H, d, J 17.4, NCH₂), 5.85

(1H, s, CHS), 6.79 (2H, d, J 8.4, Ar-H), 6.92 (1H, s, CH(OAc)₂), 7.16 – 7.27 (7H, m, Ar-H); d C (101 MHz; CDCl₃) 13.76 (CH₂CH₃), 20.20

(C(O)CH₃), 20.25 (C(O)CH₃), 35.49 (CH₂), 48.68 (CH₂), 55.18 (OCH₃), 61.65 (CHS), 62.21 (CH₂CH₃), 83.99 (CH(OAc)₂), 113.97 (Ar-C-

H), 126.79 (Ar-C-H), 127.66 (Ar-C-H), 128.48 (Ar-C-H), 128.65 (quaternary C), 130.21 (Ar-C-H), 135.72 (quaternary C), 158.87

(quaternary C), 165.76 (CO), 167.16 (CO), 168.34 (CO), 168.62 (CO); m/z 504 (100%, [M+H]⁺): Found [M+H]⁺504.1688,

C₂₅H₃₀NO₈S requires 504.1692.

1,4-Dibenzyl-3-methoxy-6-((4-methoxybenzyl)thio)piperazine-2,5-dione (10).

MeO

O

S

Benzylamine

MeOH, DMAP

S

N

Ph

OMe

O

Me

Ph

N

Ph

N

OAc

O

9

10

Chemical Formula: C₂₅H₂₉NO₈S

Molecular Weight: 503.56

Chemical Formula: C₂₇H₂₈N₂O₄S

Molecular Weight: 476.59

Benzylamine (0.21 mL, 1.93 mmol) was added to a solution of 2-(benzyl(2-ethoxy-1-((4-methoxybenzyl)thio)-2-oxoethyl)amino)-2-

oxoethane-1,1-diyl diacetate (0.65 g, 1.29 mmol) (9) in methanol (15.0 mL) and the resulting mixture stirred for 2 minutes. DMAP

(0.08 g, 0.64 mmol) was added and the resulting mixture stirred for 5 hours at RT and solvent removed under reduced pressure.

The residue was puriﬁed via Biotageꢀ Horizon (3:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 25 g) and further puriﬁed by recrys-

tallisation (petroleum spirit 40-60^ꢁC: ethyl acetate) to give 1,4-dibenzyl-3-methoxy-6-((4-methoxybenzyl)thio)piperazine-2,5-dione

(0.49 g, 79%) (10) as a colourless solid; m.p. 85-88^ꢁC; n_max/cm^-13030 (C-H), 3005 (C-H), 1671 (C=O), 1357 (CH₃), 1002 (C-O-C),

832 (C-H); d H (400 MHz, CDCl₃) 3.53 (3H, s, OCH₃), 3.62 (1H, d, J 14.5, NCH₂), 3.87 – 3.90 (4H, m, SCH₂& OCH₃), 4.09 (1H, d, J

13.8, SCH₂), 4.25 (1H, d, J 14.9, NCH₂), 4.66 (1H, s, CHO), 4.45 (1H, s, CHS), 5.18 (2H, d, J 14.7, NCH₂), 6.91 – 6.95 (2H, m,

Ar-H), 7.16 – 7.29 (6H, m, Ar-H), 7.36 (6H, dd, J 16.4 & 9.2, Ar-H); d C (101 MHz, CDCl₃) 36.29 (CH₂), 45.93 (CH₂), 48.07 (CH₂),

55.39 (OCH₃), 57.66 (OCH₃), 85.50 (CHS), 114.29 (CHO), 128.03 (Ar-C-H), 128.19 (Ar-C-H), 128.53 (Ar-C-H), 128.66 (Ar-C-H),

128.75 (Ar-C-H), 129.01 (Ar-C-H), 130.88 (Ar-C-H), 134.98 (quaternary C), 135.30 (quaternary C), 159.27 (quaternary C), 162.73

(CO), 166.29 (CO); m/z 477 (100%, [M+H]⁺); Found [M+H]⁺477.1841, C₂₇H₂₉N₂O₄S requires 477.1848.

1,3,4-Tribenzyl-6-methoxy-3-((4-methoxybenzyl)thio)piperazine-2,5-dione (11).

MeO

O

Ph

S

O

S

Benzyl bromide

N

Ph

OMe

N

Ph

OMe

Ph

N

Ph

N

LiHMDS

THF, -78 °C

O

10

11

Chemical Formula: C₂₇H₂₈N₂O₄S

Molecular Weight: 476.59

Chemical Formula: C₃₄H₃₄N₂O₄S

Molecular Weight: 566.71

LiHMDS (0.50 mL of a 1M solution in tetrahydrofuran, 0.50 mmol) was added dropwise to a solution of 1,4-dibenzyl-3-methoxy-6-

((4-methoxybenzyl)thio)piperazine-2,5-dione (0.20 g, 0.41 mmol) (10) and benzyl bromide (0.05 mL, 0.41 mmol) in tetrahydrofuran

(2.00 mL) at -78^ꢁC and the resulting mixture stirred at this temperature for 1.5 hours and 1 hour at 0^ꢁC. Saturated aqueous sodium

hydrogen carbonate (10.0 mL) was added and solvent removed under reduced pressure. The residue was partitioned between water

(10.0 mL) and dichloromethane (20.0 mL). The aqueous phase was extracted with dichloromethane (2 x 10.0 mL) and the combined

extracts were dried (MgSO₄), ﬁltered and solvent removed under reduced pressure. The residue was puriﬁed via Biotageꢀ Horizon

(3:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 10 g) to give 1,3,4-tribenzyl-6-methoxy-3-((4-methoxybenzyl)thio)piperazine-2,5-

dione (0.14 g, 59%) (11) as a pale yellow oil; n_max/cm^-13004 (C-H), 1670 (C=O), 1453 (CH₂), 1357 (CH₃), 1000 (C-O-C); d H (400 MHz,

CDCl₃) 3.18 (1H, d, J 10.9, CH₂), 3.32 (1H, d, J 14.2, CH₂), 3.38 (1H, d, J 8.8, SCH₂), 3.44 (3H, s, OCH₃), 3.73 (1H, d, J 9.1, SCH₂), 3.78

(3H, s, OCH₃), 4.05 (1H, d, J 14.2, NCH₂), 4.13 (1H, s, CHO), 4.65 (1H, d, J 14.7, NCH₂), 4.75 (1H, d, J 14.7, NCH₂), 5.30 (1H, d, J 14.7,

NCH₂), 7.01 (6H, dd, J 11.5 & 4.5, Ar-H), 7.14 – 7.17 (3H, m, Ar-H), 7.29 – 7.33 (8H, m, Ar-H), 7.54 – 7.57 (2H, m, Ar-H); d C (101 MHz,

CDCl₃) 34.61 (CH₂), 43.51 (CH₂), 47.21 (CH₂), 47.91 (CH₂), 55.41 (OCH₃), 82.93 (OCH₃), 113.98 (CHO), 127.63 (Ar-C-H), 128.13

(Ar-C-H), 128.36 (Ar-C-H), 128.60 (Ar-C-H), 128.82 (Ar-C-H), 129.03 (Ar-C-H), 129.39 (Ar-C-H), 129.56 (Ar-C-H), 130.19 (Ar-C-H),

130.48 (Ar-C-H), 130.65 (Ar-C-H), 133.87 (quaternary C), 134.70 (quaternary C), 135.44 (quaternary C), 137.89 (quaternary C),

159.02 (quaternary C), 164.23 (quaternary C), 165.06 (CO), 166.07 (CO); m/z 589 (100%, [M+Na]⁺); Found [M+Na]⁺589.2107,

C₃₄H₃₄N₂O₄SNa requires 589.2137.

Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018 e6

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( )-(3S,6S)-1,3,4-Tribenzyl-3,6-bis((4-methoxybenzyl)thio)piperazine-2,5-dione (12).

MeO

Ph

S

O

Ph

S

O

para-Methoxybenzyl mercaptan

N

Ph

N

Ph

OMe

Ph

N

Ph

N

TFA, CH₂Cl₂

S

O

OMe

11

12

Chemical Formula: C₃₄H₃₄N₂O₄S

Molecular Weight: 566.71

Chemical Formula: C₄₁H₄₀N₂O₄S₂

Molecular Weight: 688.90

TFA (1.00 mL, 13.0 mmol) was added to a solution of 1,3,4-tribenzyl-6-methoxy-3-((4-methoxybenzyl)thio)piperazine-2,5-dione

(0.18 g, 0.32 mmol) (11) in dichloromethane (5.00 mL) at RT followed by the addition of para-methoxybenzyl mercaptan (0.13 mL,

0.95 mmol) and the resulting mixture stirred at this temperature for 18 hours. After, the solution was diluted in dichloromethane

(10.0 mL) and washed with saturated aqueous sodium hydrogen carbonate (10.0 mL). The aqueous phase was extracted with di-

chloromethane (2 x 10.0 mL) and the combined extracts were dried (MgSO₄), ﬁltered and solvent removed under reduced pressure.

The residue was puriﬁed via Biotageꢀ Horizon (4:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 10 g) to give ( )-(3S,6S)-1,3,4-tri-

benzyl-3,6-bis((4-methoxybenzyl)thio)piperazine-2,5-dione (0.20 g, 91%) (12) as a pale yellow oil; n_max/cm^-13032 (C-H), 3005 (C-H),

1666 (C=O), 1356 (CH₃), 1001 (C-O-C), 833 (C-H), 730 (C-H); d H (400 MHz, CDCl₃) 3.77 – 3.78 (2H, m, CH₂), 3.79 (3H, s, OCH₃), 3.84

(3H, s, OCH₃), 3.87 – 3.89 (4H, m, SCH₂& NCH₂), 3.97 – 4.06 (2H, m, SCH₂), 4.27 (1H, s, CHS), 4.62 (1H, d, J 15.1, NCH₂), 5.28 (1H, d, J

15.1, NCH₂), 6.51 – 6.54 (3H, m, Ar-H), 6.75 – 6.81 (4H, m, Ar-H), 6.84 – 6.88 (4H, m, Ar-H), 7.00 – 7.05 (4H, m, Ar-H), 7.13 – 7.20 (4H, m,

Ar-H), 7.27 – 7.31 (2H, m, Ar-H), 7.59 – 7.63 (2H, m, Ar-H); d C (101 MHz, CDCl₃) 35.31 (CH₂), 36.93 (CH₂), 43.44 (CH₂), 46.98 (CH₂),

47.70 (CH₂), 55.38 (OCH₃), 58.20 (OCH₃), 75.78 (quaternary C), 113.96 (CHS), 127.84 (Ar-C-H), 128.50 (Ar-C-H), 128.58 (Ar-C-H),

128.62 (Ar-C-H), 128.81 (Ar-C-H), 128.90 (Ar-C-H), 129.17 (Ar-C-H), 129.86 (Ar-C-H), 130.35 (Ar-C-H), 130.65 (Ar-C-H), 130.74

(Ar-C-H), 130.81 (Ar-C-H), 130.96 (Ar-C-H), 133.96 (quaternary C), 134.27 (quaternary C), 134.41 (quaternary C), 135.74 (quaternary

C), 137.79 (quaternary C), 158.96 (quaternary C), 159.17 (quaternary C), 163.98 (CO), 165.99 (CO); m/z 711 (100%, [M+Na]⁺); Found

[M+Na]⁺711.2355, C₄₁H₄₀N₂O₄S₂Na requires 711.2327.

( )-(1S,4S)-1,2,5-Tribenzyl-7-thia-2,5-diazabicyclo[2.2.1]heptane-3,6-dione 7-sulﬁde (SOP-11).

MeO

Ph

S

O

BBr₃, CH₂Cl₂

N

Ph

N

Ph

S₂

Ph

N

-78 °C to RT,

I₂

S

O

OMe

12

SOP-11

Chemical Formula: C₄₁H₄₀N₂O₄S₂

Molecular Weight: 688.90

Chemical Formula: C₂₅H₂₂N₂O₂S₂

Molecular Weight: 446.58

Boron tribromide (0.70 mL of a 1M solution in dichloromethane, 0.70 mmol) was added dropwise to a solution of ( )-(3S,6S)-1,3,4-

tribenzyl-3,6-bis((4-methoxybenzyl)thio)piperazine-2,5-dione (0.12 g, 0.17 mmol) (12) in dichloromethane (2.00 mL) at -78^ꢁC. The re-

sulting mixture was stirred for 15 minutes whereupon aqueous sodium hydrogen carbonate (10.0 mL of a saturated aqueous solution)

and methanol (1.00 mL) were added and the biphasic mixture stirred for 15 minutes until the yellow colour had dissipated. The re-

sulting mixture was stirred at room temperature for 10 minutes, whereupon iodine was added portionwise until the colour due to

iodine just persisted and stirring was maintained for 2 minutes. Sodium thiosulfate was added and the mixture stirred for 10 minutes,

diluted with dichloromethane (10.0 mL) and water (20.0 mL). The layers were separated and the aqueous phase extracted with di-

chloromethane (2 x 20.0 mL). The combined organic extracts were dried over MgSO₄, ﬁltered and solvent removed under reduced

pressure. The residue was puriﬁed via Biotageꢀ Horizon (3:1, petroleum spirit 40-60^ꢁC: ethyl acetate; snap 10 g), followed by HPLC

separation and further puriﬁed by recrystallisation (petroleum spirit 40-60^ꢁC: ethyl acetate) to give ( )-(1S,4S)-1,2,5-tribenzyl-7-thia-

2,5-diazabicyclo[2.2.1]heptane-3,6-dione 7-sulﬁde (0.04 g, 50%) (SOP-11) as a colourless solid; m.p. 122-124^ꢁC; n_max/cm^-11641

(C=O), 1438 (CH₂), 749 (C-H); d H (400 MHz; CDCl₃) 3.62 – 3.68 (1H, m, CH₂), 3.73 – 3.79 (1H, m, CH₂), 4.23 (1H, d, J 15.2,

NCH₂), 4.34 (1H, d, J 15.0, NCH₂), 4.86 (1H, d, J 14.9, NCH₂), 5.12 (1H, d, J 15.0, NCH₂), 5.26 (1H, s, CHS), 7.12 – 7.24 (15H, m,

Ar-H); d C (101 MHz; CDCl₃) 36.39 (CH₂), 46.11 (CH₂), 48.78 (CH₂), 64.13 (CHS), 76.53 (quaternary C), 127.38 (Ar-C-H), 127.98

(Ar-C-H), 128.17 (Ar-C-H), 128.68 (Ar-C-H), 128.74 (Ar-C-H), 128.80 (Ar-C-H), 128.98 (Ar-C-H), 129.40 (Ar-C-H), 130.42 (Ar-C-H),

133.96 (quaternary C), 134.54 (quaternary C), 136.00 (quaternary C), 164.83 (CO), 165.31 (CO); m/z 469 (100%, [M+Na]⁺): Found

[M+Na]⁺469.1097, C₂₅H₂₂N₂O₂S₂Na requires 469.1101.

e7 Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018

Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

SDS-PAGE Analysis of Proteasome Activity

20 nM UbⁿGST–Wbp2 and 4 nM puriﬁed human 26S proteasome were incubated at 37^ꢁC for 2 hours in the presence of different

compounds plus 1 mM ATP. The reactions were stopped with 23 SDS sample buffer and analyzed by SDS–PAGE. The gel was

scanned using Typhoonꢀ FLA 9500 biomolecular imager (GE Healthcare life sciences) with ﬁlter for ﬂuorophore Alexa 488.

Fluorescence Polarization Assay

The ﬂuorescence polarization assay was performed in a low-volume 384-well solid black plate to which the components were added

in the following sequence: 1) 5 ml compound in 3% (v/v) DMSO or 3% DMSO control, 2) 5 ml of 26S proteasome (Enzo life science),

3) 5 ml of 5 nM UbⁿGST–Wbp2 to initiate the reaction. Fluorescence polarization was measured using a plate reader with excitation at

480 nm and emission at 520 nm (PHERAstar, BMG Labtech).

Evaluation of Ubiquitin Linkage

Substrate samples were fractionated on an SDS-polyacrylamide gel and digested in-gel with trypsin. Di-Ubs of all 7 linkages (Boston

Biotech) were used to quantify the standard heavy Ub linkages. The standard heavy Ub linkages were subsequently used as internal

standards to quantify the linkages in substrates ubiquitinated by Rsp5-WT or Rsp5-E6AP. Mass spectrometry was carried out on a

Thermo Orbitrap Velos.

Rpn11,Rpn8 di-Ub Cleavage Assay

Di-ubiquitin cleavage assay was performed in 40 mM HEPES, pH7.5, 100 mM NaCl, 100 mM KCl and 5% (v/v) glycerol. Rpn11,Rpn8

dimer (5 mM) was incubated with di-Ub^K48and different concentrations of inhibitors at 30^ꢁC. The reactions were stopped with 23 SDS

sample buffer after 2 hours and analyzed by SDS–PAGE.

hCAII Assays

hCAII was expressed and puriﬁed as previously reported (Martin et al., 2013). Human carbonic anhydrase II (hCAII) assays were car-

ried out in clear Costar 96-well plates. Each well contained a volume of 100 mL including buffer (50 mM HEPES buffer, pH 8.0), protein

(10 nM hCAII), inhibitor (100 mM), and substrate p-nitrophenyl acetate (500 mM). The protein and inhibitor were incubated in solution at

30^ꢁC for 10 min prior to the addition of the p-nitrophenyl acetate to initiate the reaction. The change in absorbance was monitored at

405 nm for 15 min. The negative control wells, containing no inhibitor, were arbitrarily set as 100% activity. Readings from back-

ground wells, which did not contain protein, were subtracted from the active assay wells to account for background hydrolysis ac-

tivity caused by the buffer. The assays were performed in triplicate. The data were normalized to values measured for uninhibited

enzyme. Assay data are reported as the mean standard deviation.

MMP-2 Assays

MMP-2 and OmniMMP ﬂuorogenic substrate were purchased from Enzo Life Sciences. The assays were carried out in black NUNC

96-well plates. Each well contained a volume of 100 mL including buffer (50 mM HEPES, 10 mM CaCl₂, 0.05% (v/v) Brij-35, pH 7.5),

human recombinant MMP (1.16 U MMP-2, Enzo Life Sciences), inhibitor (100 mM), ﬂuorogenic OmniMMP substrate (4 mM Mca-Pro-

Leu-Gly-Leu-Dpa-Ala-Arg-NH₂$AcOH, Enzo Life Sciences). The mixture of protein and inhibitor was pre-incubated in solution at

37^ꢁC for 30 min, followed by the addition of the substrate to initiate the reaction. The change in ﬂuorescence was monitored for

30 min by BioTek synergy HT ﬂuorescence plate readers with excitation and emission wavelengths at 320 and 400 nm, respectively.

The control wells, containing no inhibitor, were arbitrarily set as 100% activity. MMP activity was deﬁned as the ratio of ﬂuorescence

increase in the inhibitor wells relative to the negative control wells, expressed as a percentage. The assays were performed in trip-

licate. The data were normalized to values measured for uninhibited enzyme. Assay data are reported as the mean standard

deviation.

Ub^G76V-GFP Degradation Assay

A stable HeLa cell line with the ubiquitin fusion degradation reporter Ub^G76V-GFP were seeded on 384-well plates (5000 cells/well)

and grown for 18 hours. Cells were treated with modiﬁed DMEM (without phenol red, folic acid, riboﬂavin, and vitamin B12) containing

MG132 (4 mM) for 1 hour and washed twice with pre-warmed PBS. Modiﬁed DMEM containing FBS (2.5%), cycloheximide (CHX)

(50 mM), and DMSO or a test compound (0.1, 0.19, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 mM) was added to each well. Plates

were imaged on the ImageXpress Micro microscope (Molecular Devices) after 60 to 240 min.

IkBa Degradation Assay

Hela cells were pre-treated with DMSO or compounds as indicated for 10 minutes and then treated with 25 ng/mL of recombinant

human TNF-a (Promega) for 30 minutes. Cells were lysed with RIPA buffer with protease inhibitor cocktail, fractionated by SDS–

PAGE and immunoblotted with antibodies against IkBa or GAPDH.

Quantitative Real-Time PCR of HCT116 Cell RNA

Cells were seeded in 6-well plates at 5 310⁵cells/well in DMEM medium for 16 hours before drug treatment. Compounds were added

at 10 mM each except for actinomycin D, which was used at 1 mM as a positive control. RNA was isolated using RNeasy kit (Qiagen).

Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018 e8

Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell

Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.07.012

cDNA was synthesized with QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was performed using Roche

Lightcycler 480 and SYBR green I master kit. Sequences of primers used for qRT-PCR were as follows: c-Myc: Fwd, 5’-caccagcagc

gactctga; Rev, 5’-gatccagactctgaccttttgc; GAPDH: Fwd, 5’-acccactcctccacctttgac; Rev, 5’-ctgttgctgtagccaaattcgt.

QUANTIFICATION AND STATISTICAL ANALYSIS

Graphpad Prism 7 was used for statistical analysis of the data from proteasome activity assays, Rpn11 activity assays, cell viability

assays and quantitative real-time PCR. For each analysis, total n and S.D. are presented in the ﬁgure legend. Data were ﬁtted

to a dose-response equation (three parameters) to determine the IC₅₀or GI_50.Quantitative real-time PCR data were analyzed by

Student’s t test (two-tailed). Differences were statistically signiﬁcant when P<0.05.

DATA AND SOFTWARE AVAILABILITY

All data are available upon request to the Lead contact.

e9 Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018

Article Doi

Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11

DOI: 10.1016/j.chembiol.2018.07.012

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Article abstract of DOI:10.1016/j.chembiol.2018.07.012

Full text of DOI:10.1016/j.chembiol.2018.07.012

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