Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

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Biochem. J. (2010) 430, 461–476 (Printed in Great Britain) doi:10.1042/BJ20100383

461

Characterization of a new series of non-covalent proteasome inhibitors with

exquisite potency and selectivity for the 20S β5-subunit

Christopher BLACKBURN, Kenneth M. GIGSTAD, Paul HALES, Khristofer GARCIA, Matthew JONES, Frank J. BRUZZESE,

Cynthia BARRETT, Jane X. LIU, Teresa A. SOUCY, Darshan S. SAPPAL, Nancy BUMP, Edward J. OLHAVA¹, Paul FLEMING,

Lawrence R. DICK, Christopher TSU, Michael D. SINTCHAK and Jonathan L. BLANK²

Discovery, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, U.S.A.

pharmacological inhibition of this site in cells is sufﬁcient to

potently inhibit the degradation of a tetra-ubiquitin–luciferase

reporter, activation of NFκB (nuclear factor κB) in response

to TNF-α (tumour necrosis factor-α) and the proliferation of

cancer cells. Finally, we identiﬁed capped di-peptides that show

differential selectivity for the β5 site of the constitutively

expressed proteasome and immunoproteasome in vitro and in

B-cell lymphomas. Collectively, these studies describe the syn-

thesis, activity and binding mode of a new series of non-covalent

proteasome inhibitors with unprecedented potency and selectivity

for the β5 site, and which can discriminate between the con-

stitutive proteasome and immunoproteasome in vitro and in cells.

The mammalian 26S proteasome is a 2500 kDa multi-catalytic

complex involved in intracellular protein degradation. We

describe the synthesis and properties of a novel series of non-

covalent di-peptide inhibitors of the proteasome used on a capped

tri-peptide that was ﬁrst identiﬁed by high-throughput screening

of a library of approx. 350000 compounds for inhibitors of

the ubiquitin–proteasome system in cells. We show that these

compounds are entirely selective for the β5 (chymotrypsin-like)

site over the β1 (caspase-like) and β2 (trypsin-like) sites of

the 20S core particle of the proteasome, and over a panel of

less closely related proteases. Compound optimization, guided

by X-ray crystallography of the liganded 20S core particle,

conﬁrmed their non-covalent binding mode and provided a

structural basis for their enhanced in vitro and cellular potencies.

We demonstrate that such compounds show low nanomolar

IC₅₀values for the human 20S β5 site in vitro, and that

Key words: chymotrypsin-like, immunoproteasome, 26S protea-

some, proteasome inhibitor, β5-subunit, ubiquitin–proteasome

system (UPS).

termed the immunoproteasome exists in cells of lymphoid origin

and can be induced in most, if not all, cells in response to

the pro-inﬂammatory cytokine interferon-γ [5–7]. This enzyme

contains distinct catalytic subunits, designated β1i, β2i and

β5i, and is primarily involved in the generation of antigenic

peptides for MHC class I presentation, although a function for

the immunoproteasome in cytokine production has also been

described [8].

For each of the 20S catalytic β-subunits, the hydroxy

group of the N-terminal threonine residue (Thr^1Oγ) serves as

the nucleophile that initiates cleavage of the peptide bond

[4,5,9]. Various natural and synthetic compounds that contain

electrophilic centres or ‘warheads’ inhibit the proteasome by

forming covalent adducts with these active-site threonine residues,

including peptide aldehydes, vinyl sulfones, boronic acids,

α^ꢀβ^ꢀ-epoxyketones, 2-keto-1,3,4-oxadiazoles and β-lactones

(Figure 1A) [4–6,9–11]. The di-peptide boronic acid bortezomib

(PS-341 or VELCADE^®, Millennium Pharmaceuticals, Inc.)

is among the most potent, stable and selective of these

inhibitors [12–15], and shows nanomolar potency with respect

to cytotoxicity across a broad range of human tumour cell types

in vitro [13,14]. It is in clinical use for the treatment of multiple

myeloma [16–19] and refractory mantle cell lymphoma [20],

INTRODUCTION

The 26S proteasome is a very large (2500 kDa) multi-

subunit proteolytic complex found in high abundance in all

eukaryotic cells that is responsible for the regulated degradation

of the majority of intracellular proteins, including those

involved in signal transduction, cell-cycle control, apoptosis and

inﬂammatory responses [1–6]. It is composed of the 20S catalytic

core particle that is capped at one or both ends by the 19S

regulatory complex, also called PA700 [1–6]. The 20S core

particle is comprises four stacked heteroheptameric rings that

form a cylindrical structure with 2-fold axial symmetry. The

two outer α rings gate substrate entry through their interaction

with the 19S regulatory complex, which itself serves to bind,

unfold, de-ubiquitinate and translocate into the catalytic core

substrate proteins that have been conjugated to poly-ubiquitin

[1–6]. The two inner β rings form the central proteolytic

chamber and each contain three active sites with distinct substrate

speciﬁcities. These are located on the β1, β2 and β5 subunits

and are referred to as having caspase-like, trypsin-like and

chymotrypsin-like activities on the basis of their preference for

cleaving peptides after acidic, basic or hydrophobic amino acid

residues respectively [1–6]. A second form of the 20S proteasome

Abbreviations used: Ac, acetyl; AMC, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; HBTU, O-benzotriazole-N,N,N^ꢀ,N^ꢀ-tetramethyluronium

hexaﬂuorophosphate; HEK, human embryonic kidney; LC₅₀, half-maximal lethal concentration; MPD, 2-methyl-2,4-pentanediol; NFκB, nuclear factor

κB; IκB, inhibitory protein of NFκB; NFκB-Luc, NFκB–luciferase; PA, proteasomal activator; PDL, poly-^D-lysine; RNAi, RNA interference; siRNA, small

interfering RNA; Suc, succinyl; TEV, tobacco etch virus; TNF-α, tumour necrosis factor-α; 4xUb-Luc, tetra-ubiquitin–luciferase; UPS, ubiquitin–proteasome

system; Z, benzyloxycarbonyl.

1

Present address: Epizyme, Inc., 840 Memorial Drive, Cambridge, MA 02139, U.S.A.

To whom correspondence should be addressed (e-mail jonathan.blank@mpi.com).

2

The structural co-ordinates of the yeast 20S proteasome with the indicated ligand bound reported will appear in the PDB under accession codes: 3MG0

(bortezomib); 3MG4 (compound 1); 3MG6 (compound 6); 3MG7 (compound 8); 3MG8 (compound 16).

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462

C. Blackburn and others

Figure 1 Examples of covalent (A) and non-covalent (B) proteasome inhibitors

and is being evaluated for the treatment of other malignancies

[21–23]. Bortezomib induces cell death through a variety of

transcriptional, translational and post-translational mechanisms,

and may be preferentially cytotoxic to cancer cells by enhancing

endoplasmic reticulum stress, increasing the expression of pro-

apoptotic factors and/or inhibiting pro-survival or DNA-damage

repair pathways [4–6,21–23]. More recently, two further closely

related di-peptide boronic acids, CEP-18870 and MLN9708, have

been described that inhibit cancer cell proliferation in vitro and

show anti-tumour activity in solid and haematological preclinical

tumour models [24,25].

to contribute to the effectiveness of bortezomib as an anti-cancer

agent for the treatment of haematological malignancies, but may

limit its distribution and therefore broader utility in solid tumours

due to the high tissue abundance of the target (the proteasome has

been estimated to comprise 2% of total cellular protein [6]). More

recently, the β-lactone salinosporamide A (NPI-0052) [27] and

the epoxyketone carﬁlzomib (PR-171) (Figure 1A) [28], which

are analogues of lactacystin and epoxomicin respectively, have

demonstrated activity in preclinical models of multiple myeloma

and chronic lymphocytic leukaemia, and have entered clinical

trials for advanced solid and haematological malignancies [23].

These covalent inhibitors have comparable potency to bortezomib,

but differ in that they form essentially irreversible adducts with the

active site Thr^1Oγresidues of the proteasome [4,5,9,10,15,27–30].

Although the reactive ‘warheads’ of covalent proteasome

inhibitors can contribute to enzyme potency, they can also lack

speciﬁcity and be excessively reactive and unstable [10,11,15],

properties which may limit their efﬁcacy in vivo. Non-covalent

inhibitors that are readily reversible in their interactions with

the catalytic 20S β-subunits provide an alternative mechanism

for proteasome inhibition. Such inhibitors may offer therapeutic

Bortezomib binds with very high afﬁnity to the β5 site of

the proteasome, and to a lesser extent the β1 and β2 sites

[15], and behaves as a slowly reversible inhibitor (t _/

1

∼110 min

for dissociation from the β5 site [25]). Analysis of²the crystal

structure of the yeast 20S in complex with bortezomib indicates

that the high afﬁnity of the drug is partly mediated by the covalent

interaction of the boron atom with the nucleophilic oxygen of

Thr^1Oγ, and by the hydrogen bonding between the two acidic

boronate hydroxy groups and the amine groups of Gly^47Nand

Thr^1Nwithin each active site [5,9,26]. These properties are likely

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Novel non-covalent proteasome inhibitors

463

advantages by enabling more widespread tissue distribution

through their more rapid binding and dissociation kinetics.

TMC-95A is a complex natural cyclic tri-peptide (Figure 1B)

that competitively and non-covalently inhibits the chymotrypsin-

like activity of the proteasome with nanomolar potency, and

also inhibits the caspase-like and trypsin-like activities at higher

concentrations [4,5,9,10,31]. The high afﬁnity of TMC-95A

is due to its rigid heterocyclic ring system, which provides

speciﬁc hydrogen-bonding interactions within each 20S active site

without covalent modiﬁcation of the catalytic threonine residues

[4,5,9,32]. In addition, synthetic analogues of TMC-95A have

been described, including endocyclic biphenyl-ether macrocyles

and non-constrained linear tri-peptides that bind non-covalently

with altered afﬁnities for the trypsin- and caspase-like sites relative

to the chymotrypsin-like site [5,9,33,34]. TMC-95A shows cyto-

toxicity in the low micromolar range in cancer cells, although the

mechanism-based effects of TMC-95A or its derivatives have not

been demonstrated [4]. Other non-covalent proteasome inhibitors

include a series of 5-methoxy-1-indanone di-peptide benzamides

(e.g. CVT-659) [35,36] and 2-aminobenzylstatine derivatives

(Figure 1B) [37] that selectively inhibit the chymotrypsin-like site

with submicromolar potencies, although these compounds also

have poor cellular activity. However, homology modelling of a

2-aminobenzylstatine compound in the chymotrypsin-like

binding pocket of the proteasome has led to the design a

non-covalent tri-methoxy-L-phenylalanine-containing tri-peptide

with maintained selectivity and greatly increased cell potency

(Figure 1B) [38]. This compound is the most potent non-

covalent proteasome inhibitor described to date, inhibiting the

chymotrypsin-like activity in MDA-MB-435 cells and their

proliferation with IC₅₀values of 20 nM and 60 nM respectively

[38].

In an effort to identify cell-active inhibitors of the UPS

(ubiquitin–proteasome system) with novel chemical scaffolds,

we have screened a library of approx. 350000 compounds

using a cell-based assay that monitors accumulation of a

4xUb-Luc (tetra-ubiquitin–luciferase) reporter in response to

proteasome inhibition [15,39]. Using this approach, we report

the identiﬁcation of a C- and N-terminally capped tri-peptide

derived from the unnatural amino acid S-homo-phenylalanine that

potently and selectively inhibits the chymotrypsin-like activity of

the mammalian and yeast 20S proteasomes. Further optimization,

guided by X-ray crystallography of compounds in complex with

puriﬁed yeast 20S, has yielded a series of non-covalent di-

peptide inhibitors of the proteasome with unprecedented in vitro

and cellular potencies. The synthesis, binding mode and cellular

activity of these compounds are described in the present study.

DLCL2 and Karpas-1106P cells, RPMI 1640 medium; HCT116

and HT29 cells, McCoy’s 5a medium; and OCI-Ly-10, Iscove’s

modiﬁed Dulbecco’s medium. Clonally-derived stable MDA-

MB-231 cells expressing four tandem copies of ubiquitin fused

to ﬁreﬂy luciferase (4xUb-Luc) and HEK (human embryonic

kidney)-293 cells expressing NFκB-Luc [NFκB (nuclear factor

κB)–luciferase] were generated and maintained as described

previously [15].

Reporter assays

Cells were seeded at 10000 cells per well in white BioCoat^TM

PDL (poly-D-lysine)-coated 384-well plates (BD Biosciences) at

16–24 h prior to compound treatment. For the 4xUb-Luc assays,

MDA-MB-231 cells were incubated with compound for 8 h.

For NFκB-Luc assays, HEK-293 cells were pre-treated for 1 h

with proteasome inhibitor and then stimulated with 10 ng/ml

recombinant human TNF-α (tumour necrosis factor-α) (R&D

Systems) for a further 3 h in the continued presence of the

compound. Fireﬂy luciferase activity was measured using Bright-

Glo^TMreagents according to the manufacturer’s instructions

(Promega) in a LEADseeker^TMplate reader (GE Healthcare Life

Sciences). Inhibition of NFκB-Luc activity was calculated relative

to a no-compound (DMSO) control, whereas 4xUb-Luc reporter

accumulation was expressed as a fold increase in luciferase

activity over the DMSO control.

Cell viability assay

Calu6, HT29, MDA-MB-231 cells (each at 2000 cells/well), H460

cells (1000 cells/well) and HCT116 cells (1500 cells/well) were

plated in black clear-bottomed BioCoat^TMPDL-coated 384-well

plates (BD Biosciences). Cells were incubated with compound for

72 h, after which the medium was removed to leave 25 μl per well.

An equal volume of ATPlite^TMreagent (PerkinElmer) was then

added and luminescence was measured using a LEADseeker^TM

instrument.

siRNA (small interfering RNA) transfection and assay

MDA-MB-231 4xUb-Luc cells were transfected in a 384-

well format with 10 nM siRNAs (siGENOME SMARTpool,

Dharmacon) using DharmaFECT 1 (DH1) reagent (Dharmacon)

as follows. For the preparation of the RNAi (RNA interference)

transfection mixture for each time point, 40 μl of OptiMEM I

(Invitrogen) was dispensed into duplicate wells of a 384-well

plate each containing 9 μl/well of 0.5 μM siRNA in siRNA

buffer (Dharmacon). OptiMEM (50 μl) containing 0.53 μl of

DH2 transfection reagent (Dharmacon) was then added to each

well and the plates were incubated at room temperature (22^◦C) for

20 min. The transfection mixture (14 μl) from duplicate wells was

then transferred into six replicate wells of two separate BioCoat^TM

PDL-coated 384-well cell plates (BD Biosciences), one for 4xUb-

Luc assay and one for ATPlite assay (white and black clear-

bottomed respectively). Reverse transfection was performed by

the addition of 50 μl of MDA-MB-231 4xUb-Luc cells to each

well to give 1200 cells/well. At 48, 72 or 96 h after transfection,

one set of duplicate plates was assayed for 4xUb-Luc reporter

activity and cell viability. For each time point, six replicate wells

of cells transfected with buffer only and the non-targeting GL2

luciferase control duplex were each treated with either DMSO

or 1 μM bortezomib for 8 h prior to performing the 4xUb-Luc

assay.

EXPERIMENTAL

Cell culture

Cells were from the A.T.C.C. (Manassas, VA, U.S.A.), with the

exception of the diffuse large B-cell lymphoma lines which were

obtained from the following sources: Karpas-1106P, Deutsche

Sammlung von Mikroorganismen und Zellkulturen (DSMZ;

Braunschweig, Germany); WSU-DLCL2, Asterand (Detroit, MI,

U.S.A.); and OCI-Ly10, provided by Dr Louis M. Staudt (National

Cancer Institute, National Institutes of Health, Bethesda, MD,

U.S.A.). Cells were cultured at 37^◦C in a humidiﬁed air/6%

CO₂atmosphere in medium supplemented with 10% fetal bovine

serum, except for the medium for Karpas-1106P and OCI-Ly10

cells which contained 20% fetal bovine serum, and 100 units/ml

penicillin/100 μg/ml streptomycin (all from Invitrogen), as

speciﬁed: Calu6 cells, minimum essential medium; H460, WSU-

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464

C. Blackburn and others

Cell-based Proteasome-Glo^TMβ5 assays

state residual activity of the enzyme in the presence of inhibitor

(I) and v₀is the initial velocity in the absence of inhibitor:

Calu6 cells were plated as for the reporter assays and incubated

with compound for 1 h at 37^◦C. B-cell lymphoma subtypes

Karpas-1106P, WSU-DLCL2 and OCI-Ly10 were plated at 20000

cells per well in 384-well plates and treated with compounds

identically. The activity of the 26S proteasome was measured

in situ after compound removal by monitoring hydrolysis of

the β5 (chymotrypsin-like) substrate Suc-LLVY-aminoluciferin

(where Suc is succinyl) in the presence of luciferase using the

Proteasome-Glo^TMassay reagents according to the manufacturer’s

instructions (Promega). Luminescence was measured using a

LEADseeker^TMinstrument.

ν_s

ν₀

1

=

[I]

1 +

app

K

i

The dissociation constant, K_i, was calculated from the apparent

dissociation constant, K_i^app, using the following expression, where

[S] is the substrate concentration and K_mis the substrate binding

constant (for 20 μM Suc-LLVY-AMC):

K_i^app

K_i=

[S]

1 +

K

m

In vitro assays of puriﬁed 20S

Protein turnover assay

The peptidase activities of puriﬁed human erythrocyte and

Bulk protein turnover was measured in HCT116 cells

according to previously published procedures [41], as described

in the Supplementary material at http://www.BiochemJ.org/

bj/430/bj4300461add.htm.

peripheral blood monocyte 20S proteasomes (Boston Biochem^TM

)

were assayed using ﬂuorogenic tri-and tetra-peptide substrates

coupled to AMC (7-amino-4-methylcoumarin) [40] (AnaSpec)

in the presence of recombinant PA28α (proteasomal activator

28α; Boston Biochem^TM). The following selective substrates

were obtained from AnaSpec, with the exception of Z-LLE-

AMC (where Z is benzyloxycarbonyl), which was from Boston

Biochem^TM, and were each used at a ﬁnal concentration of 15 μM

to assay the constitutive (c) and immunoproteasome (i) sub-sites:

β1c, Z-LLE-AMC; β2c, Ac-KQL-AMC (where Ac is acetyl);

β5c, Ac-WLA-AMC; β1i, Ac-PAL-AMC; β2i, Ac-KQL-AMC;

and β5i, Ac-ANW-AMC. Reactions were performed at 37^◦C in

384-well black microtitre plates (Corning) using 0.25 nM 20S

and 12 nM PA28α in a ﬁnal volume of 50 μl of buffer containing

20 mM Hepes, pH 7.4, 0.5 mM EDTA and 0.01% BSA. Peptidase

activity was measured by monitoring the AMC liberation over

time with a Polarstar Galaxy ﬂuorimeter (BMG Labtechnologies)

using excitation and emission wavelengths of 340 nm and 460 nm

respectively. Percentage inhibition of 20S activity was calculated

relative to the controls with DMSO and 10 μM ML599698, an

analogue of bortezomib that contains a phenyl group in place of

the N-terminal pyrazine cap.

Synthesis of compound 1, the capped tri-peptide

5-methyl-N-((S)-1-((S)-1-((S)-1-(4-methylbenzylamino)-1-oxo-4-

phenylbutan-2-ylamino)-1-oxo-4-phenylbutan-2-ylamino)-1-oxo-4-

phenylbutan-2-yl)pyrazine-2-carboxamide

The trimeric compound 1 was identiﬁed by LC-MS as

an impurity in an active well identiﬁed during the high-

throughput screen [42] and its structure was conﬁrmed by full

characterization of material prepared according to Scheme 1,

as detailed in the Supplementary Experimental section at

http://www.BiochemJ.org/bj/430/bj4300461add.htm.

Synthesis of capped di-peptide proteasome inhibitors

The synthesis of di-peptide analogues was carried out using

standard peptide-coupling conditions, typically using HBTU

(O-benzotriazole-N,N,N^ꢀ,N^ꢀ-tetramethyluronium hexaﬂuorophos-

phate) to activate the carboxylic acids according to Scheme 2.

The P1 and P2 residues (R₁and R₂respectively) were installed as

described above for the preparation of compound 1 by coupling

the appropriate amine to a Boc (t-butoxycarbonyl)-protected

α-amino acid, followed by de-protection to give intermediates

II as hydrochloride salts. The three-step elaboration of II to

the di-peptide analogues of compound 1 (IV) was carried out

using automated solution-phase parallel synthesis techniques in

deep-well plates without puriﬁcation of intermediates, but with

rigorous puriﬁcation and full characterization of ﬁnal products. A

representative procedure for the parallel synthesis of compound

arrays of IV can be found in the Supplementary Experimental

section.

In vitro assays of 20S proteasomes in cell extracts

B-cell lymphoma extracts were prepared by hypotonic lysis and

assayed for proteasome activity as described previously [15] using

10 μg of protein extract per reaction in the presence of 12 nM

PA28α and the following substrates, each at 100 μM, to monitor

the catalytic activity of the c or i 20S sub-sites, as indicated: β1i,

Ac-PAL-AMC; β5i, Ac-ANW-AMC; β5c, Ac-WLA-AMC; and

β5 Suc-LLVY-AMC.

Kinetic analysis of puriﬁed 20S inhibition

Puriﬁcation of yeast 20S

Inactivation of puriﬁed human erythrocyte 20S was determined

by monitoring the hydrolysis of the β5-speciﬁc ﬂuorigenic

peptide substrate Suc-LLVY-AMC in the presence of different

concentrations of inhibitor. Substrate cleavage was continuously

monitored as a change in the ﬂuorescence emission at 460 nm

(excitation wavelength, 360 nm) and plotted as a function of time.

Assays were performed at 37^◦C in cuvettes in a ﬁnal volume

of 2 ml in a reaction buffer containing 20 mM Hepes, pH 7.5,

0.5 mM EDTA, 0.01% SDS, 0.25 nM 20S and 10 μM Suc-

LLVY-AMC, with continuous stirring. The apparent dissociation

constant, K_i^app, was determined by non-linear least-ﬁt of the

fractional velocity, v_s/v₀, as a function of [I], where v_sis the steady-

Yeast 20S wild-type and open-gate (an N-terminal tail deletion

from the α3 and α7 subunits, designated α3/7ꢀN) mutant

containing a TEV (tobacco etch virus)-protease-cleavable Protein

A-derived tag in the core particle subunit Pre1 (β4) were afﬁnity-

puriﬁed from the SDL135 and yMS159 strains respectively (gifts

from Dr M. Schmidt and Dr D. Finley, Harvard University Medical

School, Boston, MA, U.S.A.) [43,44] on IgG–Sepharose 6 Fast

Flow (GE Healthcare), according to the procedure of Leggett

et al. [45], except that ATP was omitted from the buffers and

the enzyme was eluted from the IgG resin by incubation with the

His₆–TEV protease (Invitrogen) overnight at 4^◦C. The resultant

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Novel non-covalent proteasome inhibitors

465

Scheme 1 Synthesis of compound 1

◦

(i) ArCH₂-NH₂, HBTU, NMM (N-methylmorpholine), DMF (N,N-dimethylformamide), 25 C, 24 h; (ii) 4 M HCl, dioxane; (iii) N-Boc-homo-phenylalanine, TBTU [O-(benzotriazol-

◦

1-yl)-N,N,N^ꢀ,N^ꢀ-tetramethyluronium-tetraﬂuoroborate], collidine, DMF, 25 C, 3 h; (iv) 4-methylpyrazinecarboxylic acid, TBTU, collidine, DMF, 25 C, 3 h.

Scheme 2 Synthesis of compound IV

◦

(i) R₁-NH₂, HBTU, NMM (N-methylmorpholine), DMF (N,N-dimethylformamide), 25 C, 24 h; (ii) 4 M HCl, dioxane; (iii) P3 Boc-amino acid, HBTU, NMM, DMF, 25 C, 24 h; (iv) R₄COOH, HBTU,

NMM, DMF, 25 C, 24 h.

◦

yeast 20S was puriﬁed further by gel ﬁltration on a 24 ml Superose

6 10/30 column (GE Healthcare) in 10 mM Tris/HCl, pH 7.5,

containing 1 mM EDTA, concentrated to 30 mg/ml using an

Amicon Centriplus YM-100 centrifugal ﬁlter device (Millipore)

and stored at −80^◦C.

X-ray crystallography

Crystals of puriﬁed wild-type and the open-gate mutant yeast 20S

proteasome were grown in hanging drops at room temperature,

as described previously [46], using a drop volume of 1.5 μl

of 20S (20 mg/ml in 10 mM Tris/HCl, pH 7.5, and 1 mM

EDTA) and 0.5 μl of reservoir solution containing 100 mM

Mes, pH 7.0, 40 mM Mg(CH₃COO)₂, 15% MPD (2-methyl-2,4-

pentanediol) and 10 mM EDTA. Proteasome–inhibitor complexes

were generated by soaking crystals overnight in reservoir

buffer containing 1 mM compound, 10% DMSO and 20%

MPD followed by an additional 5 h in reservoir buffer

containing 1 mM compound, 10% DMSO and 25% MPD

before being ﬂash-cooled in liquid nitrogen. Crystal data and

reﬁnement statistics are given in Supplementary Table S1 at

http://www.BiochemJ.org/bj/430/bj4300461add.htm. Data were

collected using the Structural GenomiX (SGX)-CAT beamline at

the Advanced Photon Source (APS) synchrotron of the Argonne

National Laboratory (U.S. Department of Energy Chicago, IL,

U.S.A.) and processed using the programs iMOSFLM [47] and

SCALA [48]. Starting co-ordinates for each of the proteasome–

inhibitor structures were taken from PDB entry 1G0U [43].

SigmaA-weighted F_o− F_cdifference electron density for the β5

active site was used to model inhibitor co-ordinates, starting

with a conformation generated using the small-molecule topology

generator PRODRG [49]. Model building was performed using

the program Coot [50] and reﬁnement was carried out using the

CCP4i graphical user interface [51] to the REFMAC program

[52].

Figure 2 Effects of bortezomib and depletion of 26S subunits by RNAi on

accumulation of 4xUb-Luc in cells

MDA-MB-231 cells stably expressing a 4xUb-Luc reporter were reverse-transfected with 10 nM

siRNA SMARTPools to deplete components of the 19S regulatory cap or 20S catalytic core

particleofthe26Sproteasome, asindicated. At48, 72or96 haftertransfection, theaccumulation

of the 4xUb-Luc was measured as luminescence using ﬁreﬂy luciferase reagents, as described in

theExperimentalsection. MocktransfectionswithbufferorwiththeGL2luciferasecontrolduplex

were also performed to monitor basal expression of the 4xUb-Luc reporter and its stabilization

+

by 1 μM bortezomib for the ﬁnal 8 h of transfection. Results are the means S.E.M. for six

−

assaypointsandarerepresentativeoffourindependentexperiments. PSMA, proteasomesubunit

α-type; PSMB, proteasome subunit β-type; PSMD, proteasome 26S subunit non-ATPase; rpn,

regulatory particle non-ATPase.

Luc in MDA-MB-231 cells. In this system, bortezomib and

other catalytic 20S inhibitors cause a concentration-dependent

accumulation of the constitutively expressed reporter by blocking

its intracellular degradation, as reported previously [15]. To

further validate the dependence of this reporter on the 26S

proteasome, siRNAs were used to deplete selected components of

the 20S core particle and 19S regulatory complex in MDA-MB-

231-4xUb-Luc cells. As shown in Figure 2, depletion of subunits

of either the 20S (α1 and β6) or 19S (rpn8 and rpn11) complex

resulted in a time-dependent increase in the 4xUb-Luc signal

RESULTS

Cell-based screen for inhibitors of the UPS

To identify novel cell-active small-molecule inhibitors of the UPS,

a high-throughput screen of the Millennium Pharmaceuticals,

Inc., library was performed for compounds that stabilized 4xUb-

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466

C. Blackburn and others

Figure 3 Structure of the capped tri-peptide screening hit, 5-methyl-

N-[(S)-1-{(S)-1-[(S)-1-(4-methylbenzylamino)-1-oxo-4-phenylbutan-

2-ylamino]-1-oxo-4-phenylbutan-2-ylamino}-1-oxo-4-phenylbutan-2-

yl]pyrazine-2-carboxamide, compound 1

comparable with that observed by an 8 h treatment of the cells

with a near-saturating concentration of bortezomib of 1 μM.

High-throughput screening of approx. 352500 compounds at

50 μM gave 3015 hits that stabilized 4xUb-Luc by 2-fold or

greater, representing a primary hit rate of 0.86%, and 611 of

these positives were conﬁrmed when re-tested in triplicate at

the same concentration and cut-off, representing a re-test rate

of 20.3%. The majority of these compounds were either peptide

boronic acid inhibitors of the proteasome or ubiquitin-activating

enzyme (E1) inhibitors that were part of the library generated

by proprietary medicinal chemistry and served as screening

controls. There were 191 compounds which were previously

uncharacterized as inhibitors of the UPS, for which cell-based

potency determinations were performed. Of these, 52 gave modest

2–3-fold stabilization, whereas 102 compounds gave a 3–10-fold

stabilization and 37 compounds gave >10-fold stabilization of

the reporter at concentrations up to 50 μM.

Identiﬁcation of compound 1 as a potent selective 20S β5 inhibitor

The cell-active compounds were assessed for inhibition of puriﬁed

rat brain 20S in vitro by measuring hydrolysis of the β5

(chymotrypsin-like) substrate Ac-WLA-AMC in the presence

of PA28α. Only a small minority of compounds inhibited 20S,

showing low and sub-micromolar potencies (results not shown).

Among the most potent of these were four closely related

peptidic benzyl amides synthesized by combinatorial chemistry,

of which one was subsequently identiﬁed by MS and re-synthesis

as a C- and N-terminally capped tri-peptide derived from the

unnatural amino acid S-homo-phenylalanine, and designated as

compound 1 (Figure 3) In vitro, this compound inhibited the

β5 activity of the human constitutive and immunoproteasome

with low nanomolar potencies, comparable with those of

bortezomib, but was essentially inactive against the β1 (caspase-

like) and β2 (trypsin-like) sites of either proteasome isoform

(Table 1). β5 selectivity is in contrast with that of bortezomib,

which also inhibits the β1 and, to a lesser extent, the β2

sites of the constitutive and immunoproteasome (Table 1).

Assays against a diverse panel of ten proteases indicated that

compound 1 was highly selective for the proteasome, showing

only partial (<50%) inhibitory activity against cathepsin L,

coagulation Factor β-XIIa, thrombin, tissue plaminogen activator

and trypsin at concentrations approx. 10000-fold higher than

its IC₅₀value for the 20S β5 site (Supplementary Table S2

at http://www.BiochemJ.org/bj/430/bj4300461add.htm). Kinetic

analysis of human 20S β5 inhibition by compound 1 showed

that it behaved as a rapid-equilibrium inhibitor with a K_i

Figure 4 Kinetics of proteasome inhibition by compound 1 and bortezomib

(A and B) The peptidase activity of human erythrocyte 20S (0.25 nM) was monitored over

time using the ﬂuorigenic substrate Suc-LLVY-AMC (10 μM) in the presence of 0.01% SDS

and following the addition of various concentrations of either compound 1 or bortezomib, as

indicated. (C) A non-linear least-ﬁt of the fractional velocity of the reactions, v_s/v₀, as a function

+

of inhibitor concentration, [I], was used to determine a K_iof 0.50 0.12 nM for compound 1

−

+

and 0.56 0.072 nM for bortezomib (means S.E.M., n = 3).

−

+

value of 0.50 0.12 nM (mean S.E.M., n = 3), consistent with

−

reversible non-covalent binding to the active site of the enzyme

(Figure 4). This behaviour is again in contrast with that of

bortezomib, which acts as a time-dependent slow–tight binding

inhibitor that interacts covalently with the active site Thr¹residue,

but which displays an equivalent K_ivalue for the β5 site of

+

0.56 0.072 nM (mean S.E.M., n = 3) (Figure 4 and [25]).

−

Cellular inhibition of proliferation, 4xUb-Luc degradation,

NFκB-Luc activation by compound 1

On the basis of the high afﬁnity and selectivity of compound 1 for

the 20S β5 site in vitro, its activity was characterized further in

cells as compared with that of bortezomib. Compound 1 inhibited

the proliferation of MDA-MB-231 cells in a concentration-

dependent manner with an LC₅₀(half-maximal lethal

+

concentration) value of 150 23 nM (mean S.E.M., n = 3),

−

as determined by an ATPlite assay at 72 h (see Supplementary

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Novel non-covalent proteasome inhibitors

467

Table 1 Active-site-selectivity of the peptide boronic acid, bortezomib, the capped tri-peptide screening hit, compound 1, and a series of ﬁfteen capped

di-peptides in vitro

Puriﬁed human erythrocyte 20S and peripheral blood monocyte 20S (0.25 nM each) were used as a source of constitutive (c) and immunoproteasome (i) respectively to measure the peptidase

activity of the β1 (caspase-like), β2 (trypsin-like) and β5 (chymotrypsin-like) sub-sites of each proteasome isoform. Assays were performed in the presence of 15 μM of the indicated ﬂuorigenic

peptide-AMC substrate, 12 nM PA28α and a titration of each inhibitor. As shown, and unlike bortezomib, compounds 1–16 were essentially inactive against the β1 and β2 sites of the constitutive

proteasome and immunoproteasome. The IC₅₀values of the compounds against the β5c and β5i sites are mean values for at least three independent determinations.

Human 20S–PA28 IC₅₀(nM)

Compound

β1c Z-LLE-AMC

β1i Ac-PAL-AMC

β2c Ac-KQL-AMC

β2i Ac-KQL-AMC

β5c Ac-WLA-AMC

β5i Ac-ANW-AMC

Bortezomib

1

2

3

4

5

6

7

140

5.5

1500

57000

940

8.2

16

1100

130

470

340

25

3.3

8.9

230

37

41

27

28000

83000

43000

48000

39000

>100000

70000

31000

68000

90000

>100000

89000

84000

66000

64000

>100000

65000

>100000

83000

>100000

47000

>100000

70000

44000

91000

>100000

54000

>100000

63000

>100000

95000

10

9100

13

1300

4.1

6.7

3.2

1.1

3.5

3.4

200

44

8

9

47000

>100000

6.8

4.6

2.4

4.4

3.9

11

10

11

12

13

14

15

16

99000

>100000

12

1.2

1.1

Figure S1A at http://www.BiochemJ.org/bj/430/bj4300461add.

htm). Indeed, viability assays performed across a panel

of ﬁve cancer cell lines, comprising MDA-MB-231, Calu6,

H460, HCT116 and HT29 cells, indicated that compound

1 has broad cytotoxic activity, with LC₅₀values ranging

from 50 nM to 380 nM and being approx. 10–20-fold less

potent than bortezomib (Table 2 and Supplementary Table S3

at http://www.BiochemJ.org/bj/430/bj4300461add.htm). In the

screening assay, compound 1 caused up to an approx. 100-

fold accumulation of the 4xUb-Luc reporter at 8 h [maximal

n = 7)], whereas bortezomib inhibited NFκB-Luc activity by

+

100% with an IC₅₀of 9.7 0.73 nM (mean S.E.M., n = 14)

−

(Supplementary Figure S1D and Table 2). Collectively, these data

indicate that selective inhibition of the β5 site of the proteasome

by compound 1 in cells is sufﬁcient to inhibit the degradation of

the 4xUb-Luc reporter, TNF-α-dependent NFκB activity and the

proliferation of cancer cells with potencies that are approximately

one order of magnitude lower than those of bortezomib.

X-ray crystallography of compound 1 in complex with the yeast 20S

proteasome

+

effect of 52 16-fold, with an EC₅₀(half-maximally effective

−

+

concentration) of 690 22 nM (mean S.E.M., n = 6)], showing

−

an approx. 3-fold higher EC₅₀and one-seventh of the efﬁcacy of

To determine its binding mode, compound 1 was soaked into

crystals of the yeast 20S core particle. X-ray diffraction data

obtained at 3.1 Å resolution indicated that it only occupied

the β5 sites of the proteasome (Figure 5A), consistent with

selective inhibition of this active site in vitro. The C-terminal

4-methylbenzyl group (P1) was seen to occupy a well-deﬁned S1

pocket, with the ﬁrst homo-phenylalanine residue (P2) pointing

toward solvent, the second (P3) occupying a well-deﬁned S3

pocket and the third (P4) projecting into a much larger and ill-

deﬁned S4 pocket (Figure 5B). No electron density was observed

corresponding to the N-terminal cap, presumably because this

region of the molecule is disordered. The structure of compound

1 overlaid with that of bortezomib, as determined previously [26],

suggests that, although the side chains of the two compounds

differ in their orientations, most of the backbone hydrogen-bond

interactions within the β5 site are the same (Figure 5C). Of note, in

this structure as in others, residual electron density corresponding

to Mes, the buffer in which the crystals were prepared, was

observed near Thr¹in the β5 active site (Figure 5A). The main

interaction between Mes and compound 1 involves hydrophobic

contacts between the morpholino group of Mes and the homo-

phenylalanine residue in the P2 position. For structures without

ligand in the active site, no density for the morpholino group

was observed (M.D. Sintchak, unpublished work), rather electron

density at the position of the sulfur atom of Mes was seen. This sits

+

bortezomib [maximal effect, 340 12-fold; EC₅₀= 240 21 nM

−

+

(mean S.E.M., n = 102)] (Supplementary Figure S1B and

−

Table 2). The activity of compound 1 was also assessed

in the Proteasome-Glo^TMassay that measures directly the

peptidase activity of the β5 site of the 26S proteasome in

cells using the luminogenic substrate Suc-LLVY-aminoluciferin.

Treatment of Calu6 cells for 1 h with compound 1 caused a

concentration-dependent inhibition of β5 activity in this assay

with an IC₅₀(half-maximal inhibitory concentration) value of

+

53 16 nM (mean S.E.M., n = 3). Bortezomib was approx.

−

10-fold more potent, inhibiting hydrolysis of the chymotrypsin-

+

like substrate with an IC₅₀of 3.9 0.49 nM (mean S.E.M.,

−

n = 8) (Supplementary Figure S1C and Table 2). Finally,

we assessed the effect of compound 1 on activation of the

transcription factor NFκB by TNF-α, which is dependent on

proteasomal degradation of IκBα (inhibitory protein of NFκB

α) following its rapid phosphorylation by the IκB kinase

βTrCP

complex and ubiquitination by the SCF

(E3 ubiquitin-

protein ligase complex Skp1–Cullin–F-box) [53]. Compound

1 inhibited TNFα-induced activation of NFκB-Luc in HEK-

+

293 cells with an IC₅₀of 47 7.7 nM (mean S.E.M., n = 7),

−

consistent with potent proteasome inhibition in these cells.

Interestingly, it did not fully inhibit NFκB-Luc, even at saturating

+

concentrations [maximal inhibition, 79 3.2% (mean S.E.M.,

−

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468

C. Blackburn and others

Table 2 Cell-based activity of bortezomib, compound 1 and ﬁfteen capped di-peptide proteasome inhibitors

The cell-based potencies of bortezomib and compounds 1–16 were assessed in a diverse panel of assays that monitor the short- and longer-term effects of proteasome inhibition in cells, as indicated.

In particular, inhibition of the β5 activity of the proteasome was measured directly with the Proteasome-Glo^TMassay using Suc-LLVY-aminoluciferin as substrate following incubation of Calu6 cells

(10000 per well) with compound for 1 h. Inhibition of NFκB-luciferase activity in HEK-293 cells was determined by pre-incubation of the cells (10000 per well) with compound for 1 h followed

by stimulation with 10 ng/ml TNF-α in the continued presence of compound for 3 h. The IC₅₀and percentage inhibition of NFκB-Luc activity are given. Accumulation of the 4xUb-Luc reporter in

MDA-MB-231 cells was determined by incubation of the cells (10000 per well) with compound for 8 h and expressed as the fold accumulation of luciferase activity together with an EC₅₀value

for this effect. The effects of the compounds on the viability of Calu6 cells (2000 per well) and H460 cells (1000 per well) were assessed by the ATPlite assay following incubation of the cell with

compound for 72 h. The results are mean values for at least three independent experiments.

Calu6 Proteasome-Glo^TMβ5

HEK-293 NFκB-Luc

MDA231 4xUb-Luc

Cell viability

Compound

IC₅₀(nM)

Maximal inhibition (%)

EC₅₀(nM)

Maximal induction (fold)

Calu6 LC₅₀(nM)

H460 LC₅₀(nM)

Bortezomib

1

2

3

4

5

6

7

3.9

53

9.7

47

100

79

28

84

30

16

89

86

87

78

72

93

74

77

80

92

240

690

1800

2100

6900

4700

2100

10000

930

130

47

112

71

64

650

450

31

340

52

3

5.2

13

380

130

>25000

1700

13000

2000

1200

6600

240

10

55

21

36

61

48000

610

13000

12000

310

5400

43

10

16

5.7

36

>50000

3100

24000

>50000

320

4400

29

22

6.6

9.8

10

>25000

3100

18000

2600

4300

17000

470

34

82

110

39

12

28

76

65

86

77

64

65

64

76

64

82

75

8

9

10

11

12

13

14

15

16

24

9.2

64

39

12

240

650

1000

44

520

230

9.5

210

355

9.6

at the same position as the nucleophilic water molecule referred

to as NUK in a previous publication [54]. Importantly, although

Mes was observed in the active site following crystallization, this

buffer molecule neither affects the speciﬁc activity of 20S nor the

afﬁnity of compound 1 for the active site on the basis of K_ivalue

determination (F.J. Bruzzese, unpublished work).

residue [38] at P2 gave compound 3 (Figure 7) with a β5

IC₅₀value of 130 nM (Table 1). In the next library, smaller

replacements for the P3 homo-phenylalanine residue were sought

in combination with alternative N-terminal P4 capping groups that

provided compensating hydrophobic interactions within the large

S4 binding pocket. Numerous compounds, such as compound 4

and the 4-benzyloxybenzoyl derivative compound 5, with small

polar P3 residues, in this case a threonine residue (Figure 7),

retained reasonable potency in vitro (β5 IC₅₀values of approx.

500 nM). Signiﬁcant further improvements were effected by

varying the capping group, with the indanone in compound 6

being among the optimal residues at P4 with this compound

showing a β5 IC₅₀of 25 nM (Figure 7 and Table 1) and a K_i

Optimization of capped di-peptide 20S inhibitors

The partial crystal structure of compound 1 (Figure 5) together

with the report of a potent related tri-methoxy-phenylalanine-

containing tri-peptide 20S inhibitor (Figure 1 and [38]) suggested

that one homo-phenylalanine residue could be deleted, provided

that a compensating hydrophobic N-terminal capping group could

be incorporated in place of the pyrazine. A series of di-peptide-

like libraries was therefore prepared by solution-phase parallel

synthesis as outlined in the Experimental section (Scheme 2),

denoting residues P1–P4 (Figure 6) according to the binding

mode indicated by the structure of compound 1 (Figure 5). Given

the close correlation in IC₅₀values of proteasome inhibitors

from various chemical classes for human and yeast 20S (C.

Tsu, unpublished work), compounds were optimized for in vitro

β5 potency against human 20S, whereas crystal structures of

representative examples were determined with the yeast 20S open-

gate mutant in order to guide further library design.

The ﬁrst library synthesized retained 4-methylbenzylamine as

the C-terminal capping group at P1, contained varied amino acid

residues at P2 and P3 and probed two alternative hydrophobic

N-terminal caps at P4 with 348 compounds in total. All of these

di-peptide analogues were less potent than the trimer compound

1, although several inhibited β5 activity with low and sub-

micromolar potencies. For example, the truncated analogue of

compound 1 with an N-terminal cap substitution, compound 2

(Figure 7), had a β5 IC₅₀value of 1.1 μM in vitro (Table 1).

Substitution of the homo-phenylalanine residues of compound

2 by a tryptophan at P1 and by the di-methoxy-phenylalanine

+

value of 13 1.4 nM (mean S.E.M., n = 3).

−

The crystal structure of compound 6 bound to the β5 site

of yeast 20S was determined at 2.6 Å resolution (Figure 8)

and compared with that of bortezomib determined at 2.7 Å, a

resolution comparable with that determined previously [26]. In the

structure of bortezomib, in addition to the covalent and hydrogen-

bonding interactions involving the boronate, backbone hydrogen-

bonding interactions with the β5 site can be seen (Figure 8A).

Interestingly, the same hydrogen-bonding interactions between

the backbone of compound 6 and the β5 site are observed,

including the presence of the bridging water molecule between

the P3 carbonyl group and Ala⁴⁹and Ala⁵⁰(Figure 8A).

Figures 8(B) and 8(C) show the occupancy of the binding

pockets by compound 6, whereas Supplementary Figure S2

(at http://www.BiochemJ.org/bj/430/bj4300461add.htm) shows

an alternative view to include the electron density of the water

molecule. The 4^ꢀ-methylbenzyl P1 residue occupies S1, with

the methyl group occupying a small sub-pocket at the bottom

of the cavity. The P2 homo-phenylalanine residue occupies

S2 and the P3 threonine residue projects, to a small degree,

towards the deep S3 binding pocket. The P2 homo-phenylalanine

residue does not ﬁll the S2 binding pocket, which is quite

shallow, but likely confers potency by contributing a hydrophobic

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Novel non-covalent proteasome inhibitors

469

Figure 5 Electron-density map of compound 1 and occupancy of the speciﬁcity pockets of the 20S β5 subunit

˚

(A) A 3.1 A sigmaA-weighted 2F_o-F_celectron-density map contoured at 1.0σ covering compound 1 (PDB entry 3MG4) and Mes in the β5/β6 active site of wild-type 20S is shown in stereo view,

with residue positions P1–P4 indicated. Atoms are in ball-and-stick representation with carbon in yellow (compound 1) or salmon (Mes), nitrogen in blue and oxygen in red. The side chain atoms

for Thr¹(β5) and Asp¹¹⁴(β6) are shown in ball-and-stick representation, coloured as above, except for carbon in green. Protein atoms from 20S are shown in cartoon representation coloured

by secondary structure with helices in cyan, β-strands in magenta and loops in salmon. The Figure was made using the PyMOL Molecular Graphics System (DeLano Scientiﬁc, Palo Alto, CA,

U.S.A.). (B) The β5/β6 active site with compound 1 bound (coloured as in A), showing the molecular surface of 20S. Speciﬁcity pockets S1–S4 are indicated. (C) Superposition of compound 1

and bortezomib bound to the β5/β6 active site of 20S is shown, with positions P1–P4 indicated. Colour scheme is as described above, except bortezomib carbon atoms are coloured grey.

with compound 6 was observed for the 2^ꢀ-chlorobenzyl derivative

compound 8 (β5 IC₅₀= 13 nM; Table 1 and Figure 7). A crystal

of compound 8 bound to 20S indicated an additional hydrophobic

interaction between the ortho-halogen and a second ‘sub-pocket’

within the S1 site that cannot be accessed by the less active meta-

substituted analogue ([42] and see Supplementary Figure S3 at

http://www.BiochemJ.org/bj/430/bj4300461add.htm).

In the ﬁnal library, the inﬂuence of a hydrophobic P3 residue

was assessed, with emphasis on asparagine derivatives. As with

the potency-enhancing N-terminal P4 capping groups described

above, several low nanomolar inhibitors were identiﬁed, such

as compounds 9 (β5 IC₅₀= 6.8 nM, Table 2) and 10 (β5

IC₅₀= 4.6 nM, Table 2) (Figure 7). Potent compounds such

as 11 (β5 IC₅₀= 2.4 nM, Table 1) and 12 (β5 IC₅₀= 4.4 nM,

Table 1) in which the homo-phenylalanine residue was replaced

by an alanine residue and compound 13 (β5 IC₅₀= 3.9 nM,

Table 1), in which 3-pyridyl was incorporated, were also identiﬁed

(Figure 7). Similarly, the neopentyl-asparagine residue was able to

compensate for sub-optimal interactions in S1, with compounds

14 and 15 showing good inhibitory activity (β5 IC₅₀= 11 and

interaction at a ‘ledge’ formed from Gly⁴⁷-Gly⁴⁸(Figure 8C).

Of note, the P4 indanone in compound 6 is closely related to

inhibitors reported previously [35,36], for example, CVT-659

(Figure 1B). Whereas the indanone carbonyl in these molecules

was originally designed as a ‘putative electrophilic head group’

[35,36], the crystal structure of compound 6 places this moiety

in the S4 binding pocket remote from the β5 catalytic Thr¹

residue, thereby precluding such an interaction. In addition, the

water molecule appears to be present in all higher-resolution

structures (<2.6 Å), including those without ligand bound,

such as that reported originally [54]. This water molecule was

also observed in structures with inhibitors having IC₅₀values

greater than 1 μM (M.D. Sintchak, unpublished work). Therefore

it is unlikely that this single water-mediated hydrogen bond

contributes signiﬁcantly to potency.

In the third library, optimization of the C-terminal P1

residue was explored. The requirement of the hydrogen-bonding

interaction with Gly⁴⁷can be seen from N-methylation of P1 to

give compound 7 (Figure 7), which diminished activity greatly

(β5 IC₅₀= 9.1 μM; Table 1). However, comparable potency

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470

C. Blackburn and others

The most potent proteasome inhibitor identiﬁed was compound

+

16 (Figure 7) with a β5 IC₅₀= 1.2 0.35 nM for the human

−

enzyme (Table 1) and a K_ibelow the 20S concentration of 250 pM

in the assay, indicating that it behaved as a non-covalent active-

site titrant (F.J. Bruzzese, unpublished work). The X-ray structure

of this compound bound to yeast 20S resolved at 2.6 Å is shown

in Figure 9. The potentially electrophilic α-ketoamide N-terminal

cap does not form any covalent interactions; it is positioned in

the S4 binding pocket which is located away from the catalytic

Thr¹and it also does not appear to interact with any residues

in S4 (Figure 9A). Importantly, the high potency of compound

16 and all compounds with a P3 neopentyl-asparagine group can

be accounted for by the near-optimal ﬁt of this residue in the S3

binding pocket (Figure 9B). As with compound 8 (Supplementary

Figure S3), the P1 ortho-chlorine of compound 16 is also likely

to contribute to the potency by accessing the S1 sub-pocket

(Figure 9A). Of note, however, the aromatic P2 contains only

one methylene and therefore the ledge interaction cannot take

place with the pyridyl projecting toward solvent (compare with

Figure 8C).

Figure 6 Residue assignments of di-peptide inhibitors

Optimization of compound 1 involved contracting the chain to a di-peptide of the general

structure shown and systematically modifying the central amino acids and capping groups,

as denoted by residues P1–P4 in a series of compound libraries prepared by solution-phase

parallel synthesis (see the Experimental section).

Relationship between 20S β5 potency and cellular activity of

capped di-peptides

12 nM respectively; Table 1 and Figure 7). Like compound 1, these

di-peptides at 100 μM had negligible to modest activity against

ten unrelated proteases (Supplementary Table S2), indicating that

the most potent compounds are selective for the chymotrypsin-

like site of the proteasome by 4 or 5 orders of magnitude.

The effects of the di-peptides on 26S activity in cells were

measured (Table 2) and compared with their 20S β5 IC₅₀values

determined in vitro (Table 1 and Supplementary Figure S4 at

http://www.BiochemJ.org/bj/430/bj4300461add.htm). Inhibition

of the β5 site of the 26S proteasome was measured directly in

Figure 7 Chemical structures of capped di-peptide 20S β5 proteasome inhibitors

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Novel non-covalent proteasome inhibitors

471

Figure 8 Hydrogen-bonding interactions and crystal structure of compound 6 bound to the chymotrypsin-like site of the 20S proteasome with reference to

bortezomib

(A) Schematic representation of the β5/β6 active site of 20S with compound 6 (left-hand panel) (PDB entry 3MG6) and bortezomib (right-hand panel) (PDB entry 3MG0) bound. Key hydrogen

˚

bonds between the inhibitors and the protein are shown as dashed lines coloured magenta, with distances indicated in A. Thus in the structure of bortezomib, the NH of the P1 leucine residue forms

a hydrogen bond with the carbonyl of Gly⁴⁷, whereas the carbonyl and amino groups of the P2 residue form hydrogen-bond interactions with Thr²¹. The carbonyl of the pyrazinoyl cap is involved in

a network of hydrogen-bond interactions with Ala⁴⁹, Ala⁵⁰and Asp¹¹⁴that includes a bridging water molecule. Similar hydrogen-bonding interactions between the backbone of compound 6 and the

β5 site are shown, including the presence of the bridging water molecule between the P3 carbonyl and Ala⁴⁹and Ala⁵⁰; the NH of P3 makes the hydrogen-bond interaction with Asp¹¹⁴that is formed

˚

by the pyrazine N in bortezomib. (B) 2.6 A sigmaA-weighted 2F_o-F_celectron-density map contoured at 1.0σ covering compound 6 (PDB entry 3MG6) and MES is shown in the β5/β6 active site of

yeast 20S open-gated mutant in stereo view, with positions P1–P4 indicated. The colour scheme is as described in Figure 5. (C) β5/β6 active site with compound 6 bound (coloured as described

above), showing the molecular surface of 20S. Speciﬁcity pockets S1–S4 are indicated. The P2 binding ‘ledge’ is shown by shading of the molecular surface.

Calu6 cells using the Proteasome-Glo^TMassay and corresponded

well with the inhibitory potencies determined in vitro with

puriﬁed 20S. There was also a striking correlation between

the IC₅₀values of the compounds for inhibition of NFκB-Luc

activity in cells and 20S β5 activity in vitro, with ﬁve of

the analogues, speciﬁcally compounds 10–13 and 16, showing

inhibitory NFκB potencies in the range 6.6–12 nM, equivalent to

+

(mean S.E.M., n = 6)]. Interestingly, the activity of NFκB-

−

Luc was not fully inhibited even at saturating compound

concentrations (Table 2). The di-peptides also stabilized the 4xUb-

Luc reporter in MDA-MB-231 cells with EC₅₀values that tracked

closely with their in vitro 20S β5 potencies (Table 2), although

the maximal stabilization effects of approx. 100-fold were lower

than those observed for bortezomib (Table 2), other boronates

(J.L. Blank, unpublished work) and covalent inhibitors, such as

or greater than that of bortezomib [NFκB-Luc IC₅₀= 13 2.0 nM

−

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472

C. Blackburn and others

selectively inhibiting the β5 site alone on the degradation

of short-lived protein was assessed using compounds 10 and

16, two of the most potent cell-active di-peptides. As shown

previously [41], bortezomib inhibits approx. 50% of bulk protein

turnover in HCT116 cells (see Supplementary Figure S5 at

http://www.BiochemJ.org/bj/430/bj4300461add.htm), an effect

that is comparable with that observed with ML858, a synthetic

version of salinosporamide A that irreversibly inhibits each of

the 20S active sites [15] (T.A. Soucy, unpublished work). Under

identical conditions, the di-peptides reduced protein turnover

by approx. 20% (Supplementary Figure S5), indicating that

inhibition of the β5 site alone results in partial inhibition of protein

degradation.

Constitutive and immunoproteasome selectivity of di-peptide

proteasome inhibitors

In addition to monitoring the β5 inhibitory potency of the capped

di-peptides using puriﬁed human erythrocyte constitutive 20S

proteasomes, IC₅₀values were also obtained for the β5i site of

the immunoproteasome from human peripheral blood monocytes

using the selective substrates Ac-WLA-AMC and Ac-ANW-

AMC respectively (Table 1). Bortezomib, compound 1 and the

majority of the capped di-peptides were non-selective in these

assays, inhibiting the constitutive and immunoproteasome with

similar IC₅₀values, although they tended to inhibit β5i with

slightly greater potencies (Table 1). In common with compound

1, the di-peptides were essentially inactive with respect to

inhibition of the β1i or β2i sites of the immunoproteasome.

However, a limited number of compounds showed signiﬁcantly

greater selectivity for the immunoproteasome, with compounds

4 and 5 being the best examples, showing ∼10-fold β5i

+

selectivity [compound 4, β5i IC₅₀= 41 3.3 nM and β5c

−

+

IC₅₀= 470 96 nM; compound 5, β5i IC₅₀= 27 2.7 nM and

−

+

β5c IC₅₀= 340 31 nM (means S.E.M., n = 3)]. Conversely,

−

compounds 14 and 15 showed the reverse selectivity, the

former of these two compounds being 18-fold more selective

Figure 9 Crystal structure of compound 16 bound to the chymotrypsin-like

site of the 20S proteasome

+

for the constitutive proteasome [β5c IC₅₀= 11 2.3 nM; β5i

−

+

IC₅₀= 200 62 nM (means S.E.M., n = 3)] and the latter

−

(A) β5/β6 active site with compound 16 (PDB entry 3MG8) bound to yeast open-gated 20S,

showing the molecular surface of 20S (coloured as described in Figure 5). Selectivity pockets

S1–S4 are indicated. (B) Close-up view of the S3 speciﬁcity pocket to illustrate occupancy by

the neopentyl-asparagine residue of compound 16 in the β5/β6 active site of the 20S core

particle (coloured as described above).

+

3.6-fold more selective [β5c IC₅₀= 12 2.8 nM (mean S.E.M.,

−

+

n = 4); β5i IC₅₀= 44 9.2 nM (mean S.E.M., n = 3)] (Table 1).

−

To determine whether this selectivity was maintained in cells,

three B-cell lymphoma subtypes were used that differ in their

proteasome isoform expression. As shown in Figure 10(A),

OCI-Ly10 cells do not express the immunoproteasome, as

demonstrated by the negligible hydrolysis of the β1i- and

β5i-speciﬁc substrates Ac-PAL-AMC and Ac-ANW-AMC

respectively, whereas Karpas-1106P express substantial β1i

and β5i activities, indicating considerable enrichment of the

immunoproteasome. The hydrolysis of the β5c substrate Ac-

WLA-AMC supported this expression proﬁle, although it is not

entirely selective for β5c over β5i and therefore over-represents

the amount of constitutive proteasome in Karpas-1106P cells.

The chymotrypsin-like substrate Suc-LLVY-AMC is hydrolysed

approximately equally well by the β5i and β5c sites in vitro,

although cells enriched in the immunoproteasome can show

approx. 2–3-fold higher activity with this substrate. On the basis

of these data, Karpas-1106P cells are highly enriched for the

immunoproteasome, whereas OCI-Ly10 cells only express

the constitutive proteasome. WSU-DLCL2 cells are intermediate

in this activity proﬁle, indicating that they express a mixed

population of the proteasome isoforms.

salinosporamide A [15]. Finally, the cytotoxic LC₅₀values of

these compounds also correlated well between cell lines (Table 2)

and with their 20S β5 potencies (Table 1). Interestingly, none

of the compounds were as potent as bortezomib in this assay,

although several gave LC₅₀values that were approx. 100 nM or

less in at least one cell line, speciﬁcally compounds 9–13 and

16 (Table 2). Signiﬁcantly, each of these contained the neopentyl-

asparagine substituent in P3 (Figure 8), indicating that this residue

contributes to both in vitro and cell-based potency. Furthermore,

since these compounds were also the most potent inhibitors of

NFκB-Luc activity, these data strongly suggest that their effects

on cell viability and NFκB activity are mediated by proteasome

inhibition.

Effect of 20S β5 inhibition on bulk protein turnover

The majority of intracellular proteins are degraded by the

proteasome, as demonstrated using inhibitors that irreversibly

inactivate each of the 20S active sites [4]. The effect of

We assessed the potency of the β5i- and β5c-selective

compounds

5 and 14 respectively in the three B-cell

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Novel non-covalent proteasome inhibitors

473

Table 3 Proteasome inhibition in B-cell lymphoma subtypes expressing

differing levels of immunoproteasome by β5i- and β5c-selective compounds

5 and 14 respectively compared with bortezomib

Inhibitionoftheβ5sitesoftheconstitutiveandimmunoproteasomewasassessedintheindicated

B-cell lymphoma lines (20000 cells per well) in situ using the Proteasome-Glo^TMassay. Cells

were incubated with compound for 1 h and assays were performed with the chymotrypsin-like

substrate Suc-LLVY-aminoluciferin as described in the Experimental section. The results are the

+

means S.E.M. for triplicate determinations.

−

Proteasome-Glo^TMβ5 IC₅₀(nM)

Compound

OCI-Ly10

WSU-DLCL2

Karpas-1106P

+

5

14

1300 400

39 7.6

320 120

190 76

310 150

850 240

−

+

−

+

Bortezomib

2.9 0.25

2.7 0.26

3.3 0.40

−

of compound 14 in cells that exclusively express the constitutive

proteasome as compared with those that predominantly express

the immunoproteasome. Importantly, bortezomib, which does not

discriminate substantially between β5c and β5i sites in vitro

(Table 1), gave IC₅₀values that were within 2-fold of each other

in the three B-cell lymphomas (Table 3). These data therefore

provide the ﬁrst description of non-covalent inhibitors of the

proteasome that can discriminate between the immunoproteasome

and constitutive proteasome in vitro and in cells.

DISCUSSION

The present study has identiﬁed a new series of potent

non-covalent proteasome inhibitors on the basis of a capped

trimeric peptide derived from the unnatural amino acid S-homo-

phenylalanine, ﬁrst identiﬁed by a large-scale high-throughput

cell-based screen for small-molecule inhibitors of the UPS. These

capped di-peptides, prepared by high-throughput liquid-phase

peptide synthesis methods, are entirely selective for the β5 site

over the β1 and β2 sites of the 20S core particle, and over a panel

of less closely related proteases. The most potent compounds

show IC₅₀values in the single-digit nanomolar range for the

human 20S β5 site in vitro, with the best example, compound

16, displaying an IC₅₀of 1.2 nM and a K_ibelow the enzyme

concentration in the assay (0.25 nM 20S), indicating that it

behaves as an active-site titrant. In this respect, compound 16

is remarkable in having a greater afﬁnity for the β5 site of the

proteasome than that of the covalent inhibitor bortezomib, which

displays a K_iof 0.56 nM for this site.

Compound optimization was guided by X-ray crystallography

of representative examples bound to the yeast 20S core particle,

which established their non-covalent binding mode and provided a

structural basis for potency-enhancing modiﬁcations. The crystal

structures of compounds 1, 6, 8 and 16 bound to the 20S β5

active site are shown in Figures 5, 8, S2, and 9 respectively,

and are overlaid in Supplementary Figure S6 (at http://

www.BiochemJ.org/bj/430/bj4300461add.htm). The overlay con-

ﬁrms a common binding mode in which the P1 benzyl group

occupies a well-deﬁned S1 binding pocket, whereas the P2

residue points out towards the solvent, unless it is the homo-

phenylalanine residue in which case a hydrophobic ‘ledge’

interaction can be accessed. The S3 binding pocket is also

well-deﬁned, whereas the S4 pocket is very large and can

tolerate a range of capping groups that are likely to contribute

to potency by picking up further hydrophobic interactions.

This binding mode broadly corresponds to that proposed on

the basis of a homology model [38], except that the crystal

structures obtained in the present study place the N-terminal

Figure 10 Proteasome-subtype selectivity of compounds 5 and 14 in B-cell

lymphomas expressing different levels of the immunoproteasome

(A) The activities of the β1 and β5 subunits of the constitutive (c) and immunoproteasome (i)

were assessed in diffuse large B-cell lymphoma extracts (10 μg of protein per reaction) using

recombinant PA28α (12 nM) and subtype-selective ﬂuorigenic peptide substrates (100 μM),

as indicated. As shown, the rank order of immunoproteasome expression in these cell lines

was Karpas-1106 > WSU-DLCL2 ꢂ LY10. (B and C) The B-cell lymphoma lines (20000 cells

per well) were treated with the indicated concentrations of the immunoproteasome-selective

compound 5 (B) or constitutive-selective compound 14 (C) for 1 h under cell culture conditions.

Inhibition of the β5 site of the constitutive and immunoproteasome was then assessed

in situ using the Proteasome-Glo^TMassay with the chymotrypsin-like luminogenic substrate

Suc-LLVY-aminoluciferin, as described in the Experimental section. As shown, the IC₅₀values

of compound 5 in Karpas-1106, WSU-DLCL2 and LY10 cells were 200, 380 and 620 nM

respectively; the IC50 values of compound 14 in Karpas-1106, WSU-DLCL2 and LY10 cells

+

were 870, 120 and 19 nM respectively. Results are means S.E.M. for triplicate determinations

−

and are representative of three independent experiments, the results of which are summarized

in Table 3.

lymphomas using the non-subtype-selective β5 substrate Suc-

LLVY-aminoluciferin in the Proteasome-Glo^TMassay. As shown

in Figure 10(B) and Table 3, the IC₅₀values of the β5i-selective

compound 5 correlated with immunoproteasome expression

(i.e. the rank order of potency being Karpas-1106P > WSU-

DLCL2 > OCI-Ly10), although the differences were relatively

modest (i.e. approx. ꢀ 4-fold). However, the β5c-selective

inhibitor compound 14, which was the most isoform-selective

compound in vitro, displayed substantial selectivity for the

constitutive proteasome in cells (Figure 10C). The rank order

of effect observed (i.e. OCI-Ly10 > WSU-DLCL2 > Karpas-

1106P) indicated an approx. 22-fold increase in inhibitory potency

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474

C. Blackburn and others

cap in a large S4 binding pocket rather than in small ‘accessory

hydrophobic pockets’, designated previously AS1 and AS2 [38].

Importantly, the most potent compounds identiﬁed each contained

a neopentyl-asparagine substituent in P3, which appears to

provide a near-optimal ﬁt for the S3 binding pocket of the β5

site (Figure 9B). This residue can also be found in the tri-peptide

2-keto-1,3,4-oxadiazoles (Figure 1A) that are slowly reversible

and presumably covalent in nature [11].

where the P1 and P3 residues are reversed, is preferentially

hydrolysed by the constitutive β5c subunit. The crystal structure

of the immunoproteasome has not yet been determined to provide

an explanation for these selectivity differences. However, the

primary sequences of the mature forms of the human β5c and

β5i subunits are 60.7% identical (see Supplementary Figure

S7 at http://www.BiochemJ.org/bj/430/bj4300461add.htm). We

have therefore mapped the sequence differences between the β5c

and β5i subunits on to the bovine 20S crystal structure [56] and

modelled compound 1 into the active site (see Supplementary Fig-

ure S8 at http://www.BiochemJ.org/bj/430/bj4300461add.htm).

The majority of the sequence differences between β5c and

β5i lie outside of the active site, whereas the residues that

make contacts with compound 1 appear to be mainly the same,

with some relatively minor exceptions, such as the replacement

of Ala⁴⁶and Ser⁵³with Ser⁴⁶and Gln⁵³within the S1 binding

pocket, Gly⁴⁸with Cys⁴⁸in the S2 ‘ledge’, and the reversal

of the Ala²⁷-Ser²⁸sequence with Ser²⁷-Ala²⁸in the S3 pocket.

Since only subtle differences exist between the ligand-interacting

residues that form the β5c and β5i active sites, it is not

possible to rationalize selectivity differences with respect to

substrate preference or inhibitor sensitivity based on such an

analysis. Clearly, crystal structures of the constitutive and

immunoproteasomes in complex with selective inhibitors will

help identify the structural features of each sub-site that contribute

to selectivity. Finally, although a covalent immunoproteasome-

selective inhibitor has been described recently [8], the function

of the immunoproteasome in cancer cells is unknown. The

identiﬁcation of a new class of proteasome inhibitors, such as

compounds 14 and 5, that preferentially inhibit the 20S β5c

and β5i sites respectively and can thus discriminate between the

constitutive and immunoproteasome in cells may help address

their functional roles in various cellular models of disease

including cancer.

Characterization of these compounds in cells demonstrated

that they can functionally inhibit the 26S proteasome with

potencies that correlated well with those determined in vitro

using the puriﬁed enzyme (Table 2 and Supplementary Figure

S4). Furthermore, since the analogues are essentially inactive

against the β1 (caspase-like) and β2 (trypsin-like) sites of the pro-

teasome (Table 1), these data indicate that inhibition of the β5

(chymotrypsin-like) site of the proteasome alone is sufﬁcient

to potently inhibit the degradation of the 4xUb-Luc reporter,

activation of NFκB in response to TNF-α and the proliferation of

cancer cells. Interestingly, despite the fact that the cellular IC₅₀

values of these compounds spanned three orders of magnitude,

withthe mostpotentbeing inthesingle- or double-digitnanomolar

range, neither the stabilization of 4xUb-Luc nor the inhibition of

the NFκB pathway was complete, even at saturating compound

concentrations (Table 2 and Supplementary Figure S1). These

observations suggest that non-covalent inhibition of 20S β5

activity alone is not sufﬁcient to fully inhibit the proteasomal

degradation of either 4xUb-Luc or IκBα, at least over the time-

scale of these cell-based assays. This is in contrast with the

covalent inhibitors bortezomib and salinosporamide A, which

completely block the degradation of these proteins in cells

and can minimally inhibit both the β1 and β5 sites of the

proteasome. Furthermore, estimates of bulk protein turnover in

cells suggest that the overall catalytic rate of proteasome is

reduced, but not abolished, by non-covalent inhibition of the β5

site (Supplementary Figure S5), although it remains a possibility

such inhibition completely blocks the degradation of a subset

of proteins that are only substrates of the β5 site and not the

β1 and/or β2 sites of the proteasome. Importantly, however,

prolonged inhibition of the β5 site alone appears to sufﬁcient

to inhibit cancer cell proliferation.

Proteasome inhibitors that are currently in clinical use do

not discriminate between the chymotrypsin-like activities of the

constitutive or immunoproteasome, and the potential therapeutic

beneﬁt of selective inhibition of either of these activities has not

been demonstrated. However, recent evidence from preclinical

studies with PR-957, a β5i-selective peptide epoxyketone

analogue of carﬁlzomib [28], suggests that the anti-inﬂammatory

effects of proteasome inhibitors, such as bortezomib, may be

mediated by their inhibition of the chymotrypsin-like site of the

immunoproteasome [8]. Our studies have identiﬁed compounds

that differentially inhibit the β5 sites of the constitutive and

immunoproteasome in vitro and in B-cell lymphomas. Of note,

compound 5 that preferentially inhibited the β5i site contained the

small threonine residue in P3 and the larger 4-methylbenzylamine

aromatic cap in P1. Conversely, the two β5c-selective compounds

14 and 15 each contained a smaller residue at P1 and the bulkier

potency-enhancing neopentyl-asparagine in P3 (Figure 8). This

structure–activity relationship, albeit limited, is entirely consistent

with selectivity data obtained from an approx. 6000 tri-peptide

library screen for substrates that are preferentially hydrolysed

by each active site of the constitutive and immunoproteasome

([55] and C. Tsu, unpublished work). For example, the tri-peptide

substrate Ac-ANW-AMC, where P1 is a tryptophan residue

and P3 is an alanine residue, is preferentially cleaved by the

β5i subunit of the immunoproteasome, whereas Ac-WLA-AMC,

AUTHOR CONTRIBUTION

Christopher Tsu, Paul Hales and Frank Bruzzese and designed and performed the

biochemical characterization of the compounds. Cynthia Barrett, Jane Liu and Khristofer

Garcia performed the cell-based assays and Teresa Soucy performed the protein turnover

assays. Darshan Sappal generated the MDA231-4xUb-Luc cell line and performed the

high-throughputlibraryscreen. EdwardOlhavaperformedthere-synthesisofcompound1,

and Christopher Blackburn, Kenneth Gigstad and Matthew Jones designed and performed

the chemical syntheses of the capped di-peptides. Nancy Bump puriﬁed yeast 20S

core particles for crystallization and Michael Sintchak prepared the crystals for X-ray

crystallography and analysed the data. Paul Fleming and Lawrence Dick provided project

oversight, participated in experimental design and data interpretation, and reviewed

the manuscript. Jonathan Blank performed the high-throughput screen and the initial

characterization of compound 1 in cells, contributed to experimental design and data

analysis, and wrote the manuscript with Christopher Blackburn.

ACKNOWLEDGEMENTS

We are grateful to Dr Marion Schmidt and Dr Dan Finley for providing the yeast 20S

expression strains, and to Dr Juan Gutierrez and Zhi Li their assistance with preparation

of the yeast 20S enzymes. We also thank Alejandra Raimondi for formatting the 4xUb-Luc

cell-basedassayforthehigh-throughputscreening, BenKnightforhisassistancewithdata

processing and management, and Maria Dawn-Linsley, Kim Lincoln, Charles Gauthier,

Dan Onea, Pam Ward, Ted Peters, Christina Majer and Elena Musotto for their help with

assay automation and compound management. We gratefully acknowledge the assistance

of Marjorie Solomon, David Lok, Lenny Dang and Qing Lu with the identiﬁcation of

compound 1 by MS. We thank Mingkun Fu who performed the high-resolution MS on

the synthetic compounds. We acknowledge Abe Achab and Alla Mishechkina for their

synthetic contributions towards development of the series. We are also grateful to Dr Gary

Luker (Department of Microbiology and Immunology, University of Michigan Medical

School, Ann Arbor, MI, U.S.A.) and Dr David Piwnica-Worms (Division of Biology and

Biomedical Sciences, University of Washington, St Louis, MO, U.S.A.) from whom the

4xUb-Luc expression plasmid was licenced. We are also grateful to Dr Friedrich Feuerhake

and Dr Margaret Shipp (Lymphoma Program, Dana-Faber Cancer Institute, Boston, MA,

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Novel non-covalent proteasome inhibitors

475

22 Richardson, P. G., Mitsiades, C., Hideshima, T. and Anderson, K. C. (2006) Bortezomib:

proteasome inhibition as an effective anticancer therapy. Annu. Rev. Med. 57, 33–47

23 Orlowski, R. Z. and Kuhn, D. J. (2008) Proteasome inhibitors in cancer therapy: lessons

from the ﬁrst decade, Clin. Cancer Res.14, 1649–1657

U.S.A.) for providing cell pellets derived from a panel of B-cell lymphoma subtypes to

proﬁle for immunoproteasome expression. Finally, we thank Mark Williamson and Dr

Mark Rolfe for valuable discussions.

24 Piva, R., Ruggeri, B., Williams, M., Costa, G., Tamagno, I., Ferrero, D., Giai, V., Coscia,

M., Peola, S., Massaia, M. et al. (2008) CEP-18770: a novel, orally active proteasome

inhibitor with a tumor-selective pharmacologic proﬁle competitive with bortezomib. Blood

111, 2765–2775

25 Kupperman, E., Lee, E. C., Cao, Y., Bannerman, B., Fitzgerald, M., Berger, A., Yu, J., Yang,

Y., Bruzzese, F., Liu, J. et al. (2010) Evaluation of the proteasome inhibitor MLN9708 in

preclinical models of human cancer. Cancer Res. 70, 1970–1980

FUNDING

This work was supported by Millennium Pharmaceuticals, Inc. (Cambridge, MA, U.S.A.).

All authors were employed by Millennium Pharmaceuticals at the time of their contribution

to this work.

26 Groll, M., Berkers, C. R., Ploegh, H. L. and Ovaa, H. (2006) Crystal structure of the

boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S

proteasome. Structure 14, 451–456

27 Chauhan, D., Catley, L., Li, G., Podar, K., Hideshima, T., Velankar, M., Mitsiades, C.,

Mitsiades, N., Yasui, H., Letai, A. et al. (2005) A novel orally active proteasome inhibitor

induces apoptosis in multiple myeloma cells with a mechanism distinct from bortezomib.

Cancer Cell 8, 407–419

28 Kuhn, D. J., Chen, Q., Voorhees, P. M., Strader, J. S., Shenk, K. D., Sun, C. M., Demo,

S. D., Bennett, M. K., van Leeuwen, F. W. B., Chanan-Khan, A. A. and Orlowski, R. Z.

(2007) Potent activity of carﬁlzomib, a novel, irreversible inhibitor of the ubuquitin–

proteasome pathway, against preclinical models of multiple myeloma. Blood 110,

3281–3290

29 Groll, M., Huber, R. and Potts, B. C. M. (2006) Crystal structures of salinoporamide A

(NPI-0047) in complex with the 20S porteasome reveal important consequences of

β-lactone ring opening and a mechanism for irreversible binding. J. Am. Chem. Soc.

128, 5136–5141

30 Groll, M., Kim, K. B., Kairies, N., Huber, R. and Crews, C. M. (2000) Crystal structure of

expoxomicin:20S proteasome reveals a molecular basis for selectivity of

α^ꢀ,β^ꢀ-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 122, 1237–1238

31 Koguchi, Y., Kohno, J., Nishio, M., Takahashi, K., Okuda, T., Ohnuki, T. and Komatsubara,

S. (2000) TMC-95A, B, C, and D, novel proteasome inhibitors produced by Apiospora

montagnei Sacc. TC 1093. Taxonomy, production, isolation, and biological activities.

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Received 15 March 2010/2 July 2010; accepted 15 July 2010

Published as BJ Immediate Publication 15 July 2010, doi:10.1042/BJ20100383

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Biochem. J. (2010) 430, 461–476 (Printed in Great Britain) doi:10.1042/BJ20100383

SUPPLEMENTARY ONLINE DATA

Characterization of a new series of non-covalent proteasome inhibitors with

exquisite potency and selectivity for the 20S β5-subunit

Christopher BLACKBURN, Kenneth M. GIGSTAD, Paul HALES, Khristofer GARCIA, Matthew JONES, Frank J. BRUZZESE,

Cynthia BARRETT, Jane X. LIU, Teresa A. SOUCY, Darshan S. SAPPAL, Nancy BUMP, Edward J. OLHAVA¹, Paul FLEMING,

Lawrence R. DICK, Christopher TSU, Michael D. SINTCHAK and Jonathan L. BLANK²

Discovery, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, U.S.A.

Figure S1 Activities of compound 1 and bortezomib in cells

(A) The concentration-dependent effects of compound 1 and bortezomib on the proliferation of MDA-MB-231-4xUb-Luc reporter cell line were determined by the ATPlite assay at 72 h.

(B) Accumulation of the 4xUb-Luc reporter in the stable MDA-MB-231 cell line was determined by incubation with the indicated concentrations of compounds for 8 h followed by luciferase assay.

(C) The β5 activity of the 26S proteasome was measured in Calu6 cells by the Proteasome-Glo^TMassay using Suc-LLVY-aminoluciferin as a substrate following incubation of the cells with the

indicated concentration of compounds for 1 h. (D) Inhibition of NFκB–luciferase activity in the stable HEK-293 cell line was determined by pre-incubation of the cells with the indicated concentration

+

of compounds for 1 h followed by stimulation with 10 ng/ml TNF-α in the continued presence of compound for a further 3 h. Results are means S.E.M. from replicate determinations and are

representative of several independent experiments.

−

SUPPLEMENTARY EXPERIMENTAL

of buffer containing 50 mM Tris/HCl, pH 8.0, 100 mM NaCl,

5 mM CaCl₂, 0.01% Tween 20 and their respective substrates

Boc-QGR-AMC (50 μM), Suc-AAPF-AMC (40 μM), MeOSuc-

AAPV-AMC (50 μM), H-AFK-AMC (65 μM), Bz-FVR-AMC

(20 μM), Boc-E(OBzl)GR-AMC (50 μM) and Z-FR-

AMC (12 μM). The activities of cathepsin B and L were

measured using 2 nM enzyme in 50 μl of 100 mM sodium

acetate, pH 5.5, containing 5 mM DTT (dithiothreitol), 1 mM

EDTA, 0.01% Brij 35 and the respective substrates Z-RR-AMC

(60 μM) and Z-FR-AMC (37.5 μM). Calpain I activity was

measured using 200 nM enzyme in 50 μl of 50 mM Tris/HCl,

pH 7.4, containing 75 μM CaCl₂, 1.5 mM DTT and 100 μM

Suc-LY-AMC. Percentage inhibition of the proteases was

Protease selectivity assays

Protease selectivity of the compounds was assessed using a

panel of puriﬁed human enzymes (all from Calbiochem, EMD

Chemicals) by monitoring hydrolysis rates of peptide-AMC

substrates (all from Bachem) using ﬂuorescence measurements

as described for ‘In vitro assays of puriﬁed 20S’ in the main

paper, with modiﬁcations as follows. To measure the activities

of coagulation Factor β-XIIa (200 nM), chymotrypsin (2 nM),

elastase (4 nM), plasmin (4 nM), thrombin (4 nM), tissue

plaminogen activator (25 nM) and trypsin (4 nM) (ﬁnal

concentrations), reactions were performed at 30^◦C in 50 μl

1

Present address: Epizyme, Inc., 840 Memorial Drive, Cambridge, MA 02139, U.S.A.

To whom correspondence should be addressed (e-mail jonathan.blank@mpi.com).

2

The structural co-ordinates of the yeast 20S proteasome with the indicated ligand bound reported will appear in the PDB under accession codes: 3MG0

(bortezomib); 3MG4 (compound 1); 3MG6 (compound 6); 3MG7 (compound 8); 3MG8 (compound 16).

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C. Blackburn and others

calculated relative to DMSO and either 2–20 μM E64 [trans-

epoxysuccinyl-L-leucylamido(4-guanadino)butane; Sigma] for

the cysteine proteases (cathepsins and calpain) or complete

EDTA-free protease inhibitor cocktail (1 tablet/ml diluted 1:10

(v/v) in assay buffer, Roche) for the remaining serine proteases.

Protein turnover assay

Bulk protein turnover was measured in HCT-116 cells as described

previously [1]. Brieﬂy, HCT116 cells (100000 cells/well in 12-

well plates) were labelled for 20 min under cell culture incubation

conditions in methionine- and cysteine-free DMEM (Dulbecco’s

modiﬁed Eagle’s medium; Invitrogen), supplemented with 10%

dialysed FBS (fetal bovine serum), 2 mM L-glutamine and

50 μCi/well of EasyTag^TMEXPESS ³⁵S-labelled methion-

ine/cysteine mix (NEN Radiochemicals, PerkinElmer). The cells

were then washed three times with the same medium containing

the unlabelled 2 mM methionine and 0.2 mM cysteine, and then

incubated with 3 μM compound in this medium for the indicated

times. Medium (50 μl) was removed from the cells and subjected

to liquid scintillation counting to monitor the release of ³⁵S-

labelled peptides. At the end of the experiment, cells were washed

in PBS and then solubilized in 1 ml of 0.2 M NaOH and subjected

to liquid scintillation counting. Percentage protein turnover at

each time point was calculated as: [(total counts released into

medium)/(total counts released into medium + total counts in

NaOH-solubilized cells)]×100.

Figure S2 Electron density of compound 6 and the water molecule involved

in peptide binding

˚

A 2.6 A sigmaA-weighted F_o−F_celectron-density omit map contoured at 2.5σ covering

compound 6 and an associated water molecule in the β5/β6 active site of 20S is shown, with

positions P1–P4 indicated. After removal of all ligand atoms and water molecules from the

model, random co-ordinate displacements were added to remove model bias prior to several

cycles of reﬁnement and omit map calculation. For clarity, the portion of the omit map covering

the water molecule is coloured magenta and hydrogen bonds are indicated by dashed lines.

Atoms are shown in ball-and-stick representation with carbon in yellow, nitrogen in blue, oxygen

in red, and sulfur in orange. The side-chain atoms for Asp¹¹⁴(β6) are shown in ball-and-stick

representation coloured as above, except for carbon in green. Protein atoms from 20S are shown

in cartoon representation coloured by secondary structure with helices in cyan, β-strands in

magenta and loops in salmon. The ﬁgure was made using the PyMOL Molecular Graphics

System (DeLano Scientiﬁc, Palo Alto, CA, U.S.A.).

Synthesis of compound 1, the capped tri-peptide 5-methyl-N-((S)-

1-((S)-1-((S)-1-(4-methylbenzylamino)-1-oxo-4-phenylbutan-

2-ylamino)-1-oxo-4-phenylbutan-2-ylamino)-1-oxo-4-phenylbutan-

2-yl)pyrazine-2-carboxamide

To a mixture of (S)-2-(tert-butoxycarbonylamino)-4-phenyl-

butanoic acid (0.634 g, 2.26 mmol) and 4-methylbenzylamine

(0.306 ml, 2.40 mmol) in dichloromethane (10.0 ml) was

added HBTU (O-benzotriazole-N,N,N^ꢁ,N^ꢁ-tetramethyluronium-

hexaﬂuorophosphate) (0.984g, 2.6 mmol) and N-methylmor-

pholine (0.50 ml, 4.54 mmol). The reaction mixture was

stirred at room temperature overnight and then washed with

saturated aqueous NaHCO₃. The organic layer was dried over

anhydrous MgSO₄and evaporated and the residue dissolved in

tetrahydrofuran (20 ml), treated with 4.0 M HCl in 1,4-dioxane

(20 ml) and stirred for 16 h at ambient temperature. The solvents

were removed in vacuo to give (S)-2-amino-N-(4-methylbenzyl)-

4-phenylbutanamide hydrochloride as an oil (0.70 g, 98%).

ESI (electrospray ionization)–MS + : calculated for C₁₈H₂₂N₂O

[M + H] 283; found 283.

Figure S3 Comparison of the structures of compounds 8 and 6 bound to the

chymotrypsin-like site of the 20S proteasome

Close-up view of the S1 sub-pocket being ﬁlled by the ortho-chloro substituent in P1

for compound 8 (left-hand panel), compared with compound 6 that lacks this substituent

(right-handpanel). The colour scheme is as described in Figure S2.

A solution of (S)-2-amino-N-(4-methylbenzyl)-4-phenylbu-

tanamide hydrochloride (0.50 g, 1.57 mmol) and (S)-2-(tert-

butoxycarbonylamino)-4-phenylbutanoic acid (0.439 g, 1.57

mmol) and 2,4,6-collidine (0.571 mg, 4.71 mmol) in DMF (10 ml)

was treated with TBTU [O-(benzotriazol-1-yl)-N,N,N^ꢁ,N^ꢁ-

tetramethyluronium tetraﬂuoroborate] (0.556 g, 1.73 mmol) and

stirred at ambient temperature for 3 h. The reaction mixture was

diluted with water and extracted with dichloromethane. The

extracts were dried over MgSO₄and evaporated. The residue

was re-dissolved in dichloromethane containing 1% MeOH

and hexane was added to precipitate tert-butyl (S)-1-[(S)-1-(4-

methylbenzylamino)-1-oxo-4-phenylbutan-2-ylamino]-1-oxo-4-

phenylbutan-2-ylcarbamate (0.546 g, 90%).

treated with 4 M HCl in dioxane (2.1 ml, 10.4 mmol) and

stirred at room temperature for 4 h. The solvent was evaporated

and the residue triturated with ether to give the corresponding

amine hydrochloride (0.65 g, 100%) which was used without

further puriﬁcation. The next S-homo-Phe residue was coupled

as described for the previous step and the Boc protecting

group removed as described above to give (S)-2-amino-

N-{(S)-1-[(S)-1-(4-methylbenzylamino)-1-oxo-4-phenylbutan-

2-ylamino]-1-oxo-4-phenylbutan-2-yl}-4-phenylbutanamide as

the hydrochloride salt (81% yield). The amine described above

was coupled to 5-methylpyrazine-2-carboxylic acid using the

coupling conditions described above and worked up in the usual

way. The crude product was puriﬁed by ﬂash chromatography

A solution of tert-butyl (S)-1-[(S)-1-(4-methylbenzylamino)-1-

oxo-4-phenylbutan-2-ylamino]-1-oxo-4-phenylbutan-2-ylcarba-

mate (0.754 g, 1.4 mmol) in dichloromethane (10 ml) was

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Novel non-covalent proteasome inhibitors

Figure S4 Correlation between in vitro and cellular potencies of bortezomib and compounds 1–16

Data presented in Tables 1 and 3 in the main paper are expressed in μM concentrations and compared to show the correlation between in vitro potency of the compounds against the β5 site of the

constitutive 20S proteasome determined with Ac-WLA-AMC as substrate and Calu6 cell Proteasome-Glo^TMβ5 IC₅₀, HEK293-NFκB-Luc IC₅₀, MDA-MD-231-4xUb-Luc EC₅₀and Calu6 cell ATPlite

viability LC₅₀. The viability effects of the compounds in Calu6 cells and H460 cells, as shown in Table 3 in the main paper, shown as LC₅₀values are expressed in μM concentrations and correlated.

The open symbol represents bortezomib and the closed symbols are numbered to indicate compounds 1–16.

on silica gel eluting with a gradient of 0 to 5% methanol in

dichloromethane to give 5-methyl-N-[(S)-1-{(S)-1-[(S)-1-(4-

methylbenzylamino)-1-oxo-4-phenylbutan-2-ylamino]-1-oxo-4-

phenylbutan-2-ylamino}-1-oxo-4-phenylbutan-2-yl]pyrazine-2-

carboxamide (compound 1) in 73% yield.

Compound 1

¹H NMR (300 MHz, d6 DMSO) δ: 1.91 (m, 5H), 2.10 (m, 2H),

2.21 (s, 3H), 2.59 (m, 8H), 3.31 (s, 1H), 4.19 (m, 2H), 4.25 (m,

1H), 4.66 (m, 1H), 7.01 (d, 2H, J = 7.8 Hz), 7.17 (m, 17H), 8.21

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C. Blackburn and others

Figure S5 Effect on intracellular protein turnover of potent di-peptide

proteasome inhibitors 11 and 16 compared with bortezomib

HCT116 cells were metabolically labelled for 20 min with [³⁵S]methionine/cysteine and then

chased with excess unlabelled amino acids in the presence of compound 11, 16 or bortezomib

(each at 3 μM) for the times indicated. Intracellular degradation of ³⁵S-labelled proteins was

then determined as described in the Experimental section in the main paper and expressed as a

+

percentage of total label incorporated into the proteins. The results are means S.E.M. (n = 6)

−

and are representative of two similarly performed experiments.

(d, 1H, J = 7.9 Hz), 8.34 (d, 1H, J = 7.1 Hz), 8.45 (d, 1H, J = 7.8

Hz), 8.65 (m, 1H), 8.81 (d, 1H, J = 8.3 Hz), and 9.05 (d, 1H,

J = 11.6 Hz). HRMS (high-resolution MS; ESI + ): calculated

for C₄₄H₄₈N₆O₄[M + H] 725.3815; found 725.3799.

Figure S6 Common occupancy of the chymotrypsin-like site of the 20S

proteasome by compounds 1, 6, 8 and 16

Superposition of the structures of compounds 1, 6, 8, and 16 bound to the β5/β6 active site of

20S, showing the molecular surface of 20S with positions P1–P4 indicated. The colour scheme

is as described in Figure S2.

Synthesis of (S)-2-(tert-butoxycarbonylamino)-4-

(neopentylamino)-4-oxobutanoic acid (compound 18)

(s, 9H), 0.91 (s, 9H). MS (ESI + ): calculated for C₁₄H₂₆N₂O₅

[M + H] 303; found 303.

Compound 18 was prepared according to Scheme S1. A solution

of Boc-Asp-OBn (0.937g, 2.9 mmol), neopentylamine (0.267 g,

2.9 mmol), HBTU (O-benzotriazole-N,N,N^ꢁ,N^ꢁ-tetramethyl-

uronium-hexaﬂuorophosphate) (1.33 g, 3.5 mmol) and

N-methylmorpholine (0.44 g, 4.4 mmol) in anhydrous methylene

chloride (15.0 ml) was stirred at ambient temperature for 16 h.

The solvent was evaporated and the residue obtained was

partitioned between ethyl acetate (35 ml) and saturated aqueous

NaHCO₃(25 ml). The organic layer was dried over anhydrous

MgSO₄, concentrated and puriﬁed by ﬂash chromatography on

silica gel to afford (S)-benzyl 2-(tert-butoxycarbonylamino)-4-

(neopentylamino)-4-oxobutanoate (17) as a white solid (0.84 g,

96%) which was dissolved in methanol (3 ml) and stirred under

20 psi (1 psi = 6.9 kPa) H₂for 2 h in the presence of 10%

palladium on carbon (10 mol%). The Pd/C was removed via

ﬁltration through celite and the solvent evaporated to afford 18 in

quantitative yield.

Synthesis of capped di-peptide proteasome inhibitors

A representative procedure for the parallel synthesis of compound

arrays (denoted IV in Scheme 2 in the main paper) follows: a

deep-well polypropylene synthesis plate was charged with the

appropriate amine salts II (0.25 mmol) in one dimension and

a solution of the amino acid (0.25 mmol) constituting the P3

coupling partner in DMF (2.0 ml) added in the second dimension

by use of a Tecan liquid handler. HBTU (0.5 ml, 0.5 M solution

in DMF) and N-methylmorpholine (0.9 mmol) were then added

to each well by means of the liquid handler. The plate was shaken

at room temperature for 16 h. The residue in each well obtained

after evaporation of the solvent in a Genevac was partitioned

between CHCl₃/THF (3:1) (3 ml per well) and saturated aqueous

NaHCO₃(2 ml) with each solution delivered by the liquid handler.

The phases were separated by centrifugation and the aqueous

layer was extracted with additional CHCl₃/THF (3:1) (3 ml) and

the combined organic layers were evaporated. The residue thus

obtained was treated with 4 M HCl in dioxane (2 ml), shaken

at ambient temperature for 5 h and concentrated in a Genevac

evaporator to remove solvent and residual HCl. The N-terminal

cap was then coupled and the ﬁnal products isolated as in the

Compound 18

1H NMR (400 MHz, C²HCl₃) δ 6.59 (s, 1H), 5.90 (d, J = 5.2 Hz,

1H), 4.41–4.27 (m, 1H), 3.48 (s, 1H), 3.22 (dd, J = 13.3, 7.1 Hz,

1H), 3.02–2.94 (m, 2H), 2.76 (dd, J = 15.6, 9.0 Hz, 1H), 1.44

Scheme S1 Synthesis of compound 18

◦

(i) Neopentyl amine, HBTU, NMM, DCM, 25 C, 24h; (ii) H₂, Pd/C, MeOH.

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Novel non-covalent proteasome inhibitors

Figure S7 Sequence alignment of the human β5 subunits of the constitutive and immunoproteasome

PSB5_HUMAN (PSMB5, β5 constitutive subunit, upper sequence) is compared with PSB8_HUMAN (PSMB8, β5 immunoproteasome subunit, lower sequence), with regions of sequence identity

shaded black. Residues forming the inhibitor-binding pockets are indicated by circles below the aligned sequences and coloured as follows: S1 pocket in blue, S2 pocket in magenta and S3 pocket

in green. Note that Ala⁴⁹sits between the S1 and S3 pockets and has been assigned to both. Residues from the β6 subunit that form the S4 pocket, as well as a portion of the S3 pocket, are not

shown because this subunit does not differ between the constitutive and immunoproteasome.

with a water–MeCN gradient (0.1% formic acid) optimized by

the A2Prep Agilent software. Compounds 2–16 described herein

were further characterized by 1H NMR spectroscopy and HRMS

(see below).

HRMS

Molecular mass measurements were performed using a QSTAR^®

XL quadrupole TOF (time-of-ﬂight) mass spectrometer (Applied

Biosystems) coupled to an 1100 series HPLC system (Agilent

Technologies). An isocratic ﬂow of 50% solvent A (20%

CH₃CN/80% H₂O with 0.1% CH₃COOH) and 50% solvent

B (100% CH₃CN with 0.1% CH₃COOH) at 200 μl/min was

used to deliver each sample to the electrospray source of the

mass spectrometer, tuned in ESI TOF-MS positive mode with a

3 min acquisition time for each analysis. The mass spectrometer

was externally calibrated between m/z 400 and 800 by using API

Calibration (NaCsI) Solution (Waters Corp). For each analysis,

Figure S8 Residue conservation between the β5/β6 active site of the

approx. 5 scans were summed and the centroid m/z value of the

protonated monoisotopic molecular ion [M + H]⁺was recorded.

The relative deviations from the calculated exact masses based on

the expected formulae were also calculated. For each compound,

+

constitutive and immunoproteasome

Surface representation of the β5 (light blue) and β6 (grey) subunits of bovine 20S

proteasome (PDB entry 1IRU [56]). Residues that are non-identical between constitutive and

immunoproteasome are coloured red. The structure of compound 1 has been modelled into the

active site on the basis of a superposition of the β5 subunit structure from yeast 20S, reported

in the present study, with the corresponding subunit of the bovine 20S structure.

the experimental and calculated [M + H] values were within

4

−

ppm, supporting their expected elemental composition (see the

Supplementary Results section).

SUPPLEMENTARY RESULTS

1H NMR and HRMS data for synthetic compounds

Compound 2

previous step. All ﬁnal compounds IV (some 1500 in total) were

puriﬁed by preparative reverse phase HPLC using mass-directed

fraction collection and their purities and identities established

by LC-MS. Preparative HPLC was conducted on an Agilent 1100

series LC/MSD instrument using a Waters SunFire C18 5 μm Prep

OBD column (19 mm×150 mm). The compounds were eluted

1H NMR (400 MHz, d6 DMSO) δ: 1.82 (m, 2H), 1.92 (m, 2H),

2.22 (s, 3H), 2.56 (m, 2H), 3.45 (d, 1H), 3.55 (d, 1H), 4.20 (d,

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C. Blackburn and others

Table S1 Crystallographic data and reﬁnement statistics

rmsd, root mean square deviation.

Compound

Bortezomib

1

6

8

16

PDB ID

Space group

Unit cell dimensions (A,

Wavelength (A)

Resolution (A)

R_sym* (%)

Total observations

Unique reﬂections

Average redundancy

<I/σ>

3MG0

P2₁

3MG4

P2₁

3MG6

P2₁

3MG7

P2₁

3MG8

P2₁

◦

˚

)

a=136.1, b=300.5, c=145.8, β=113.2 134.6, 300.8, 144.7, 112.7 137.3, 299.6, 145.8, 113.7 136.0, 300.4, 145.8, 113.7 136.3, 299.2, 145.7, 113.1

˚

0.97947

50 - 2.7

9.8 (32.8)†

1017555

276539

3.7

9.7 (2.5)

92.9 (71.6)

50 - 2.7

272720/2818

22.6/25.3

49541

0.97931

50 - 3.1

11.6 (30.5)

723147

177491

4.1

7.1 (2.8)

94.1 (61.2)

50 - 3.1

173803/3565

20.5/25.2

49541

90

0.97958

50 - 2.6

9.2 (35.3)

2255995

315914

7.1

11.1 (2.7)

96.3 (76.9)

50 - 2.6

310987/3189

21.5/24.9

49298

0.97958

50 - 2.8

12.3 (44.2)

950351

257519

3.7

8.0 (2.2)

97.2 (85.7)

50 - 2.8

251281/5191

21.7/25.9

49298

88

0.97969

50 - 2.6

7.3 (32.7)

1034821

315407

3.3

11.1 (2.9)

95.6 (85.9)

50 - 2.6

308947/6368

22.4/25.3

49298

92

Completeness (%)

˚

Reﬁnement resolution (A)

Reﬂections (working/test)

R_cryst/R_free(%)‡

Protein atoms

Ligand atoms

168

86

Mes atoms

0

24

10

24

10

24

10

24

10

Mg²⁺atoms

Water atoms

rmsd bond lengths (A)

rmsd bond angles ( )

1037

0.008

1.11

0

858

0.011

1.31

4

5

˚

0.008

1.13

0.008

1.14

0.008

1.12

◦

Ramachandran analysis

Most-favoured (%/number)

Additional allowable (%/number) 10.7/594

Generously allowed (%/number) 0.5/29

88.5/4896

89.8/4969

9.5/526

0.5/27

90.4/4997

8.9/491

0.4/23

90.5/4993

8.6/475

0.6/31

91.3/5041

8.1/445

0.4/21

Disallowed (%/number)

0.3/14

0.2/13

0.3/16

0.2/13

*R_sym= (ꢀ_hklꢀ_i|I_i(hkl)−<I(hkl)>|)/ꢀ_hklꢀ_iI_i(hkl) for n independent reﬂections and i observations of a given reﬂection. <I(hkl)> is the average intensity of the i^thobservation.

†Numbers in parenthesis are for highest resolution shell.

‡R_cryst= ꢀ_h||F_o(h)|−|F_c(h)||/ꢀ_h|F_o(h)|, where F_oand F_care the observed and calculated structure factors respectively.

2H), 4.28 (m, 3H), 6.84 (m, 1H), 6.96 (m, 2H), 7.01 (m, 3H), 7.10

(m, 8H), 7.17 (m, 2H), 7.25 (m, 5H), 7.33 (m, 3H), 8.16 (d, 1H,

J = 6.7 Hz), 8.32 (t, 1H, J = 6.1 Hz), and 8.40 (d, 1H, J = 8 Hz).

HRMS (ESI + ): calculated for C₄₂H₄₃N₃O₄[M + H] 654.3332;

found 654.3346.

Compound 5

1H NMR (400 MHz, DMSO) δ 8.32 (t, J = 6.0 Hz, 1H), 8.15 (d,

J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.9 Hz,

2H), 7.43–7.36 (m, 4H), 7.24 (t, J = 7.3 Hz, 2H), 7.19–7.03 (m,

9H), 5.14 (s, 2H), 5.02 (d, J = 6.5 Hz, 1H), 4.42 (dd, J = 8.0, 4.9

Hz, 1H), 4.30 (td, J = 8.7, 4.8 Hz, 1H), 4.20 (dt, J = 12.5, 6.2 Hz,

2H), 4.11 (dd, J = 11.5, 6.3 Hz, 1H), 2.65–2.52 (m, 2H), 2.25 (s,

3H), 2.03–1.93 (m, 1H), 1.92–1.75 (m, 1H), 1.27 (s, 9H), 1.12

(d, J = 6.3 Hz, 3H). HRMS (ESI+): calculated for C₄₀H₄₇N₃O₅

[M + H] 650.3596; found 650.3594.

Compound 3

1H NMR (300 MHz, d6 DMSO) δ: 2.25 (s, 3H), 2.67 (m, 1H),

2.90 (m, 1H), 3.01 (m, 1H), 3.12 (m, 1H), 3.42 (m, 2H), 3.65

(s, 3H), 3.67 (s, 3H), 4.18 (m, 2H), 4.51 (m, 1H), 4.57 (m, 1H),

6.65 (m, 1H), 6.71 (m, 1H), 6.81 (m, 4H), 6.97 (m, 5H), 7.05 (d,

3H, J = 8.3 Hz), 7.12 (m, 2H), 7.20 (t, 2H, J = 7.8 Hz), 7.35 (m,

4H), 7.59 (d, 1H, J = 7.7 Hz), 8.19 (d,d, 1H, J = 14.0, 7.9 Hz),

and 8.33 (m, 1H). HRMS (ESI + ): calculated for C₄₄H₄₄N₄O₆

[M + H] 725.3339; found 725.3336.

Compound 6

1H NMR (400 MHz, DMSO) δ 8.38 (dt, J = 11.9, 6.0 Hz, 1H),

8.03 (dd, J = 8.1, 3.8 Hz, 2H), 7.53 (dd, J = 8.5, 2.6 Hz, 1H), 7.26

(dd, J = 10.1, 4.8 Hz, 2H), 7.21–7.04 (m, 8H), 6.96 (dd, J = 8.5,

2.0 Hz, 1H), 5.03 (s, 1H), 4.34 (dd, J = 8.1, 4.5 Hz, 1H), 4.29

(td, J = 8.6, 4.7 Hz, 1H), 4.22 (dd, J = 5.5, 2.8 Hz, 2H), 4.03 (dd,

J = 6.3, 4.7 Hz, 1H), 3.84 (d, J = 2.2 Hz, 3H), 3.69–3.59 (m, 1H),

2.85 (dd, J = 14.3, 5.1 Hz, 1H), 2.76–2.67 (m, 1H), 2.65–2.51 (m,

2H), 2.46–2.35 (m, 2H), 2.24 (s, 3H), 2.06–1.93 (m, 1H), 1.90–

1.78 (m, 1H), 1.06 (d, J = 6.3 Hz, 2H), 1.00 (d, J = 6.3 Hz, 1H).

HRMS (ESI+): calculated for C₃₄H₃₉N₃O₆[M + H] 586.2917;

found 586.2931.

Compound 4

1H NMR (400 MHz, d6 DMSO) δ: 0.99 (d, 3H, J = 6.3 Hz), 1.83

(m, 1H), 1.98 (m, 1H), 2.25 (s, 3H), 2.57 (m, 2H), 3.56 (d, 1H,

J = 14.1 Hz), 3.60 (d, 1H, J = 13.8 Hz), 4.00 (m, 1H), 4.22 (m,

4H), 5.05 (d, 1H, J = 5.1 Hz), 6.84 (m, 1H), 6.98 (m, 3H), 7.12

(m, 9H), 7.27 (m, 3H), 7.36 (m, 2H), 8.03 (d, 1H, J = 8.1 Hz), 8.10

(d, 1H, J = 8.3 Hz), and 8.36 (t, 1H, J = 5.9 Hz). HRMS (ESI + ):

calculated for C₃₃H₃₉N₃O₅[M + H] 594.2968; found 594.2981.

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Novel non-covalent proteasome inhibitors

Table S2 Protease selectivity of compounds

Protease selectivity of compounds was assessed using ﬂuorigenic peptide-AMC substrates and a panel of puriﬁed human enzymes, as described in the Supplementary Experimental section.

Percentage inhibition of the proteases was determined in the presence of 100 μM compound and calculated relative to DMSO and the inhibitor control E64 or complete EDTA-free protease inhibitor

cocktail. Results are means obtained from two independent experiments, each performed in duplicate.

Enzyme inhibition at 100 μM compound (%)

Compound

Cathepsin B

Cathepsin L

Coagulation Factor β-XIIa

Chymotrypsin

Elastase

Plasmin

Thrombin

Tissue plasminogen activator

Trypsin

Calpain I

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

−15

15

1

12

5

22

16

4

45

37

34

38

36

26

31

36

34

32

40

38

33

35

42

49

−4

−3

1

−8

−6

−7

−3

−11

1

−20

−12

−15

−9

22

12

21

17

12

21

29

13

30

15

12

9

21

14

16

19

12

14

18

13

26

17

8

12

0

−9

17

−12

29

5

−1

−3

1

3

20

−8

84

3

−18

−11

−12

−11

−13

−18

5

−2

−6

2

1

3

−1

15

2

−2

−4

1

3

20

3

8

−9

33

3

18

−6

21

28

0

−11

55

25

22

−40

7

50

15

32

6

−3

−14

−13

17

−7

−6

−8

−6

10

9

−1

−6

−5

−4

12

−4

2

36

2

0

−10

−4

23

−1

1

−10

−18

−15

11

−10

7

−3

2

15

34

12

34

14

20

Table S3 Anti-proliferative effects of compound 1 and bortezomib in cancer

cell lines

Compound 9

1H NMR (400 MHz, d6 DMSO) δ: 0.78 (s, 9H), 1.83 (m, 1H), 2.01

(m, 1H), 2.25 (s, 3H), 2.72 (m, 5H), 4.21 (m, 3H), 4.76 (d, 1H),

5.17 (s, 2H), 7.16 (m, 11H), 7.39 (m, 6H), 7.84 (m, 3H), 8.21 (d,

1H, J = 8.1 Hz), 8.42 (m, 1H), and 8.53 (d, 1H). HRMS (ESI+):

calculated for C₄₁H₄₈N₄O₅[M + H] 677.3703; found 677.3729.

The indicated cell lines (1000–2000 cells per well) were incubated with a range of concentrations

of compound 1 or bortezomib for 72 h under cell culture conditions. Cell viability was

assessed my monitoring cellular ATP levels using the luminescence-based ATPlite assay as

+

described in the Experimental section of the main paper. IC₅₀data are means S.E.M. for 3–11

determinations.

−

ATPlite cell viability LC₅₀(nM)

Cell line

Compound 1

Bortezomib

Compound 10

+

MDA-MB-231

Calu6

H460

HCT116

HT29

150 23

130 10

8.6 1.0

5.2 0.76

1H NMR (400 MHz, d6 DMSO) δ: 0.78 (s, 9H), 1.82 (m, 1H),

2.03 (m, 1H), 2.24 (s, 3H), 2.44 (m, 3H), 2.63 (m, 3H), 2.80 (m,

4H), 4.14 (m, 2H), 4.25 (m, 1H), 4.60 (m, 1H), 6.98 (m, 3H), 7.11

(m, 7H), 7.25 (m, 3H), 7.85 (t, 1H, J = 6.1 Hz), 8.22 (d,d, 2H,

J = 5.4, 7.7 Hz), and 8.45 (t, 1H, J = 6.1 Hz). HRMS (ESI+):

calculated for C₃₆H₄₅FN₄O₄[M + H] 617.3503; found 617.3527.

−

+

−

+

380 110

13 2.0

−

+

50 8.1

4.6 0.68

−

+

55 8.9

5.4 1.1

−

Compound 7

Compound 11

1H NMR (400 MHz, d6 DMSO) δ: 1.12 (d, 3H, J = 6.3 Hz), 1.90

(m, 2H), 2.60 (m, 2H), 2.75 (s, 1H), 2.84 (s, 2H), 4.09 (m, 1H),

4.48 (m, 3H), 4.78 (d, 1H, J = 6.4 Hz), 4.91 (m, 1H), 5.19 (s,

2H), 7.20 (m, 11H), 7.33 (t, 2H, J = 7.4 Hz), 7.40 (t, 2H, J = 6.1

Hz), 7.47 (d, 2H, J = 7 Hz), 7.89 (m, 2H), 7.97 (m, 1H), and

8.29 (m, 1H). HRMS (ESI+): calculated for C₃₆H₃₉N₃O₅[M + H]

594.2968; found 594.2967.

1H NMR (400 MHz, d6 DMSO) δ: 0.75 (s, 9H), 1.25 (d, 3H,

J = 7.4 Hz), 2.71 (m, 2H), 2.80 (d, 2H, J = 6.2 Hz), 4.27 (m, 3H),

4.67 (m, 1H), 7.02 (m, 1H), 7.19 (m, 1H), 7.26 (m, 2H), 7.35 (m,

1H), 7.53 (m, 1H), 7.89 (t, 1H, J = 6.3 Hz), 8.22 (m, 1H), 8.32

(d, 1H, J = 7.3 Hz), 8.46 (t, 1H, J = 6.1 Hz), 8.71 (d, 1H, J = 8.1

Hz), 8.75 (s, 1H), and 12.25 (s, 1H). HRMS (ESI+): calculated

for C₂₉H₃₃F₂N₅O₅[M + H] 570.2528; found 570.2510.

Compound 8

Compound 12

1H NMR (300 MHz, d6 DMSO) δ: 1.12 (d, 3H, J = 6.3 Hz), 1.89

(m, 1H), 2.01 (m, 1H), 2.63 (m, 2H), 3.31 (s, 1H), 4.13 (m, 1H),

4.38 (m, 2H), 4.43 (m, 1H), 5.00 (d, 1H, J = 6.5 Hz), 5.19 (s,

2H), 7.10 (d, 2H, J = 9.1 Hz), 7.16 (m, 3H), 7.36 (m, 11H), 7.87

(d, 2H, J = 8.98 Hz), 7.99 (d, 1H, J = 8 Hz), 8.22 (d, 1H, J = 8

Hz), and 8.39 (t, 1H, J = 5.9 Hz). HRMS (ESI+): calculated for

C₃₅H₃₆ClN₃O₅[M + H] 614.2422; found 614.2404.

1H NMR (400 MHz, d6 DMSO) δ: 0.75 (s, 9H), 1.27 (d, 3H,

J = 6.9 Hz), 2.41 (m, 3H), 2.71 (m, 5H), 4.24 (t, 1H, J = 7.4 Hz),

4.32 (m, 2H), 4.58 (d, 1H, J = 6.8 Hz), 7.18 (m, 3H), 7.27 (m,

5H), 7.41 (m, 1H), 7.84 (t, 1H, J = 5.7 Hz), 8.14 (d, 1H, J = 8.1

Hz), 8.33 (d, 1H, J = 6.9 Hz), and 8.51 (t, 1H, J = 5.3 Hz). HRMS

(ESI+): calculated for C₂₈H₃₇ClN₄O₄[M + H] 529.2582; found

529.2602.

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C. Blackburn and others

10H), 5.77 (ddt, J = 17.2, 10.3, 5.1 Hz, 1H), 5.10 (dq, J = 17.2,

1.7 Hz, 1H), 5.03–4.98 (m, 1H), 4.60 (dd, J = 14.5, 7.4 Hz, 1H),

4.15–4.07 (m, 1H), 3.67 (dd, J = 12.1, 5.2 Hz, 2H), 2.94–2.75 (m,

4H), 2.69–2.57 (m, 2H), 2.56–2.51 (m, 2H), 2.42 (dd, J = 9.2, 6.5

Hz, 2H), 2.08–1.96 (m, 1H), 1.90–1.72 (m, 1H), 0.81 (s, 9H).

HRMS (ESI+): calculated for C₃₁H₄₂N₄O₄[M + H] 535.2384;

found 535.328.

Compound 13

1H NMR (400 MHz, d6 DMSO) δ: 0.75 (s, 9H), 2.36 (m, 4H),

2.56 (m, 1H), 2.74 (m, 3H), 2.89 (m, 1H), 3.18 (m, 1H), 4.35 (m,

2H), 4.54 (m, 2H), 7.17 (d, 3H, J = 6.5 Hz), 7.28 (m, 6H), 7.42

(m, 1H), 7.72 (d, 1H, J = 7.9 Hz), 7.84 (t, 1H, J = 3.9 Hz), 8.08 (d,

1H, J = 7.7 Hz), 8.36 (d, 1H, J = 7.9 Hz), 8.44 (m, 2H), and 8.60

(t, 1H, J = 5.4 Hz). HRMS (ESI+): calculated for C₃₃H₄₀ClN₅O₄

[M + H] 606.2847; found 606.2866.

Compound 16

Compound 14

1H NMR (300 MHz, d6 DMSO) δ: 0.74 (s, 9H), 2.66 (m, 2H),

2.79 (m, 2H), 2.91 (m, 1H), 3.10 (m, 1H), 4.34 (d, 2H, J = 5.8

Hz), 4.64 (m, 2H), 7.27 (m, 7H), 7.42 (m, 1H), 7.54 (m, 1H), 7.87

(t, 1H, J = 6.2 Hz), 8.23 (m, 1H), 8.37 (d, 2H, J = 5.93 Hz), 8.44

(d, 1H, J = 8.24 Hz), 8.62 (t, 1H, J = 5.84 Hz), 8.72 (m, 2H),

and 12.26 (m, 1H). HRMS (ESI+): calculated for C₃₄H₃₇ClN₆O₅

[M + H] 645.2592; found 645.2586.

1H NMR (400 MHz, DMSO) δ 8.20 (d, J = 7.8 Hz, 1H), 8.08

(d, J = 7.9 Hz, 1H), 7.88 (dd, J = 13.3, 7.2 Hz, 1H), 7.77 (d,

J = 7.8 Hz, 1H), 7.22 (dd, J = 13.2, 6.5 Hz, 4H), 7.13 (t, J = 7.3

Hz, 6H), 4.55 (dd, J = 14.2, 7.5 Hz, 1H), 4.05 (s, 1H), 3.80 (dd,

J = 13.9, 6.5 Hz, 2H), 2.89–2.83 (m, 2H), 2.80–2.73 (m, 2H),

2.70 (t, J = 2.7 Hz, 2H), 2.44–2.36 (m, 3H), 1.97 (s, 1H), 1.74 (s,

1H), 1.02 (dd, J = 18.7, 6.6 Hz, 6H), 0.80 (s, 9H). HRMS (ESI+):

calculated for C₃₁H₄₄N₄O₄[M + H] 537.3441; found 537.3442.

REFERENCE

Compound 15

1

Soucy, T. A., Smith, P. G., Milhollen, M. A., Berger, A. J., Gavin, J. M., Adhikari, S.,

Brownell, J. E., Burke, K. E., Cardin, D. P., Critchley, S. et al. (2009) An inhibitor of

NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–737

1H NMR (400 MHz, DMSO) δ 8.20 (dd, J = 7.9, 3.2 Hz, 2H),

8.11 (t, J = 5.7 Hz, 1H), 7.87 (t, J = 6.2 Hz, 1H), 7.33–7.07 (m,

Received 15 March 2010/2 July 2010; accepted 15 July 2010

Published as BJ Immediate Publication 15 July 2010, doi:10.1042/BJ20100383

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