Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7

Article

Structure-Guided Development of a Potent and

Selective Non-covalent Active-Site Inhibitor of USP7

Graphical Abstract

Authors

Ilaria Lamberto, Xiaoxi Liu,

Hyuk-Soo Seo, ...,

Dharminder Chauhan,

Sirano Dhe-Paganon, Sara J. Buhrlage

Correspondence

dhepag@crystal.harvard.edu (S.D.-P.),

saraj_buhrlage@dfci.harvard.edu (S.J.B.)

In Brief

Lamberto et al. report the structure-

guided development of inhibitors of the

deubiquitinating enzyme (DUB) USP7.

The studies provide optimized and well-

characterized probes for studying USP7

in normal and disease biology and,

furthermore, lend validation to the notion

that potent and selective active-site

inhibitors of DUBs can be achieved.

Highlights

d

Functional and structural characterization of USP7 inhibitors

Inhibitors bind the S4-S5 pocket of the enzyme

Inhibitors exhibit a high degree of selectivity for USP7 relative

to 40 other DUBs

Lamberto et al., 2017, Cell Chemical Biology 24, 1–11

December 21, 2017 ª 2017 Elsevier Ltd.

http://dx.doi.org/10.1016/j.chembiol.2017.09.003

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

Cell Chemical Biology

Article

Structure-Guided Development of a Potent

and Selective Non-covalent Active-Site

Inhibitor of USP7

Ilaria Lamberto,¹^,⁵Xiaoxi Liu, Hyuk-Soo Seo, Nathan J. Schauer, Roxana E. Iacob, Wanyi Hu, Deepika Das,

1,5

1

3

1

2

4

2

3

2

Tatiana Mikhailova, Ellen L. Weisberg, John R. Engen, Kenneth C. Anderson, Dharminder Chauhan,

Sirano Dhe-Paganon, * and Sara J. Buhrlage

1,

1,4,6,

*

1

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA

²Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA

³Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA

⁴Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA

⁵These authors contributed equally

⁶Lead Contact

*Correspondence: dhepag@crystal.harvard.edu (S.D.-P.), saraj_buhrlage@dfci.harvard.edu (S.J.B.)

http://dx.doi.org/10.1016/j.chembiol.2017.09.003

SUMMARY

quitinating enzymes (DUBs). The ﬁrst recognized role of the ubiq-

uitin system was controlling protein turnover (Ciechanover et al.,

Deubiquitinating enzymes (DUBs) have garnered 1980; Hershko et al., 1980). Ubiquitin tags are also responsible

signiﬁcant attention as drug targets in the last 5–10 for signaling a wide range of non-degradative functions. Ubiqui-

tination can affect protein activity by modulating conformational

changes, complexation with other proteins (Ea et al., 2006; Wu

years. The excitement stems in large part from the

powerful ability of DUB inhibitors to promote degra-

dation of oncogenic proteins, especially proteins

that are challenging to directly target but which are

stabilized by DUB family members. Highly optimized

and well-characterized DUB inhibitors have thus

become highly sought after tools. Most reported

DUB inhibitors, however, are polypharmacological

et al., 2006), susceptibility to addition of other post-translation

modiﬁcations including phosphorylation and acetylation (Hunter,

2007; Zhang et al., 2008; Zhao et al., 2008), and cellular localiza-

tion (Li et al., 2003). Through combined degradative and non-

degradative functions, ubiquitination coordinates a wide range

of cellular processes including proteolysis (Ciechanover et al.,

2000), DNA repair (Jackson and Durocher, 2013), chromatin

agents possessing weak (micromolar) potency to- remodeling (Weake and Workman, 2008), receptor signaling

ward their primary target, limiting their utility in target (Haglund and Dikic, 2012), and immunity (Malynn and Ma,

validation and mechanism studies. Due to a lack of 2010; Zinngrebe et al., 2014), among others. Not surprisingly,

aberrant ubiquitin system activity is linked to disease, including

high-resolution DUB,small-molecule ligand complex

structures, no structure-guided optimization efforts

cancer (Hoeller and Dikic, 2009; Pinto-Fernandez and Kessler,

2

016), infection (Isaacson and Ploegh, 2009; Maculins et al.,

016), and neurodegeneration (Ciechanover and Brundin,

003; Ciechanover and Kwon, 2015). The relationship between

have been reported for a mammalian DUB. Here,

we report a small-molecule,ubiquitin-speciﬁc prote-

ase (USP) family DUB co-structure and rapid design

of potent and selective inhibitors of USP7 guided

by the structure. Interestingly, the compounds are

non-covalent active-site inhibitors.

ubiquitin and cancer biology has been clinically validated by

Food and Drug Administration approval of the proteasome inhib-

itor bortezomib for multiple myeloma (Kane et al., 2003).

There are approximately 100 human DUBs belonging to six

distinct families, ﬁve of which (ubiquitin-speciﬁc protease

[USP], ubiquitin C-terminal hydrolase [UCH], ovarian tumor

INTRODUCTION

protease [OTU], Josephin, and Mindy) are cysteine proteases,

while the sixth (JAB/MPN/MOV34 [JAMM/MPN]) is composed

Ubiquitin is a 76-amino-acid protein attached to substrate of zinc metalloproteases (Abdul Rehman et al., 2016; Clague

proteins post-translationally via isopeptide bond formation et al., 2013; Komander et al., 2009; Komander and Rape,

between ubiquitin’s C-terminal glycine and a substrate lysine 2012). Many DUBs have been linked to physiological and/or

side chain (Komander and Rape, 2012); linear and branched pathophysiological functions. For example, USP1 and USP4

polyubiquitin chains are assembled via attachment of another are involved in DNA-damage repair (Kee and Huang, 2015),

molecule of ubiquitin to one of seven lysines or the N-terminal USP22 and BAP1 have a role in chromatin function (Atanassov

methionine of ubiquitin (Pickart and Fushman, 2004). Ubiquitin et al., 2011), and USP2 and USP8 are reported to stabilize

is attached to substrate proteins by the coordinated action of oncogenic proteins cyclin D1 (Shan et al., 2009) and mutant

ubiquitin-activating (E1), conjugating (E2), and ligating (E3) epidermal growth factor receptor (Byun et al., 2013), respec-

enzymes and removed by a family of proteases known as deubi- tively. While dozens of apo- and ubiquitin-bound structures

Cell Chemical Biology 24, 1–11, December 21, 2017 ª 2017 Elsevier Ltd.

1

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

have been solved (Hu et al., 2002; Johnston et al., 1997; poor solubility and general toxicity (Chen et al., 2017). Additional

Komander et al., 2009), very few have been achieved with USP7 inhibitors (shown in Figure S1B) have been identiﬁed,

non-ubiquitin-based compounds (Davies et al., 2012; Ratia although none possess features superior to P5091/P22077 and

et al., 2008; Schlierf et al., 2016). Notably, small-molecule,DUB signiﬁcant optimization efforts have not been undertaken (Aleo

complex structures are lacking for the largest 56-member et al., 2006; Colland et al., 2009; El-Desoky et al., 2017; Nichol-

mammalian USP family. son et al., 2008; Reverdy et al., 2012; Tanokashira et al., 2016;

The ﬁrst DUB inhibitor, the dual USP14/UCHL5 inhibitor Yamaguchi et al., 2013).

VLX1570, entered clinical trials in 2015 (Wang et al., 2016b).

Here we report the structure-guided development of next-

Overall, however, DUB inhibitor development is still in its early generation small-molecule probes of USP7. High-resolution

stages. Approximately 40 DUB inhibitors have been reported, USP7,small-molecule crystal structures enabled us to rapidly

although most are weak, multi-targeted agents (D’Arcy et al., develop XL188, a highly selective 90 nM inhibitor of USP7,

2

015; Ndubaku and Tsui, 2015). Given the current dearth of from a 7.2-mM lead, as a probe of USP7. Furthermore, we

potent and selective inhibitors, skepticism remains as to whether show that XL203C, the enantiomer of XL188, is more than

or not this enzyme class will be druggable in a manner analogous 80-fold less potent against USP7, and thus serves as an inactive

to that of protein kinases, for example. A signiﬁcant hindrance to control compound. In contrast to P22077/P5091, which target

the generation of potent and selective DUB inhibitors is a lack of the invariant catalytic cysteine of USP DUBs, XL188 is a non-

structure-guided optimization efforts. One example of structure- covalent active-site inhibitor. We demonstrate that the

guided development of a DUB inhibitor, which targeted the XL188/XL203C active/inactive inhibitor pair is a powerful combi-

SARS DUB PLPro (Baez-Santos et al., 2015), generated com- nation for studying USP7 function in cellular models.

pounds with half-maximal inhibitory concentrations (IC50) below

500 nM and exhibiting a high degree of selectivity relative to RESULTS

mammalian DUBs. In this case, selectivity was explained by

signiﬁcant structural differences between viral and mammalian XL188 Is a Potent and Selective Inhibitor of USP7

DUBs. Breakthroughs in X-ray crystallography of small-molecule As part of an effort to identify chemical starting points for devel-

DUB inhibitor complexes have the potential to enable rapid opment of DUB inhibitors by proﬁling the inhibitory activity of

development of potent and selective inhibitors of mamma- compounds reported in peer-reviewed and patent literature

lian DUBs.

for activity against large panels of DUBs (Ritorto et al., 2014),

The DUB USP7 has been shown to be involved in regulation of we identiﬁed a highly selective inhibitor of USP7 (1, structure

a myriad of cellular processes, including epigenetics, cell cycle, in Figure 1A) reported in a 2013 patent from Hybrigenics (Col-

DNA repair, immunity, viral infection, and tumorigenesis. USP7, land and Gourdel, 2013; Kessler, 2014). When screened for

also known as HAUSP (herpes virus-associated ubiquitin- inhibitory activity across a panel of 38 puriﬁed DUBs at a con-

speciﬁc protease), was ﬁrst discovered as a protein that plays centration of 100 mM, USP7 was the only DUB substantially

a role in viral lytic growth (Everett et al., 1997). Interest in the inhibited (Figure S1C and Table S1). Dose-response analysis

enzyme intensiﬁed when USP7 was implicated in regulating using USP7 catalytic domain (amino acids 208–560) or full-

degradation of the tumor suppressor p53 (Li et al., 2002) by length enzyme (1–1,102) and ubiquitin-aminomethylcoumarin

stabilizing the major E3 ligase for p53, MDM2 (Cummins et al., (Ub-AMC) as substrate conﬁrmed USP7 inhibitory activity,

2

004; Li et al., 2004). Recently several epigenetic modiﬁers, although potency was weak with IC50s in the double-digit

including the methyltransferase PHF8 (Wang et al., 2016a), micromolar range (Figures 1B and S1D). Isothermal titration

demethylase DNMT1(Du et al., 2010; Felle et al., 2011; Qin calorimetry (ITC), using the catalytic domain, conﬁrmed binding

et al., 2011), and acetyltransferase Tip60 (Dar et al., 2013), as with a dissociation constant (K

D

) of 8 mM (Figure S1E and Table

well as H2B itself (van der Knaap et al., 2005), have been identi- S2). We solved the structure of USP7 bound by 1, which

ﬁed as direct targets of USP7. Other notable targets of USP7 enabled rapid structure-guided development of XL188 (Fig-

include the transcription factors FOXP3, which in T-regulatory ure 1A), a highly potent and selective inhibitor of USP7.

(

Treg) cells links this DUB enzyme to immune response (van XL188 inhibited USP7 catalytic domain and full-length enzyme

Loosdregt et al., 2013), and N-Myc, which is stabilized in with IC₅₀values of 193 and 90 nM, respectively (Figure 1B). The

neuroblastoma cells (Tavana et al., 2016). Consistent with its interaction of XL188 with USP7 was conﬁrmed using ITC and

regulation of diverse substrates and biological processes, differential scanning ﬂuorimetry (DSF) (Figures 1C and S1F;

USP7 has emerged as a drug target in a wide range of malig- Table S2). Consistent with the 100-fold improvement in

nancies including multiple myeloma (Chauhan et al., 2012), biochemical inhibition of USP7 by XL188 compared with 1, a

breast cancer (Wang et al., 2016a), neuroblastoma (Tavana

et al., 2016), glioma (Cheng et al., 2013), and ovarian cancer ITC (Figure 1C, Table S2). The selectivity of XL188 was as-

Zhang et al., 2016).

sessed against a panel of 41 puriﬁed DUBs, using ubiquitin-

P22077 and its close analog P5091 are the inhibitors most rhodamine (Ub-Rho) as substrate. XL188 retained the excellent

D

K of 104 nM was measured for USP7 catalytic domain using

(

frequently utilized to probe USP7 functions (for structures see selectivity for USP7 observed with 1; at a concentration of

Figure S1A). P22077 exhibits modest potency against USP7 10 mM, XL188 exhibited little to no inhibition of any DUBs other

(

IC50 = 8.0 mM) and equipotent inhibition of two additional than USP7 (Figure 1D and Table S1). In contrast, the enan-

DUBs, USP10 and USP47 (Altun et al., 2011; Ritorto et al., tiomer of XL188, XL203C (Figure 1A), showed 80-fold less

014). In addition to modest potency and selectivity, reported potent inhibition of USP7 (IC₅₀= 7.18 mM, Figure 1B) and no

drawbacks of these nitrothiophene-based compounds include signiﬁcant inhibition of other DUBs (Figure S1G and Table S1).

2

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Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

A

O

N

Structure-guided

design

O

N

O

N

OH

N

O

N

O

N

Cl

>100-fold potency

improvement

N

H

N

H

O

N

XL188

O

N

XL203C

O

1

B

C

USP7 catalytic domain

USP7 full-length

Time (s)

1

0

.2

.0

.8

1.2

1.0

0.8

0.6

0.4

0.2

1

XL188

0

.02

0

XL203C

0.6

0.4

0.2

-0.02

-

0.04

0.06

-0.08

-0.10

-0.12

-0.14

-0.16

0.0

0

0.01 0.1

1

10 100

0

0.01 0.1

1

10 100

Compound (μM)

2

0

-2

-4

Protein

Compound

IC₅₀(μM) ± SEM (n)

12.3 ± 0.9 (18)

0.193 ± 0.006 (4)

10.7 ± 1.3 (2)

1

USP7 catalytic domain

aa 208-560)

XL188

XL203C

1

(

-6

-8

10

12

-14

K_d= 104 ± 15 nM

n = 1.06 ± 0.01

ΔH = -15.11 ± 0.13

kcal/mol

-

10.2 ± 3.1 (3)

USP7 full length

aa 1-1102)

XL188

XL203C

0.090 ± 0.016 (3)

7.18 ± 2.18 (3)

ΔS = -19.60 cal/mol•K

(

-

16

0

0.5 1.0 1.5 2.0 2.5 3.0

Mole ratio

D

1

40

20

00

1

80

60

40

20

0

Deubiquitinating enzyme

Figure 1. Structure and Selectivity of XL188

(

A) Structure-guided optimization of 1 led to USP7 inhibitor XL188. The enantiomer of XL188, XL203C, is 80-fold less active.

B) Dose-dependent inhibition of USP7 catalytic domain (amino acids 208–560) and full-length USP7 (amino acids 1–1,102) by 1, XL188, and XL203C using

Ub-AMC as substrate.

(

C) Assessment of XL188 binding to USP7 using isothermal calorimetry.

D) Inhibitory activity of XL188 across a panel of 41 puriﬁed DUBs using ubiquitin-rhodamine (Ub-Rho) as substrate.

See also Figure S5.

USP7 catalytic domain and Ub-AMC as substrate. 1 inhibited quinazolinone via a short carbon chain and amide bond linkage,

isolated USP7 catalytic domain with an IC50 of 12.3 mM and improved potency 2- to 4-fold relative to the parent compound 9.

full-length protein with an IC50 of 10.2 mM. Compounds 2 and With several analogs exhibiting submicromolar USP7 IC50s, we

3

, in which the hydroxypiperidine -OH group was removed or considered metabolic stability as an additional parameter for

replaced with -CN, respectively, exhibited little inhibition of compound prioritization, since a probe suitable for in vivo studies

USP7 at concentrations up to 100 mM. Similarly, removal of the would be highly valuable for pharmacological validation of USP7

amide carbonyl, as in 4, abrogated USP7 activity as did contrac- in animal disease models. XL188 exhibited the greatest stability

tion of the 6-membered piperidine to a 5-membered ring (5). in the presence of mouse liver microsomes with a half-life of

Occupancy of the S4 hydrophobic pocket by the phenyl ring 31 min (Figure 4B). Competitive activity-based protein proﬁling

was conﬁrmed to be required for activity, as removal of the moi- using the DUB-targeting activity-based probe HA-Ub-Vs

ety (6) or shortening the linker between the hydroxypiperidine (Hewings et al., 2017) conﬁrmed that XL188 bound native

and phenyl (7) resulted in biochemical IC50s > 100 mM. In USP7 (Figure 4C). Treatment of HEK293T lysates with XL188

contrast, installation of a racemic methyl group on the methylene signiﬁcantly blocked labeling of USP7 by HA-Ub-Vs with an

adjacent to the phenyl ring (8) improved biochemical potency IC50 of approximately 0.9 mM, a value that represents a signiﬁ-

approximately 10-fold, consistent with data reported in the cant improvement compared with 1 (at a concentration of

patent exemplifying 1. To explore the importance of stereo- 500 mM, 1 competes for 50% of probe labeling [Figure S5A]).

chemistry of the methyl group we prepared both enantiomers, Because in this experiment an irreversible probe (HA-Ub-Vs) is

which revealed that the (R)-stereoisomer (9) was approximately competing for occupancy of USP7 with a reversible inhibitor,

1

00-fold more potent than the (S)-stereoisomer (10). Unfortu- the measured cell-based IC50s may underestimate binding. A

nately, investigation of 9’s ability to bind and inhibit USP7 in cells hemagglutinin blot of the same treated lysates (Figure S5B)

was hampered by poor solubility in aqueous buffer. To improve conﬁrmed the high degree of selectivity for USP7 observed in

this property we focused on installation of polar moieties in place the 41-member puriﬁed enzyme panel. As a measure of general

of the solvent-exposed chloro atom (Figure 4B). Installation of toxicity, we treated peripheral blood mononuclear cells (PBMCs)

different groups, including an N-methyl-piperazine, piperidine, with XL188 and observed no growth suppression at concentra-

dimethylamine, and imidazole, linked to the 7-position of the tions up to 10 mM following 72 hr of treatment (Figure S5C). To

6

Cell Chemical Biology 24, 1–11, December 21, 2017

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

Figure 5. The USP7 Inhibitor XL188

A

B

+

-

+

1

-

+

5

-

+

-

1

+

-

5

+

-

+

-

cycloheximide

XL188 (μM)

10 20 XL203C (μM)

XL188 (μM)

10 20

XL203C (μM)

10 20

Promotes Loss of HDM2 and Accumulation

of p53 and p21

10 20

-

1

5

1

5

-

HDM2

p53

(

A) Analysis of HDM2, p53, and p21 protein levels

HDM2

in MCF7 cells treated with XL188 or XL203C at the

indicated concentration for 16 hr.

p53

p21

(B) Analysis of HDM2, p53, and p21 protein levels

in MCF7 cells following 16 hr of treatment with

XL188 or XL203C at the indicated concentration

with addition of cycloheximide for the last 2 hr.

p21

GAPDH

MCF7

(

C) Analysis of HDM2, p53, and p21 protein levels

C

D

in MM.1S cells treated with XL188 or XL203C at

the indicated concentration for 6 hr.

XL188 (μM)

10 20

XL203C (μM)

10 20

+

-

+

1

-

+

5

-

+

-

5

+

-

+

-

cycloheximide

XL188 (μM)

10 20 XL203C (μM)

-

1

5

1

5

10 20

-

(

D) Analysis of HDM2, p53, and p21 protein

HDM2

p53

-

levels in MM.1S cells following 6 hr of treatment

with XL188 or XL203C at the indicated concen-

tration with addition of cycloheximide for the

last 2 hr.

HDM2

p53

p21

GAPDH

MM.1S

achieve an ideally matched negative control compound to use in basic and disease biology have increased in recent years, these

conjunction with XL188, we prepared its enantiomer, XL203C. efforts have produced limited numbers of well-characterized and

XL203C showed 80- to 100-fold less potent inhibition of USP7 optimized inhibitors. The DUB enzyme USP7 is one of the most

relative to XL188 in both biochemical (Figures 1B and 4B) and well-studied DUBs and is reported to regulate the abundance

cellular target engagement assays (Figure 4C).

of proteins involved in DNA-damage repair, cell-cycle regulation,

and epigenetic and transcription factor control of gene expres-

sion. Notably, USP7 has been implicated in a range of different

cancer types, controlling some of the most important proliferative

XL188 Promotes USP7-Dependent Loss of HDM2 and

Increase of p53 and p21

To further examine XL188’s on-target effects, we evaluated its processes. To identify the most promising disease contexts in

impact on the USP7-HDM2-p53-p21 axis. The role of USP7 in which to deploy USP7 drugs and to more accurately catalog

promoting degradation of HDM2 resulting in increased levels the activities of USP7 as it pertains to both the promise and liabil-

of p53 and p21 is documented in multiple models (Chauhan ities associated with blockade of USP7 activity, highly potent and

et al., 2012; Kon et al., 2010). In MCF7 cells treated with well-characterized probe compounds have been sought after.

XL188, levels of p53 and p21 increased following 16 hr of treat- Here, we present an inhibitor of USP7, XL188, with double-digit

ment with concentrations in line with the IC50 of native USP7 nanomolar potency toward USP7 and exquisite selectivity rela-

(

Figure 5A). Consistent with the effects being USP7 depen- tive to the largest available panel of puriﬁed DUBs. We combined

dent, no observable change in levels of the same proteins XL188 with a negative control compound, XL203C, to present a

across the same range of concentrations with the compara- set of tools for interrogating USP7 biology. Thus, we conﬁrmed

tively inactive enantiomer XL203C was observed (Figure 5A). USP7 as a key regulator of HDM2-p53-p21 signaling by showing

Degradation of HDM2 by XL188 was observed in the same that XL188, but not XL203C, promoted degradation of HDM2, re-

system when new protein synthesis was blocked by addition sulting in increased levels of p53 and p21.

of cycloheximide (Figure 5B). The necessity to block protein

Our observation that the IC50s for minimum and full-length

synthesis to observe degradation of HDM2 is consistent with USP7 are similar (190 versus 90 nM), in light of the fact that

the established negative feedback loop involving these USP7 catalytic domain efﬁciency is augmented by the presence

proteins in which p53 transcriptionally upregulates HDM2 of the C-terminal Ubl domains (Faesen et al., 2011), may suggest

(

Wu et al., 1993). Similarly, treatment of multiple myeloma that the kinetics of the equilibrium between active and inactive

MM.1S cells with XL188, but not XL203C, led to loss of states of the full-length enzyme is rapid. Alternatively, if we pro-

HDM2 accompanied by an increase in downstream tumor sup- pose an additional stable intermediate toward the activated

pressors p21 and p53, as has previously been reported for the state, represented by Phe409 ﬂipping, our results suggest that

USP7 inhibitor P5091 (Figures 5C and 5D) (Chauhan the full-length construct stabilizes this intermediate to a greater

et al., 2012).

extent compared with the minimal construct. Although prema-

ture, we might further suggest that the mechanism by which

the C-terminal domains upregulate the catalytic domains is by

stabilizing Phe409 ﬂipping.

DISCUSSION

Through their role in reversing ubiquitination and coordinating a

XL188 represents one of only a small set of mammalian

wide range of cellular processes including proteolysis, DNA DUB inhibitors with low nanomolar potency and a high degree

repair, receptor signaling, transcription, and immunity, DUBs of selectivity relative to other DUBs (Dexheimer et al., 2010;

are emerging as a new class of drug targets. Although efforts to Ndubaku and Tsui, 2015). On comparing the state of the DUB

develop small-molecule probes of DUBs to study their role in ﬁeld with the established kinase ﬁeld, the ﬁrst publication on

Cell Chemical Biology 24, 1–11, December 21, 2017

7

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

phosphorylation came 23 years prior to the ﬁrst publication on

ubiquitination, and the current state of modulating ubiquitylation

via DUBs is roughly where drugging phosphorylation via kinases

was 20 years ago (Cohen and Tcherpakov, 2010). In this

project, rapid optimization of XL188 from 1 was enabled largely

by our success in achieving a high-resolution crystal structure

of the lead compound in complex with USP7. We established a

series of biochemical, biophysical, and selectivity assays to iden-

tify promising compounds for crystallographic and medicinal

chemistry studies. By applying similar triaging assays, we believe

that similar success in crystallography and small-molecule probe

development can be achieved for other DUB family members.

Overall, our studies provide optimized and well-characterized

small-molecule probes for use in studying the roles of USP7 in

normal and disease biology and, furthermore, lend considerable

validation to the notion that DUBs are a class of enzymes for

which potent and selective inhibitors can be achieved.

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Cell Lines

B Primary Cell Cultures

d METHOD DETAILS

B Chemical Synthesis

B USP7 Cloning, Expression, and Puriﬁcation

B Ub-AMC Assay

B Differential Scanning Fluorimetry

B Isothermal Titration Calorimetry

B Selectivity Proﬁling

B Mouse Liver Microsome Stability

B Competitive Activity Based Protein Proﬁling

B Cell Treatments

B Peripheral Blood Mononuclear Cell Testing

B Crystallization, Data Collection and Structure Determi-

nation

B Hydrogen Exchange Experiments

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND SOFTWARE AVAILABILITY

SIGNIFICANCE

SUPPLEMENTAL INFORMATION

DUBs are a 100-member family of proteases that control

levels and activation of proteins through removal of post-

translational ubiquitin moieties. Of particular interest to

drug discovery, DUBs stabilize disease-linked proteins,

including many targets that remain undruggable. Thus,

DUB-targeting drugs can promote degradation of disease-

causing proteins, representing a novel strategy for modu-

lating the activity of challenging targets in cancer and other

diseases. Progress in the development of potent and selec-

tive DUB inhibitors for use as chemical probes and transla-

tion to the clinic has been slow, however. Of note, struc-

ture-guided medicinal chemistry campaigns have been

lacking for mammalian DUBs, suggesting that success in

crystallography of small-molecule DUB co-structures

enabling such efforts could signiﬁcantly affect the ﬁeld.

We succeeded in solving high-resolution X-ray crystal

structures of USP7 bound by a small-molecule ligand series.

The co-structures enabled us to rapidly improve the potency

of the lead inhibitor 100-fold to 90 nM. Furthermore, we show

that the newly developed inhibitor exhibits exquisite selec-

tivity for USP7 relative to broad panels of puriﬁed and

cellular DUBs and that the compound, but not a negative

control stereoisomer, promotes degradation of a known

USP7 substrate. The developed compounds are thus vali-

dated as high-quality probes for pharmacological interroga-

tion of the diverse reported functions of USP7. Importantly,

this work provides proof of concept that potent and selec-

tive DUB inhibitors can be achieved and that structural char-

acterization of chemical leads with their cognate DUB target

could accelerate probe and drug discovery programs.

Supplemental Information includes NMR spectra, ﬁve ﬁgures, and four tables

and can be found with this article online at http://dx.doi.org/10.1016/j.

chembiol.2017.09.003 .

AUTHOR CONTRIBUTIONS

S.J.B., S.D.-P., D.C., and K.C.A. initiated the project, and S.J.B. and

S.D.-P. oversaw all aspects of the project. I.L. ran biochemical assays,

designed and generated USP7 mutants, and designed and performed

cell experiments. X.L. designed and synthesized reported compounds.

H.-S.S. and S.D.-P. carried out crystallographic studies and ITC and DSF

experiments. T.M. generated USP7 mutants and performed biochemical

assays. W.H. synthesized reported compounds. N.J.S. and D.D. performed

cell experiments. R.E.I. and J.R.E. are responsible for the HDX MS studies.

D.C. and K.C.A. oversaw cell studies. E.L.W. tested compounds on

PBMCs. S.J.B. and S.D.-P. wrote the manuscript with input from all

authors.

ACKNOWLEDGMENTS

Crystallographic work was based upon research conducted at the Advanced

Photon Source on the Northeastern Collaborative Access Team beamlines

(NIGMS P41 GM103403). Funding was also provided by NIH (R01

CA211681) (S.J.B., S.D., K.C.A), Dana-Farber/Northeastern University Joint

Program in Cancer Drug Development (S.J.B., R.E.I., E.L.W.), Ellison Founda-

tion (S.J.B.), Chleck Family Foundation (N.J.S.), and an award from TWD

Kemtech J. Wang (S.J.B.).

Received: May 29, 2017

Revised: August 9, 2017

Accepted: September 5, 2017

Published: October 19, 2017

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

Antibodies

SOURCE

IDENTIFIER

MDM2 (SMP14)

Santa Cruz

Cat# sc-965; RRID: AB_627920

Cat# 9282; RRID: AB_331476

Cat# 2947; RRID: AB_823586

Cat# 2118; RRID: AB_561053

Cat# 4833; RRID: AB_10557113

Cat# ab49969; RRID: AB_880330

P53

Cell Signaling Technology

Abcam

P21

GAPDH (14C10)

HAUSP (USP7) (D17C6)

HA-tag (HA-7)

Bacterial and Virus Strains

E.coli BL21DE3

EMD Millipore

Cat# 69450

E.coli 10beta

New England Biolabs

Cat# C3020K

Chemicals, Peptides, and Recombinant Proteins

Ubiquitin-AMC

Boston Biochem

Cat# U-550

Cat# U-212

HA-Ubiquitin-vinyl sulfone

Deposited Data

USP7/1 structure

USP7/XL188 structure

USP7/8 structure

Experimental Models: Cell Lines

Human: MM.1S

Protein databank

PDB: 5VSB

PDB: 5VS6

PDB: 5VSK

Kenneth C. Anderson’s laboratory

Jean Zhao’s laboratory

N/A

Human: MCF7

Human: 293T

James E. Bradner’s laboratory

Oligonucleotides

Primer for USP7 WT and mutant proteins,

see Table S4

This paper

Recombinant DNA

USP7 residues 208-560 in pET28aLIC

USP7 residues 1-1102 in pET28PP

Software and Algorithms

GraphPad Prism

This paper

GraphPad Software Inc

https://www.graphpad.com/

scientiﬁc-software/prism/

XDS

Kabsch, W. (2010) Acta Cryst. D66, 125-132

Winter, (2010) J. Appl. Cryst. 43, 186-190

Evans (2006) Acta Cryst. D62, 72-82

Emsley et al. (2010) Acta Cryst. D66,

http://xds.mpimf-heidelberg.mpg.de/

https://xia2.github.io

XIA2

POINTLESS

COOT

http://www.ccp4.ac.uk

https://www2.mrc-lmb.cam.ac.uk/

personal/pemsley/coot/

4

86-501

PYMOL

PHASER

PHENIX

The PyMOL Molecular Graphics System,

Schr o¨ dinger, LLC

https://pymol.org/

McCoy et al. (2007)

http://www.ccp4.ac.uk

https://www.phenix-online.org-

J. Appl. Cryst. 40, 658-674

Adams et al., 2010

CONTACT FOR REAGENT SHARING AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Sara

Buhrlage (saraj_buhrlage@dfci.harvard.edu).

e1 Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017

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EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell Lines

HEK293TcellswereobtainedfromtheJamesE.Bradner’slaboratoryandwerenotfurtherauthenticated.MM.1Scellswereobtainedfrom

Kenneth C. Anderson’s laboratory and were not further authenticated. MCF7 cells were obtained from Jean Zhao’s laboratory and were

not further authenticated. 293T cells were grown in DMEM with high glucose (Gibco 11965-118) supplemented with 10% heat inactivated

ꢀ

fetalbovineserum (FBS)(Gibco16140-071), andpenicillin/streptomycin(Gibco15140-122)andmaintainedinahumidiﬁed37 C/5% CO

2

incubator. MCF7 and MM.1S cells were grown in RPMI (Gibco 11875-119) supplemented with 10% heat inactivated fetal bovine serum

ꢀ

(

FBS) (Gibco 16140-071), and penicillin/streptomycin (Gibco 15140-122) and maintained in a humidiﬁed 37 C/5% CO

2

incubator.

Primary Cell Cultures

Primary cells were obtained through written consent under approval of the Dana-Farber Cancer Institute Institutional Review Board.

Peripheral blood mononuclear cells (PBMCs) from normal individuals were isolated by density gradient centrifugation through Ficoll-

Plaque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden) at 400xg for 25 minutes, followed by two washes in PBS. Cells

ꢀ

were then maintained in RPMI media, supplemented with 10% FBS, and maintained in a humidiﬁed 37 C/5% CO

2

incubator.

METHOD DETAILS

Chemical Synthesis

General Methods and Materials

All commercially available starting materials were purchased from Sigma Aldrich, Fisher Scientiﬁc, Oakwood Chemical and Combi

Block. All reagents were used as received without further puriﬁcation. Known compounds were synthesized according to published

literature procedures and any modiﬁcations are noted. Anhydrous solvents, such as tetrahydrofuran (THF), diethyl ether, dichlorome-

thane (DCM), dimethyl formamide (DMF), dimethylsulfoxide (DMSO), 1,4-dioxane, and toluene (PhMe) were purchased from Fisher Sci-

entiﬁc, and used as received. If necessary, air or moisture sensitive reactions were carried out under an inert atomsphere of nitrogen.

Removal of solvents was accomplished on a B u€ chi R-300 rotary evaporator and further concentration was done under a Welch

1

chromatography using Teledyne CombiFlash chromatography system, and/or reversed phase chromatography on Waters

400B-01 vacuum line, and Labconco FreeZone 6 plus system. Puriﬁcation of compounds was performed by normal phase column

ꢀ

TM

Micromass ZQ preparative system with SunFire Prep C18 OBD

5mM column. The purity was analyzed on Waters Acquity

UPLC system. Analytical thin layer chromatography (TLC) plates were purchased from Fisher Scientiﬁc (EMD Millipore TLC Silica

Gel60 F254). Visualization was accomplished by irradiation under UV light (254 nm).

1

13

All H-NMR spectra were recorded at 298K on a Bruker ARX 500 (500 MHz) spectrometer. C-NMR spectra were recorded on a

Bruker ARX 500 (126 MHz) spectrometer. Samples were dissolved in CDCl3, DMSO-d6, or CD OD. The spectra were referenced to

3

1

the residual solvent peak (chloroform-d: 7.26 ppm for H-NMR and 77.16 ppm for C-NMR; DMSO-d6: 2.50 ppm for H-NMR and

13

1

3

1

13

3

9.25 ppm for C-NMR, CD

standard. Chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad peak), coupling constants

Hz), and number of protons. Mass spectrometry (LCMS) data were obtained on Waters Acquity UPLC system in positive ESI mode.

Synthetic Procedures

3

OD: 3.31 ppm for H NMR and 49.00 ppm for C NMR or tetramethylsilane (TMS) as the internal

(

S1. R=Cl; S2. R=NO

-aminobenzoic acids (10.0mmol) and formamide (1.8g, 40.0mmol) were mixed in pressure tube, which was heated at 150 C

overnight. Then the reaction was cooled to room temperature. The solid was suspended in cold water, then collected by vacuum

2

ꢀ

2

ﬁltration, and dried on high vacuum line. The products 1.6g (-Cl) and 1.8g (-NO

2

) were isolated as light brown solid in 88% (-Cl)

2

and 95% (-NO ) yields with no further puriﬁcation.

S3

ꢀ

Sodium hydride (60% dispersion in mineral oil) (0.88g, 22.0mmol) was dissolved in 40mL anhydrous DMSO at 0 C under N

2

.

Trimethylsulfoxonium iodide (4.84g, 22.0mmol) was added into the solution portionwise. When addition completed, the mixture

Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017 e2

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

was warmed up to room temperature, and stirred for 40min. Then 1-Boc-4-piperidone (3.98g, 20.0mmol) was added portionwise.

ꢀ

The reaction mixture was then stirred at room temperature for 1 hour, then at 65 C for another hour. Then the mixture was poured

on 100mL ice. Aqueous phase was extracted using EtOAc (50mL32). Combined organic phase was washed with brine, dried over

MgSO

4

, ﬁltered, and evaporated under reduced pressure. The crude material was puriﬁed by ﬂash column chromatography (50%

EtOAc in hexanes) to afford 2.98g product in 70% yield.

S4. R=Cl; S5. R=NO

2

Into the solution of S1 (2.13g, 11.8mmol) in 50mL DMF was added S3 (2.78g, 13.0mmol) and Cs

2

CO

mixture was heated at 80 C overnight. Then the reaction was cooled to room temperature, and diluted with EtOAc. The solution

3

(11.54g, 35.4mmol). The

ꢀ

4

was washed with saturated NH Cl (50mL32) Aqueous phase was extracted with more EtOAc. Combined organic phase was washed

with brine, dried over MgSO4, followed by ﬁltration and evaporation under reduced pressure. The crude material was puriﬁed by ﬂash

column chromatography (40% to 100% EtOAc in hexanes) to afford 3.94g product in 85% yield.

S4 was taken up in triﬂuoroacetic acid (TFA) as 1M solution, which was stirred at room temperature for 2 hours. The solution was

concentrated under reduced pressure, further on high-vac overnight. S6 was directly used as starting material for the following

synthesis without further puriﬁcation.

S6. R=Cl; S7. R=NO

Amide formation by HATU-catalyzed coupling reaction: S6 was taken up in DMF as 1M solution, and 3 equivalence of Et

added. The carboxylic acid (1.2eq) was pre-mixed with HATU (2eq) and Et N (5eq) in DMF at the same concentration, which was

2

3

N was

3

stirred at room temperature for 10min. Two solutions were then mixed together and further stirred at room temperature for 5 hours.

The reaction was directly subjected to prep. HPLC puriﬁcation. The isolated product was then further puriﬁed by normal phase ﬂash

chromatography to afford product with desired purity for following biological tests.

Amide formation by acylation using acid chloride: S6 (0.04g, 0.1mmol) was taken up in 2mL dichloromethane. Et

3

N (0.07mL,

.5mmol) was added, followed by addition of acetyl chloride (0.015mL, 0.2mL). The reaction was kept at 0 C stirring for 2 hours.

ꢀ

0

Then the reaction was quenched by adding drops of water, followed by immediate puriﬁcation by ﬂash column chromatography.

The isolated product was further puriﬁed by HPLC to afford 19mg product in 57% yield.

e3 Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017

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USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

Reduction of aromatic nitro group: S7 (0.49g, 1.08mmol) was dissolved in 10mL AcOH/EtOH (1:1). Iron powder (0.25g, 4.39mmol)

ꢀ

was added in one portion. The reaction was then stirred at 50 C for 1 hour. The iron powder was removed by ﬁltration. Filtrate was

concentrated under reduced pressure. The crude material was then puriﬁed by normal phase ﬂash column chromatography (10% to

4

0% MeOH in EtOAc), followed by reverse phase HPLC to afford 0.22g product S8 in 53% yield

S8

Installation of solubilizing groups: S8 (0.11g, 0.25mmol) was dissolved in 5mL dichloromethane. Et

3

N (0.035mL, 0.25mmol) was

ꢀ

added at -20 C.3-bromopropionyl chloride (0.03mL, 0.25mmol) in 1mL DCM was added dropwisely. The reaction was stirred at

ꢀ

0

C for 3h. Then it was quenched by addition of drops of water, then concentrated under reduced pressure. The crude product

was used for the next step without further puriﬁcation.

Crude material from last step (0.06g, 0.1mmol) was dissolved in 1mL DMF. Into the solution was added N-methylpiperazine

ꢀ

(

0.016mL, 0.12mmol) and Et

3

N(0.028mL, 0.2mmol). The reaction was stirred at 80 C for 3 hours. The solution was directly subjected

to reverse phase HPLC puriﬁcation, followed by normal phase ﬂash column chromatography (20% to 60% MeOH in EtOAc with 0.5%

Et N) to afford 0.043g product XL188 in 75% yield.

3

Compound Characterization

2

1

(

7-chloro-3-((1-(3-phenylpropanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 33% yield) H NMR (500 MHz, CDCl

d 8.21 (d, J = 8.6 Hz, 1H), 7.93 (s, 1H), 7.70 (d, J = 1.9 Hz, 1H), 7.46 (dd, J = 8.6, 2.0 Hz, 1H), 7.31 – 7.25 (m, 2H), 7.22 – 7.18 (m, 3H), 4.69

d, J = 13.4 Hz, 1H), 3.88 – 3.73 (m, 3H), 3.00 – 2.92 (m, 2H), 2.91 – 2.82 (m, 1H), 2.60 (dp, J = 14.3, 7.3 Hz, 2H), 2.48 (td, J = 13.1, 2.4 Hz,

3

)

(

1

5

H), 2.17 – 2.04 (m, 1H), 1.70 (d, J = 12.9 Hz, 1H), 1.62 (d, J = 12.8 Hz, 1H), 1.17 (qd, J = 12.5, 4.3 Hz, 1H), 0.97 (qd, J = 12.5, 4.2 Hz,

1

H). C NMR (126 MHz, DMSO) d 169.56, 159.76, 149.58, 148.95, 141.43, 138.88, 128.37, 128.18, 127.28, 126.30, 125.79, 120.37,

3

+

0.79, 44.55, 40.74, 38.22, 35.01, 33.98, 30.87, 29.54, 28.84. LCMS (ESI) m/z 410.29 [(M+H) ; calcd for C23

H

25ClN

3

O

2

: 410.16].

3

4-((7-chloro-4-oxoquinazolin-3(4H)-yl)methyl)-1-(3-phenylpropanoyl)piperidine-4-carbonitrile: white solid, commercial com-

1

pound) H NMR (500 MHz, DMSO) d 8.44 (s, 1H), 8.17 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.62 (dd, J = 8.6, 2.1 Hz, 1H),

7

3

2

1

.25 (ddd, J = 13.3, 7.9, 4.0 Hz, 4H), 7.20 – 7.12 (m, 1H), 4.46 (d, J = 13.8 Hz, 1H), 4.36 – 4.24 (m, 2H), 3.97 (d, J = 14.3 Hz, 1H),

.05 (t, J = 12.5 Hz, 1H), 2.80 (t, J = 7.7 Hz, 2H), 2.73 – 2.57 (m, 3H), 1.88 (t, J = 14.6 Hz, 2H), 1.61 (dtd, J = 17.1, 13.0, 3.9 Hz,

1

3

H). C NMR (126 MHz, DMSO) d 170.41, 160.54, 149.90, 149.20, 141.81, 139.78, 128.99, 128.89, 128.70, 128.13, 126.94,

+

26.34, 121.06, 120.79, 50.64, 42.38, 40.40, 38.51, 34.30, 32.87, 32.28, 31.22. LCMS (ESI) m/z 435.29 [(M+H) ; calcd for

+

C

24

H24ClN

4

O

2

: 435.16].

4

1

7-chloro-3-((4-hydroxy-1-(3-phenylpropyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 13% yield) H NMR (500 MHz,

(

MeOD) d 8.29 (s, 1H), 8.21 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 7.54 (dd, J = 8.6, 2.0 Hz, 1H), 7.26 (q, J = 7.1 Hz, 2H), 7.21 – 7.13

Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017 e4

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

13

(

MeOD) d 161.13, 149.97, 148.77, 140.38, 140.25, 128.24, 128.13, 128.00, 127.52, 126.00, 120.22, 67.60, 56.16, 53.81, 48.21, 32.27,

m, 3H), 4.11 (s, 2H), 2.97 (d, J = 12.0 Hz, 2H), 2.76 – 2.59 (m, 6H), 1.98 – 1.81 (m, 4H), 1.63 (d, J = 13.4 Hz, 2H). C NMR (126 MHz,

+

3

2.00, 25.90. LCMS (ESI) m/z 412.39 [(M+H) ; calcd for C23

H

27ClN

3

O

2

: 412.18].

5

1

(7-chloro-3-((3-hydroxy-1-(3-phenylpropanoyl)pyrrolidin-3-yl)methyl)quinazolin-4(3H)-one: white solid, 8% yield)

H NMR

500 MHz, DMSO) d 8.29 (s, 1H), 8.17 (dd, J = 8.6, 4.3 Hz, 1H), 7.76 (t, J = 2.3 Hz, 1H), 7.59 (ddd, J = 8.7, 7.5, 2.1 Hz, 1H), 7.29 –

(

7

3

5

1

3

.19 (m, 4H), 7.16 (t, J = 6.8 Hz, 1H), 5.27 (s, 1H), 4.17 (s, 1H), 4.14 (d, J = 4.0 Hz, 1H), 3.51 (s, 2H), 3.32 (dt, J = 23.6, 11.7 Hz,

H), 2.84 – 2.74 (m, 2H), 2.59 – 2.51 (m, 2H), 2.47 – 2.40 (m, 1H), 1.95 (ddt, J = 39.5, 12.7, 9.3 Hz, 1H), 1.76 (ddd, J = 12.9, 11.0,

1

.7 Hz, 1H). C NMR (126 MHz, DMSO) d 169.93, 169.76, 160.22, 160.13, 150.33, 149.06, 149.04, 141.55, 141.51, 138.95,

3

28.45, 128.40, 128.35, 128.22, 127.24, 126.27, 125.81, 120.46, 78.33, 76.84, 55.97, 55.57, 51.10, 50.87, 44.46, 43.77, 36.06,

+

5.63, 35.06, 34.54, 30.29, 30.23. LCMS (ESI) m/z 412.29 [(M+H) ; calcd for C22

H23ClN

3

O

3

: 412.14].

6

1

3-((1-acetyl-4-hydroxypiperidin-4-yl)methyl)-7-chloroquinazolin-4(3H)-one: white solid, 57% yield) H NMR (500 MHz, DMSO)

(

d 8.40 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.6, 2.1 Hz, 1H), 4.09 – 3.96 (m, 4H), 3.58 (d, J =

13

1

3.4 Hz, 1H), 3.33 – 3.20 (m, 1H), 2.97 – 2.85 (m, 1H), 1.98 (s, 3H), 1.56 (td, J = 13.3, 4.3 Hz, 1H), 1.49 – 1.34 (m, 3H). C NMR

126 MHz, DMSO) d 167.66, 159.89, 150.25, 148.62, 138.65, 128.19, 126.92, 125.88, 120.05, 68.98, 53.39, 41.40, 36.44, 34.61,

(

+

3

3.93, 20.98. LCMS (ESI) m/z 336.18 [(M+H) ; calcd for C16

H

19ClN

3

O

3

: 336.11].

7

(

7-chloro-3-((4-hydroxy-1-(2-phenylacetyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, commercial compound)

1

H NMR (500 MHz, DMSO) d 8.27 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.6, 2.1 Hz, 1H), 7.30 –

.24 (m, 2H), 7.24 – 7.15 (m, 3H), 4.07 – 4.03 (m, 1H), 3.97 (dd, J = 38.6, 13.8 Hz, 2H), 3.74 – 3.64 (m, 3H), 3.29 – 3.20 (m, 1H),

7

1

.03 – 2.92 (m, 1H), 1.51 – 1.27 (m, 4H). C NMR (126 MHz, DMSO) d 168.31, 159.88, 150.12, 148.71, 138.66, 135.76, 128.57,

3

+

28.19, 127.99, 126.93, 126.00, 125.95, 120.07, 68.97, 53.45, 41.15, 39.35, 36.86, 34.61, 33.99. LCMS (ESI) m/z 412.29 [(M+H) ;

1

+

calcd for C22

H

23ClN

3

O

3

: 412.14].

8

(7-chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, commercial compound)

H NMR (500 MHz, DMSO) d 8.29 (s, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.56 (dd, J = 8.6, 2.1 Hz, 1H), 7.30 –

1

7

1

.22 (m, 4H), 7.18 – 7.13 (m, 1H), 4.12 – 3.55 (m, 4H), 3.41 – 2.85 (m, 3H), 2.62 (dd, J = 14.9, 6.6 Hz, 1H), 2.54 (dd, J = 14.9,

13

.6 Hz, 1H), 1.40 (t, J = 15.9 Hz, 4H), 1.25 (d, J = 7.0 Hz, 3H). C NMR (126 MHz, DMSO) d 168.89, 159.94, 159.88, 150.11,

48.74, 146.42, 146.30, 138.68, 128.20, 127.97, 127.94, 126.94, 126.66, 126.63, 125.98, 125.72, 125.67, 120.10, 69.04, 68.99,

e5 Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

+

3.57, 40.82, 40.71, 39.99, 36.67, 35.99, 35.77, 34.78, 34.64, 34.09, 33.95, 21.81, 21.61. LCMS (ESI) m/z 440.39 [(M+H) ; calcd for

5

+

C

24

H27ClN

3

O

3

: 440.17].

9

1

(R)-7-chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 50% yield) H NMR

(

500 MHz, DMSO) d 8.27 (d, J = 12.8 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 1.7 Hz, 1H), 7.58 (dd, J = 8.6, 2.0 Hz, 1H), 7.26

dd, J = 13.8, 6.7 Hz, 4H), 7.20 – 7.08 (m, 1H), 4.93 (d, J = 5.9 Hz, 1H), 4.04 (d, J = 13.6 Hz, 2H), 3.92 (q, J = 13.9 Hz, 1H), 3.65

t, J = 12.5 Hz, 1H), 3.28 – 3.06 (m, 2H), 2.86 (ddd, J = 13.9, 8.6, 3.2 Hz, 1H), 2.68 – 2.50 (m, 2H), 1.62 – 1.26 (m, 3H), 1.26 – 1.12

1

3

m, 3H). C NMR (126 MHz, DMSO) d 168.92, 159.90, 150.16, 148.76, 146.43, 146.31, 138.70, 128.22, 127.99, 127.96, 126.98,

1

3

26.68, 126.64, 125.99, 125.74, 125.69, 120.12, 69.05, 69.00, 53.58, 40.82, 40.71, 39.99, 36.68, 36.01, 35.78, 34.78, 34.65,

+

3 3

4.09, 33.95, 21.83, 21.63. LCMS (ESI) m/z 440.29 [(M+H) ; calcd for C24H27ClN O : 440.17].

1

0

1

(S)-7-chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 57% yield) H NMR

(

500 MHz, DMSO) d 8.27 (d, J = 12.8 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 1.6 Hz, 1H), 7.58 (dd, J = 8.6, 2.0 Hz, 1H), 7.26

dd, J = 13.7, 6.6 Hz, 4H), 7.21 – 7.09 (m, 1H), 4.93 (d, J = 5.8 Hz, 1H), 3.99 (dd, J = 43.9, 13.7 Hz, 2H), 3.91 (s, 1H), 3.65 (t, J =

(

1

3

1

3

2.2 Hz, 1H), 3.28 – 3.09 (m, 2H), 2.85 (d, J = 13.2 Hz, 1H), 2.68 – 2.55 (m, 2H), 1.60 – 1.25 (m, 3H), 1.20 (dd, J = 6.9, 1.5 Hz,

1

H). C NMR (126 MHz, DMSO) d 168.89, 159.94, 159.88, 150.11, 148.74, 146.42, 146.30, 138.68, 128.20, 127.97, 127.94,

3

26.94, 126.66, 126.63, 125.98, 125.72, 125.67, 120.10, 69.04, 68.99, 53.57, 40.82, 40.71, 39.99, 36.67, 35.99, 35.77, 34.78,

+

4.64, 34.09, 33.95, 21.81, 21.61. LCMS (ESI) m/z 440.29 [(M+H) ; calcd for C24

H27ClN

3

O

3

: 440.17].

XL188

((R)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)

1

propanamide: off-white solid, 75% yield) H NMR (500 MHz, DMSO) d 10.57 (s, 1H), 8.20 (d, J = 13.1 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H),

8

4

2

.02 (d, J = 1.6 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.25 (dd, J = 12.4, 6.2 Hz, 4H), 7.13 (dd, J = 18.7, 10.4 Hz, 1H), 4.96 (d, J = 5.8 Hz, 1H),

.02 (d, J = 13.6 Hz, 1H), 3.90 (q, J = 14.0 Hz, 2H), 3.63 (dd, J = 29.0, 16.2 Hz, 1H), 3.27 – 3.12 (m, 2H), 2.86 (dd, J = 17.7, 14.8 Hz, 1H),

13

.69 – 2.61 (m, 2H), 2.61 – 2.52 (m, 3H), 2.48 – 2.22 (m, 8H), 2.16 (s, 3H), 1.58 – 1.26 (m, 4H), 1.20 (d, J = 6.4 Hz, 3H). C NMR

(

126 MHz, DMSO) d 170.76, 168.83, 159.92, 159.87, 149.10, 148.73, 146.41, 146.28, 144.06, 127.93, 127.90, 126.97, 126.62,

1

3

26.60, 125.69, 125.63, 118.04, 116.26, 114.45, 69.03, 68.98, 54.37, 53.22, 51.96, 45.30, 40.81, 40.71, 39.97, 36.67, 35.95,

+

43 6 4

5.74, 34.77, 34.64, 34.09, 34.01, 33.95, 21.79, 21.61. LCMS (ESI) m/z 575.32 [(M+H) ; calcd for C32H N O : 575.33].

Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017 e6

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

XL203C

(

(S)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)

1

propanamide: off-white solid, 26% yield) H NMR (500 MHz, DMSO) d 10.52 (s, 1H), 8.19 (d, J = 13.1 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H),

.02 (d, J = 2.0 Hz, 1H), 7.62 (dd, J = 8.8, 1.4 Hz, 1H), 7.25 (dd, J = 12.6, 6.3 Hz, 4H), 7.19 – 7.08 (m, 1H), 4.93 (s, 1H), 4.08 – 3.97

m, 1H), 3.90 (q, J = 14.0 Hz, 2H), 3.64 (t, J = 12.9 Hz, 1H), 3.30 – 3.09 (m, 2H), 2.95 – 2.81 (m, 1H), 2.69 – 2.60 (m, 2H), 2.61 –

8

(

13

2

1

6

5

.52 (m, 3H), 2.47 – 2.25 (m, 8H), 2.15 (s, 3H), 1.58 – 1.26 (m, 4H), 1.24 – 1.14 (m, 3H). C NMR (126 MHz, DMSO) d 170.76,

68.82, 159.88, 149.11, 148.74, 146.41, 146.29, 144.04, 127.94, 127.91, 127.00, 126.63, 125.69, 125.64, 118.04, 116.28, 114.46,

8.99, 54.43, 53.24, 52.02, 45.38, 40.82, 40.71, 39.96, 36.68, 35.96, 35.75, 34.77, 34.64, 34.03, 21.80, 21.61. LCMS (ESI) m/z

+

43 6 4

75.32 [(M+H) ; calcd for C32H N O : 575.33].

O

N

OH

O

N

H

N

O

1

N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)

(

propenamide: white solid, 16% yield) H NMR (500 MHz, DMSO) d 10.84 (s, 1H), 8.25 (d, J = 13.2 Hz, 1H), 8.15 – 7.99 (m, 2H), 7.69

1

(

d, J = 8.7 Hz, 1H), 7.28 – 7.25 (m, 4H), 7.19 – 7.10 (m, 1H), 4.05 – 3.88 (m, 3H), 3.65 (t, J = 12.8 Hz, 1H), 3.28 – 3.14 (m, 10H), 2.92 – 2,85

13

m, 3H), 2.79 (s, 3H), 2.64 – 2.53 (m, 4H), 1.53 – 1.28 (m, 4H), 1.20 (d, J = 6.0, 3H). C NMR (126 MHz, DMSO) d 169.60, 160.66,

1

60.61, 158.82, 158.57, 149.95, 149.34, 147.15, 147.02, 144.63, 128.69, 128.66, 127.69, 127.38, 127.35, 126.44, 126.39, 118.90,

17.14, 115.37, 69.76, 69.71, 53.94, 41.57, 41.46, 40.88, 40.70, 37.42, 36.71, 36.49, 35.50, 35.37, 34.81, 34.66, 22.53, 22.34.

+

43 6 4

LCMS (ESI) m/z 575.32 [(M+H) ; calcd for C32H N O : 575.33].

1

2

N-(3-((4-hydroxy-1-(4-methyl-3-phenylpentanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-

(

1

-yl)propenamide: off-white solid, 40% yield) H NMR (500 MHz, DMSO) d 10.65 (s, 1H), 8.21 (d, J = 16.9 Hz, 1H), 8.11 (dd, J = 8.7,

.9 Hz, 1H), 8.05 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.23 (td, J = 7.6, 2.9 Hz, 2H), 7.15 (t, J = 6.5 Hz, 2H), 7.13 – 7.06 (m, 1H), 4.04 – 3.93

1

(

m, 2H), 3.85 (s, 1H), 3.75 – 3.61 (m, 1H), 3.58 – 2.98 (m, 10H), 2.91 – 2.55 (m, 10H), 1.88 –1.80 (m, 1H), 1.53 – 1.25 (m, 3H), 1.12 – 1.06

13

m, 1H), 0.89 (t, J = 7.2 Hz, 3H), 0.70 – 0.61 (m, 3H). C NMR (126 MHz, DMSO) d 169.94, 169.86, 160.61, 160.53, 149.95, 149.29,

1

4

44.51, 144.09, 144.03, 128.82, 128.79, 128.24, 128.16, 127.81, 126.31, 118.89, 117.21, 115.33, 69.76, 69.66, 54.04, 50.83, 49.24,

9.08, 48.75, 42.48, 41.51, 37.43, 37.33, 36.01, 35.94, 35.55, 35.31, 34.72, 34.65, 32.83, 32.63, 32.39, 21.24, 21.19, 20.76, 20.56.

+

47 6 4

LCMS (ESI) m/z 603.43 [(M+H) ; calcd for C34H N O : 603.37].

1

3

(R)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-morpholinopropanamide:

(

1

off-white solid, 65% yield) H NMR (500 MHz, DMSO) d 10.52 (s, 1H), 8.20 (d, J = 13.3 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 8.04 (d, J =

.8 Hz, 1H), 4.96 (d, J = 6.4 Hz, 1H), 4.02 (d, J = 13.7 Hz, 1H), 3.90 (q, J = 14.0 Hz, 2H), 3.73 – 3.60 (m, 1H), 3.59 – 3.55 (m, 4H), 3.18

ddd, J = 21.0, 18.3, 9.2 Hz, 2H), 2.93 – 2.80 (m, 1H), 2.65 (t, J = 6.9 Hz, 2H), 2.61 – 2.54 (m, 3H), 2.41 (m, 4H), 1.40 (dddd, J = 41.2, 26.0,

1

(

e7 Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

1

6.8, 9.5 Hz, 4H), 1.19 (dd, J = 6.9, 1.7 Hz, 3H). C NMR (126 MHz, DMSO) d 170.62, 168.81, 160.00, 149.15, 148.76, 146.44, 146.31,

3

1

3

44.09, 118.13, 116.29, 114.51, 69.06, 65.93, 53.73, 52.79, 40.85, 40.75, 39.98, 36.71, 36.00, 35.78, 34.79, 34.66, 34.11, 33.98,

+

40 5 5

3.81, 21.83, 21.64. LCMS (ESI) m/z 562.32 [(M+H) ; calcd for C31H N O : 562.30].

1

4

(R)-3-(dimethylamino)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)propanamide:

(

off-white solid, 36% yield) H NMR (500 MHz, DMSO) d 10.47 (s, 1H), 8.18 (dd, J = 13.0, 7.3 Hz, 1H), 8.06 (d, J = 8.7 Hz, 1H), 8.02 (d, J =

1

2

.0 Hz, 1H), 7.63 – 7.59 (m, 1H), 7.24 (dd, J = 12.6, 6.3 Hz, 4H), 7.17 – 7.09 (m, 1H), 4.92 (s, 1H), 4.07 – 3.95 (m, 1H), 3.89 (q, J = 14.0 Hz,

H), 3.63 (t, J = 13.0 Hz, 1H), 3.18 (ddd, J = 20.8, 18.0, 9.1 Hz, 2H), 2.86 (ddd, J = 13.8, 10.5, 5.5 Hz, 1H), 2.60 – 2.54 (m, 3H), 2.54 – 2.47

2

13

(

m, 2H), 2.17 (s, 6H), 1.56 – 1.23 (m, 4H), 1.24 – 1.15 (m, 3H). C NMR (126 MHz, DMSO) d 170.76, 168.83, 159.89, 149.09, 148.74,

1

4

46.42, 146.29, 144.07, 127.94, 127.92, 126.97, 126.61, 125.70, 125.64, 118.06, 116.26, 114.46, 68.99, 54.59, 53.29, 44.63, 40.82,

+

0.72, 39.98, 39.96, 36.68, 35.97, 35.75, 34.78, 34.65, 34.61, 34.11, 33.96, 21.80, 21.61. LCMS (ESI) m/z 520.31 [(M+H) ; calcd for

+

C

29

H

38

N

5

O

4

: 520.29].

1

5

N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(1H-imidazol-1-yl)propenamide:

(

1

white solid, 19% yield) H NMR (500 MHz, DMSO) d 10.62 (s, 1H), 9.16 (s, 1H), 8.22 (d, J = 12.9 Hz, 1H), 8.09 (d, J = 8.7 Hz, 1H), 8.00

s, 1H), 7.80 (s, 1H), 7.67 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.28 – 7.25 (m, ,4H), 7.15 (d, J = 3.6 Hz, 1H), 4.03 – 3.87 (m, 3H), 3.65 (t, J =

(

1

3

1

4

2.4 Hz, 1H), 3.26 – 3.14 (m, 2H), 3.08 (t, J = 6.3 Hz, 2H), 2.90 – 2.85 (m, 1H), 2.64 – 2.49 (m, 4H), 1.54 – 1.25 (m, 4H), 1.20 (d, J = 5.4 Hz,

1

H). C NMR (126 MHz, DMSO) d 169.60, 169.43, 160.60, 160.55, 158.88, 158.60, 149.99, 149.25, 147.15, 147.03, 144.33, 136.33,

3

28.68, 128.66, 127.85, 127.37, 127.35, 126.44, 126.39, 122.58, 120.32, 118.86, 117.25, 115.33, 69.77, 69.71, 54.04, 44.94,

+

1.55, 41.44, 40.71, 37.41, 36.70, 36.57, 36.49, 35.52, 35.39, 34.80, 34.66, 22.53, 22.35. LCMS (ESI) m/z 543.22 [(M+H) ; calcd

+

for C30

H

35

N

6

O

4

: 543.27].

O

N

OH

O

N

H

O

1

6

((N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(piperidin-1-yl)propenamide:

1

white solid, 18% yield) H NMR (500 MHz, DMSO) d 10.69 (s, 1H), 9.32 (s, 1H), 8.23 (d, J = 12.8 Hz, 1H), 8.11 (dd, J = 8.7, 1.9 Hz, 1H),

8

3

6

1

4

.05 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.19 – 7.31 (m, 4H), 7.15 (s, 1H), 4.03 – 3.89 (m, 3H), 3.65 (t, J = 12.4 Hz, 1H), 3.48 – 3.40 (m, 4H),

.26 – 3.15 (m, 2H), 2.98 – 2.89 (m, 5H), 2.64 – 2.51 (m, 2H), 1.84 (d, J = 13.3 Hz, 2H), 1.68 – 1.62 (m, 3H), 1.55 – 1.31 (m, 5H), 1.21 (d, J =

1

.6 Hz, 3H). C NMR (126 MHz, DMSO) d 169.60, 169.22, 160.61, 160.56, 158.87, 158.59, 150.01, 149.29, 147.15, 147.03, 144.41,

3

28.68, 128.66, 127.87, 127.37, 127.35, 126.44, 126.39, 118.89, 117.27, 115.38, 69.77, 69.72, 54.05, 52.89, 52.05, 41.55, 41.45,

+

0.69, 37.41, 36.71, 36.49, 35.53, 35.40, 34.81, 34.67, 31.22, 23.00, 22.53, 22.35, 21.65. LCMS (ESI) m/z 560.22 [(M+H) ; calcd

+

for C32

H

42

N

5

O

4

: 560.32].

NMR spectra are provided as Supplemental Information.

Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017 e8

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

USP7 Cloning, Expression, and Puriﬁcation

USP7 full length (amino acids 1-1102) and catalytic domain (208-560) were ampliﬁed using Addgene plasmid #16655(Cummins and

Vogelstein, 2004) as a template and primers listed in Table S4 and cloned into pET28PP and pET28aLIC, respectively, using InFusion

HD EcoDry Cloning Kit (Takara Bio Cat# 121416). Amino acid mutations of catalytic domain were introduced by PCR using

QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following manufacturer’s protocol using primers listed in Table

S4. All DNAs were handled in E.coli 10beta cells (New England Biolabs Cat# C3020K) and transformed in E.coli BL21(DE3) for protein

expression.

A construct of human USP7 covering residues 208-560 in the pET28aLIC vector was overexpressed in E. coli BL21 (DE3) in terriﬁc

ꢀ

broth (TB) medium in the presence of 50 ug/ml of kanamycin. Cells were grown at 37 C to an OD of 0.8, cooled to 17 C, induced with

5

ꢀ

00 mM isopropyl-1-thio-D-galactopyranoside (IPTG), incubated overnight at 17 C, collected by centrifugation, and stored at -80 C.

Cell pellets were sonicated in buffer A (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 10 mM Imidazole, and 3 mM BME) and the

resulting lysate was centrifuged at 30,000 xg for 40 min. Ni-NTA beads (Qiagen) were mixed with lysate supernatant for 30 min and

washed with buffer A. Beads were transferred to an FPLC-compatible column and the bound protein was washed with 15% buffer B

(50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 300 mM imidazole, and 3 mM BME) and eluted with 100% buffer B. Thrombin

ꢀ

was added to the eluted protein and incubated at 4 C overnight. The sample was then concentrated and passed through a Superdex

200 16/60 column (GE healthcare) in a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, and 1 mM TCEP.

ꢀ

Fractions were pooled, concentrated and frozen at -80 C.

USP7 full length (amino acids 1-1102) in pET28aLIC was transformed in BL21(DE3) cells. An overnight culture was used to inoc-

ꢀ

ulate one liter of TB supplemented with 50 ug/ml kanamycin. Cells were grown at 37 C until they reached optical density (OD) ꢁ0.6 at

ꢀ

600 nm. Protein expression was initiated by the addition of 0.4 mM IPTG. Cells were then grown for 16-20 hours at 17 C prior collec-

tion by centrifugation. Cell pellets were washed in PBS and resuspended in 25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol and

ꢀ

1

mM TCEP, 10 mM Imidazole, 0.1% IGEPAL sonicated and incubated with Ni-Nta beads (Quiagen) for 30 min at 4 C. Beads were

washed with 10% buffer B (25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol and 1mM TCEP, 250 mM Imidazole,) and eluted with

1

2

00% buffer B. Protein containing fractions were concentrated and loaded on Superdex 200 10/300 GL column in a buffer containing

ꢀ

0 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, and 1 mM TCEP. Fractions were pooled, concentrated and frozen at -80 C.

Ub-AMC Assay

USP7 and mutants were tested for their activity in Ubiquitin-AMC assay in presence or absence of inhibitors. For this assay

USP7 catalytic domain WT or mutant was used at the following concentrations: 250nM USP7 WT, M407K, M407K/M410S or

Q351S, 125 nM H461A, 600 nM Y514A and 10 nM M410S. For the same assay USP7 full length WT and Q351 mutant were used

at 50 nM. USP7 variants were pre-incubated with different concentrations of inhibitors or DMSO as a control in 50 mM HEPES

pH7.6, 0.5 mM EDTA, 11 uM ovalbumin, 5 mM DTT. The reaction was incubated 30 min at room temperature prior to the addition

of 2 uM Ubiquitin-AMC (Boston Biochem) substrate. The initial rate of the reaction was measured by collecting ﬂuorescence data

at one minute intervals over 30-minute period using a Clariostar ﬂuorescence plate reader at excitation and emission wavelength

of 345 and 445 nm respectively. The calculated initial rate values were plotted against inhibitor concentrations to determine IC50s.

All the experimental data were plotted using Prism GraphPad.

Differential Scanning Fluorimetry

Differential Scanning Fluorimetry (DSF) experiments were carried out in a RTPCR 7500 Real-Time System (LifeTech) in 96 well plates,

a total volume of 20 ul, and with an optimized SYPRO Orange dye concentration (5000x concentration in DMSO, Invitrogen).

Compound dilutions in assay buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) were ﬁrst prepared by an NT8 liquid handler

ꢀ

(

Formulatrix) and then addition of dye was performed also by the same liquid handler. Sealed plates were heated at 1 C/min from

ꢀ ꢀ ꢀ

25 C to 95 C with ﬂuorescence readings every 0.5 C. Tm values were determined as the minimum of the ﬁrst derivative of the

recorded ﬂuorescence intensity versus temperature plot.

Isothermal Titration Calorimetry

The binding afﬁnity of protein/ligand was measured by adding 0.02 mM protein in cell and titrating with 0.2 mM ligand in the syringe

ꢀ

using an Auto-ITC200 microcalorimeter (Malvern) at 20 C. Proteins and ligands were prepared within ITC buffer containing 20 mM

HEPES pH 7.5, 150 mM NaCl, and 2% DMSO. The data were ﬁt using Origin 7.0 software. All ITC results were summarized in

Table S2.

Selectivity Proﬁling

Selectivity proﬁling (DUBProﬁler) was performed by Ubiquigent using the manufacturer’s protocols.

Mouse Liver Microsome Stability

Mouse liver microsome (MLM) half-life measurements were performed by Scripps Florida Pharmacology Core according their

protocols.

e9 Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

Competitive Activity Based Protein Proﬁling

HEK 293T cells were pelleted, washed with PBS, lysed on ice (50 mM Tris pH 7.6, 150 mM NaCl, 5 mM MgCl

NP-40, 10% glycerol, 1 mM TCEP, phosphatase inhibitor cocktails (Sigma P5726 and Calbiochem 524624), and protease inhibitors

pepstatin, leupeptin, PMSF, and aprotinin), and clariﬁed by centrifugation. Protein content was quantiﬁed by BCA, and 50 ug of

lysate was diluted into 30 uL labeling buffer (50 mM Tris pH 7.6, 5 mM MgCl , 0.5 mM EDTA, 250 mM sucrose, 1 mM TCEP) and

incubated at room temperature with shaking with the indicated inhibitors for 30 minutes. Samples were then supplemented with

2

, 0.5 mM EDTA, 0.5%

(

2

1

uM HA-Ub-VS and incubated at room temperature with shaking for 15 minutes. Reactions were quenched with 4x LDS sample

ꢀ

buffer (Thermo Fisher B0007) supplemented with 10% BME, vortexed vigorously, and heated to 95 C for 5 minutes. Samples

were resolved by SDS-PAGE and analyzed by Western blot with the indicated antibodies.

Cell Treatments

Cells were treated with DMSO or different concentrations of XL-188 or XL-203 for 6 (MM1S) or 16 (MCF7) hours in presence or

absence of cycloheximide. For the experiments in which cycloheximide was used, cells were treated with compounds for 4

(

MM1S) or 14 (MCF7) hours prior to the addition of 50 ug/ml of cycloheximide. At 6 or 16h time point cells were washed in PBS

and lysed in modiﬁed RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM Tris, 150 mM NaCl, 1 mM

EDTA) containing phosphatase inhibitor cocktails 1 and 2 (Sigma), and protease inhibitors. Protein concentrations were quantiﬁed

using the BCA protein assay kit (Pierce) and samples were probed by immunoblotting using mdm2 (Santa cruz sc-965), p53 (Cell

signaling 9282), p21 (Cell signaling 2947), GAPDH (Cell signaling 2118), USP7 (Cell signaling 4833) antibodies.

Peripheral Blood Mononuclear Cell Testing

Peripheral blood mononuclear cells (PBMCs) were generously provided by Dr. Steven Treon and Dr. Guang Yang. PBMCs from

normal individuals were isolated by density gradient centrifugation through Ficoll-Plaque Plus (Amersham Pharmacia Biotech AB,

Uppsala, Sweden) at 400xg for 25 minutes, followed by two washes in PBS. Cells were then maintained in RPMI media, supple-

mented with 10% FBS. Primary cells were obtained through written consent under approval of the Dana-Farber Cancer Institute Insti-

tutional Review Board. The trypan blue exclusion assay has been previously described(Weisberg et al., 2002) and was used for quan-

tiﬁcation of PBMCs prior to seeding for CellTiter-Glo Luminescent Cell Viability assays (Promega, Madison, WI). These assays were

used for proliferation studies and carried out according to manufacturer instructions. Cell viability is reported as percentage of control

(

untreated) cells, and error bars represent the standard deviation for each data point.

Crystallization, Data Collection and Structure Determination

One equivalence of compound (from a 100 mM DMSO stock) was mixed with 1 mM protein and crystallized by sitting-drop vapor

ꢀ

diffusion at 20 C in the following crystallization buffer: 30% PEG3350, 0.2 M NaFormate, 0.1 M NaCitrate-pH 5.5, and 10 mM

DTT. Crystals were transferred brieﬂy into crystallization buffer containing 25% glycerol prior to ﬂash-freezing in liquid nitrogen.

Diffraction data from complex crystals were collected at beamline 24ID-C of the NE-CAT at the Advanced Photon Source (Argonne

National Laboratory). Data sets were integrated and scaled using XDS.(Kabsch, 2010) Structures were solved by molecular replace-

ment using the program Phaser(McCoy et al., 2007) and the search model PDB entry 1NB8. The ligand was positioned and

preliminarily reﬁned using Buster and Rhoﬁt.(Smart et al., 2012) Iterative manual model building and reﬁnement using Phenix(Adams

et al., 2010)and Coot(Emsley and Cowtan, 2004) led to a model with excellent statistics.

Hydrogen Exchange Experiments

Hydrogen exchange experiments were performed essentially as described in Iacob et al(Iacob et al., 2009). A stock solution of USP7

catalytic domain at 50 pmol/mL in 20mM Hepes (pH 7.5), 200 mM NaCl, 1 mM TCEP, 5% glycerol H

exchange in USP7 alone was initiated by dilution with 15-fold D O buffer (pD 7.5), at room temperature. At each deuterium exchange

time point (from 10 s to 4 hours) an aliquot from the exchange reaction was removed and labeling was quenched by adjusting the pH

2

O was prepared. Deuterium

2

to 2.5 with an equal volume of quench buffer (0.8% Formic Acid and 0.8M Guanidine Hydrochloride, H

immediately injected into the LC/MS system.

2

O). Quenched samples were

For the HDX MS experiments of USP7 bound to compounds, each compound was individually incubated with USP7 as follows:

XL188 was incubated at RT for 30 min with USP7 at a protein: compound ratio of 1:10, ensuring that 99.65 % was bound after dilution

2 2

with D O. 9 was mixed at a protein: compound ratio of 1:10, ensuring that 99.02 % was bound after dilution with D O. 1 was mixed at

2

a protein: compound ratio of 1:20, ensuring that 88.19% was bound after dilution with D O. The same time course as for the

protein alone was implemented for the compounds work (10sec-4h). Mass analysis: Each sample was analyzed as previously descri-

bed.(Wales et al., 2008) Brieﬂy, the samples were digested online using a Poroszyme immobilized pepsin cartridge (2.1 mm x 30 mm,

ꢀ

TM

Applied Biosystems) at 15 C for 30 s, then injected into a custom Waters nanoACQUITY UPLC HDX Manager and analyzed on a

XEVO G2 mass spectrometer (Waters Corp., USA). The average amount of back-exchange using this experimental setup was

2

0-30%, based on analysis of highly deuterated peptide standards. Deuterium levels were not corrected for back-exchange and

are therefore reported as relative.(Wales and Engen, 2006) All experiments were performed in duplicate. The error of measuring

the mass of each peptide averaged ± 0.12 Da in this experimental setup (4). The HDX-MS data were processed using PLGS 3.0

and DynamX 3.0 (Waters Corp., USA).

Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017 e10

Please cite this article in press as: Lamberto et al., Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of

USP7, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.09.003

QUANTIFICATION AND STATISTICAL ANALYSIS

All biochemical curves and statistical analyses were produced using Prism 6 (GraphPad Software).

DATA AND SOFTWARE AVAILABILITY

Structure ﬁles and coordinates have been deposited to PDB under these accession numbers: 5VSB for USP7/1, 5VSK for USP7/8

5

VS6 for USP7/XL188.

e11 Cell Chemical Biology 24, 1–11.e1–e11, December 21, 2017

Article Doi

Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7

DOI: 10.1016/j.chembiol.2017.09.003

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.chembiol.2017.09.003

Full text of DOI:10.1016/j.chembiol.2017.09.003

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