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
`
`d Inhibitors bind the S4-S5 pocket of the enzyme
`
`d 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
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1022
`Page 1
`
`EXHIBIT 10
`DELANSORNE DECLARATION
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`214LT:20700:449509:1:ALEXANDRIA
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`

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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
`
`Cell Chemical Biology
`
`Article
`
`Structure-Guided Development of a Potent
`and Selective Non-covalent Active-Site
`Inhibitor of USP7
`
`Ilaria Lamberto,1,5 Xiaoxi Liu,1,5 Hyuk-Soo Seo,1,5 Nathan J. Schauer,1 Roxana E. Iacob,3 Wanyi Hu,1 Deepika Das,2
`Tatiana Mikhailova,4 Ellen L. Weisberg,2 John R. Engen,3 Kenneth C. Anderson,2 Dharminder Chauhan,2
`Sirano Dhe-Paganon,1,* and Sara J. Buhrlage1,4,6,*
`1Department 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
`
`Deubiquitinating enzymes (DUBs) have garnered
`significant attention as drug targets in the last 5–10
`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
`agents possessing weak (micromolar) potency to-
`ward their primary target, limiting their utility in target
`validation and mechanism studies. Due to a lack of
`high-resolution DUB,small-molecule ligand complex
`structures, no structure-guided optimization efforts
`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.
`
`INTRODUCTION
`
`Ubiquitin is a 76-amino-acid protein attached to substrate
`proteins post-translationally via isopeptide bond formation
`between ubiquitin’s C-terminal glycine and a substrate lysine
`side chain (Komander and Rape, 2012); linear and branched
`polyubiquitin chains are assembled via attachment of another
`molecule of ubiquitin to one of seven lysines or the N-terminal
`methionine of ubiquitin (Pickart and Fushman, 2004). Ubiquitin
`is attached to substrate proteins by the coordinated action of
`ubiquitin-activating (E1), conjugating (E2), and ligating (E3)
`enzymes and removed by a family of proteases known as deubi-
`
`quitinating enzymes (DUBs). The first recognized role of the ubiq-
`uitin system was controlling protein turnover (Ciechanover et al.,
`1980; Hershko et al., 1980). Ubiquitin tags are also responsible
`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
`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
`remodeling (Weake and Workman, 2008), receptor signaling
`(Haglund and Dikic, 2012), and immunity (Malynn and Ma,
`2010; Zinngrebe et al., 2014), among others. Not surprisingly,
`aberrant ubiquitin system activity is linked to disease, including
`cancer (Hoeller and Dikic, 2009; Pinto-Fernandez and Kessler,
`2016), infection (Isaacson and Ploegh, 2009; Maculins et al.,
`2016), and neurodegeneration (Ciechanover and Brundin,
`2003; Ciechanover and Kwon, 2015). The relationship between
`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
`protease [OTU], Josephin, and Mindy) are cysteine proteases,
`while the sixth (JAB/MPN/MOV34 [JAMM/MPN]) is composed
`of zinc metalloproteases (Abdul Rehman et al., 2016; Clague
`et al., 2013; Komander et al., 2009; Komander and Rape,
`2012). Many DUBs have been linked to physiological and/or
`pathophysiological functions. For example, USP1 and USP4
`are involved in DNA-damage repair (Kee and Huang, 2015),
`USP22 and BAP1 have a role in chromatin function (Atanassov
`et al., 2011), and USP2 and USP8 are reported to stabilize
`oncogenic proteins cyclin D1 (Shan et al., 2009) and mutant
`epidermal growth factor receptor (Byun et al., 2013), respec-
`tively. While dozens of apo- and ubiquitin-bound structures
`
`Cell Chemical Biology 24, 1–11, December 21, 2017 ª 2017 Elsevier Ltd. 1
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1022
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`

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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
`
`have been solved (Hu et al., 2002; Johnston et al., 1997;
`Komander et al., 2009), very few have been achieved with
`non-ubiquitin-based compounds (Davies et al., 2012; Ratia
`et al., 2008; Schlierf et al., 2016). Notably, small-molecule,DUB
`complex structures are lacking for the largest 56-member
`mammalian USP family.
`The first DUB inhibitor, the dual USP14/UCHL5 inhibitor
`VLX1570, entered clinical trials in 2015 (Wang et al., 2016b).
`Overall, however, DUB inhibitor development is still in its early
`stages. Approximately 40 DUB inhibitors have been reported,
`although most are weak, multi-targeted agents (D’Arcy et al.,
`2015; Ndubaku and Tsui, 2015). Given the current dearth of
`potent and selective inhibitors, skepticism remains as to whether
`or not this enzyme class will be druggable in a manner analogous
`to that of protein kinases, for example. A significant hindrance to
`the generation of potent and selective DUB inhibitors is a lack of
`structure-guided optimization efforts. One example of structure-
`guided development of a DUB inhibitor, which targeted the
`SARS DUB PLPro (Baez-Santos et al., 2015), generated com-
`pounds with half-maximal inhibitory concentrations (IC50) below
`500 nM and exhibiting a high degree of selectivity relative to
`mammalian DUBs. In this case, selectivity was explained by
`significant structural differences between viral and mammalian
`DUBs. Breakthroughs in X-ray crystallography of small-molecule
`DUB inhibitor complexes have the potential to enable rapid
`development of potent and selective inhibitors of mamma-
`lian DUBs.
`The DUB USP7 has been shown to be involved in regulation of
`a myriad of cellular processes, including epigenetics, cell cycle,
`DNA repair, immunity, viral infection, and tumorigenesis. USP7,
`also known as HAUSP (herpes virus-associated ubiquitin-
`specific protease), was first discovered as a protein that plays
`a role in viral lytic growth (Everett et al., 1997). Interest in the
`enzyme intensified when USP7 was implicated in regulating
`degradation of the tumor suppressor p53 (Li et al., 2002) by
`stabilizing the major E3 ligase for p53, MDM2 (Cummins et al.,
`2004; Li et al., 2004). Recently several epigenetic modifiers,
`including the methyltransferase PHF8 (Wang et al., 2016a),
`demethylase DNMT1(Du et al., 2010; Felle et al., 2011; Qin
`et al., 2011), and acetyltransferase Tip60 (Dar et al., 2013), as
`well as H2B itself (van der Knaap et al., 2005), have been identi-
`fied as direct targets of USP7. Other notable targets of USP7
`include the transcription factors FOXP3, which in T-regulatory
`(Treg) cells links this DUB enzyme to immune response (van
`Loosdregt et al., 2013), and N-Myc, which is stabilized in
`neuroblastoma cells (Tavana et al., 2016). Consistent with its
`regulation of diverse substrates and biological processes,
`USP7 has emerged as a drug target in a wide range of malig-
`nancies including multiple myeloma (Chauhan et al., 2012),
`breast cancer (Wang et al., 2016a), neuroblastoma (Tavana
`et al., 2016), glioma (Cheng et al., 2013), and ovarian cancer
`(Zhang et al., 2016).
`P22077 and its close analog P5091 are the inhibitors most
`frequently utilized to probe USP7 functions (for structures see
`Figure S1A). P22077 exhibits modest potency against USP7
`(IC50 = 8.0 mM) and equipotent inhibition of two additional
`DUBs, USP10 and USP47 (Altun et al., 2011; Ritorto et al.,
`2014). In addition to modest potency and selectivity, reported
`drawbacks of these nitrothiophene-based compounds include
`
`2 Cell Chemical Biology 24, 1–11, December 21, 2017
`
`poor solubility and general toxicity (Chen et al., 2017). Additional
`USP7 inhibitors (shown in Figure S1B) have been identified,
`although none possess features superior to P5091/P22077 and
`significant optimization efforts have not been undertaken (Aleo
`et al., 2006; Colland et al., 2009; El-Desoky et al., 2017; Nichol-
`son et al., 2008; Reverdy et al., 2012; Tanokashira et al., 2016;
`Yamaguchi et al., 2013).
`Here we report the structure-guided development of next-
`generation small-molecule probes of USP7. High-resolution
`USP7,small-molecule crystal structures enabled us to rapidly
`develop XL188, a highly selective 90 nM inhibitor of USP7,
`from a 7.2-mM lead, as a probe of USP7. Furthermore, we
`show that XL203C, the enantiomer of XL188, is more than
`80-fold less potent against USP7, and thus serves as an inactive
`control compound. In contrast to P22077/P5091, which target
`the invariant catalytic cysteine of USP DUBs, XL188 is a non-
`covalent active-site inhibitor. We demonstrate that
`the
`XL188/XL203C active/inactive inhibitor pair is a powerful combi-
`nation for studying USP7 function in cellular models.
`
`RESULTS
`
`XL188 Is a Potent and Selective Inhibitor of USP7
`As part of an effort to identify chemical starting points for devel-
`opment of DUB inhibitors by profiling the inhibitory activity of
`compounds reported in peer-reviewed and patent literature
`for activity against large panels of DUBs (Ritorto et al., 2014),
`we identified a highly selective inhibitor of USP7 (1, structure
`in Figure 1A) reported in a 2013 patent from Hybrigenics (Col-
`land and Gourdel, 2013; Kessler, 2014). When screened for
`inhibitory activity across a panel of 38 purified DUBs at a con-
`centration of 100 mM, USP7 was the only DUB substantially
`inhibited (Figure S1C and Table S1). Dose-response analysis
`using USP7 catalytic domain (amino acids 208–560) or full-
`length enzyme (1–1,102) and ubiquitin-aminomethylcoumarin
`(Ub-AMC) as substrate confirmed USP7 inhibitory activity,
`although potency was weak with IC50s in the double-digit
`micromolar range (Figures 1B and S1D). Isothermal titration
`calorimetry (ITC), using the catalytic domain, confirmed binding
`with a dissociation constant (KD) of 8 mM (Figure S1E and Table
`S2). We solved the structure of USP7 bound by 1, which
`enabled rapid structure-guided development of XL188 (Fig-
`ure 1A), a highly potent and selective inhibitor of USP7.
`XL188 inhibited USP7 catalytic domain and full-length enzyme
`with IC50 values of 193 and 90 nM, respectively (Figure 1B). The
`interaction of XL188 with USP7 was confirmed using ITC and
`differential scanning fluorimetry (DSF)
`(Figures 1C and S1F;
`Table S2). Consistent with the 100-fold improvement
`in
`biochemical inhibition of USP7 by XL188 compared with 1, a
`KD of 104 nM was measured for USP7 catalytic domain using
`ITC (Figure 1C, Table S2). The selectivity of XL188 was as-
`sessed against a panel of 41 purified DUBs, using ubiquitin-
`rhodamine (Ub-Rho) as substrate. XL188 retained the excellent
`selectivity for USP7 observed with 1; at a concentration of
`10 mM, XL188 exhibited little to no inhibition of any DUBs other
`than USP7 (Figure 1D and Table S1). In contrast, the enan-
`tiomer of XL188, XL203C (Figure 1A), showed 80-fold less
`potent inhibition of USP7 (IC50 = 7.18 mM, Figure 1B) and no
`significant inhibition of other DUBs (Figure S1G and Table S1).
`
`Post-Grant Review Petition for US 9,840,491
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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
`
`O
`
`N
`
`OH
`
`N
`XL203C
`
`N
`
`O
`
`Time (s)
`
`NH
`
`O
`
`N
`
`N
`
`0.02
`0
`-0.02
`-0.04
`-0.06
`-0.08
`-0.10
`-0.12
`-0.14
`-0.16
`
`N
`
`O
`
`C
`
`Corrected Heat Rate (μcal/s)
`
`Kd = 104 ± 15 nM
`n = 1.06 ± 0.01
`ΔH = -15.11 ± 0.13
`kcal/mol
`ΔS = -19.60 cal/mol•K
`
`0
`
`0.5
`
`2.0
`1.5
`1.0
`Mole ratio
`
`2.5
`
`3.0
`
`02
`
`-2
`-4
`-6
`-8
`-10
`-12
`-14
`-16
`
`1.2
`1.0
`0.8
`0.6
`0.4
`0.2
`
`0.0
`
`Normalized ν0
`
`USP7 catalytic domain
`
`0
`
`1
`10 100
`0.01 0.1
`Compound (μM)
`
`1.2
`1.0
`0.8
`
`0.6
`0.4
`0.2
`
`0.0
`
`A
`
`Cl
`
`B
`
`Normalized ν0
`
`NH
`
`O
`
`N
`
`N
`
`Structure-guided
`design
`>100-fold potency
`improvement
`
`O
`
`N
`
`N
`
`OH
`
`1
`
`N
`
`O
`
`USP7 full-length
`
`OH
`
`O
`
`N
`
`N
`
`XL188
`
`1
`XL188
`XL203C
`
`0
`
`1
`10 100
`0.01 0.1
`Compound (μM)
`
`Normalized Fit (kcal/mol)
`
`Protein
`
`Compound
`
`USP7 catalytic domain
`(aa 208-560)
`
`USP7 full length
`(aa 1-1102)
`
`1
`
`XL188
`
`XL203C
`
`1
`
`XL188
`
`XL203C
`
`IC50 (μM) ± SEM (n)
`12.3 ± 0.9 (18)
`
`0.193 ± 0.006 (4)
`
`10.7 ± 1.3 (2)
`
`10.2 ± 3.1 (3)
`
`0.090 ± 0.016 (3)
`
`7.18 ± 2.18 (3)
`
`140
`120
`100
`80
`60
`40
`20
`0
`
`D
`
`Activity (% control)
`
`10 μM XL188
`
`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 S1; Tables S1 and S2.
`
`XL188 Binds the S4-S5 Pocket of USP7
`We determined co-crystal structures of 1 and XL188 in complex
`with purified, recombinant USP7 catalytic domain (Figures 2A,
`2B, S2A, and S2B) to 1.9 A˚ and 2.2 A˚ , respectively. These
`high-resolution structures revealed that the catalytic cysteine
`and switching loop were in the unproductive conformation, as
`seen in apo structures (Hu et al., 2002), but with significantly
`different unit cell dimensions. A notable interaction in apo struc-
`tures (represented by PDB: 1NF8) was the face-to-face (or active
`site-to-active site) contact, highlighted by the mutual and
`
`complete insertion of Leu288’s side chain within a hydrophobic
`pocket near the catalytic cysteine, formed by helix a4, and loops
`a1 and a4-a5. This interaction was not present in our complex
`structures; instead, the active site in our complex structure
`was nearly free of crystal contacts. Our results therefore further
`confirm the relevance of the proposed USP7 autoinhibited
`conformation; namely, that it is not a crystal contact artifact.
`Importantly, our complex structures revealed unambiguous
`electron density (Figures S2A and S2C) for the inhibitors in the
`substrate binding cleft leading to the active site.
`Inhibitors
`
`Cell Chemical Biology 24, 1–11, December 21, 2017 3
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1022
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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
`
`Figure 2. Characterization of XL188 Binding to USP7
`(A) Ribbon diagram of USP7 with XL188.
`(B) Stereo view of USP7 (light blue) bound to XL188 (yellow). Hydrogen bonds are indicated by dashed lines.
`(C) Molecular surface representation of the USP7dXL188 co-structure. Highlighted regions indicate regions of altered HDX in the presence of XL188. Darker
`colors correspond to significant changes, whereas lighter colors correspond to regions with subtle changes.
`See also Figures S2 and S3; Table S3.
`
`occupied the S4 and S5 subsites, about 5 A˚ removed from the
`catalytic triad (Figures 2A and S2A), involving multiple hydrogen
`bonds with four inhibitor hetero-atoms (Figures 2A and S2B)
`common to both compounds. Specifically, the quinazolinone
`ketone formed hydrogen bonds with peptide backbone nitro-
`gens of Arg408 and Phe409, and the quinazolinone cyclic nitro-
`gen formed a hydrogen bond with the amide side chain of
`Gln297. The tertiary hydroxy group was stabilized by hydrogen
`bonds with the carboxylic group of Asp295 as well as the peptide
`backbone nitrogen of Val296. Asp295 is highly conserved
`among the USP family of deubiquitinating enzymes (Quesada
`et al., 2004), as it hydrogen bonds with ubiquitin’s backbone
`P4 position, an interaction presumed to be important for sub-
`strate stabilization (Hu et al., 2002). The oxygen atom of the
`piperidine amide was within 3 A˚ of the hydroxyl group of
`Tyr465, a strictly conserved DUB family side chain (Figure S4A).
`In addition, the phenyl ring of 1 and XL188 was buried in the S4
`pocket, which was bounded by the aromatic rings of Tyr514,
`His456, Phe409, and the aliphatic chains of Lys420 and
`Arg408. Notably, the side chain of Phe409 flips to reveal the
`hydrophobic pocket, a conformational
`rearrangement also
`observed upon binding of ubiquitin (Hu et al., 2002). The addi-
`tional methyl group of XL188 present at the carbon a to the
`phenyl ring was involved in multiple van der Waals interactions
`including with the backbone of Asn460 and the phenyl side chain
`
`4 Cell Chemical Biology 24, 1–11, December 21, 2017
`
`of Phe409, and was associated with lower B factors in the BL2
`loop. All atoms of 1 and XL188 were buried except the chlorine
`atom and N-methyl-piperazine side chain, respectively (Figures
`S2D and S2E). Compared with 1, XL188 was associated with a
`
`rotation of the fingers by about 5
`counterclockwise around an
`axis through the piperazine group.
`Crystallographic studies were complemented with hydrogen-
`deuterium exchange mass spectrometry (HDX MS) to monitor
`changes in protein dynamics. Exchange of backbone amide hy-
`drogens with bulk solvent can be accurately measured upon
`inhibitor binding (Iacob et al., 2009; Wales and Engen, 2006). On-
`line digestion of USP7 was performed and 85 peptic peptides
`covering 85% of USP7 catalytic domain were investigated with
`HDX MS in the free and bound states (Figures S3A and S3B).
`Both XL188 and 1 protected the BL1 and a-4/5 loops (Figure 2C),
`confirming that the observed crystal structure interactions are
`also relevant in solution. While the locations of the major changes
`were the same, XL188 protected USP7 from exchange more
`than 1, consistent with increased affinity (Figures S3B and
`S3C). Moreover, HDX MS results showed ligand-induced
`conformational changes and stabilizations distant
`from the
`active site. Inhibitors also stabilized/protected the palm region
`including helices a-3/4,
`that is near the catalytic cysteine,
`consistent with a previously proposed allosteric regulatory
`mechanism for USP7 (Faesen et al., 2011). Furthermore, a
`
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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
`
`acids lining the ligand binding pocket are conserved among
`the USP family of DUBs. Figure 3A shows a detailed ligand
`interaction diagram of XL188 with USP7; in the diagram residues
`that are conserved, defined as >80% of 52 other USPs possess-
`ing an equivalent residue according to MView classification
`(Figure S4A) (Brown et al., 1998), are indicated by a red box. Pre-
`vious site-directed mutagenesis studies show that amino acid
`substitutions at several of these positions abrogate the ability
`of the enzyme to cleave DUB substrates (Hu et al., 2002).
`Thus, with the goals of gaining insight
`into the observed
`compound selectivity and identifying the most productive ligand
`interactions in our crystal structures, we primarily focused our
`mutagenesis studies on non-conserved residues contained
`within the ligand binding pocket. Seven USP7 mutants with a
`single amino acid substitution and one with double substitution
`were generated (Figure 3B). Gln351, Met407, and Met410 were
`substituted with Ser, Lys, and Ser, respectively, selected based
`on the prevalence of the amino acid at equivalent positions in
`other USPs (Figure S4A); all other mutated amino acids were
`replaced with alanine. Six of the eight mutant proteins retained
`the ability to cleave the DUB substrate Ub-AMC, although two
`with significantly reduced activity relative to wild-type (Fig-
`ure 3B). The mutagenesis studies were carried out in parallel to
`compound optimization studies; thus, 1 was utilized to assess
`the inhibitory activity of the chemical series toward the mutants.
`1 inhibited four of the active mutants with IC50 values within
`several-fold of its IC50 for wild-type enzyme (Figure 3B). How-
`ever, USP7Q351S and USP7Y514A were highly resistant to 1,
`with 1 exhibiting no inhibitory effect at concentrations up to
`100 mM compound (Figures 3B and S4B). Gln351 is unique to
`USP7, with only one other USP DUB, USP14, containing this
`residue at the equivalent position and 80% of USPs containing
`a residue with a small side chain (Figure S4A). Tyr514, on the
`other hand, is highly conserved among DUBs. Thus, Gln351
`may be an important determinant of selectivity for the hydroxypi-
`peridine-based inhibitors. Initial mutagenesis studies were car-
`ried out using the catalytic domain; full-length USP7Q351S
`was confirmed to be resistant to both 1 and XL188 (Figures
`3C, 3D, and S4C). Gln351 hydrogen bonds with Gln297 only in
`the autoinhibited apo form and may be required for stabilization
`of the conformation bound by these inhibitors. Although a
`complete understanding of the molecular-level contribution of
`Gln351S to selectivity awaits further structural studies, these
`studies support that the binding mode observed in crystallo-
`graphic studies accurately represents the binding mode in
`solution.
`
`Structure-Activity Relationship Investigation
`The high-resolution structure of USP7 bound by 1 enabled rapid
`optimization of potency, solubility, and pharmacological pro-
`perties of the compound leading to XL188. As detailed above,
`four compound hetero-atoms were involved in hydrogen-
`bonding interactions with USP7, the phenyl ring was buried in
`the S4 hydrophobic pocket normally filled by the Leu73 side
`chain of substrate ubiquitin, and the chloro atom was solvent
`exposed.
`Initial structure-activity relationship investigations
`(Figure 4A)
`focused on establishing the importance of the
`hydrogen-bonding and hydrophobic interactions observed in
`IC50s were measured using
`the structure. All biochemical
`
`Cell Chemical Biology 24, 1–11, December 21, 2017 5
`
`A
`
`B
`
`Mutation
`WT
`Q351S
`M407K
`M410S
`M407K/M410S
`K420A
`H456A
`H461A
`Y514A
`
`1
`IC50 (uM)
`12
`> 100
`13
`48
`9
`N/A
`N/A
`16
`> 100
`
`Activity relative to WT
`in Ub-AMC assay
`=
`=
`--
`++
`=
`inactive
`inactive
`=
`--
`
`USP7 full-length
`
`USP7 WT
`USP7Q351S
`
`0
`
`1
`10
`0.1
`XL188 (μM)
`
`100
`
`1.2
`1.0
`0.8
`
`0.6
`0.4
`0.2
`
`0.0
`
`USP7 full-length
`
`D
`
`Normalized ν0
`
`USP7 WT
`USP7Q351S
`
`0
`
`0.1
`
`10
`
`100
`
`1
`1 (μM)
`
`1.2
`1.0
`0.8
`
`0.6
`0.4
`0.2
`
`0.0
`
`C
`
`Normalized ν0
`
`Figure 3. Analysis of USP7 Mutant Proteins
`(A) Detailed ligand interaction diagram of XL188 with USP7. Residues for
`which >80% of other USPs contain an amino acid belonging to the same class
`are boxed red.
`(B) Summary of activity against Ub-AMC and inhibition by 1 for USP7 mutant
`catalytic domain proteins.
`(C) Dose-response inhibition of full-length USP7WT and USP7Q351 (amino
`acids 1–1,102) by 1.
`(D) Dose-response inhibition of full-length USP7WT and USP7Q351 (amino
`acids 1–1,102) by XL188.
`See also Figure S4.
`
`disordered loop between a8/b14 was protected from deuterium
`incorporation, suggesting that this region becomes ordered
`upon inhibitor binding.
`
`Mutagenesis Studies Reveal Determinants of Selectivity
`Given the high degree of selectivity of this chemical series for
`USP7, we were surprised to discover that nearly every residue
`directly interacting with inhibitor and several additional amino
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1022
`Page 6
`
`EXHIBIT 10
`DELANSORNE DECLARATION
`
`214LT:20700:449509:1:ALEXANDRIA
`
`

`

`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
`
`O
`
`N
`
`N
`
`OH
`
`N
`
`R1
`
`(rac)-Me
`
`(R)-Me
`
`(S)-Me
`
`O
`
`R1
`USP7
`IC50 (μM)
`0.56
`
`0.19
`
`11
`
`MLM t1/2
`(min)
`
`33.7
`
`31.1
`
`19
`
`13.6
`
`O
`
`NH
`
`R
`
`R
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`B
`
`USP7
`IC50 (μM)
`
`> 100
`
`> 100
`
`1.3
`
`ID
`
`11
`
`XL188
`
`XL203C
`
`A
`
`ID
`
`Structure
`
`USP7
`IC50 (μM)
`
`ID
`
`Structure
`
`1
`
`2
`
`3
`
`Cl
`
`Cl
`
`Cl
`
`O
`
`N
`
`O
`
`N
`
`O
`
`N
`
`N
`
`N
`
`N
`
`OH
`
`CN
`
`N
`
`O
`
`N
`
`O
`
`N
`
`O
`
`12
`
`> 100
`
`> 100
`
`6
`
`7
`
`8
`
`Cl
`
`Cl
`
`Cl
`
`O
`
`N
`
`O
`
`N
`
`O
`
`N
`
`N
`
`N
`
`N
`
`OH
`
`OH
`
`N
`
`O
`
`N
`
`O
`
`OH
`
`N
`
`(rac)-iPr
`
`(R)-Me
`
`(R)-Me
`
`(rac)-Me
`
`(rac)-Me
`
`0.27
`
`0.48
`
`0.13
`
`0.24
`
`0.35
`
`Densitometry
`
`7.3
`
`21.1
`
`3.9
`
`7.3
`
`XL188
`XL203C
`
`0
`
`1
`10
`0.1
`Compound (μM)
`
`100
`
`N
`
`N
`
`N
`
`O
`
`NN
`
`N
`
`120
`100
`80
`
`60
`40
`
`20
`0.0
`
`% USP7 labeling
`
`4
`
`5
`
`Cl
`
`Cl
`
`O
`
`N
`
`O
`
`N
`
`N
`
`N
`
`OH
`
`N
`
`OH
`
`N
`
`O
`
`> 100
`
`> 100
`
`9
`
`10
`
`Cl
`
`Cl
`
`O
`
`N
`
`O
`
`N
`
`O
`
`OH
`
`OH
`
`O
`
`N
`
`O
`
`N
`
`N
`
`N
`
`12
`
`13
`
`14
`
`15
`
`16
`
`0.42
`
`40
`
`115
`
`GAPDH
`
`---
`
`+-
`
`0.05
`
`+-
`
`0.5
`
`+-5
`
`+-5
`
`0
`
`+--
`
`---
`
`+
`0.5
`-
`
`+
`0.05
`-
`
`+5-
`
`+
`50
`-
`
`+--
`
`HA-Ub-VS
`XL188 (μM)
`XL203C (μM)
`
`C
`
`HA-Ub-USP7
`USP7
`
`Figure 4. Structure-Activity Relationship Studies
`(A and B) Structures, USP7 inhibitory activity, and mouse liver microsome (MLM) stability of synthesized compounds.
`(C) Analysis of the ability of XL188 and XL203C to bind native USP7 across multiple doses in HEK293T lysates using competitive activity-based protein profiling.
`Error bars represent SD (n = 2).
`See also Figure S5.
`
`USP7 catalytic domain and Ub-AMC as substrate. 1 inhibited
`isolated USP7 catalytic domain with an IC50 of 12.3 mM and
`full-length protein with an IC50 of 10.2 mM. Compounds 2 and
`3, in which the hydroxypiperidine -OH group was removed or
`replaced with -CN, respectively, exhibited little inhibition of
`USP7 at concentrations up to 100 mM. Similarly, removal of the
`amide carbonyl, as in 4, abrogated USP7 activity as did contrac-
`tion of the 6-membered piperidine to a 5-membered ring (5).
`Occupancy of the S4 hydrophobic pocket by the phenyl ring
`was confirmed to be required for activity, as removal of the moi-
`ety (6) or shortening the linker between the hydroxypiperidine
`IC50s > 100 mM.
`(7) resulted in biochemical
`In
`and phenyl
`contrast, installation of a racemic methyl group on the methylene
`adjacent to the phenyl ring (8) improved biochemical potency
`approximately 10-fold, consistent with data reported in the
`patent exemplifying 1. To explore the importance of stereo-
`chemistry of the methyl group we prepared both enantiomers,
`which revealed that the (R)-stereoisomer (9) was approximately
`100-fold more potent than the (S)-stereoisomer (10). Unfortu-
`nately, investigation of 9’s ability to bind and inhibit USP7 in cells
`was hampered by poor solubility in aqueous buffer. To improve
`this property we focused on installation of polar moieties in place
`of the solvent-exposed chloro atom (Figure 4B). Installation of
`different groups, including an N-methyl-piperazine, piperidine,
`dimethylamine, and imidazole, linked to the 7-position of the
`
`6 Cell Chemical Biology 24, 1–11, December 21, 2017
`
`quinazolinone via a short carbon chain and amide bond linkage,
`improved potency 2- to 4-fold relative to the parent compound 9.
`With several analogs exhibiting submicromolar USP7 IC50s, we
`considered metabolic stability as an additional parameter for
`compound prioritization, since a probe suitable for in vivo studies
`would be highly valuable for pharmacological validation of USP7
`in animal disease models. XL188 exhibited the greatest stability
`in the presence of mouse liver microsomes with a half-life of
`31 min (Figure 4B). Competitive activity-based protein profiling
`using the DUB-targeting activity-based probe HA-Ub-Vs
`(Hewings et al., 2017) confirmed that XL188 bound native
`USP7 (Figure 4C). Treatment of HEK293T lysates with XL188
`significantly blocked labeling of USP7 by HA-Ub-Vs with an
`IC50 of approximately 0.9 mM, a value that represents a signifi-
`cant improvement compared with 1 (at a concentration of
`500 mM, 1 competes for 50% of probe labeling [Figure S5A]).
`Because in this experiment an irreversible probe (HA-Ub-Vs) is
`competing for occupancy of USP7 with a reversible inhibitor,
`the measured cell-based IC50s may underestimate binding. A
`hemagglutinin blot of the same treated lysates (Figure S5B)
`confirmed the high degree of selectivity for USP7 observed in
`the 41-member purified enzyme panel. As a measure of general
`toxicity, we treated peripheral blood mononuclear cells (PBMCs)
`with XL188 and observed no growth suppression at concentra-
`tions up to 10 mM following 72 hr of treatment (Figure S5C). To
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1022
`Page 7
`
`EXHIBIT 10
`DELANSORNE DECLARATION
`
`214LT:20700:449509:1:ALEXANDRIA
`
`

`

`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
`
`C
`
`XL188 (μM)
`5 10 20
`
`XL203C (μM)
`5
`10
`20
`
`1
`
`-
`
`1
`
`MCF7
`
`XL188 (μM)
`5 10 20
`
`XL203C (μM)
`5
`10
`20
`
`1
`
`-
`
`1
`
`B
`
`D
`
`HDM2
`
`p53
`
`p21
`
`GAPDH
`
`HDM2
`
`p53
`
`p21
`
`GAPDH
`
`+
`-
`-
`
`+
`1
`-
`
`+
`5
`-
`
`+
`+
`10 20
`-
`-
`
`+
`-
`1
`
`+
`-
`5
`
`+
`-
`10
`
`+
`-
`20
`
`cycloheximide
`XL188 (μM)
`XL203C (μM)
`HDM2
`
`MCF7
`
`+
`-
`-
`
`+
`1
`-
`
`+
`5
`-
`
`+
`+
`10 20
`-
`-
`
`+
`-
`5
`
`+
`-
`10
`
`+
`-
`20
`
`p53
`
`p21
`
`GAPDH
`
`cycloheximide
`XL188 (μM)
`XL203C (μM)
`HDM2
`p53
`
`p21
`
`GAPDH
`
`Inhibitor XL188
`5. The USP7
`Figure
`Promotes Loss of HDM2 and Accumulation
`of p53 and p21
`(A) Analysis of HDM2, p53, and p21 protein levels
`in MCF7 cells treated with XL188 or XL203C at the
`indicated concentration for 16 hr.
`(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.
`(C) Analysis of HDM2, p53, and p21 protein levels
`in MM.1S cells treated with XL188 or XL203C at
`the indicated concentration for 6 hr.
`(D) Analysis of HDM2, p53, and p21 protein
`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.
`
`MM.1S
`
`MM.1S
`
`achieve an ideally matched negative control compound to use in
`conjunction with XL188, we prepared its enantiomer, XL203C.
`XL203C showed 80-