Expert Opinion on Therapeutic Patents
`
`ISSN: 1354-3776 (Print) 1744-7674 (Online) Journal homepage: http://www.tandfonline.com/loi/ietp20
`
`Selective and reversible inhibitors of ubiquitin-
`specific protease 7: a patent evaluation
`(WO2013030218)
`
`Benedikt M Kessler
`
`To cite this article: Benedikt M Kessler (2014) Selective and reversible inhibitors of ubiquitin-
`specific protease 7: a patent evaluation (WO2013030218), Expert Opinion on Therapeutic Patents,
`24:5, 597-602, DOI: 10.1517/13543776.2014.882320
`To link to this article: http://dx.doi.org/10.1517/13543776.2014.882320
`
`Published online: 24 Jan 2014.
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`Date: 02 November 2017, At: 01:43
`
`EXHIBIT 4
`
`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1016
`Page 1
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`

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`1.
`
`2.
`
`3.
`
`4.
`
`Introduction
`
`Chemistry
`
`Biology and action
`
`Expert opinion
`
`EXHIBIT 4
`
`Patent Evaluation
`
`Selective and reversible inhibitors
`of ubiquitin-specific protease 7:
`a patent evaluation
`(WO2013030218)
`
`Benedikt M Kessler
`University of Oxford, Target Discovery Institute, Nuffield Department of Medicine, Oxford, UK
`
`The invention described in this review (WO2013030218) relates to compounds
`based on the quinazolin-4-one scaffold, their process of preparation and
`applications to inhibit the ubiquitin-specific protease 7 (USP7), a deubiquiti-
`nating enzyme (DUB), which is considered a potentially important new drug
`target for treating cancer and immunological disorders. Data are presented
`indicating that these small-molecule compounds are useful as selective and
`reversible inhibitors of USP7 in vitro and also in a cellular context, although
`the panel of other enzymes tested was limited. The synthesis strategy allows
`for the generation of a considerable variety of compounds, although similar
`properties of selective USP7 inhibition were reported for other related com-
`pound classes, thereby increasing the complexity of the patenting process.
`However, structural patterns that contribute to the selectivity of USP7 and
`other DUB enzyme inhibition are starting to emerge. Practical implications
`involve the treatment of cancer, neurodegenerative diseases, immunological
`disorders, diabetes, bone and joint diseases, cardiovascular diseases and viral
`and bacterial infections. The quality of these findings and a comparison to
`other compound classes with similar properties, as well as the potential for
`further development toward clinical exploitation are discussed.
`
`Keywords: anticancer treatment, anti-inflammatory, protease, small-molecule inhibitor,
`ubiquitin, ubiquitin-specific protease 7
`
`Expert Opin. Ther. Patents (2014) 24(5):597-602
`
`1. Introduction
`
`Pharmacological inhibition of molecular targets within the ubiquitin-proteasome
`system to modulate ‘proteostasis’, a term for protein turnover, has received increased
`attention in recent years. The reason behind this development is the fact that basic
`scientific knowledge about molecular aspects of ubiquitin processing and conjugat-
`ing enzymes and their involvement in human diseases has been accumulated to an
`extent to justify the investment for pharmacological intervention strategies from
`both academic and pharma.
`Most proteins within eukaryotic cells are turned over by the ubiquitin-
`proteasome system [1]. Ubiquitin, a small 76 amino acid protein, is covalently
`attached to protein substrates via lysine side chains and the ubiquitin C terminus
`via isopeptide bonds to form ubiquitin--protein or poly-ubiquitin--protein conju-
`gates, thereby influencing their biological fate [2,3]. Therefore, enzymes involved in
`either attaching or removing ubiquitin molecules are critically involved in control-
`ling lifespan, function or localization of cellular proteins. Enzymes that cleave poly-
`ubiquitin chains, mono-ubiquitin conjugates and ubiquitin proproteins are referred
`to as deubiquitinating enzymes (DUBs), and there are ~ 100 genes within the
`human genome that are believed to exert DUB enzyme activity [4], 79 of which
`
`10.1517/13543776.2014.882320 © 2014 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674
`All rights reserved: reproduction in whole or in part not permitted
`
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`EXHIBIT 4
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`B. M. Kessler
`
`are putatively functional [5]. Many of them are implicated in
`human diseases, and for one of these enzymes, ubiquitin-
`specific protease 7 (USP7), connections to cancer is quite
`likely [6]. Initially, it was thought that cell cytotoxic (and
`therefore antitumor) effects of USP7 are mediated through
`modulating the stability of p53 and its ubiquitin E3 ligases
`HDM2/HDMX [7,8]. However, USP7 also appears to stabi-
`lize a number of other protein targets, including phosphatase
`and tensin homolog (PTEN), claspin, Chk1 kinase and the
`transcription factor forkhead box protein O4 (FOXO4), all
`of which are linked to tumorigenesis [9]. In addition to this,
`USP7 was reported to also stabilize protein targets in immu-
`nological pathways, such as the signalling molecules IKKg,
`TRAF6 and Foxp3, the latter of which controls activation of
`regulatory T cells [10,11]. USP7 directly interacts with the viral
`proteins ICP0 and EBNA1 [12] and was also shown to modu-
`late ubiquitination of the epigenetic targets H2B, BMI1,
`MEL-18 and DNMT1 and to be involved in DNA damage
`response pathways [13]. More recently, USP7 was shown to
`be implicated in adipocyte differentiation by deubiquitylating
`the acetyltransferases TIP60 [14]. The diverse nature of sub-
`strates apparently modulated by USP7 renders its biology
`more complex and potentially links USP7 to several human
`diseases, and it is therefore predicted that USP7 inhibition
`by small molecules will have a variety of different effects in
`cellular physiology.
`Despite this complexity, early studies on USP7 inhibition
`show antitumor properties
`in multiple myeloma
`cells
`[15]), neuroblastoma cells (P22077:
`[16]) and p53
`(P5091:
`wild-type and isogenic cancer cells (HBX41108: [7]). More
`recent evidence suggests that based on USP7s effect on mole-
`cules involved in immune signaling [11,17], pharmacological
`interference with USP7 activity will have immunomodulatory
`consequences.
`These findings raise the opportunity to exploit USP7
`inhibitor development commercially for different disease
`types, in particular cancer [18] and inflammation [19].
`The present patent [20] reports on another chemical scaffold
`that has been exploited to obtain USP7 selective inhibitors
`and their biochemical characterization.
`
`2. Chemistry
`
`The chemistry described in this patent application is a novel
`extension of already existing patents and publications cover-
`ing small molecule USP7 inhibitors on the basis of the ami-
`dotetrahydroacridine and cyano-indenopyrazine scaffolds,
`which include HBX 41,108 HBX 19,818 (Figure 1 and
`Table 1) [7,21,22]. There are already several patents that have
`been created around these compounds
`from the same
`authors [23,24], rendering the exact interpretation and alloca-
`tion of intellectual property rights complex. The currently
`described class of compounds have a quinazolin-4-one
`structural basis, and variations are predominantly described
`on the L1 site using linear or branched (Ci -- C6) alkylenes
`
`optionally substituted by one or more chemical groups (see,
`e.g., Figure 1A). The patent application describes 30 example
`compounds that were synthesized based on these schemes,
`from which two (examples 2 and 5) were tested for their
`biochemical properties of inhibiting USP7 using recombi-
`nant and biochemical assays (Figure 1B and C). For these
`compounds, the IC50 value for USP7 inhibition in cell
`lysates is in the range of 25 -- 50 µM [20]. Interestingly,
`the same authors previously described very similar com-
`pounds with selective USP7 inhibition properties on the
`basis of
`the amidotetrahydroacridine core structure that
`included HBX 19,818 and HBX 28,258 (Table 1), which
`in addition to a different core, the chlorine has been substi-
`tuted with an oxygen and thereby do not fall under this pat-
`ent structure [21]. The IC50 values for USP7 inhibition
`in vitro and in cells are similar to HBX 19,818, which is
`28.1 µM and also in the low micromolar range when tested
`in cells [21], which is not unexpected as they share overlap-
`ping structural properties with the ones described in the
`patent application. The structural features governing selec-
`tivity for USP7 inhibition as compared to other DUBs or
`different protease species are not documented in detail in
`this patent application, but are characterized in previous
`studies reported by the same authors. A structural feature
`that contributes to the selectivity of USP7 DUB inhibition
`in the compound HBX41,108 is the chlorine substitution.
`When this moiety was
`replaced by a hydroxyl group
`(HBX 91,490), the USP7 inhibitory capacity was lost [7].
`Also, a comparison of HBX 19,818 and HBX 28,258
`with the inactive inhibitor derivative HBX 28,364 revealed
`that the basic alkyl amide side chain (R) is also an impor-
`tant specificity element (Table 1) [21]. The compounds cov-
`ered under
`the patent application appear
`to have the
`characteristics of reversible inhibitors as tested by measuring
`enzyme activity recovery using gel filtration, large dilution
`assays and native electrospray ionization mass spectrometry.
`These compounds behaved in a similar fashion to HBX
`41,108, for which an Eadie--Hofstee analysis indicated that
`this compound was an uncompetitive inhibitor of USP7
`activity [10]. This is in contrast to the HBX 19,818 inhibitor
`that was
`shown to react with the USP7 catalytic site
`Cys223 directly [21]. Thus, it is thought that the compounds
`described in the patent application, similar to HBX 41,108,
`preferentially inhibited USP7 after formation of the enzy-
`me--substrate complex rather than preventing the interaction
`of the substrate with USP7. Other small molecules with the
`capacity to inhibit USP7 have been described in the litera-
`ture and include PR-619, a general DUB inhibitor that
`inhibits USP7 (Life Sensors, Inc., PA, USA) [25,26], and
`the
`selective USP7 inhibitors P5091, P045204 and
`P22077 (structural analogs of P5091) (Progenra, Inc., PA,
`USA) (Table 1) [6,15,25]. More recently, the natural product
`Spongiacidin C was also shown to have USP7 inhibitory
`activity [27]. All these small molecules described appear to
`inhibit USP7 in the high nanomolar/low micromolar range
`
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`Expert Opin. Ther. Patents (2014) 24(5)
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`Selective and reversible inhibitors of USP7 (WO2013030218)
`
`B.
`
`A.
`
`C.
`
`Figure 1. Illustration of the structural scaffold and small molecules targeting ubiquitin-specific protease 7 (USP7) enzyme
`activity. (A) Inhibitors based on the quinazolin-4-one scaffold as described [20]; (B,C) example compounds 2 (B) and 5 (C) that
`were synthesized based on the quinazolin scaffold and that exert selectivity for USP7 inhibition are shown (Table 1).
`
`(Table 1). The selectivity toward USP7 against other
`DUBs has been tested either by using a panel of recombi-
`nant DUB enzymes or by using ubiquitin-based active
`site-directed probes to profile DUB activity in cellular
`extracts obtained from cancer cells treated with various
`inhibitor concentrations and does vary considerably between
`the different compounds reported (Table 1) [15,21,25]. Inter-
`estingly,
`claims
`are made
`for
`the
`compounds HBX
`19,818, HBX 28,258 and P5091 to be USP7-specific
`(> 30-fold over other DUBs), whereas
`the compound
`P22077 has been reported to also inhibit USP47 within a
`similar range [25,28], a trait that has not been examined for
`any of the other small compounds reported to be selective
`for USP7 (Table 1).
`
`3. Biology and action
`
`The initial goal that stimulated the development of small mol-
`ecules targeting USP7 was to interfere with tumorigenesis and
`cancer [8,10]. Indeed, this has been observed in several studies
`using different sources of small-molecule inhibitors [7,15,16],
`although compounds with DUB and USP7 inhibition prop-
`erties appear to be generally cytotoxic, which requires a careful
`evaluation and titration to determine potential therapeutic
`windows [15,25,29].
`Since USP7 functions in p53 biology, DNA damage path-
`ways, transcription factor regulation, interacts with viral pro-
`teins
`and also appears
`to play a
`role
`in regulating
`inflammatory pathways, it is generally expected that pharma-
`cological inhibition of USP7 will have a multitude of effects.
`Most studies so far, including this patent application, has
`been limited to biological studies using cell lines to predomi-
`nantly characterize the inhibitory state of USP7 upon small-
`molecule treatment (this patent) and subsequent functional
`effects on cell viability, cytokine expression, transcription
`
`and protein interactions. There are only a few cases reported
`yet where in vivo studies were performed, indicating poten-
`tially promising results demonstrating antitumor effects in
`xenograft mice [15].
`
`4. Expert opinion
`
`The current patent on small-molecule USP7 inhibitors is in
`principle an extension of previously reported patents from
`the same institution. The molecular scaffold described here
`is very similar, and little pharmacological data has been
`described with these derivatives directly, but
`the patent
`attempts to cover possible applications in a variety of human
`diseases in a broad fashion. The chemistry around these novel
`compound inhibitors is described thoroughly, but the charac-
`terization of biological effects and applications are scarce.
`Other recent patents related to the one described here
`[24] (USP7 inhibitor
`are [19] (treatment of inflammation),
`compounds
`based
`on
`the
`1,5-dihydro-pyrrol-2-one
`skeleton -- Hybrigenics), [23] (amidotetrahydroacridine-based
`compounds -- Hybrigenics) and [18] (anti-neoplastic com-
`pounds, compositions and methods -- Progenra). These are
`the two main producers of small-molecule USP7 selective
`compounds, but other organizations such as Mission Thera-
`peutics, the DUB-Alliance and other pharma companies are
`currently also actively developing novel compounds [30,31].
`The Hybrigenics compounds have been used most widely,
`but also the Progenra inhibitors have proven to be useful as
`research tools to target DUB activity. Current efforts are
`ongoing on multiple sites to further develop their compounds
`for drug development.
`Generally, it is difficult to evaluate whether the compounds
`described in this or other previous patents are truly
`USP7-‘specific’, as no biochemical characterization to date is
`capable of screening all ~ 90 human DUBs and enzymes
`
`Expert Opin. Ther. Patents (2014) 24(5)
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`
`Table 1. Pharmacological properties of USP7 selective small-molecule inhibitors.
`
`Name
`
`Example 1*
`
`Structure
`
`IC50 for USP7
`z
`
`25 -- 50 µM
`
`DUB Selectivity
`
`USP7 selectivity
`Tested in cell extracts and against
`USP5,8,10, CYLD, UCH-L3
`
`Ref.
`
`[20]
`
`z
`28.1 µM
`z
`22.6 µM
`
`424 nM
`
`USP7 selectivity
`Tested against USP2,5,8,20
`UCH-L1, L3, SENP1, caspase 3
`
`Some USP7 selectivity, only tested
`against UCH-L1, cathepsins, HIV
`protease
`
`8.6 µM
`z
`~20 µM
`420 nM
`
`USP7, USP47
`Tested against a panel of 26 DUBs,
`cathepsins, 20S proteasome
`
`HBX 19,818
`HBX 28,258
`
`HBX 41,108
`
`P22077
`
`P45204
`
`P5091
`
`PR619
`
`Spongiacidin C
`
`4.2 µM
`z
`5 -- 10 µM
`
`4.3 µM
`
`3.8 µM
`
`[21]
`
`[7]
`
`[25]
`[26]
`[6]
`
`[15]
`
`USP7 selectivity
`Tested against USP2,5,8,20,21,28
`UCH-L1, caspase 3, cathepsin K,
`calpain 1, MMP13
`
`Not selective for USP7
`
`[25,26]
`
`USP7 selectivity demonstrated, but
`against a small panel of DUBs only
`
`[27]
`
`*Example compound 1 from [20].
`z
`Tested against either cell extracts or intact cells.
`CYLD: Cylindromatosis (turban tumour syndrome); DUB: Deubiquitinating; MMP: Matrix metalloproteinase; SENP: Sentrin-specific protease;
`UCH: Ubiquitin C-terminal hydrolase; USP: Ubiquitin-specific protease.
`
`derived from other species. It is likely that many of such com-
`pounds may have cross-reactivities to other related DUBs
`once their inhibitory activities can be assessed on a wider
`range. For instance, an attempt toward this was described
`using differential DUB activity profiling experiments compar-
`ing cells treated or not with USP7 inhibitor by quantitative
`proteomics, capable of screening ~ 50 cellular DUBs [25].
`
`Besides this, there may be a potential therapeutic benefit of
`pharmacologically targeting multiple DUBs at once that can
`result in synergistic effects, as indicated for P22077 targeting
`USP7 and USP47 [28].
`The therapeutic potential of small molecules with selectiv-
`ity toward USP7 is considerable and may not be restricted
`to anticancer effects, but most likely will include applications
`
`600
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`Expert Opin. Ther. Patents (2014) 24(5)
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`Selective and reversible inhibitors of USP7 (WO2013030218)
`
`as anti-inflammatory agents. However, we are at an early stage
`of making accurate predictions, but it is likely that these two
`areas will become a major focus of future academic and
`pharma-based efforts of drug development targeting USP7
`in human diseases.
`
`Declaration of interest
`
`The author has an affiliation with the DUB Alliance (Cancer
`Research Technologies/Forma Therapeutics) and has been
`supported by the Biomedical Research Centre (NIHRI), UK.
`
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`Cancer Res Technol 2013; Available from:
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`cancer-news/press-release/forma-
`therapeutics-and-cancer-research-
`technology-to-discover-cancer-drugs-
`targeting
`
`31. Delivering highly innovative cancer
`therapeutics targeting ubiquitin pathways
`involved in the DNA damage response.
`Mission Ther 2013;
`
`Available from: http://www.
`missiontherapeutics.com
`
`Affiliation
`Benedikt M Kessler
`University of Oxford, Target Discovery Institute,
`Nuffield Department of Medicine,
`Roosevelt Drive, Oxford OX3, 7BN, UK
`Tel: +01 865 631 921;
`E-mail: Benedikt.kessler@ndm.ox.ac.uk
`
`602
`
`Expert Opin. Ther. Patents (2014) 24(5)
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`Post-Grant Review Petition for US 9,840,491
`EXHIBIT 1016
`Page 7
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