Identification and validation of selective deubiquitinase inhibitors

Article abstract of DOI:10.1016/j.chembiol.2021.05.012

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.

Full text of DOI:10.1016/j.chembiol.2021.05.012

Resource
Identification and validation of selective
deubiquitinase inhibitors
Graphical abstract
Authors
Anthony C. Varca, Dominick Casalena,
Wai Cheung Chan, ..., Jarrod A. Marto,
Douglas Auld, Sara J. Buhrlage
Panel of 8 Deubiquitinases (DUBs)
~50k compound library
OTUD3
UCHL1
USP7
USP8
USP10
USP30
USP17
USP28
Correspondence
saraj_buhrlage@dfci.harvard.edu
Ub
Rho
Ub
Rho
Ub
DUB
DUB
Ub
Rho
DUB
Rho
Primary screen
In brief
Ub
Rho
Deubiquitinases (DUBs) have emerged as
therapeutically interesting targets due to
their involvement in protein turnover in
cells. Varca et al. describe a multi-DUB
high-throughput screen and orthogonal
validation campaign to identify new tool
compounds for elucidating DUB function
and provide a roadmap for future DUB
inhibitor screens.
Dose-response
Confirmation
Log [inhibitor], μM
Ub
S
S
...
SH
DUB1
Hit ID +
Validation
DUB1
DUB2
SH
Ub
DUB2
DUB2
Ub
DUB3
DUB3
Expanded Selectivity Profiling
Activity-based protein profiling
DUB Hits/Leads
USP7
USP8
USP10
USP28
USP30
Highlights
d
d
d
d
High-throughput screen of ꢀ50k compound library against 8
deubiquitinases
Identification and validation of selective inhibitors for
individual DUBs
Study demonstrates importance of validation experiments as
part of hit triage
Generation of robust dataset of compound activities against
selected DUBs
Varca et al., 2021, Cell Chemical Biology 28, 1–14
August 19, 2021 ª 2021 Published by Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2021.05.012
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Identification and validation
of selective deubiquitinase inhibitors
Anthony C. Varca,1,2 Dominick Casalena,3 Wai Cheung Chan,1,2,4,6 Bin Hu,1,2 Robert S. Magin,1,2 Rebekka M. Roberts,1,2
Xiaoxi Liu,1,2 He Zhu,1,4,5,6 Hyuk-Soo Seo,1 Sirano Dhe-Paganon,1 Jarrod A. Marto,1,4,6 Douglas Auld,3
and Sara J. Buhrlage1,2,7,
*
1Department of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
3FAST Lab, Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
4Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
5Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
6Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA 02215, USA
7Lead contact
*Correspondence: saraj_buhrlage@dfci.harvard.edu
https://doi.org/10.1016/j.chembiol.2021.05.012
SUMMARY
Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through cat-
alytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB
biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no
approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a
broader framework for future inhibitor development across the gene family, we performed high-throughput
screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-
characterized DUB families. Promising hit compounds were validated in a series of counter-screens and
orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through
these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly
identifying and validating selective inhibitors of related enzymes.
INTRODUCTION
DUBs are isopeptidases that catalyze the removal of ubiquitin
from their target proteins. There are currently around 100 re-
Since its discovery over 40 years ago, the ubiquitin-proteasome ported mammalian DUBs divided broadly into 2 classes based
system (UPS) has been the subject of extensive research inter- on their catalytic mechanism: cysteine proteases and zinc metal-
est. The UPS mediates ubiquitination, a process whereby ubiq- loproteases. In addition, the approximately 90 cysteine protease
uitin, a small 76 amino acid protein, is added to substrate pro- DUBs can be further subdivided into 6 families based on
teins by the concerted action of ubiquitin activating (E1), sequence homology (Mevissen and Komander, 2017; Abdul Reh-
conjugating (E2), and ligating (E3) enzymes and removed by deu- man et al., 2016; Kwasna et al., 2018; Haahr et al., 2018; Her-
biquitinases (DUBs) (Kerscher et al., 2006). The most common manns et al., 2018; Hewings et al., 2018; Gopinath et al., 2016).
functional outcome of polyubiquitination from UPS-mediated DUBs play significant roles in physiology, including growth factor
attachment of multiple ubiquitin molecules to a single substrate signaling, genome stability, redox regulation, cell fate, apoptosis,
is proteasomal degradation of the substrate protein (Kleiger and and others (Turcu et al., 2009; Amerik and Hochstrasser, 2004;
Mayor, 2014; Ciechanover, 2005). This process is critical for Cotto-Rios et al., 2012; Ramakrishna et al., 2011). A myriad of
regulating protein turnover and homeostasis. Therefore, pro- studies have nominated DUBs as an emergent class of drug tar-
cesses such as oncogenic transformation affect the UPS and gets to multiple human pathologies, such as cancer (Fraile et al.,
render molecular players involved in the UPS potential drug tar- 2012; D’Arcy et al., 2015; Pinto-Fernandez and Kessler, 2016),
gets of interest (Ding et al., 2009; Senft et al., 2018). The approval neurodegeneration (Das et al., 2020; Lim et al., 2020; Leroy
of bortezomib (Richardson et al., 2003), a proteasome inhibitor, et al., 1998), and inflammation/immune response (Hu and Sun,
as a cancer therapeutic as well as the recent discovery that 2016; Zinngrebe et al., 2014; Wertz et al., 2004). In some in-
immunomodulatory drugs (IMiDs) target E3 ligases to promote stances, genomic alteration renders DUBs as primary drivers of
targeted degradation of specific neo-substrates (Petzold et al., pathogenesis (Oliveira et al., 2004; Reincke et al., 2015; Chen
2016; Matyskiela et al., 2016; Winter et al., 2015; Sakamoto et al., 2018). In other contexts, DUBs can be therapeutically tar-
et al., 2001) have heightened the interest in UPS drug discovery geted to mediate degradation of protein drivers of a disease
and development. In this context, DUBs have also emerged as state. In a growing number of examples, DUBs were shown to
an enzyme family of growing interest for therapeutic targeting.
stabilize ‘‘undruggable’’ proteins, including transcription factors,
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such as Myc, Np63, and HIF1a (Prieto-Garcia et al., 2020; Popov UCHL1, and OTUD3 (Figure 1B). Selection of these DUB en-
et al., 2007; Bingol et al., 2014; Flugel et al., 2012; Boselli et al., zymes was based on multiple considerations. First was ease of
2017). The ability to promote the selective degradation of these access to ensure enough quantity of high-purity enzyme (Fig-
proteins through DUB inhibition highlights the immense potential ure S1A). After taking this into account, we sought the inclusion
of DUBs as therapeutic targets. Despite this broad array of func- of multiple members of the largest DUB family (the USP subfam-
tion and therapeutic potential, there is a significant lack of well- ily) in addition to representatives from the two other most well-
validated probe compounds for a majority of DUBs hindering studied cysteine protease DUB families (UCHL and OTU). In
the ability to further study the biological outcomes of DUB addition, we selected DUBs that were associated with inter-
inhibition.
esting biology and/or were lacking in chemical probes to further
Recent studies focused on one of the best studied DUBs, explore their biological function. By using this assortment of
USP7, demonstrated that developing potent and selective DUBs, we were able to assess targetability and cross-reactivity
DUB inhibitors is achievable, and that pharmacological DUB in- within and across DUB families from hits from the initial screen.
hibition can promote degradation of disease-relevant proteins The approximately 50k compound library was comprised of a
for therapeutic benefit (Lamberto et al., 2017; Turnbull et al., curated small-molecule diversity set as well as a smaller natural
2017; Kategaya et al., 2017; Gavory et al., 2018; Stolte et al., product collection. Both compound sets demonstrate good
2018; Schauer et al., 2020a; Leger et al., 2020). However, selec- drug-like properties with reasonable lipophilicity, polar surface
tive compounds have been identified for only a very limited num- area, and molecular weight (Waring, 2010; Lipinski et al., 2001;
ber of DUBs beyond USP7 (Ndubaku and Tsui, 2015; Schauer Navia and Chaturvedi, 1996; Leeson and Springthorpe, 2007)
et al., 2020b; Ritorto et al., 2014). A majority of the reported with the natural product set showing a wider range of properties
DUB inhibitors exhibit weak inhibitory activity (double-digit as shown in Figure 1C.
micromolar range), contain undesirable chemical features, and/
Our screening and compound triage cascade is summarized
or are now known to possess poor selectivity across the DUB in Figure 1D. For the primary assay, all compounds were tested
enzyme family (Ritorto et al., 2014; Schauer et al., 2020b; Ndu- against each DUB at concentrations of 20, 25, or 50 mM.
baku and Tsui, 2015; Farshi et al., 2015). Therefore, despite the Selected actives were subsequently tested in dose-response
accumulating evidence that DUBs represent promising thera- against a minimum of two DUBs to confirm activity and selec-
peutic targets, the number of high-quality chemical probes and tivity. To assess the ability of parallel DUB screening to accel-
lead compounds with appropriate potency and selectivity con- erate identification of high-quality DUB hit and lead compounds,
tinues to be limited.
we applied filters to the dose-response dataset to identify potent
Here, we describe generation of a dense dataset of DUB- and selective scaffolds for each DUB. The selectivity and po-
ligand interactions that enabled rapid generation of small-mole- tency filters, discussed in more detail later on, enabled us to
cule inhibitors and chemical probes for multiple DUBs as well as identify actives with selectivity for a single DUB among the entire
a wealth of data to facilitate future inhibitor development studies. library as well as rank these compounds by potency against the
To achieve this, we employed high-throughput, parallel target DUB. These top hits were selected for resynthesis and
screening of eight DUBs against the same library of 47.48k small confirmation via the Ub-Rho screening assay along with other
molecules, comprised of well-curated diversity compounds as purified enzyme- and cell-based orthogonal assays.
well as natural products. Unlike previous efforts in this area
We initiated our screening campaign by performing compre-
that prioritized screening hits based on potency, we placed a hensive assay development for USP7 and UCHL1, two well-
strong emphasis on selectivity as a strategy to identify and prior- studied DUBs with well-characterized positive control com-
itize hits. Validation studies confirmed this approach facilitated pounds. A Design of Experiment that investigated buffer, pH,
rapid confirmation of hits as bona fide selective DUB inhibitors. salt, BSA, EDTA, detergent, and reducing agent was performed
Indeed, we credentialed best-in-class probes of USP28 as well (Figure S1B) and allowed us to select a single buffer that pro-
as identifying selective starting points for medicinal chemistry duced robust signal for both DUBs. Notably, the conditions al-
optimization to achieve chemical probe or clinical compound lowed us to reduce enzyme and substrate concentration up to
development for other DUBs.
over 200-fold compared with starting conditions (Figure S1C);
reagent consumption was an important consideration for this
study given the large number of enzymes and the expense of
the substrate. We next performed a time course titration of
both enzyme and substrate to identify reagent concentrations
and a single time point for assay readout in which substrate turn-
RESULTS
Assay development and optimization provides robust
screening assay
This effort was conducted in collaboration with the Novartis Insti- over was in the linear range with a signal-to-noise ratio of at least
tute for Biomedical Research’s (NIBR) FAST Lab program which 53 (Figure S2A). Using the optimized assay conditions, we
enables academic labs to access a nonproprietary compound confirmed that known inhibitors of each DUB displayed the ex-
collection assembled at NIBR and work side-by-side with pected inhibitory activity (Figure S2B).
NIBR scientists to perform screens. For our screening campaign,
As an initial assessment of assay performance and potential
we employed recombinant DUB enzymes and a fluorogenic DUB for identifying selective DUB inhibitors from the compound
substrate, ubiquitin-rhodamine110 (Ub-Rho) (Figure 1A). This is collection, we screened the entire compound collection at a con-
a robust assay that is adaptable to most DUBs and proved centration of 20 mM against USP7 and UCHL1 followed by dose-
amenable to high-throughput screening. The eight DUBs response analysis of all actives against both DUBs. The primary
screened were USP7, USP8, USP10, USP17, USP28, USP30, assays performed well with Z0 values of 0.89 and 0.92 for USP7
2
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A
B
C
D
Figure 1. Overview of DUB HTS campaign
(A) Schematic of Ub-Rho screening assay. An uninhibited DUB cleaves rhodamine from ubiquitin (top) resulting in a fluorescent signal. In the presence of an
inhibitor (bottom), the DUB cannot cleave the substrate, and fluorescence is unchanged.
(B) DUBs included in the screen along with constructs used (cat denotes catalytic domain, FL denotes full-length protein, cat + UBA denotes the catalytic domain
plus UBA domain). All proteins are human isoforms except USP28, which is from mouse (Mus musculus).
(C) Summary of drug-like properties, partition coefficient (logP) to polar surface area (PSA) and molecular weight (MW), among library members.
(D) Diagram of screening cascade workflow.
and UCHL1, respectively (Table S1). Using >20% inhibition as (IC50 < 25 mM and R20% inhibition) that were inactive against
criteria for calling actives, we observed 289 hits for USP7 and USP7 (IC50 > 50 mM) (Figure S3). Given the excellent assay per-
175 hits for UCHL1. Hit compounds exhibiting inhibition based formance and initial indication of selective inhibitors, we pro-
on our cutoff for each enzyme were tested in dose-response ceeded to screening the other six DUBs. Before screening the
against both DUBs in triplicate starting at 25 mM for UCHL1 additional DUBs, we confirmed that each DUB was active in
and 50 mM for USP7. Based on compound availability, 128 of the same buffer utilized for USP7 and UCHL1 and performed
the 289 USP7 hits and 101 of the 175 UCHL1 hits were retested titration and time course experiments to select screening condi-
at dose. As was the case for the primary assay, the confirmatory tions (Figure S4). The use of the optimized buffer for all DUBs
assays performed well with Z0 values greater than 0.8 for each further aided comparison of the datasets by preventing any
DUB. We classified any compounds that exhibited R20% inhibi- buffer-specific artifacts from complicating the analysis.
tion at the highest tested dose in the dose-response testing as
active. Using these criteria, the confirmation rate was 83% for Primary screen reveals multiple hits across all DUBs
USP7 (106 of 128 compounds) and 57% for UCHL1 (58 of 101 The same set of compounds screened against USP7 and UCHL1
compounds). Notably, we found 34 compounds that demon- were screened against the six additional DUBs at a concentra-
strated inhibition of USP7 (half-maximal inhibitory concentration tion of 20 mM (USP8 and USP10), 25 mM (USP28, USP30, and
[IC50] < 50 mM and R20% inhibition) that were inactive against OTUD3), or 50 mM (USP17). All screens performed well with Z0
UCHL1 (IC50 > 25 mM) and 12 compounds inhibiting UCHL1 values between 0.49 and 0.92 (Table S1). We classified
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Tanimoto Similarity
A
UCHL1
OTUD3
USP10
USP17
USP28
USP8
USP7
USP30
Activation
Inhibition
B
1500
1000
500
0
97
142
334
509
UCHL1
USP7
OTUD3
USP10
USP28
USP8
1413
2010
2500
USP30
USP17
3075
3000
2000
1000
0
Set Size
Figure 2. Primary screening data
(A) Heatmap of primary screen hits. Maximal inhibition of each compound is plotted for each DUB. Compounds are clustered on the x axis by structural similarity
(Tanimoto score). Patches of blue in each of the rows show compound clusters that demonstrate inhibition of the corresponding DUB.
(B) UpSet plot summarizing primary screening data. Compounds with activity against at least one DUB, as defined by maximal inhibition R30%, are clustered by
activity against each DUB in the panel. The bar chart represents the number of compounds in a particular cluster, the dot underneath the bar chart correlates that
cluster with one or more DUBs (the presence of a single dot correlates to a specific DUB on the left, the presence of multiple dots means those compounds are hits
against those particular DUBs). Single DUB hits are colored in red to highlight potentially selective hits in the primary screen for each DUB. The bar chart on the
lower left represents the total number of hits for each DUB regardless of selectivity. The top 40 clusters of compounds are shown.
compounds exhibiting R30% inhibition of target as active. Hit Tanimoto score. Visual inspection of the heatmap indicates
rates were variable, with 0.4% for UCHL1 on the low end and several qualitative conclusions: (1) selective inhibitors for each
6.5% for USP17 on the high end. Among the USP family, DUB, (2) multi-targeted inhibitors, and (3) examples of multiple
USP7 had the lowest hit rate of 0.6% (Table S1). The heatmap structurally related hits. To further demonstrate our findings of
in Figure 2A depicts all actives from the primary, single-point selective and multi-targeted compounds we plotted the hits in
screen across all eight DUBs. Each DUB is indicated along the an UpSet plot (Figure 2B) (Conway et al., 2017). Gratifyingly,
y axis and hits, defined as R30% inhibition of target, are high- the analysis revealed selective hits for each DUB. The number
lighted and clustered by structural similarity as determined by of selective compounds for each DUB was quite varied, ranging
4
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A
B
Figure 3. Dose-response screening data with selectivity and potency filters
(A) Number of compounds that qualify as hits for each DUB based on filter criteria listed in the table below. The arrow denotes increasing potency and/or
selectivity of hit compounds for each DUB. The * denotes that, for UCHL1, 25 mM was the highest tested dose in the dose-response and thus this value was used
for calling hits as active against UCHL1.
(B) The violin plot shows the distribution of IC50 values for DUBs that had qualifying hits in section IV in (A).
from 12 for UCHL1 and USP7 to 1,403 for USP17. In general, the the DUBs screened and supported proceeding to dose-
USP family seemed to have more hits compared with UCHL1 (12 response confirmation of the screening hits.
selective hits) and OTUD3 (34 selective hits), although there were
notable exceptions, such as USP7 (12 selective hits) and USP10 Dose-response screen leads to identification of potent
(70 selective hits). Encouragingly, the selective hits for USP28 and selective hits
(671 selective hits), USP8 (968 selective hits), USP30 (1198 se- For the dose-response confirmation assay with the additional six
lective hits), and USP17 (1,403 selective hits) represented the DUBs, we screened USP8 and USP10 together, followed by
largest clusters of compounds observed in the UpSet plot. As USP17, OTUD3, USP28, and USP30 as these primary screens
anticipated, we also observed multi-targeted inhibitors. In the di- were completed in different time frames. All hits against USP8
versity set, we observed 127 multi-targeted inhibitors, defined as and USP10 were tested in eight-point dose-response in triplicate
hitting 5 of the 8 DUBs included in the screen. In the natural starting at 40 mM for USP8 and 50 mM for USP10 against both
product set we observed a higher percentage of multi-targeted DUBs to confirm inhibitory activity against the target and provide
inhibitors (Figure S5A). Examination of these multi-targeted com- further evidence of selectivity over other DUBs. All USP17,
pounds revealed structures or substructures that have been an- USP28, USP30, and OTUD3 active compounds were evaluated
notated in the literature as potential PAINS compounds (Baell starting at 50 mM in four-point dose-response against all four
and Holloway, 2010; Daina et al., 2017); however, some of the DUBs. All assays performed well with Z0 values ranging from
compounds contained structures that do not contain obvious 0.58 to 0.75 and confirmation rates ranging from 33% to 91%
flags (PAINS/electrophiles) underscoring the importance of (Table S1), using R30% inhibition at the highest concentration
selectivity profiling up-front. Some of these compounds from tested for calling a compound active.
the diversity set are depicted in Figure S5B as a resource for
Next, we analyzed the dose-response data of the active com-
others conducting DUB HTS, but we note that we have not per- pounds through the application of a series of selectivity and po-
formed additional experiments to confirm the compounds as tency filters. These different filters were implemented individually
pan-inhibitors or investigated whether they are real multi-tar- and in combination to allow us to assess the impact of each
geted binders or false positives. Overall, the data from the pri- parameter intriagingcompounds and narrowinonthemostprom-
mary screen indicated potential selective hit compounds for all ising actives for each DUB. Section I in Figure 3A depicts the
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A
HN
O
USP7
NH
USP8
N
N
NH
S
N
N
O
O
N
Spiro indolinone
expansion set
O
N
AV-9606-27
O
NH
USP7
USP8
N
F
S
O
H2N
O
O2
N
X
AV-9606-41
AD-10942-8
N
USP7 expansion screening hit
USP7 IC50 = 9 μM
USP8 expansion screening hit
USP8 IC50 = 2.1 μM
N
O
USP8 IC50 > 50 μM
USP7 IC50 > 50 μM
O
NH
USP8 primary screening hit
B
USP7
USP10
USP28
HN
N
O
O
O
O
N
N
N
O
N
N
O
S
S
N
NH
O
N
NH
O
NH
O2N
HO
S
O
NH
O
O
Br
AV-9606-27
Z56965384
AV-9606-99
bin-01-07-07
Cl
O
USP30
O
N
O
O
NH
N
Cl
N
H
S
S
N
O
O
N
Cl
O
AV-9606-129
AV-11324-75
O
H
N
N
H
O
AV-11324-5
C
USP7 Ub-Rho
USP8 Ub-Rho
USP10 Ub-Rho
150
100
50
150
100
50
150
100
50
AV-9606-27
AV-9606-41
AD-10942-8
Z56965384
0
0
0
-2
-1
0
1
2
-2
-1
0
1
2
-2
-1
0
1
2
3
Log [inhibitor], μM
Log [inhibitor], μM
Log [inhibitor], μM
USP28 Ub-Rho
USP30 Ub-Rho
150
100
50
150
100
50
AV-9606-99
AV-9606-129
bin-01-07-07
AV-11324-75
AV-11324-5
0
0
-4
-3
-2
-1
0
1
2
-4
-3
-2
-1
0
1
2
Log [inhibitor], μM
Log [inhibitor], μM
(legend on next page)
6
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number of active compounds for each DUB in the dose-response the expansion set. We thus screened the expansion set against
dataset using the initial criteria of maximum inhibition R30% and USP8 to potentially identify additional USP8 hits while also
an IC50 below 40 mM for the target DUB (or below 25mM for UCHL1 providing a counter screen for USP7. The compounds were as-
as this was the highest dose for UCHL1). With these criteria, we sessed in eight-point dose-response in triplicate against both
observed a disparate number of confirmed hits among the DUBs USP7 and USP8. Gratifyingly, we identified a different spiro indo-
that was in line with what was observed for the primary screen. linone scaffold active against USP7 and inactive against USP8
DUBs, such as UCHL1 and USP7, were on the low end with 54 (AV-9606-41, Figure 4A). In addition, we identified compounds
and 53, respectively, whereas DUBs on the other end of the spec- with a spiro indolinone group sharing the same fused ring as
trum had >500 hits confirmed in the dose-response (USP17, the initial USP8 hit with increased activity over the screening
USP28, and USP30). To focus in on the top compounds for each hit that were inactive against USP7. AD-10942-8 represents
DUB we applied a series of filters to the data. First, we applied a the best hit from among those compounds (Figure 4A).
selectivity filter which removed compounds with an IC50 at or
below 40 mM (25 mM for UCHL1) against any other DUB evaluated Resynthesis and biochemical assays confirm activity of
(Figure3A, section II). Filtering thedatasetinthismannerproduced selected hits
a marked reduction in the number of hits for most DUBs, including We focused on the most stringently triaged group of compounds
eliminating>90%ofhitsforsomeDUBs. Tothissetofcompounds, for each DUB for validation studies and to assess the effective-
we then applied a potency filter where we only included com- ness of our approach in yielding highly selective DUB inhibitors.
pounds with an IC50 below 10 mM with maximum inhibition We initially selected two of the USP7 hits (AV-9606-27 [Pub-
R30% and this produced a reduction in the number of hits Chem CID: 2998850] and AV-9606-41 [PubChem CID:
downtothetenstohundredsrangeformostDUBs(Figure3A,sec- 3624054]) as our lab has significant experience with this DUB
tion III). In some cases, in fact, no qualifying hits remained while for (Lamberto et al., 2017; Schauer et al., 2020a), as well as one
other DUBs there were anywhere from 1 to 160 hits that qualified. USP10 hit (Z56965384 [PubChem CID: 3908773]), one USP8
We applied one last filter of increasing the maximum inhibition hit (AD-10942-8), four USP28 hits (AV-9606-99 [PubChem CID:
to R80% to help us further focus in on the most promising com- 3487576], AV-9606-129 [PubChem CID: 3985183], AV-11324-
pounds (Figure 3A, section IV). This ultimately narrowed the num- 75 [PubChem CID: 136087907], and bin-01-07-07 [PubChem
ber of hits down to around 80 overall, but they were not split evenly CID: 2019582]) and the single USP30 hit (AV-11324-5 [PubChem
among DUBs. There were 74 hits for USP28 while there were none CID: 4383255]) (Figure 4B). The lone USP17 hit was not included
for USP8, USP17, UCHL1, or OTUD3, and only 1 each for USP7 in validation studies as it contained a core scaffold similar to one
and USP30 and 2 for USP10. Visual inspection of all the hits re- with reported redox activities (Lor et al., 2007).
vealed that the vast majority contained synthetically tractable
Fresh aliquots of each compound were obtained by chemical
drug-like structures. In addition, examination of the datasets for synthesis or from commercial vendors and tested in 12-point
each DUB, particularly in the case of USP28, revealed multiple an- dose-response in the Ub-Rho assay. For USP28, as we had em-
alogs of the same core structure that scored as hits leading to ployed the mouse isoform of the protein in the screen, the cata-
increased confidence that these compounds represented real lytic domain of the human protein was utilized to confirm activity
inhibitors. Interestingly, the IC50 values of the most potent com- against the human isoform. All the compounds confirmed upon
pound for each DUB was quite varied, with some in the low nano- retest with IC50 values against the respective DUB target ranging
molar range (Figure 3B).
from 50 nM to 30 mM (Table 1), which was in good agreement
with the screening IC50 values. The USP30 compound and
several of the USP28 inhibitors were the most potent with low
nanomolar IC50 values. The USP30 hit, AV-11324-5, was found
Expansion library leads to identification of additional
USP7 and USP8 hit compounds
We noted that one of the hits for USP7, AV-9606-27, contained a to have an IC50 value of 125 nM (Figure 4C, Table 1). The four
core motif that was present in a significant number of members USP28 inhibitors we selected inhibited the DUB with IC50 values
of a larger 250k screening deck, so we proceeded to screen an from the low nanomolar to low micromolar: AV-9606-99
expansion set containing compounds with structural similarity to (4.33 mM), AV-9606-129 (402 nM), AV-11324-75 (176 nM) and
this hit (Figure 4A). The key structural element of this collection bin-01-07-07 (46 nM) (Figure 4C; Table 1). The USP7 (AV-
was the presence of a spiro indolinone group with various fused 9606-27 and AV-9606-41), USP8 (AD-10942-8), and USP10
ring cores and substituents. Upon examination of the USP8 hits (Z56965384) hits all exhibited micromolar inhibitory activity in
in the selective group (Figure 3A, section II), we observed a hit the dose-response confirmation with IC50 values of 6.77, 3.92,
that shared structural similarity with some of the compounds in 9.28, and 26.6 mM, respectively (Figure 4C; Table 1).
the USP7 spiro indolinone expansion set. While the USP8 hit
As DUBs are susceptible to inactivation by oxidation of the
did not contain a spiro indolinone group, it did contain a similar catalytic cysteine residue by redox active compounds, we
fused ring system that appeared on numerous derivatives in analyzed all confirmed actives using a resazurin-based redox
Figure 4. Conformation of screening hits
(A) Structures of USP7 and USP8 screening hits prompted further screening of a spiro indolinone expansion set leading to the identification of new USP7 and
USP8 hits for validation studies.
(B) Structures of screening hits for each corresponding DUB that were selected for validation studies.
(C) Dose-response data for screening hits in (A and B). Compounds were screened in a 12-point dose-response using a kinetic Ub-Rho biochemical assay. Data
are represented as mean SEM, n R 2.
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confirm to inhibit USP7, or any other DUBs in the panel (Fig-
ure S7B). One possibility this result raised was that AV-9606-41
Table 1. IC50 data for selected screening hits
DUB
Compound
IC50 SEM mM (n)
inhibits the catalytic domain construct of USP7 used in our
studies but not full-length enzyme, although the compound ex-
hibited autofluorescence in the control experiment at Ubiquigent
rendering it difficult to confidently draw a conclusion. We evalu-
ated the inhibitory activity of AV-9606-41 against full-length
USP7 in dose-response using a kinetic Ub-Rho assay, conditions
less sensitive to assay interference, and observed no inhibition of
full-length protein (Figure S7D). Based on this finding we dropped
AV-9606-41 from further validation experiments, although the
compound may be a useful tool for studying conformation differ-
ences between catalytic domain and full-length USP7. The USP8
compound, AD-10942-8, inhibited multiple DUBs in the panel;
however, the compound also exhibited autofluorescence in the
control experiment (Figure S7B). These results, coupled with
USP7
AV-9606-27
AV-9606-41
AD-10942-8
Z56965384
AV-9606-99
AV-9606-129
bin-01-07-07
AV-11324-75
AV-11324-5
4.68 1.05 (3)
3.83 1.60 (3)
6.01 0.18 (4)
26.6 4.69 (14)
4.33 1.08 (8)
0.402 0.197 (6)
0.046 0.017 (3)
0.176 0.073 (5)
0.275 0.070 (6)
USP8
USP10
USP28
USP30
IC50 values for selected screening hits. Data are represented as mean
SEM with the number of replicates for each compound (n).
assay in dose-response (Lor et al., 2007). Most of the com- the apparent redox activity of the compound observed previ-
pounds exhibited little to no redox activity with those that did ously, led to us excluding the USP8 hit from further validation
only exhibiting redox behavior at concentrations well above their studies. The USP10 compound, Z56965384, also showed inhibi-
IC50 values (Figure S6). The exception to this was the USP8 hit, tion of multiple DUBs within the panel (Figure S7B). However, the
AD-10942-8, which generated a significant signal at lower con- compound exhibited assay interference consistent with quench-
centrations. Based on this finding, AD-10942-8 may inhibit ing fluorescence based on a control experiment in the absence of
USP8 through oxidation of the catalytic cysteine residue rather enzyme. We confirmed inhibition of an ‘‘off-target’’ DUB, USP8,
than inhibiting the enzyme through a direct binding interaction.
using our kinetic assay (Figure S7C). We also tested the com-
pound against both USP8 and USP10 in dose-response using
ubiquitin-AMC, a different substrate containing a fluorophore
that emits at a different wavelength. In this experiment we
Selectivity of hit compounds confirmed against an
expanded DUB panel
While our screening results indicated that these compounds are observed a 3- to 10-fold increase in IC50 value indicating the com-
selective toward a single DUB over those included in the screen, pound is affecting rhodamine fluorescence (Figure S7C). These
we next sought to confirm this result by profiling the compounds results exclude Z56965384 as a potent or selective starting point
against a comprehensive library of DUBs. The USP7, USP8, for USP10 inhibitor development.
USP10, USP28, and USP30 hits were assessed for inhibitory ac-
tivity against a panel of 41 purified DUBs using Ubiquigent’s Orthogonal assays validate USP28 and USP30 hits
DUBprofiler platform. This assay measured cleavage of Ub- We focused further validation studies on the UPS28 and USP30
Rho using an endpoint assay. In addition to the activity assay, hits given these DUBs had sub-micromolar hits with good selec-
compounds are also evaluated in two separate control experi- tivity. To assess target binding to native enzyme and selectivity in
ments for autofluorescence and for modulation of the product a cellular environment, we performed competitive activity-based
signal to determine potential compound interference with assay protein profiling (ABPP) coupled with quantitative mass spec-
readout. Five of the eight compounds tested were confirmed to trometry (Hemelaar et al., 2004; Pinto-Fernandez et al., 2019;
inhibit their intended target with excellent selectivity. Figure 5 Borodovsky et al., 2002) (Figure 6A). In brief, HEK293T lysates
shows the profiling data for these compounds with each DUB were treated with inhibitor followed by co-incubation with a 1:1
in the panel shown on the x axis, while percent activity of the mixture of DUB activity-based probes, biotin-ubiquitin-propar-
DUB with compound treatment is plotted on the y axis. Com- gylamine (Ub-PA) and biotin-ubiquitin-vinyl methyl ester (Ub-
pounds that inhibit the DUB will show a reduced percent activity VME). The probes will irreversibly label the active site cysteine
remaining. The USP30 inhibitor and all four USP28 compounds of DUBs not already bound by inhibitor and contain a biotin moi-
exhibited a high degree of selectivity across the entire panel for ety for streptavidin enrichment. Following affinity purification, the
the target DUBs identified from the screen. The USP30 inhibitor samples are subjected to tryptic digest and peptide sequencing
AV-11324-5 showed remarkable selectivity for USP30 over all by liquid chromatography-tandem mass spectrometry enabled
other DUBs in the panel. All four of the USP28-targeting com- identification of DUBs that are bound and pulled down by the
pounds significantly inhibited two DUBs, USP28 and USP25, probe. Based on comparison to a DMSO control sample, we
with no other DUBs inhibited. USP25 and USP28 share high can identify which DUB(s) the compounds are binding to and
sequence homology within the DUB domain as well as identical thus preventing labeling with the covalent probe. Figure 6B
domain structure compared with all other USP family members shows the results of the target engagement experiments. For
(Valero et al., 2001; Liu et al., 2018; Gersch et al., 2019; Sauer the USP30 compound we see robust inhibition of USP30 with lit-
et al., 2019). To independently confirm USP25 inhibition, we ex- tle to no off-target activity further highlighting the selectivity of
pressed and purified USP25 and tested the hit compounds in this compound. For the dual USP25/USP28, we observed strong
dose-response in the Ub-Rho assay and confirmed the com- binding to native USP25 with all four compounds but only the two
pounds demonstrated comparable activity against USP25 and most potent compounds bin-01-07-7 and AV-9606-180 (a close
USP28 (Figure S7A). The USP7 inhibitor, AV-9606-41, did not derivative of screening hit AV-11324-75 with similar potency in
8
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80
AV-9606-99
60
AV-9606-129
40
20
0
AV-11324-75
Bin-01-07-07
AV-11324-5
Figure 5. Selectivity profiling
Compounds were assessed for selectivity against an expanded panel of purified recombinant DUBs using DUBprofiler (Ubiquigent). Compounds were tested at
100 mM, except bin-01-07-07 which was tested at 10 mM, and the percent activity of each DUB remaining following compound treatment is reported for each
compound.
the Ub-Rho dose-response assay, Figure S7E) exhibited strong 2010; Schauer et al., 2020a; Boselli et al., 2017). While there is
inhibition of USP28. To further confirm the ABPP results we per- great interest in DUBs as a way to provide a high-precision
formed a dose-response treatment of the most potent com- mechanism to target oncogenic ‘‘undruggable’’ proteins for
pounds (AV-9606-180, AV-11324-75, and bin-01-07-07) in degradation, the number of selective small-molecule probes re-
cellular lysate followed by addition of the covalent activity-based mains limited (Ndubaku and Tsui, 2015; Schauer et al., 2020b).
probes and readout by western blot for USP28 (Figure 6C). Com- One of the main challenges in developing inhibitors selective
pound binding prevents probe labeling resulting in the presence for a given DUB is the similarity between DUB family members,
of the native USP28 band. Probe binding results in a higher-mo- especially USPs, which ultimately leads to compounds with
lecular-weight adduct (DUB + probe) that is resolvable by west- poor selectivity profiles and limits their utility in elucidating
ern blot as a band above the native protein. Results demonstrate DUB function (Ritorto et al., 2014). Recent reports in the literature
good dose-dependent inhibition and prevention of probe label- validate the plausibility of developing selective inhibitors of sin-
ing in line with expected compound activity.
gle DUBs (Kluge et al., 2018; Lee et al., 2010; Schlierf et al.,
A dual USP25/USP28 inhibitor AZ1 has been reported in the 2016; Kategaya et al., 2017; Lamberto et al., 2017; Turnbull
literature so we sought to benchmark our inhibitors against this et al., 2017). These results encouraged us to perform HTS with
compound (Wrigley et al., 2017). We observed biochemical inhi- the focus on using selectivity, rather than potency, as a way to
bition of USP25 and USP28 by AZ1 with IC50 values of 3.47
identify and prioritize hits. We screened 8 DUBs (USP7, USP8,
0.73 mM (n = 4) and 0.98 0.02 mM (n = 2), respectively, in line USP10, USP17, USP28, USP30, UCHL1, and OTUD3) spanning
with reported values for the compound. In the ABPP assay, 3 of the DUB families against a 50,000-member compound li-
AZ1 strongly blocked labeling of USP25 by DUB ABP; however, brary to identify selective inhibitors for each of the DUBs at the
it had little impact on USP28. The compound, as previously re- earliest stages of inhibitor development.
ported, did not exhibit activity toward any other DUBs detected
We screened 47.26k compounds against each DUB that pro-
in the experiment. These results nominate bin-01-07-07 and AV- duced variable hit rates in the primary screen ranging from 0.4%
9606-180 as best-in-class probes of USP28. Both compounds to 6.9%. These data may also indicate a difference in ‘‘druggabil-
inhibit the DUB with IC50 values around or below 100 nM, ity’’ among the different DUB families as we had much lower hit
approximately 10-fold more potent that AZ1, and exhibit sub- rates for UCHL1 and OTUD3 compared with the USP family on
stantially stronger cellular target engagement (Wrigley et al., average. Follow-up hit validation studies that comprised profiling
2017). The combination of two structurally distinct scaffolds in dose-response against target DUBs and one to three of the other
studies that probe USP28 function will be highly valuable.
DUBs included in the screen resulted in confirmation of inhibitors
Overall, the generated dataset also provides a number of scaf- for five of the eight DUBs screened providing starting points for
folds that may retain activity and selectivity against single DUBs further medicinal chemistry optimization for probe development.
but were not selected for follow-up studies based on our initial In this study, USP28 was a clear outlier with 73 hits satisfying the
focusonthe morepotenthits. Theseweakercompounds may pro- most stringent criteria. Inspection of the structures of these hits
videstartingpointsformedicinal chemistrycampaignstogenerate showed multiple compounds with the same core scaffold. This
more potent analogs that retain selectivity against a single DUB.
accounts for the larger number of hits observed as well as
gave us confidence in these compounds as inhibitors of
USP28 and provided some initial SAR. In the case of USP30,
the lone hit that was discovered was reported as a potent and se-
DISCUSSION
DUBs control diverse processes within the cell and have been lective USP30 inhibitor during the course of our screening by
implicated in many different diseases including cancer, neurode- another group validating the ability of our approach to identify
generation, and inflammation/immune response (Harrigan et al., selective DUB inhibitors (Kluge et al., 2018).
2018). Studies have demonstrated that pharmacologic inhibition
To further map the selectivity profiles of our hit compounds, we
of specific DUBs can lead to degradation of their substrate pro- employed a larger DUB panel, which disqualified some of the hits
teins, and result in a beneficial therapeutic effect (Kapuria et al., based on their activity against other DUBs not included in the
Cell Chemical Biology 28, 1–14, August 19, 2021
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A
ubiquitin
DUB3
SH
SH
S
DUB3
ubiquitin
DUB1
biotin
Streptavidin pull down
Trypsin digest
DUB1
electrophile
S
ubiquitin
Lyse cells
inhib
Treat with activity-based
probe
Treat with inhibitor
or DMSO
TMT labeling
LC-MS/MS
DUB2
inhib
DUB
DUB2
Data analysis
B
D 4
1
1
1 9
2
2 4
2 5
2 7
2 8
3
3 0
3 1
3 2
3
3 4
3 5
3 6
3 7
3 8
3 9
4
4 0
4 2
4 3
4 6
4 7
4 8
5
5 1
5 4
7
8
9
I
N
A T X N 3
B A P
C Y L
F
F
J
J
O
O
O
O
O T U
O
O
O
O
U
U
U
U S P 1 0
U S P
U S P 1 4
U S P 1 5
U S P 1 6
U S P
U S P 2
U S P 2 0
U S P 2 1
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
U S P
V C P
Y O D
Z R A
Z
AZ1
Bin-01-01-07
AV-9606-99
AV-9606-129
AV-9606-180
AV-11324-5
XL188
60
80
100
% blockage of probe labeling
C
AV-9606-180
AV-11324-75
bin-01-07-07
Ub-PA/Ub-VME
[Compound], μM
+
+
+
+
1
+
+
+
+
1
+
+
+
+
1
-
+
0
100
50
10
100
50
10
100
50
10
0
USP28
GAPDH
AZ1
AV-9606-99
AV-9606-129
Ub-PA/Ub-VME
[Compound], μM
+
+
+
+
1
+
+
+
+
1
+
+
+
+
1
-
+
0
100
50
10
100
50
10
100
50
10
0
USP28
GAPDH
Figure 6. Validation of USP28 and USP30 hits by quantitative activity-based protein profiling
(A) Schematic for quantitative activity-based protein profiling.
(B) Heatmap showing results of screening compounds profiled in ABPP assay. Compounds demonstrate blockage of probe labeling of target DUBs compared
with all other DUBs detected in cell lysate. AZ1 is a literature reported USP25/28 inhibitor (Wrigley et al., 2017). XL-188 is a USP7 inhibitor included as a positive
control (Lamberto et al., 2017). See also Table S2.
(C) Western blots show blockage of USP28 labeling by covalent probe (Ub-PA/Ub-VME) in cellular lysate after 1 h pre-treatment with indicated compounds in
dose response.
screen. In the case of the USP28 inhibitors, all of the compounds for medicinal chemistry efforts to tune selectivity toward one of
displayed activity against USP25 in addition to USP28, which is the two DUBs. Our finding with the USP7 inhibitor, AV-9606-41,
not unsurprising given the high sequence homology shared by where the hit displayed potent and selective activity in the screen
both DUBs (Liu et al., 2018; Valero et al., 2001; Sauer et al., done withcatalyticdomainbut noactivity againsta full-lengthpro-
2019;Gerschetal., 2019). Indeed, thisisborneoutbytheliterature tein, provides a cautionary note to anyone using isolated domains
given that reported USP28 inhibitors, AZ1 and vismodegib, have for screening campaigns. Our results with the USP8 inhibitor,
equipotent activity against USP25 (Wrigley et al., 2017; Wang where the hit displayed significant redox activity, demonstrates
et al., 2021). Some of the compounds discovered through our the importance of performing this type of analysis for large
screening efforts demonstrate roughly 10-fold better potency screens in general and for DUB inhibitors specifically. USP8 has
than AZ1 and offer multiple different scaffolds that can be utilized been a difficult DUB to target as demonstrated by the lack of
as probe compounds for USP25/28 biology or as starting points well-validated, selective inhibitors in the literature. These results
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d KEY RESOURCES TABLE
may indicate that some DUBs, such as USP8, may be more sus-
ceptible to redox inactivation (Kulathu et al., 2013; Lee et al., 2013;
Cotto-Rios et al., 2012). The Ubiquigent profiling results for the
USP7, USP8, and USP10 hits, where we saw compound interfer-
ence with assay readout by autofluorescence or fluorescence
quenching, emphasize the importance of these types of analysis
as part of an effort to validate compound activity. Taken together,
our results illustrate the power of taking selectivity into account at
averyearlystageinthescreening process, whenconducting work
on a large protein family. Keeping selectivity in mind, we were able
to identify potential probes along with many viable starting points
for probe development for a multitude of DUBs from a library of
approximately 50,000 compounds, which is modest compared
with some of the larger screening collections deployed for HTS.
Indeed, it is entirely possible and even likely that this compound
collection is too small of a sample to identify viable hits for some
of the morerecalcitrant DUBs as we observed in our screen. While
our focus has been on the identification of selective inhibitors, our
method for screening has produced a robust dataset that can be
mined for other compounds of interest. For example, although we
have identified a number of potential pan-DUB inhibitors, the pan-
DUB activity of these compounds remains to be formally
validated. In addition, while our focus was on the most potent
and selective inhibitors, the data generated by these experiments
includes many selective but weaker inhibitors that could serve as
starting points for medicinal chemistry campaigns. Collectively,
the work described here represents the most comprehensive
DUB inhibitor screening and profiling effort to date. As such, it of-
fers a number of future opportunities for development of pan- or
selective DUB inhibitors as chemical tool compounds and/or
drug leads to both further explore the biological functions of these
DUBs and examine their value as therapeutic targets.
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODELS AND SUBJECT DETAILS
B Microbe strains
B Cell lines
d METHOD DETAILS
B Recombinant protein expression and purification
B Design of experiments (DOE) operation
B Ubiquitin rhodamine 110 endpoint assay (for screening
USP7 and UCHL1)
B Ubiquitin rhodamine 110 endpoint assay (for screening
other DUBs)
B Ubiquitin rhodamine 110 kinetic assay
B Ubiquitin-AMC kinetic assay
B Redox assay
B In vitro DUB selectivity profiling
B Quantitative activity-based protein profiling
B Competitive activity based protein profiling
B UpSet plot
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Chemical synthesis
B General methods and materials
B Compound synthesis
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
chembiol.2021.05.012.
SIGNIFICANCE
ACKNOWLEDGMENTS
S.J.B. and J.A.M. acknowledge generous support from the Mark Foundation
ASPIRE program, NIH CA233800, and NIH CA247671.
Deubiquitinating enzymes (DUBs) are an integral part of the
ubiquitin-proteasome system and have been reported to
regulate many key biological pathways. DUBs represent an
emerging target class that have been implicated in many
different diseases. The lack of well-validated and selective
probe compounds for specific DUBs has presented an
obstacle for full elucidation of their role in biological pro-
cesses and diseases. In this study, we optimized and de-
ployed a high-throughput screen of a 50,000 compound
library against a panel of 8 DUBs spanning 3 of the DUB fam-
ilies, with an emphasis on identifying selective hits for each
DUB early on in the process. We demonstrate that this
approach has led to the identification of selective inhibitors
for a subset of the DUBs included in the screen and has
generated a robust dataset that will be useful in DUB inhibitor
development. Identification of these selective compounds
can serve as the basis of medicinal chemistry campaigns
geared toward the optimization of potency for the develop-
ment of high-quality DUB inhibitors.
AUTHOR CONTRIBUTIONS
A.C.V., D.C., D.A., and S.J.B. conceived of and designed the study. A.C.V. and
D.C. carried out the assay optimization, high-throughput screening, and initial
dose-response validation which were reviewed under the supervision of D.A.
and S.J.B. A.C.V., R.S.M., R.M.R., H.-S.S., and S.d.P. generated recombinant
proteins used in the screen and validation studies. A.C.V., B.H., and X.L. resyn-
thesized compounds for validation studies. W.C.C. and H.Z. prepared ABPP
and chemoproteomic samples and analyzed MS samples. J.A.M. oversaw
all MS experiments. The paper was written by A.C.V. and S.J.B. and reviewed
by all co-authors.
DECLARATION OF INTERESTS
J.A.M. serves on the SAB of 908 Devices and receives sponsored research
funding from Vertex and AstraZeneca. S.J.B. serves on the SAB of Adenoid
Cystic Carcinoma Foundation. The authors have submitted patent applica-
tions related to compounds in this manuscript.
STAR+METHODS
Received: September 18, 2020
Revised: March 11, 2021
Accepted: May 19, 2021
Published: June 14, 2021
Detailed methods are provided in the online version of this paper
and include the following:
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STAR+METHODS
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IRDyeꢀ 800CW Goat anti-Rabbit IgG
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(2’R,3R,3’S)-2’-(4-fluorobenzoyl)-3’-
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(S)-N-(1-((5-(N-isopropylsulfamoyl)
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Ubiquitin-Rhodamine 110 (Ub-Rho)
Ubiquitin-propargylamine (Ub-PA)
Ubiquitin-vinyl methyl ester (Ub-VME)
R&D Systems
UbiQ
Cat#U-555-050
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UbiQ
Critical commercial assays
DUBprofilerꢁ
Ubiquigent
N/A
(Continued on next page)
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Continued
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Deposited data
SOURCE
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Mass Spectrometry Interactive Virtual
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Dataset Identifier: MSV000087089
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PubChem
https://pubchem.ncbi.nlm.nih.gov/
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Experimental models: cell lines
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Cat# CRL-3216; RRID:CVCL_0063
Recombinant DNA
pOPINK-OTUD3 (OTU+UBA, aa 52-275)
pET28aLIC-USP7 (His-tagged, aa 207-532)
Mevissen et al., 2013
This paper
Addgene plasmid# 61411
N/A
N/A
pET28aLIC-USP8 (His-tagged, aa
742-1110)
This paper
pET28PP-USP10 (His-tagged, aa 376-798)
pET28b-USP17 (His-tagged, aa 1-530)
pET28a USP25 (His-tagged, aa 207-532)
This paper
This paper
This paper
This paper
N/A
N/A
N/A
N/A
pET28aLIC-USP28 (His-tagged, aa
207-532)
pET28PP-USP30 (His-tagged, aa 207-532)
pGEX6P1-UCHL1 (GST-tagged, aa 1-223)
This paper
This paper
N/A
N/A
Software and algorithms
Prism 8
GraphPad
LiCOR
https://www.graphpad.com/scientific-
software/prism/;
RRID: SCR_002798
Image Studio Lite
https://www.licor.com/bio/image-studio-
lite/;
RRID:SCR_013715
Other
UpSetR code
Multiplierz v2.0
Mascot 2.6.1
Conway et al., 2017
Alexander et al., 2017
Matrix Science
https://github.com/hms-dbmi/UpSetR
RRID:SCR_012058
RRID:SCR_014322
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Sara Buhr-
lage (saraj_buhrlage@dfci.harvard.edu).
Materials availability
The materials generated in this study will be distributed upon request to the lead contact. There are restrictions to availability due to a
Material Transfer Agreement (MTA).
Data and code availability
The accession number for the primary and dose-response screening datasets reported in this paper is PubChem: AID1645869.
https://pubchem.ncbi.nlm.nih.gov/bioassay/1645869.
The accession number for the raw massspectrometrydata files for TMT 6-Plex activity-based protein profilingreported inthispaper is
MassIVE:MSV000087089(MassspectrometryInteractiveVirtualEnvironment,https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp).
EXPERIMENTAL MODELS AND SUBJECT DETAILS
Microbe strains
Recombinant proteins were expressed in E. coli BL21(DE3) bacteria, which were grown in Luria Broth (LB) at 37ꢀC with shaking at
200 rpm and then induced at 16ꢀC with shaking at 200 rpm.
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Cell lines
HEK293T (female human origin) cell lines were used in this study. HEK293T cells (ATCC, Cat# CRL-3216) are a clonal isolate of
HEK293 cells transformed with the SV40 large T antigen. For growth, HEK293T cells were maintained in DMEM containing 4 mM
L-glutamine (Gibco) supplemented with 10% (v/v) 10% heat inactivated fetal bovine serum (FBS) (Gibco), and penicillin/streptomycin
(100 U/mL penicillin and 0.1 mg/mL streptomycin) (Gibco). Cells were grown at 37ꢀC with 5% CO2 in a water-saturated incubator. For
passaging, cells were incubated with trypsin/EDTA at 37ꢀC to detach cells.
METHOD DETAILS
Recombinant protein expression and purification
The constructs encoding USP7 catalytic domain (AA 208–560), USP7 full-length (AA 1–1102), USP8 catalytic domain (AA 743–1110),
USP10 catalytic domain (AA 376–798), USP30 catalytic domain (AA 65–517), USP28 catalytic domain (AA 140–697 for mouse, AA
149–703 for human), and USP25 (AA 157–714) were cloned into pET28 expression vectors with an N-terminal 6xHis tag in NEB
10-beta Competent E. coli (High Efficiency) cells. The construct encoding UCHL1 full length (AA 1–223) was cloned into a pGEX6P1
expression vector with an N-terminal GST tag in NEB 10-beta Competent E. coli (High Efficiency) cells. Plasmids containing desired
constructs were then isolated, transfected into E. coli BL21 (DE3) cells and overexpressed. Cells were grown at 37ꢀC to an OD of 0.9,
cooled to 16ꢀC, induced with 500 mM isopropyl-1-thio-D-galactopyranoside (IPTG), incubated overnight at 16ꢀC, collected by centri-
fugation, and stored at ꢁ80ꢀC. Cell pellets were sonicated in lysis buffer (25 mM Tris pH 8, 1 M NaCl, and 10 mM BME) supplemented
with 10 mg/mL phenylmethanesulfonylfluoride (PMSF) and the resulting lysate was centrifuged at 30,000 xg for 40 min. Ni-NTA beads
(Qiagen) were mixed with lysate supernatant for 2 hr, and washed with lysis buffer supplemented with 25 mM imidazole. The bound
protein was eluted with lysis buffer supplemented with 300 mM imidazole. The sample was then concentrated to 1 mL (30 kDa
concentrator; Amicon Ultra, Millipore), and run on a Superdex 200 (GE healthcare) in buffer containing 25 mM HEPES pH 7.5,
200 mM NaCl, and 1 mM DTT. Fractions were pooled, concentrated, and frozen at ꢁ80ꢀC. A plasmid expressing USP17 (1–530)
was synthesized by BioBasic in a pET28 expression vector, and expressed and purified as described previously (Hjortland and Me-
secar, 2016). A plasmid expressing OTUD3 (52–275) was purchased from Addgene (Cat# 61,411) and was expressed and purified as
described(Mevissen et al., 2013). As judged by gel all proteins were at or above 90% purity, except for USP17 and USP30 which were
approximately 70% pure. Yield ranged from 0.5–2 mg/L.
Design of experiments (DOE) operation
To test for optimal buffer conditions a design of experiments (DOE) approach was used. This involves first deciding on relevant buffer
components to vary, taking from literature sources or baseline buffer conditions. These components are then varied at different con-
centrations and typically to cover a full permutation of all possible combinations yields several thousand experiments. Therefore,
once all the factors are chosen the experimental design is created in JMP statistical software (developed by SAS Institute) which pro-
vides the optimal combination of factors to achieve ꢂ2% coverage of all conditions allowing adequate modeling of each compo-
nent’s effect on the assay. We used the output of the JMP design to create a dispense list to deliver the buffer components to
the assay plate using a Formulatrix Tempest liquid handler that is capable of dispensing up to 12 reagents in varying volumes and
combinations. Once the data is collected the results are imported into JMP to make a model of the assay response to all the factors
and the quality of the model is considered good when the actual vs predicted plot of the data achieves a r2 ꢂ 0.9. Profiles of the buffer
effects on the assay signal can be used to make decisions on buffer composition and the top buffers are also ranked based on overall
signal so that the top scoring buffers can be chosen for confirmation testing. In the DUB assay DOE, HEPES and Tris buffers were
prepared at three pH values (7, 7.5, and 8) and NaCl was varied a 0, 25, and 50mM. BSA was varied at 0 and 1% and the detergents
CHAPS, Tween 20, PF127, NP-40, and Triton X-, were varied at 0, 0.25, and 0.5 of their CMC values. As well, the reducing reagents
DTT and TCEP were varied at 0 and 1mM and EDTA was varied at 0 and 1mM. For this DOE, full permutation of all conditions would
test 4,320 different buffers and the experimental design employed tested 8.6% of these. Following the JMP modeling the top buffers
were prepared and tested in the assay to confirm performance (Figure S1).
Ubiquitin rhodamine 110 endpoint assay (for screening USP7 and UCHL1)
2.5 mL of a solution of DUB in assay buffer (50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween 20, 5 mM DTT) at 2x the final concentration
in the assay was added to columns 1–46 on a Greiner medium binding 1536 well plate (catalog number: 782076). Compounds were
acoustically transferred to columns 1–46 (columns 45–46 contained only DMSO as a neutral control). 2.5 mL of assay buffer was
dispensed to columns 47–48. Plates were then incubated for 1 hr. 2.5 mL of Ub-Rho110 at 2x the final concentration in the assay
was then added to all wells on the plate. Plates were incubated at room temperature for 10 min and the fluorescence was recorded
using a PheraStar fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm respectively.
Ubiquitin rhodamine 110 endpoint assay (for screening other DUBs)
2.5 mL of a solution of DUB in assay buffer (50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween 20, 5 mM DTT) at 2x the final concentration
in the assay was added to columns 1–46 on a Greiner medium binding 1536-well plate (catalog number: 782076). Compounds were
acoustically transferred to columns 1–46 (columns 45–46 contained only DMSO as a neutral control). 2.5 mL of assay buffer was
dispensed to columns 47–48. Plates were then incubated for 10 min. 2.5 mL of Ub-Rho110 at 2x the final concentration in the assay
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was then added to all wells on the plate. Plates were incubated at room temperature for 30 min–3 hr depending on the DUB. After
incubation, reactions were quenched with the addition of 2.5 mL of stop buffer (assay buffer +0.2% trifluoroacetic acid) and fluores-
cence was recorded using a PheraStar fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm
respectively.
Analysis of screening data. Normalization of raw data to positive and negative control wells, systematic pattern models, and
curve fitting were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis(Gubler
et al., 2018). For measuring compound potency, a four-parameter sigmoid Hill curve model was fitted to the data (Gubler et al., 2018)
and the absolute IC50 was used for potency measurements, that is the concentration of compound where the data showed 50% in-
hibition. If no data points achieved 50% inhibition, then the absolute IC50 was reported as > X mM (where X is the highest tested
concentration).
Ubiquitin rhodamine 110 kinetic assay
DUBs from the high-throughput screen were tested for their respective activity in the Ub-Rho110 assay in the presence or absence of
inhibitors using Nunc 384-well plates (Thermo Scientific catalog number: 2262260). Individual DUBs were pre-incubated with
different concentrations of inhibitors in assay buffer (50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween 20, 5 mM DTT). Compounds
and protein were incubated for 30 min at room temperature prior to the addition of Ub-Rho110 (Boston Biochem) substrate. The initial
rate of the reaction was measured by collecting fluorescence data at one-minute intervals over 30-min period using a Clariostar fluo-
rescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm respectively. The calculated initial rate values
were normalized to no compound control wells and were plotted against inhibitor concentrations to determine IC50s. All the exper-
imental data were plotted using GraphPad Prism (log(inhibitor) vs. normalized response - Variable slope, least squares fit). All assays
were performed at least twice for each compound and error bars are reported as SEM.
Ubiquitin-AMC kinetic assay
The same assay protocol as the Ubiquitin rhodamine 110 kinetic assay was followed, substituting Ub-AMC for the Ub-Rho110 and
reading at the specified wavelengths for AMC (excitation and emission wavelengths of 345 nm and 445 nm respectively).
Redox assay
50 mL of redox assay buffer (50 mM Tris pH 7.5, 50 mM NaCl, 5 mM DTT, and 25 mM resazurin) was added to column 1 of an Axygen 96-
well Polypropylene PCR Microplate (product number: PCR-96-FS-C). 25 mL of redox assay buffer was added to remaining wells in
columns 2–11. Column 12 received 25 mL of redox assay buffer with 0.5 mL DMSO. 0.5 mL of 10 mM compound was then added to
each row in column 1 for an initial concentration of 100 mM. A serial two-fold dilution of each compound was then performed down
each row, omitting column 12. The plate was then incubated for 30 min in darkness, transferred to a Nunc 384-well plate (Thermo Sci-
entific catalog number: 2262260) and then read on a Clariostar fluorescence plate reader at excitation and emission wavelengths of
545 nm and 600 nm respectively. The baseline signal based on the averaged DMSO control wells was then subtracted from all
data points. Fluorescence values for each compound in dose were plotted as a function of compound dose using GraphPad Prism
(log(agonist) vs. response - Variable slope (four parameters), least squares fit). All assays were performed at least twice for each
compound.
In vitro DUB selectivity profiling
Compounds were screened using the Ubiquigent DUBProfiler SPT system (www.ubiquigent.com/drug-discovery-services/
dubprofiler/). Each of 41 purified DUBs was incubated with compound for 15 min, then Ubiquitin rhodamine 110 (Ub-Rho110)
was added and percent inhibition was determined based on fluorescence relative to a DMSO control. The autofluorescence control
experiment determines the fluorescence signal generated by the compound at screening concentration in absence of enzyme rela-
tive to the enzyme and no enzyme control for each of the DUBs in the panel. The product signal modulation (PSM) control experiment
determines the fluorescence signal generated by the compound (at screening concentration) in the presence of synthetic rhodamine
110 glycine (the product of enzymatic cleavage of the DUBprofiler Ub-Rho substrate) and DUBprofiler substrate (at standard assay
concentration) expressed as a percentage relative to the fluorescence signal of synthetic rhodamine 110 glycine and DUBprofiler
substrate (at standard assay concentration). Autofluorescence and PSM data can indicate autofluorescence (positive %) or quench-
ing (negative %) of background signal and/or assay signal.
Quantitative activity-based protein profiling
DUB activity based protein profiling was performed using conditions modified from those in Schauer et al.(Schauer et al., 2020a),
based on work by Lawson et al.(Lawson et al., 2017). HEK 293T cells were lysed (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM
MgCl2, 0.5 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM TCEP, protease and phosphatase inhibitors) and the lysate was clarified
by centrifugation, then diluted to 10 mg/mL. 200 mL aliquots were incubated with 250 mM compound or DMSO for 30 min at RT. Se-
lective USP7 inhibitor XL188 was included in each run as a positive control(Lamberto et al., 2017). Afterward, the treated lysates were
incubated with 1 mM each of Biotin-Ub-PA and Biotin-Ub-VME for 15 min at RT. 125 mL magnetic streptavidin sepharose slurry was
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added to each sample, followed by incubation at RT for 30 min with end-to-end rotation. After immobilizing the beads using a mag-
netic rack, the beads were washed (33 0.2% SDS, 3x PBS, 2x ddH2O). After the final wash, supernatant was removed, and the resin
was flash frozen and stored at ꢁ80ꢀC.
Streptavidin beads were resuspended in 95 mL 100 mM Tris pH 8.0. Each sample was denatured with 0.1% rapigest, reduced
(10 mM dithiothreitol), alkylated (22.5 mM iodoacetamide), and digested with trypsin at 37ꢀC overnight. The next day, beads were
captured using a magnetic rack, and supernatants were acidified with 10% TFA, incubated at 37ꢀC for 30 min, and centrifuged at
14,000 rpm for 15 min at 4ꢀC to remove rapigest. Peptides were then desalted by C18 and dried by vacuum centrifugation.
Dried peptides were reconstituted in 40mL 50mM pH 8.0 TEAB, and 1/4 unit of TMT reagent was added, and reactions incubated at
RT for 1 hr. TMT reactions were pooled and treated with hydroxylamine according to the manufacturer’s instructions. Peptide mix-
tures were then dried, reconstituted in 100 mM ammonium bicarbonate and desalted by SP3(Hughes et al., 2014). Eluted TMT
labeled peptides were dissolved in 5% acetonitrile with 100 mM ammonium formate, pH 10, and analyzed by our DEEP-SEQ multi-
dimension peptide fractionation platform (Zhou et al., 2011, 2013; Ficarro et al., 2011) built around a NanoAcquity UPLC system (Wa-
ters, Milford, MA) and coupled directly to a timsTOF Pro ion mobility mass spectrometer (Bruker, Billerica, MA) (Meier et al., 2015,
2018). As described (Fu et al., 2020), each of 11 peptide fractions was eluted from the first dimension reversed phase column
(5 cm 3 150 mm packed with 5 mm XBridge C18, Waters, Milford, MA) and second dimension anion exchange column (5 cm 3
150 mm I.D. fused silica packed with 10 mm POROS 10HQ Applied Biosystems, Foster City, CA) using injections of acetonitrile or
ammonium formate at pH 10.0. Peptide fractions were diluted on-line with solvent A (0.1% formic acid in water) to reduce pH and
organic concentration and facilitate retention on the third dimension precolumn (5 cm 3 100 mm I.D. fused silica packed with
7 mm Symmetry C18, Waters). Peptides were eluted with an HPLC gradient (6.5–35% B in 120 min, A = 0.1% formic acid in water,
B = 0.1% formic acid in acetonitrile), resolved on an analytical column (30 mm I.D. 3 50 cm Monitor C18, Orochem, Naperville, IL) and
introduced to the mass spectrometer by electrospray ionization using a captive spray ion source (spray voltage = 1.8 kV). The mass
spectrometer acquired ion mobility MS spectra over a mass range of m/z 100–1700 and 1/k0 of 0.6–1.6, and then performed 10 cy-
cles of PASEF MS/MS with a target intensity of 20k, a threshold of 2500 and a quadrupole isolation width of 1 Da. Active exclusion
was enabled with a release time of 0.4 min. For TMT reporter ion detection, TIMS stepping function (Ogata and Ishihama, 2020) was
used. Briefly, the collision energy was ramped stepwise against the ion mobility: 39.88 eV for 1/k0 0.6–0.83, 46.25 eV for 1/k0 0.83–
1.0, 52.50 eV for 1/k0 1.0–1.18, 58.75 eV for 1/k0 1.18–1.35, and 64.63 eV for 1/k0 1.35–1.6. The collision energy for the second step is
decreased by 20%. Other settings corresponding to the two-step collision energy include collision RF of 500 and 1500, transfer time
of 25 ms and 60 ms, and pre pulse storage time of 8 ms and 12 ms, respectively.
Raw data files were converted to mgf using multiplierz scripts (Alexander et al., 2017), and searched using Mascot 2.6.1 against a
forward and reversed human refseq database (NCBI). Search parameters specified a precursor mass tolerance of 20 ppm, a product
ion tolerance of 50 mmu, fixed carbamidomethylation of cysteine, TMT 6-plex labeling of lysine and N-termini, and variable oxidation
of methionine. For TMT quantitation, identified peptides were filtered to a peptide score no less than 13 and a false discovery rate of
1%. We used Pep2gene (Askenazi et al., 2010) to reduce the set of peptides to those which mapped to unique gene identifiers, and
then aggregated reporter ions to derive a protein-level quantification value (Table S2).
Competitive activity based protein profiling
HEK293T cells were pelleted, washed with PBS, lysed on ice with lysis buffer (20 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 10% glyc-
erol, 1 mM TCEP, phosphatase inhibitor cocktails (Sigma P5726 and Calbiochem 524624), and protease inhibitors (pepstatin, leu-
peptin, PMSF, and aprotinin), and clarified by centrifugation. Protein content was quantified by BCA and diluted to 2 mg/mL in lysis
buffer. 40 mL samples of lysate were incubated with compounds or DMSO at desired concentrations and incubated at room temper-
ature for 1 hr. Samples were then supplemented with 1:1 Ub-Pa/Ub-VME probe at a final concentration in the sample of 2 mM and
incubated at room temperature with shaking for 30 min. Reactions were quenched with 4x LDS sample buffer (Thermo Fisher B0007)
supplemented with 10% BME, vortexed vigorously, and heated to 95ꢀC for 5 min. Samples were resolved by SDS-PAGE and
analyzed by Western blot with the indicated antibodies (Primary antibodies used: USP28 rabbit monoclonal antibody from Abcam,
catalog number: ab126604. GAPDH rabbit monoclonal antibody from Cell Signaling, catalog number: 2118S. Secondary antibody
used: IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody from LI-COR, catalog number: 926–32,211)
UpSet plot
UpSet plot was generated using UpSetR code from GitHub.
QUANTIFICATION AND STATISTICAL ANALYSIS
All biochemical curves and associated statistical analyses were produced using GraphPad Prism Software. Details of replicates and
data analysis for specific experiments can be found in figure legends or the methods section.
Chemical synthesis
List of abbreviations: DCM (dichloromethane); DMSO (dimethyl sulfoxide); Et2O (diethyl ether); EtOAc (ethyl acetate); EtOH (ethanol);
Et3N (triethylamine); g (gram); HCl (hydrochloric acid); LC/MS (liquid chromatography-mass spectrometry); MeCN (acetonitrile);
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MeOH (methanol); MHz (megahertz); mg (milligram); mL (mL); mmol (millimole); NaHCO3 (sodium bicarbonate); Na2SO4 (sodium sul-
fate); NH4Cl (ammonium chloride); NMR (nuclear magnetic resonance); THF (tetrahydrofuran); mL (mL).
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. Anhydrous solvents, such as tetrahydrofuran (THF), dichloro-
methane (DCM), diethyl ether (Et2O), dimethyl formamide (DMF), dimethylsulfoxide (DMSO), 1,4-dioxane, and toluene were pur-
chased from Fisher Scientific, and used as received. If necessary, air or moisture sensitive reactions were carried out under an inert
€
atmosphere of nitrogen. Removal of solvents was accomplished on a Buchi R-300 rotary evaporator and further concentration was
done under a Welch 1400B-01 vacuum line, and Labconco FreeZone 6 plus system. Purification of compounds was performed by
normal phase column chromatography using Teledyne CombiFlash chromatography system, and/or reversed phase chromatog-
raphy on Waters 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).
All 1H-NMR spectra were recorded at 298K on a Bruker ARX 500 (500 MHz) spectrometer. Samples were dissolved in CDCl3,
DMSO-d6, or CD3OD obtained from Cambridge Isotope Laboratories. The spectra were referenced to the residual solvent peak
(chloroform-d: 7.26 ppm for 1H-NMR and 77.16 ppm for 13C-NMR; DMSO-d6: 2.50 ppm for 1H-NMR and 39.25 ppm for
13C-NMR, CD3OD: 3.31 ppm for 1H NMR and 49.00 ppm for 13C NMR or tetramethylsilane (TMS) as the internal 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.
Z56965384 was purchased directly from Enamine at 85% purity. The compound was purified via HPLC to afford the desired prod-
uct. LC/MS (ESI) m/z 486.57 [M + H]+; calcd for C22H19N2O9S+: 487.08. 1H NMR (500 MHz, DMSO-d6) d 10.26 (s, 1H), 8.27 (d, J =
2.4 Hz, 1H), 8.01–7.98 (m, 2H), 7.97 (dd, J = 7.9, 1.6 Hz, 1H), 7.85 (dd, J = 8.9, 2.4 Hz, 1H), 7.65 (ddd, J = 8.9, 7.4, 1.6 Hz, 1H), 7.52 (dd,
J = 8.3, 1.1 Hz, 1H), 7.28 (td, J = 7.6, 1.2 Hz, 1H), 7.23 (d, J = 8.9 Hz, 1H), 7.16–7.08 (m, 2H), 5.69 (s, 2H), 3.88 (s, 3H).
Final compounds submitted for testing were prepared and stored as 10 mM stock solutions in DMSO. Stocks were stored at ꢁ20ꢀC
and aliquoted as needed for specific experiments to reduce the number of freeze thaw cycles. We observed no apparent solubility
issues with the compounds either in DMSO or aqueous buffer conditions at the concentrations used.
Compound synthesis
3-Methyl-1-phenyl-1H-spiro[benzo[4,5]thiazolo[3,2-a]pyrazolo[3,4-d]pyrimidine-4,30-indolin]-20-one (AV-9606-27)
Step 1: N-(3-methyl-1-phenyl-1H-pyrazol-5-yl)benzo[d]thiazol-2-amine (AV-9606-26)
2-chlorobenzo[d]thiazole (173.9 mg, 1.0 mmol) was suspended in THF (5 mL). To the reaction was added sodium hydride (37.9 mg,
1.5 mmol), and the reaction was stirred at room temperature for 30 min under nitrogen. 3-methyl-1-phenyl-1H-pyrazol-5-amine
(130.2 uL, 1.0 mmol) was then added to the reaction and the reaction was heated to 60ꢀC for 18 hr. The reaction mixture was diluted
with EtOAc and washed with water and brine. The organics were combined, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The resulting crude was purified via silica gel chromatography (0–100% EtOAc: hexanes) to afford the
desired product (25.9 mg, 8.5%). LC/MS (ESI) m/z 306.87 [M + H]+; calcd for C17H15N4S+: 307.10.
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Step 2: 3-methyl-1-phenyl-1H-spiro[benzo[4,5]thiazolo[3,2-a]pyrazolo[3,4-d]pyrimidine-4,30-indolin]-20-one (AV-9606-27)
AV-9606-26 (25.9 mg, 0.08 mmol) and isatin (14.2 mg, 0.10 mmol) were combined and suspended in DMF (2 mL). To the reaction
was then added TMS-Cl (32.3 uL, 0.24 mmol). The reaction was heated at 100ꢀC for 16 hr. The reaction was diluted with water and
placed in a sonication bath for 90 min. The reaction was then filtered to isolate the resulting precipitate. The precipitate was then
purified via silica gel chromatography (0–100% EtOAc: hexanes) to afford the desired product (2.1 mg, 6.0%). LC/MS (ESI) m/z
1
435.87 [M + H]+; calcd for C25H18N5OS+: 436.12. H NMR (500 MHz, Methanol-d4) d 7.86–7.77 (m, 1H), 7.59 (d, J = 7.7 Hz, 1H),
7.48 (q, J = 7.6 Hz, 2H), 7.38–7.27 (m, 2H), 7.17 (dd, J = 12.5, 7.8 Hz, 2H), 7.09 (s, 1H), 6.55 (d, J = 8.3 Hz, 1H), 1.70 (s, 3H).
60-amino-30-isopropyl-2-oxo-20H-spiro[indoline-3,40-pyrano[2,3-c]pyrazole]-50-carbonitrile (AV-9606-41)
Methyl isobutyrylacetate (96.7 mL, 0.69 mmol) and hydrazine hydrate (21.2 mL, 0.68 mmol) were combined and diluted in EtOH
(3 mL). The reaction was heated to 65ꢀC for 15 min at which point isatin (102.0 mg, 0.69 mmol), DMAP (8.6 mg, 0.07 mmol), and ma-
lononitrile (48.4 mg, 0.73 mmol) were added. The reaction was stirred at 65ꢀC for 1 hr. The reaction mixture was concentrated under
reduced pressure and resuspended in EtOAc then washed with water and brine. The organics were combined and dried over anhy-
drous Na2SO4 then filtered and concentrated under reduced pressure. The crude residue was then purified via silica gel chromatog-
raphy (0–100% EtOAc: hexanes) to afford the desired product (43.9 mg, 20.1%). LC/MS (ESI) m/z 322.07 [M + H]+; calcd for
C
(d, J = 7.2 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H), 4.10 (q, J = 5.1 Hz, 1H), 3.17 (d, J = 4.8 Hz, 2H), 2.14–2.01
17H16N5O2+: 322.13. 1H NMR (500 MHz, DMSO-d6) d 12.30 (s, 1H), 10.60 (s, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.17 (s, 2H), 7.04
(m, 1H), 0.95 (d, J = 7.1 Hz, 3H), 0.66 (d, J = 7.0 Hz, 3H).
(20R,3R,30S)-2’-(4-fluorobenzoyl)-3’-(thiophene-2-carbonyl)-2’,3’,8’,8a’-tetrahydro-7’H-spiro[indoline-3,1’-indolizin]-
2-one (AD-10942-8)
Step 1: (Z)-3-(2-(4-fluorophenyl)-2-oxoethylidene)indolin-2-one (AV-9606-80)
Isatin (149.0 mg, 1.0 mmol) was suspended in EtOH (1 mL) and 4’-fluoroacetophenone (121.4 mL, 1.0 mmol) was added. The re-
action was stirred at room temperature for 6 hours. The reaction was filtered to isolate the resulting precipitate in the reaction mixture.
The precipitate was resuspended in EtOH (5 mL) and concentrated HCl (0.5 mL) was added, and the reaction was stirred at 60ꢀC for 2
hours. The reaction was filtered again to afford the desired product (139.1 mg, 52.0%). LC/MS (ESI) m/z 268.07 [M+H]+; calcd for
C16H11FNO2+: 268.08. 1H NMR (500 MHz, Chloroform-d) d 8.30 (d, J = 7.8 Hz, 1H), 8.15 (ddd, J = 10.9, 6.6, 3.8 Hz, 3H), 7.82
(s, 1H), 7.33 (td, J = 7.7, 1.2 Hz, 1H), 7.21 (t, J = 8.6 Hz, 2H), 7.02 (td, J = 7.7, 1.0 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H).
Step 2: 2-bromo-1-(thiophen-2-yl)ethan-1-one (AV-9606-87)
Copper(II) Bromide (338.1 mg, 1.5 mmol) was suspended in EtOH (5 mL). To the reaction was then added 2-acetylthiophene
(108.1 mL, 1.0 mmol) and the reaction was heated to 60ꢀC for 24 hours. The reaction was concentrated under reduced pressure
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and the crude residue (AV-9606-87) was used in the subsequent reaction without further purification. LC/MS (ESI) m/z 204.98 [M+H]+;
calcd for C6H6BrOS+: 204.93.
Step 3: 2-(2-oxo-2-(thiophen-2-yl)ethyl)isoquinolin-2-ium bromide (AV-9606-91)
AV-9606-87 (102.0 mg, 0.5 mmol) was dissolved in toluene (2 mL). To the reaction was then added isoquinoline (58.7 mL, 0.5 mmol).
The reaction was then heated to reflux for 30 minutes. The reaction was cooled to room temperature and filtered then washed with
hexanes. The oily residue on the bottom of the reaction vessel was dissolved in DCM and concentrated. The crude residue was sus-
pended in 1,4-dioxane and sonicated to precipitate out the desired product from solution. The reaction was filtered to afford the
desired product (92.4 mg, 55.3%). The crude material was used in the next step without further purification. LC/MS (ESI) m/z
253.87 [M]+; calcd for C15H12NOS+: 254.06.
Step 4: (2’R,3R,3’S)-2’-(4-fluorobenzoyl)-3’-(thiophene-2-carbonyl)-2’,3’,8’,8a’-tetrahydro-7’H-spiro[indoline-3,1’-indolizin]-2-
one (AD-10942-8)
AV-9606-91 (92.4 mg, 0.28 mmol) and AV-9606-80 (74.8 mg, 0.28 mmol) were combined and suspended in EtOH (2.25 mL). Et3N
(46.8 mL, 0.34 mmol) was then added and the reaction was stirred at room temperature for 4.5 hours. The reaction was filtered, and
the precipitate was washed with EtOH. The precipitate was recrystallized from EtOH/DCM to afford the desired product (52.2 mg,
43.6%). LC/MS (ESI) m/z 521.18 [M+H]+; calcd for C31H22FN2O3S+: 521.13. 1H NMR (500 MHz, DMSO-d6) d 10.52 (s, 1H), 8.10
(dd, J = 4.9, 1.2 Hz, 1H), 8.03 (dd, J = 3.9, 1.2 Hz, 1H), 7.42 – 7.34 (m, 2H), 7.28 (dd, J = 4.9, 3.9 Hz, 1H), 7.18 (dd, J = 7.6,
1.2 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H), 6.88 (dd, J = 7.4, 1.3 Hz, 1H), 6.82 (dt, J = 9.0, 7.0 Hz, 2H), 6.70 (ddt, J = 21.5, 7.6, 6.4 Hz,
3H), 6.53 (d, J = 7.6 Hz, 1H), 6.27 (d, J = 7.7 Hz, 1H), 6.14 (d, J = 5.3 Hz, 1H), 5.34 (s, 1H), 5.14 (d, J = 7.5 Hz, 1H), 4.73 (d, J =
5.3 Hz, 1H).
5-bromo-3’-phenyl-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (AV-9606-99)
5-bromoisatin (112.4 mg, 0.50 mmol) and isatoic anhydride (122.6 mg, 0.75 mmol) were combined and suspended in acetic acid
(2 mL). Aniline (68.0 mL, 0.75 mmol) was added and the reaction mixture was stirred at room temperature for 15 minutes. The reaction
mixture was then transferred to a microwave vial, sealed and heated to 160ꢀC for 30 min. The reaction mixture was then diluted with
EtOAc and washed with water and brine. The organics were collected, dried over anhydrous Na2SO4, filtered and concentrated under
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reduced pressure. The resulting crude was purified via silica gel chromatography (0-100% EtOAc: hexanes) to afford the desired
product (65.2 mg, 31.0%). LCMS (ESI) m/z = 420.07 [M+H]+; calcd for C21H15BrN3O2+: 420.03. 1H NMR (500 MHz, DMSO-d6)
d 10.56 (s, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.67 (d, J = 6.2 Hz, 2H), 7.33 (td, J = 8.5, 1.9 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.19 (t, J =
7.3 Hz, 1H), 7.03 (d, J = 8.1 Hz, 2H), 6.78 (t, J = 7.5 Hz, 1H), 6.70 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H).
2-(3,4-dichlorophenyl)-N-(5-(N,N-dimethylsulfamoyl)-2-methylphenyl)acetamide (AV-9606-129)
3,4-dichlorophenylacetic acid (100.4 mg, 0.49 mmol) was combined with HATU (226.4 mg, 0.60 mmol) and suspended in DMF
(3 mL). Et3N (204.9 mL, 1.47 mmol) was added and the reaction was stirred at room temperature for about 15 minutes. To the reaction
was then added 3-amino-N,N,4-trimethylbenzene-1-sulfonamide (108.8 mg, 0.51 mmol). Stir at room temperature for 42 hours. The
reaction was diluted with EtOAc and washed with water and brine. The organic layer was collected, dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The crude was purified via silica gel chromatography (0-100% EtOAc:hexanes) to
afford the pure compound as a light brown powder (41.7 mg, 21%). LC/MS (ESI) m/z 400.87 [M+H]+; calcd for C17H19Cl2N2O3S+:
401.05. 1H NMR (500 MHz, Methanol-d4) d 7.86 (d, J = 1.9 Hz, 1H), 7.57 (d, J = 2.1 Hz, 1H), 7.55 – 7.45 (m, 3H), 7.32 (dd, J = 8.3,
2.1 Hz, 1H), 3.78 (s, 2H), 2.67 (s, 6H), 2.31 (s, 3H).
5-(5-(3-chlorobenzyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one (AV-11324-75)
Step 1: 6-chloro-N-hydroxynicotinimidamide (AV-11324-22)
2-chloro-5-cyanopyridine (1542.1 mg, 11.1 mmol) and hydroxylamine HCl (897.0 mg, 12.9 mmol) were combined and suspended
in EtOH (25 mL). Et3N (7735.6 uL, 55.5 mmol) was then added and the reaction was heated to 65ꢀC for 6 hours. White precipitate was
observed in the reaction. Filtration afforded the desired product as a white powder (1378.1 mg, 72%). LC/MS (ESI) m/z 171.99
[M+H]+; calcd for C6H7ClN3O+: 172.03.
Step 2: 5-(3-chlorobenzyl)-3-(6-chloropyridin-3-yl)-1,2,4-oxadiazole (AV-11324-74)
3-chlorophenylacetic acid (173.2 mg, 1.02 mmol) and CDI (255.5 mg, 1.68 mmol) were combined and suspended in MeCN (3 mL).
The reaction was stirred at room temperature for 2 hours. The reaction was then filtered and 6-chloro-N-hydroxynicotinimidamide
(197.0 mg, 1.15 mmol) was then added and the reaction was stirred at room temperature for 30 minutes. DBU (305.1 uL,
2.04 mmol) was then added and the reaction was heated to 60ꢀC for 1 hour. The reaction was concentrated under reduced pressure
and resuspended in EtOAc and washed with water and brine. The organic layer was collected, dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude was purified via silica gel chromatography (0-100% EtOAc:hexanes) to afford
the pure compound (194.5 mg, 62%). LC/MS (ESI) m/z 305.77 [M+H]+; C14H10Cl2N3O+: 306.02.
Step 3: 5-(5-(3-chlorobenzyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one (AV-11324-75)
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5-(3-chlorobenzyl)-3-(6-chloropyridin-3-yl)-1,2,4-oxadiazole (192.1 mg, 0.63 mmol) was dissolved in concentrated HCl (3 mL) and
heated to reflux for 2h. The reaction was removed from heat and quenched with water and neutralized with saturated NaHCO3. The
aqueous solution was extracted with DCM. The organic layer was collected and washed with brine. The organic layer was then iso-
lated, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude was then purified via silica gel
chromatography (0-10% MeOH:DCM) to afford the desired product as a white powder (5.7 mg, 2%). LC/MS (ESI) m/z 287.87
1
[M+H]+; calcd for C14H11ClN3O2+: 288.05. H NMR (500 MHz, DMSO-d6) d 7.97 (d, J = 2.6 Hz, 1H), 7.87 (dd, J = 9.6, 2.7 Hz, 1H),
7.50 (d, J = 2.1 Hz, 1H), 7.45 – 7.33 (m, 2H), 6.48 (d, J = 9.6 Hz, 1H), 4.43 (s, 2H).
N-(3,4-dimethoxyphenethyl)-4-propyl-2,3-dihydro-1H-cyclopenta[4’,5’]pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin-7-
amine (bin-01-07-07)
Step 1: 2-butyrylcyclopentan-1-one (bin-01-07-01)
To a dried flask charged with DCM/Et2O (9/1; 25 mL) was added bismuth(III) chloride (0.5 g, 1.6 mmol, 0.05 eq) and zinc iodide (0.77
g, 2.4 mmol, 0.075 eq) rapidly (under dryer). This is followed by the addition of butyryl chloride (3.66 mL, 35.2 mmol, 1.1 eq) and the
suspension was stirred for 5 minutes at room temperature. After that, the 1-(trimethylsiloxy)cyclopentane was added at once under
rapid stirring. After 40 minutes at room temperature, the reaction was quenched by saturated NaHCO3. After the organic layers were
separated, the aqueous layer was washed by DCM (3 3 30 mL). The organic layers were combined, dried over Na2SO4, and concen-
trated to give the crude product bin-01-07-01 which was used directly without further purification. LC/MS (ESI) m/z 155.10 [M+H]+;
calcd for C9H15O2+: 155.11.
Step 2: 3-chloro-1-propyl-6,7-dihydro-5H-cyclopenta[c]pyridine-4-carbonitrile (bin-01-07-03)
The reaction mixture of bin-01-07-01 (32 mmol, crude reaction mixture obtained directly from last step), cyano acetamide (2.5 g,
30 mmol), diethyl amine (3.1 mL, 30 mmol) in EtOH (50.0 mL) was stirred at room temperature for 24 hours. The reaction mixture was
then concentrated and washed with cooled EtOH to give pure product bin-01-07-02 that was used in the next step directly. The re-
action mixture of bin-01-07-02 (2.0 g, 10 mmol) in anhydrous 1,4-dioxane (10.0 mL) and POCl3 (5.0 mL) was heated at 90ꢀC overnight.
The reaction mixture was then poured into ice water and adjusted to pH = 7. The resulting precipitate was then filtered, dried to give
+
pure product as white solid bin-01-07-03, (30% yield over three steps). LC/MS (ESI) m/z 221.28 [M+H]+; calcd for C12H14ClN2
221.08.
:
Step 3: ethyl 1-amino-5-propyl-7,8-dihydro-6H-cyclopenta[d]thieno[2,3-b]pyridine-2-carboxylate (bin-01-07-04)
To a solution of bin-01-07-03 (220 mg, 1 mmol, 1.0 eq) in EtOH (5.0 mL) was added sodium ethoxide (75 mg, 1.1 mmol, 1.1 eq) and
ethyl thioglycolate (120 mL, 1.1 mmol, 1.1 eq.). The reaction mixture was stirred under reflux for 5 hours. The reaction mixture was then
cooled down to room temperature and concentrated under reduced pressure. The resulting yellow brown solid was then dissolved
with water and EtOAc (combined 100 mL). The organic layer was then washed with saturated brine and concentrated. The crude
material was purified through silica gel chromatography (0-15% EtOAc:hexanes) to give pure product bin-01-07-04 as white solid,
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(85% yield). LC/MS (ESI) m/z 305.07 [M+H]+; calcd for C16H21N2O2S+: 305.13.
Step 4: 7-chloro-4-propyl-2,3-dihydro-1H-cyclopenta[4’,5’]pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidine (bin-01-07-06)
A mixture of bin-01-07-04 (220 mg, 1 mmol, 1.0 eq) in formamide (5 mL) was refluxed for 4 hours. After that, the reaction mixture
was poured into water and extracted with EtOAc. The combined organic phase was then concentrated and purified through silica gel
chromatography (0-10% MeOH:DCM) to give pure product bin-01-07-05. LC/MS (ESI) m/z 286.07 [M+H]+; calcd for C15H16N3OS+:
286.10. To a solution of bin-01-07-05 (1 mmol) in 1,4-dioxane (1.0 mL) was added POCl3 (1.0 mL). The reaction was heated at 95ꢀC
overnight. The reaction was poured into icy water and adjusted to pH around 7. The resulting precipitate was isolated via filtration to
give the desired product as a pure white solid. The filtrate was extracted with EtOAc, concentrated, and purified via silica gel chro-
matography (0-10% EtOAc:hexanes) to afford additional product. The product from both conditions were combined to afford bin-01-
07-06 (60% yield over two steps).
Step 5: N-(3,4-dimethoxyphenethyl)-4-propyl-2,3-dihydro-1H-cyclopenta[4’,5’]pyrido[3’,2’:4,5] thieno[3,2-d]pyrimidin-7-amine
(bin-01-07-07)
A solution of bin-01-07-05 (28.5 mg, 0.1 mmol, 1.0 eq) and 3,4-dimeoxyphenethylamine (36 mg, 0.2 mmol) in EtOH (5 mL) was
heated to reflux overnight. The reaction mixture was then concentrated and the crude material was purified via silica gel chromatog-
raphy (0-60% EtOAc:hexanes) to give pure product bin-01-07-07 (85% yield). LC/MS (ESI) m/z 449.18 [M+H]+; calcd for
C25H29N4O2S+: 449.20. 1H NMR (500 MHz, DMSO-d6) d 8.61 (s, 1H), 7.89 (t, J = 5.5 Hz, 1H), 6.87 ‒ 6.84 (m, 2H), 6.76 (dd, J =
8.2, 1.9 Hz, 1H), 3.75 ‒ 3.72 (m, 2H), 3.71 (s, 3H), 3.70 (s, 3H), 3.47 (t, J = 7.5 Hz, 2H), 2.98 (t, J = 7.5 Hz, 2H), 2.91 ‒ 2.86 (m, 2H),
2.84 ‒ 2.78 (m, 2H), 2.18 (Pent, J = 7.5 Hz, 2H), 1.75 (Sextet, J = 7.5 Hz, 2H), 0.96 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 159.26, 158.61, 156.59, 155.10, 153.51, 150.57, 148.59, 147.23, 135.73, 131.93, 122.81, 120.52, 112.79, 112.59, 111.92, 55.50,
55.34, 42.10, 37.44, 34.34, 32.04, 29.57, 24.24, 21.21, 13.92.
(S)-N-(1-((5-(N-isopropylsulfamoyl)naphthalen-1-yl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide (AV-11324-5)
Step 1: sodium 5-acetamidonaphthalene-1-sulfonate
5-Aminonaphthalene-1-sulfonic acid (1.12 g, 5.0 mmol) was dissolved in 5 mL pyridine, and acetic anhydride (0.76 g, 7.5 mmol)
was added dropwise. The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure.
The crude was then dissolved in 2N NaOH solution and stirred at room temperature for 2 hours. The reaction mixture was then poured
into 100% EtOH. Solid precipitate was collected by filtration and washed with additional cold EtOH, and dried under high vacuum to
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afford the desired product (0.74 g, 52%). LC/MS (ESI) m/z 265.97 (sulfonic acid form +H) [M+H]+; calcd for C12H11NNaO4S+: 288.03.
Step 2: 5-acetamidonaphthalene-1-sulfonyl chloride
A solution of sodium 5-acetamidonaphthalene-1-sulfonate (0.61 g, 2.1 mmol) in chlorosulfuric acid (10 mL) was stirred at 65ꢀC for 1
hour. Then the reaction mixture was carefully poured onto ice, and the crude product was precipitated and collected by filtration. The
solid material was further washed with ice-cold water and dried under vacuum to afford crude product which was used directly
without further purification.
Step 3: N-(5-(N-isopropylsulfamoyl)naphthalen-1-yl)acetamide
To the solution of 5-acetamidonaphthalene-1-sulfonyl chloride (0.28 g, 1.0 mmol) in 5 mL DCM was added isopropylamine (0.08 g,
1.2 mmol) and Et3N (0.42 mL, 3.0 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated under
reduced pressure. The crude was purified by silica gel chromatography (0-100% EtOAc:hexanes) to afford the desired product (0.08
g, 26%). LC/MS (ESI) m/z 306.87 [M+H]+; calcd for C15H19N2O3S+: 307.11.
Step 4: 5-amino-N-isopropylnaphthalene-1-sulfonamide (XL-9872-143)
N-(5-(N-isopropylsulfamoyl)naphthalen-1-yl)acetamide (0.08 g, 0.27 mmol) was dissolved in 5N NaOH aqueous solution (15.0 eq)
and 3 mL MeOH. The mixture was stirred at 65ꢀC then 80ꢀC for 48 hours. Then the mixture was diluted with DCM and washed with
saturated NH4Cl and brine. The organic phase was concentrated under reduced pressure and purified by silica gel chromatography
using (0-100% EtOAc:hexanes) to afford desired product (0.04 g, 56%). LC/MS (ESI) m/z 264.87 [M+H]+; calcd for C13H17N2O2S+:
265.10.
Step 5: tert-butyl benzoyl-L-phenylalaninate (AV-9606-205)
Benzoic acid (205.4 mg, 1.68 mmol) and HATU (857.0 mg, 2.25 mmol) were combined and suspended in DMF (4 mL). To the re-
action was then added L-phenylalanine tert-Butyl ester hydrochloride (332.6 mg, 1.5 mmol) followed by Et3N (1045.4 mL, 7.5 mmol).
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The reaction was stirred at room temperature 1 hour. The reaction was then heated to 100ꢀC for an additional 30 minutes at which
point the reaction became fully soluble. The reaction was then diluted with EtOAc and washed with water and brine. The organics
were collected and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was pu-
rified via silica gel chromatography (0-100% EtOAc:hexanes) to afford the desired product (385.9 mg, 79.1%). LC/MS (ESI) m/z
269.87 [M-tert-Butyl+H]+; calcd for C16H16NO3+: 270.11.
Step 6: benzoyl-L-phenylalanine
AV-9606-205-1 (385.9 mg, 1.19 mmol) was then suspended in 4N HCl in 1,4-dioxane (5 mL) and stirred at room temperature for 24
hours. The reaction was filtered to isolate the precipitate observed in the reaction as the desired product (287.2 mg, 89.6%). LC/MS
(ESI) m/z 269.97 [M+H]+; calcd for C16H16NO3+: 270.11. The resulting material was used in the next step without further purification.
Step 7: (S)-N-(1-((5-(N-isopropylsulfamoyl)naphthalen-1-yl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide (AV-11324-5)
AV-9606-205-2 (41.4 mg, 0.15 mmol) was suspended in DMF (1 mL) and placed in an ice bath. To the reaction was then added EDC
hydrochloride (30.7 mg, 0.16 mmol) followed by HOBt (17.6 mg, 0.11 mmol). The reaction was stirred at room temperature for 1 hour.
To the reaction was then added XL-9872-143 (19.9 mg, 0.075 mmol) and the reaction was stirred at room temperature for 72 hours.
The reaction was then purified directly via silica gel column chromatography to afford the desired product (13.3 mg, 34%). LC/MS
(ESI) m/z 515.39 [M+H]+; calcd for C29H30N3O4S+: 516.20. 1H NMR (500 MHz, DMSO-d6) d 10.32 (s, 1H), 8.83 (d, J = 7.7 Hz, 1H), 8.53
(d, J = 8.5 Hz, 1H), 8.22 – 8.11 (m, 2H), 7.94 – 7.84 (m, 3H), 7.72 – 7.60 (m, 3H), 7.57 – 7.51 (m, 1H), 7.50 – 7.43 (m, 4H), 7.33 (t, J =
7.5 Hz, 2H), 7.24 (t, J = 7.4 Hz, 1H), 5.03 (ddd, J = 9.6, 7.6, 5.6 Hz, 1H), 3.33 – 3.17 (m, 4H), 0.87 (dd, J = 8.1, 6.5 Hz, 6H).
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