Using sulfuramidimidoyl fluorides that undergo sulfur(vi) fluoride exchange for inverse drug discovery

Article abstract of DOI:10.1038/s41557-020-0530-4

Drug candidates that form covalent linkages with their target proteins have been underexplored compared with the conventional counterparts that modulate biological function by reversibly binding to proteins, in part due to concerns about off-target reactivity. However, toxicity linked to off-target reactivity can be minimized by using latent electrophiles that only become activated towards covalent bond formation on binding a specific protein. Here we study sulfuramidimidoyl fluorides, a class of weak electrophiles that undergo sulfur(vi) fluoride exchange chemistry. We show that equilibrium binding of a sulfuramidimidoyl fluoride to a protein can allow nucleophilic attack by a specific amino acid side chain, which leads to conjugate formation. We incubated small molecules, each bearing a sulfuramidimidoyl fluoride electrophile, with human cell lysate, and the protein conjugates formed were identified by affinity chromatography–mass spectrometry. This inverse drug discovery approach identified a compound that covalently binds to and irreversibly inhibits the activity of poly(ADP-ribose) polymerase 1, an important anticancer target in living cells. [Figure not available: see fulltext.].

Full text of DOI:10.1038/s41557-020-0530-4

Articles
https://doi.org/10.1038/s41557-020-0530-4
Using sulfuramidimidoyl fluorides that undergo
sulfur(vi) fluoride exchange for inverse drug
discovery
Gabriel J. Brighty1,2,10, Rachel C. Botham1,2,6,10, Suhua Liꢀ ꢀ1,7,10, Luke Nelson1, David E. Mortenson1,2,8
,
Gencheng Li1, Christophe Morisseau3,4, Hua Wang1,9, Bruce D. Hammock3,4, K. Barry Sharplessꢀ ꢀ1,5
ꢀ✉
and Jeffery W. Kellyꢀ ꢀ1,2,5
ꢀ✉
Drug candidates that form covalent linkages with their target proteins have been underexplored compared with the conven-
tional counterparts that modulate biological function by reversibly binding to proteins, in part due to concerns about off-target
reactivity. However, toxicity linked to off-target reactivity can be minimized by using latent electrophiles that only become
activated towards covalent bond formation on binding a specific protein. Here we study sulfuramidimidoyl fluorides, a class of
weak electrophiles that undergo sulfur(vi) fluoride exchange chemistry. We show that equilibrium binding of a sulfuramidimi-
doyl fluoride to a protein can allow nucleophilic attack by a specific amino acid side chain, which leads to conjugate formation.
We incubated small molecules, each bearing a sulfuramidimidoyl fluoride electrophile, with human cell lysate, and the protein
conjugates formed were identified by affinity chromatography–mass spectrometry. This inverse drug discovery approach iden-
tified a compound that covalently binds to and irreversibly inhibits the activity of poly(ADP-ribose) polymerase 1, an important
anticancer target in living cells.
n conventional drug discovery approaches, a large number of perturb the reactive side chain’s pKa, and so enhance its nucleophi-
small molecules are screened for their ability to modify the func- licity4. The potential of SuFEx reactions employing sulfonyl fluo-
I
tion of an isolated target protein or a particular cellular pheno- rides and arylfluorosulfates has been demonstrated in the fields of
type1. These approaches can yield hits that exert their action by both material science7–9 and late-stage drug functionalization to enhance
covalent and non-covalent mechanisms. Drugs that form covalent the pharmacologic activity10.
conjugates with their targets have historically been avoided due
Here we apply the IDD approach4,6 to another family of
to off-target toxicity concerns, but they can offer potential bene- SuFEx-derived electrophiles, the sulfuramidimidoyl fluorides
fits over non-covalent inhibitors in terms of increased duration of (SAFs)11, whose proteome reactivity has not been previously
action, lower dosing requirements and the possibility of decreased assessed. We explored the hypothesis that the unique chemical
development of resistance2,3. Therefore, we have developed an environments within proteins should allow the use of less-reactive
inverse drug discovery (IDD) strategy, which involves individually SuFEx-derived SAFs for IDD. The SAFs were prepared by a
reacting a small collection of diverse organic compounds that har- two-step SuFEx reaction process (Fig. 1a)11. First, an alkyl or aryl
bour a weak, but activatable, electrophile with the human proteome primary amine was reacted with thionyl tetrafluoride (SOF4) at a
in cell lysate to identify the protein(s) targeted4. This method identi- standard temperature and pressure in the presence of two equiva-
fies numerous nucleophilic sites within the human proteome using lents of an organic base to produce the corresponding iminosulfur
only a handful of mildly reactive compounds.
oxydifluoride. Next, a secondary amine was used to displace one
An earlier IDD study using arylfluorosulfates activated towards of the remaining fluorides in the presence of triethylamine to pro-
covalent bond formation by the geometry and composition of the duce the corresponding SAF (Fig. 1a). The reduced electrophilic-
complementary protein binding sites afforded both enzyme and ity of the SAFs compared with arylfluorosulfates in acetonitrile
non-enzyme conjugates4. From this study, we learned that the pres- probably arises from the replacement of a S=O bond with a S=NR
ence of a suitable protein binding pocket allows the electrophilic bond, which increases the electron density around the sulfur centre
sulfur centre to be placed proximal to a tyrosine or lysine side-chain in SAFs to afford an even stronger bond between the fluorine and
nucleophile, which enables the sulfur(vi) fluoride exchange (SuFEx) sulfur11. Although the sulfur centres in both SAFs and arylfluoro-
reaction to proceed4–6. The additional presence of a nearby cationic sulfates are tetrahedral, in SAFs this centre is also chiral11.
arginine and/or lysine side chain seems to be critical to lower the
In this study, we matched SAFs with the human protein(s) that
barrier for the SuFEx reaction, as these residues probably facilitate they react with using affinity chromatography–mass spectrom-
extraction of the fluoride ion. These cationic residues may also etry. Selected conjugation reactions were then validated using
1Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA. 2Department of Molecular Medicine, The Scripps Research Institute,
La Jolla, CA, USA. 3Department of Entomology and Nematology, University of California Davis, Davis, CA, USA. 4UC Davis Comprehensive Cancer
Center, University of California Davis, Davis, CA, USA. 5The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA.
6Present address: Codexis, Redwood City, CA, USA. 7Present address: School of Chemistry, Sun Yat-Sen University, Guangzhou, P. R. China. 8Present address:
Gilead Sciences Inc., Foster City, CA, USA. 9Present address: Sorrento Therapeutics, Inc., San Diego, CA, USA. 10These authors contributed equally:
✉
Gabriel J. Brighty, Rachel C. Botham, Suhua Li. e-mail: sharples@scripps.edu; jkelly@scripps.edu
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
a
b
H
N
N
N
1: R =
N
O
N
2: R =
3: R =
N
R'
R'
R''
SOF4 (g)
NH2
O
N
MeO2C
(1.2 or 2.0 equiv.)
F
N
R''
N
N
R
S
R
S
R
NEt3 (2 equiv.)
MeCN, r.t.
NEt3 (1.5 equiv.
or none)
NH
O
O
O
F
F
OH
O
4: R =
MeCN, r.t.
O
F
c
O
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
N
250 kDa
150
S
5: R =
N
1–5
O
R
100
75
O
F
50
F
S
O
37
N
S
N
N
N
25
20
6
7
15
10
O
O
S
F
F
N
N
H
N
S
N
O
F
S
O
N
F
O
H
O
S
F
O
N
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
F
8
9
10
250
O
N
150
100
75
O
S
N
O
N
F
O
O
F
O
50
S
H
N
N
37
N
N
25
20
N
S
O
O
H
S
F
N
O
N
15
F
10
11
12
13
O
O
N
N
O
S
N
O
S
F
N
N
NH
F
F
O
N
S
O
N
N
14
15
16
Fig. 1 | SaFs 1–16 react with proteins in HEK293t cell lysate. a, Stepwise synthetic scheme for the synthesis of SAF-containing compounds. A primary
amine was reacted with SOF4 to form an iminosulfur oxydifluoride. This intermediate was then reacted with a secondary amine to form as SAF.
b, Structures of SAF compounds 1–16 used in this study. c, Top: SDS-PAGE/rhodamine analysis of HEK293T lysate incubated (18ꢀh) with the SAF
compound (50ꢀµM), after the addition of TMR-N3 via CuAAC (see Methods for detailed conditions). Each SAF appears to react with a different set of
proteins, as indicated by the different banding patterns in each lane. Bottom: Coomassie staining of the SDS-PAGE gel indicates an equal loading of protein
in each lane. The fluorescence intensity of each band is a product of SAF reactivity towards that protein rather than a difference in protein abundance
between treated HEK293T lysates. This experiment was conducted twice (nꢀ=ꢀ2) with similar results. r.t., room temperature.
recombinant proteins in vitro. We demonstrate that structurally between a given SAF and the targeted proteins increased linearly
distinct SAFs react with different sets of human proteins. Lastly, over 24hours (Supplementary Fig. 1). Therefore, we chose an incu-
we matched a thymidine-based SAF with poly(ADP-ribose) poly- bation period of 18hours as a practical experimental time point to
merase 1 (PARP1), and demonstrated covalent inhibition of PARP1 capture sufficient quantities of SAF-conjugated proteins. The 16
both in vitro and in live cells, potentially complementing the conjugation reactions were then subjected to a copper(i)-catalysed
non-covalent PARP1 inhibitors used to ameliorate cancer.
azide–alkyne cycloaddition (CuAAC) reaction12,13 with tetrameth-
ylrhodamine azide (TMR-N3) and the conjugates were separated in
a denaturing gel (SDS–polyacrylamide gel electrophoresis (SDS–
Results
SAFs react with human proteins. We set out to survey the human PAGE)) visualized by tetramethylrhodamine fluorescence (Fig. 1c).
proteome for its reactivity with 16 SAF-containing compounds Notably, compounds 1–16 displayed different reactivity profiles
(Fig. 1b). Compounds 1–16 comprise largely unpublished SAFs towards the human proteome detectable by SDS–PAGE, which indi-
harbouring terminal acetylenes available at the outset of this study11. cates that the equilibrium binding fragments of these compounds
We purposefully did not try to select for motifs that would bind are critical determinants of selectivity for the conjugation process.
to a particular protein family, although we did use our intuition to To more comprehensively survey the conjugates formed and to
maximize the structural diversity. Each SAF was individually incu- detect lower-abundance conjugates, we utilized quantitative liquid
bated with HEK293T cell lysate for 18hours. The extent of reaction chromatography–tandem mass spectrometry (LC–MS/MS)4.
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
a
b
DMSO
SAF X
SAF Y
SAF number
10 11 12 13 14 15 16
1
2
3
4
5
6
7
8
9
MIF
PARP1
PARP2
EPHX2
BCAT1
NME1
(1)–(3)
Ratio
12
HACD3
CRABP2
TK1
TMT
126
TMT
127
TMT
128
TMT
129
TMT
130
TMT
131
(4)
(5)
9
6
3
FAH
RAE1
P4HB
PDIA6
PIK3C3
RPL37A
RPL38
TIGAR
GSTP1
VDAC2
CYP51A1
LC–MS/MS
Protein
identification
Protein
quantification
Fig. 2 | Proteins targeted by SaFs 1–16 identified using isobaric MS/MS tagging in conjunction with proteomic mass spectrometry. a, TMT LC–MS/MS
workflow. After the HEK293T lysates were treated with a particular probe (50ꢀµM) for 18ꢀh, (1) they were (subject to a CuAAC reaction with biotin-N3.
Next, (2) the protein–probe conjugates were enriched using streptavidin resin and the bound proteins were eluted from the resin using Na2S2O4. (3)
The enriched conjugates were then digested with trypsin and (4) the resulting peptides were labelled with the respective TMT reagent. (5) The tagged
peptides were pooled and subjected to LC–MS/MS based on multidimensional protein identification technology, which allows for protein identification and
quantification compared with DMSO controls. b, Heat map displaying enrichment ratios of probes 1–16 for selected proteins by quantitative LC–MS/MS.
The displayed proteins have enrichment ratios >1.50 (treated samples versus DMSO control), and P values <0.25 (calculated by a two-tailed, unpaired
t-test). Grey squares indicate a failure to make this cutoff. Data are the result of a single experiment that contained two replicate treatment conditions
(nꢀ=ꢀ2). See Supplementary Information for the precise enrichment ratios and exact P values.
Proteomics identifies the targets of SAFs 1–16. To identify (HEK293T) revealed substantial differences in the set of protein
the human proteins targeted by SAFs 1–16, we subjected the 16 conjugates formed from distinct SAFs (Fig. 2b depicts a sam-
proteome-labelling reactions (18 hours of incubation at 25°C) pling of SAF-reactive proteins). The SAFs appear to target pro-
to CuAAC reactions with a reductively cleavable biotin azide teins with a variety of functions, which include both enzymes
(biotin-N3) (refs. 12–14). The SAF-conjugated proteins were subse- and non-enzymes (Supplementary Fig. 2a,b). Structural proteins
quently enriched via streptavidin affinity chromatography. Sodium and proteins that bind RNA were just a couple of the molecu-
dithionite (Na2S2O4) was used to elute the bound proteins from the lar functions enriched for (enrichment over DMSO >1.5 and
streptavidin resin. Enriched proteins were then digested with tryp- P value <0.25) by SAFs 1–16. Among the identified proteins
sin and subjected to tandem-mass tag (TMT) labelling15 to enable were GSTP1, NME1 and CRABP2, all of which have previously
the quantification of the SAF–protein conjugates relative to a pro- demonstrated reactivity towards arylfluorosulfates4,6. Notably,
teome cell lysate sample treated with dimethylsulfoxide (DMSO) we found strong enrichment of several therapeutically impor-
(as the vehicle, but otherwise identically treated) by LC–MS/MS tant proteins that were not previously targeted by arylfluoro-
(Fig. 2a). We defined the enrichment ratio for each protein as the sulfates. These included PARP1 and PARP2, critical members
‘average of the TMT reporter ion intensity for a given protein in of DNA damage-repair pathways and validated therapeutic tar-
the SAF-treated samples’/‘average of the TMT reporter ion inten- gets for BRCA-mutant-associated breast and ovarian cancers16,17
.
sity for that same protein in the vehicle-treated samples’. Proteins Also targeted were the macrophage migration inhibitory factor
were ranked according to this enrichment ratio. Duplicate mea- (MIF), which enhances the metastatic potential of certain cancers
surements of each treatment condition (SAF treated versus DMSO and acts as a pro-inflammatory signal in both chronic and acute
treated) were compared by their enrichment ratios and statistical inflammatory diseases18,19, soluble epoxide hydrolase (EPHX2),
significance to assess the reliability of each protein target identifica- which has garnered attention as a pharmacological target for the
tion. We prioritized protein ‘hits’ that exhibited a strong enrichment treatment of certain cardiovascular diseases20, and branched chain
relative to that of the vehicle treated (enrichment ratios >1.5) and amino acid transaminase 1 (BCAT1), which is a therapeutic tar-
a reasonable statistical agreement between duplicate measurements get for myeloid leukaemia, carcinoma, glioblastoma and certain
(P values <0.25). Additionally, we noted the strong enrichment of breast cancers21–23. Although we observed enrichment for 137
several proteins with different SAFs, which further indicates that proteins that have been targeted by other SuFEx-derived electro-
these proteins are reactive towards SAFs.
philes, more than 72% of the 491 proteins identified by SAFs 1–16
As observed in the SDS–PAGE-based comparisons (Fig. 1c), are distinct from those identified using arylfluorosulfates4,6 or sul-
LC–MS/MS analysis of SAFs 1–16 reacting with human cell lysate fonyl fluorides24–27 (Supplementary Fig. 2c).
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
a
b
250 kDa
150
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
P = 0.0002
100
75
***
50
37
P = 0.0030
**
25
20
P = 0.0015
P = 0.0059
15
10
**
**
Standard curve (µM)
c
d
e
Lys903
Tyr907
Tyr466
Tyr96ʹ
Tyr383
Olaparib
TPPU
Lys33
Pro2
BITC
Asp335
Tyr889
f
g
h
BITC
–
+
TPPU
–
+
Olaparib
–
+
Fig. 3 | Validation of reactions between SaFs and selected recombinant proteins. a, Fluorescence-based analysis of reactions (24ꢀh) between selected
SAFs (30ꢀµM) and selected proteins (3ꢀµM). The standard curve comprises four threefold dilutions of fluorescently labelled MIF (3.00–0.037ꢀµM). This
gel is representative of three independent experiments (nꢀ=ꢀ3). b, Quantification of the fluorescence bands in a. Error bars represent s.e.m. for three
independent experiments (nꢀ=ꢀ3). P values were calculated using a two-tailed, unpaired t-test. **Pꢀ≤ꢀ0.01, ***Pꢀ≤ꢀ0.001; exact P values are shown. c, X-ray
crystal structure of MIF conjugated to BITC, with Tyr96′ from the adjacent monomer (PDB ID 3WNT). d, X-ray crystal structure of EPHX2 bound to
TPPU (PDB ID 4OD0). e, X-ray crystal structure of the PARP1cat domain bound to olaparib (PDB ID 5DS3). Residues highlighted in yellow denote the
SAF-reactive nucleophiles identified by LC–MS/MS. Other labelled residues denote other potential binding sites and/or residues that may be important
for reaction with the SAFs (c–e). f, Fluorescence-based analysis of the reaction of MIF (5ꢀµM) and 13 (50ꢀµM) for 24ꢀh in the presence or absence of BITC
(50ꢀµM). This experiment was conducted once (nꢀ=ꢀ1). g, Fluorescence-based analysis of the reaction of EPHX2 (3ꢀµM) and 9 (50ꢀµM) for 24ꢀh in the
presence or absence of TPPU (10ꢀµM). This experiment was conducted once (nꢀ=ꢀ1). h, Fluorescence-based analyses of the reaction of PARP1cat (3ꢀµM)
with 2 (100ꢀµM) for 24ꢀh in the presence or absence of olaparib (10ꢀµM). This experiment was conducted once (nꢀ=ꢀ1).
Validation of selected SAF–protein reactions. To validate PARP1cat, respectively, and compounds 10, 1, 15 and 11 were
SAF-reactive proteins identified by affinity chromatography–MS/ selected as negative hits for the same proteins, respectively. After a
MS, we expressed and purified four therapeutically important pro- reaction period of 18hours, each protein (3μM) was subjected to
teins and reacted each purified protein with either a SAF that exhib- CuAAC reactions with TMR-N3 and the conjugates were separated
ited a high enrichment ratio (a ‘positive’ hit) or one that afforded a by SDS–PAGE and visualized by fluorescence (Fig. 3a). In all four
low or no enrichment (a ‘negative’ hit). For PARP1, we expressed cases, the positive hits reacted more than the negative ones with their
only the catalytic domain (PARP1cat). Compounds 13, 11, 9 and 2 respective proteins, and exhibited statistical significance (Fig. 3b).
were selected as the positive hits for MIF, BCAT1, EPHX2 and It appears that neither BCAT1 nor EPHX2 were particularly reactive
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
a
b
O
F
NAD+
+
–
+
+
+
+
+
+
+
+
+
+
+
+
18: R =
N
N
O
O
NH
Automodified
PARP1
NH
O
F
N
O
19: R =
20: R =
NHBoc
O
HN
PARP1
N
N
S
Automodified
PARP1
O
R
N
F
S
21: R =
22: R =
N
O
PARP1
18–22
N
O
18
1
19
2
20
3
21
4
5
22 23
23
c
d
P = .0011
120
100
80
60
40
20
0
P =*.0*020
**
P = .0018
**
P =*.0*032
F
O
P = .0130
*
NH
Lys903
Tyr907
HN
Rucaparib
Tyr896
NH
18
1
19
2
20
3
21
4
5
22 23
Tyr889
Rucaparib
20 min incubation
18 h incubation
e
f
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
20 min incubation
18 h incubation
Olaparib
–
+
–
+
–
+
–
+
2
2
20
20
5
5
23
23
Fig. 4 | analysis of PaRP1 inhibitory activity of a subset of SaFs. a, Structures of SAFs 18–23 used in a PARP1 automodification assay. Left: the structures
of 18–22, which all share a common thymidine core structure, with the affixed 2° amine differing between them. Right: the structure of compound 23. None
of these compounds were subjected to the IDD workflow (Fig. 2a) used to identify SAF-reactive proteins. b, SDS–PAGE analysis of an in vitro assay of PARP1
activity. PARP1 was incubated with the indicated compound for either 20ꢀmin (top) or 18ꢀh (bottom), and then NAD+ (100ꢀµM) was added. Automodification
reactions were run for 1ꢀh at room temperature before analysis. c, Quantification of SDS–PAGE in b. Error bars represent s.e.m. for three independent
experiments (nꢀ=ꢀ3). P values were determined using a two-tailed, paired t-test. *Pꢀ≤ꢀ0.05, **Pꢀ≤ꢀ0.01 compared with samples treated with DMSO (see
Source Data for the analysis and exact P values). The dashed line at 53.5% indicates the approximate baseline of unmodified PARP1 in vitro (average
between vehicle treatments at 20ꢀmin and 18ꢀh). d, Left: X-ray crystal structure of rucaparib bound in the NAD+ binding site of PARP1cat (PDB ID 4RV6).
Tyr907 (the SAF-reactive residue) is highlighted in yellow, and proximal Tyr889 is highlighted in green. Rucaparib is highlighted in pink. Right: the structure
of the FDA-approved PARP1 inhibitor rucaparib. e, In-gel fluorescence analysis of reactions between PARP1cat (3ꢀµM) and selected SAF probes (30ꢀµM).
Olaparib (30ꢀµM) was added as a control to negate the reaction within the NAD+ binding site of PARP1cat. PARP1cat was incubated with the indicated
compound for either 20ꢀmin (top) or 18ꢀh (bottom) before the reactions were subjected to CuAAC conditions. f, Quantification of in-gel fluorescence in e.
Error bars represent s.e.m. for three independent experiments (nꢀ=ꢀ3). Results from reactions that contain olaparib are omitted for clarity.
towards their SAF probes. This may be explained by the loss of a induce conformational changes that affect the binding and/or
critical post-translational modification(s) in the recombinant pro- reactivity of the SAF. Compound 17 (not included in our library
tein and/or different conditions experienced in lysate versus those of 16 compounds), was found to react quantitatively and stoichio-
experienced in buffer alone. Both EPHX2 and BCAT1 are known metrically with MIF after 16hours at 25°C in vitro, as determined
to be sensitive to the redox potential of their media28,29, which may by LC–electrospray ionization (ESI) MS (Supplementary Fig. 3).
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
a
b
2 (µM)
Ola (µM)
100 100 0.1 0.1
1
1
10 10
DMSO
2 (µM)
5 (µM)
1
+
10 50 100
1
+
10 50 100
DMSO Ola
–
–
+
–
+
–
+
+
+
–
+
+
+
–
+
+
+
–
+
+
H2O2
H2O2
–
+
+
+
+
+
+
+
+
Wash out
kDa 250
kDa 250
150
IB:
PAR
IB:
PAR
150
100
100
75
75
50
50
250
250
150
IB:
PARP1
IB:
PARP1
150
PARP1
100
PARP1
100
IB:
IB:
GAPDH
GAPDH
Fig. 5 | SaF 2 irreversibly inhibits the activity of PaRP1 in HeLa cells. a, Immunoblot (IB) analysis of PARylation in HeLa cells treated with SAFs. The cells
were incubated with olaparib (ola, positive control), 2 or 5 at increasing concentrations for 24ꢀh before the addition of H2O2 (10ꢀmM). Blots were analysed
with antibodies that targeted either PAR (top) or PARP1 (middle). The reduction of both PAR and PAR-modified PARP1 (that is, a higher molecular weight
smear in the middle panel) indicates the inhibition of PARP1’s enzymatic activity. This experiment was conducted once (nꢀ=ꢀ1). b, Western blot analysis
of PARylation after HeLa cells were treated with either 2 (100ꢀµM) or olaparib (0.1–10ꢀµM) for 24ꢀh and the compounds removed by washing cells twice
with PBS (pH 7.4). Blots were analysed with antibodies that targeted either PAR (top) or PARP1 (middle). The reduction of both PAR and PAR-modified
PARP1 in cells treated with 2 and washed indicate that 2 inhibited PARP1 irreversibly. This experiment was conducted once (nꢀ=ꢀ1). GAPDH, glyceraldehyde
3-phosphate dehydrogenase.
Therefore, a standard curve that comprises serial dilutions from Subsequent analysis of the resulting conjugate by LC–MS/MS con-
3 µM to 37nM in threefold decrements of recombinant MIF fully firmed that the reaction between EPHX2 and 9* takes place at
conjugated to 17 and tetramethylrhodamine was included on each Tyr466, the same residue that reacts with 9 (Supplementary Fig. 7).
denaturing gel to quantify the extent of each reaction (Fig. 3a and A labelling reaction between PARP1cat and 2 in the presence of the
Supplementary Fig. 4).
PARP1 non-covalent inhibitor olaparib33 (Fig. 3e) abolished cova-
lent conjugate formation (Fig. 3h), which indicates that 2 modifies
SAF-reactive sites on three target proteins. To gain further PARP1 in the nicotinamide adenine dinucleotide (NAD+) binding
insights into the SAF conjugation sites, we reacted three of the site34. By subjecting the reaction of 2 with PARP1cat to LC–MS/MS
purified proteins with the SAF used to identify it from the human analysis, we determined that 2 reacts with Tyr907 in this site to
proteome in the presence of known active site inhibitors (Fig. 3). form the PARP1·2 conjugate (Supplementary Fig. 8). This was fur-
Conjugation was again confirmed by subjecting these in vitro reac- ther confirmed by a strongly attenuated reaction between 2 and the
tions to a CuAAC reaction with TMR-N3 and examining the results Y907F mutant of PARP1cat (as compared with that of wild-type
via SDS-PAGE. Addition of the validated MIF-reactive compound PARP1cat) (Supplementary Fig. 9). Although we cannot rule out
benzyl isothiocyanate (BITC; 50μM), which forms a bond with the allosteric mechanisms of competition, the previously reported
N-terminal proline residue (Pro1) of MIF30 (Fig. 3c), to the reaction ligands BITC, TPPU and olaparib appear to bind proximal to the
of recombinant MIF (5μM) and 13 (50μM) strongly attenuates the SAF-reactive residues in MIF, EPHX2 and PARP1cat, respectively.
conjugate formation (Fig. 3f). This suggests that SAF probe 13 reacts
with Pro1 or a nearby residue. This result was further confirmed by SAFs inhibit PARP1 activity in vitro. PARP1 executes a critical
LC–MS/MS analysis of the MIF·13 conjugate (Supplementary Fig. 5). signalling post-translation modification in response to single-
Inasimilarfashion,additionofthedocumentedEPHX2non-covalent stranded DNA breaks. Using NAD+, it mediates polymerization
inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) of ADP-ribose onto itself and other target proteins35. The newly
urea (TPPU; 10μM)31 (Fig. 3d) to the reaction of recombinant synthesized poly(ADP-ribose) (PAR) chains act as a signal for
EPHX2 (3μM) with 9 (50μM) largely ablates the fluorescence sig- the recruitment of diverse families of proteins to the sites of DNA
nal (Fig. 3g), which indicates that 9 occupies the enzyme’s active damage36. Given the status of PARP1 as a validated pharmacologi-
site. This site contains two tyrosine residues, Tyr383 and Tyr466, cal target, we sought to confirm that the reaction with SAF probes
that carry out the hydrolysis of certain lipid epoxides29. Either of inhibits the enzyme’s activity. We examined the activity of PARP1
these two catalytic residues appear to be a likely site of reaction in the presence of selected SAFs, of which all shared a common
between the SAFs and EPHX2, as both of them are susceptible to propargyl thymidine structure except 23 (Fig. 4a). Preincubation of
nitration32. Analysis of the EPHX2·9 conjugate by LC–MS/MS PARP1 with activated damaged DNA and the SAF compound for
implicates Tyr466 as the reactive residue in EPHX2 (Supplementary 20minutes before the addition of NAD+ resulted in a limited inhibi-
Fig. 6). Note that 9 also contains an arylfluorosulfate; however, the tion of PARP1, with the notable exception of 23, which exhibited
reaction between 9 and EPHX2 occurs at the SAF electrophilic cen- substantial inhibition (Fig. 4b). However, notable inhibition was
tre, as this protein is enriched by other SAFs (1 and 12) that do observed with 2 and 5 after a reaction period of 18hours (Fig. 4b,c).
not harbour arylfluorosulfates. To support the hypothesis that this Compound 23 shares considerable structural similarity with ruca-
reaction occurs at the SAF sulfur centre and not the fluorosulfate, parib, another Food and Drug Administration (FDA)-approved
we subjected EPHX2 to reaction with an additional compound, 9*, PARP1 inhibitor37,38. Examination of the co-crystal structure of
that shares a structural similarity with 9 but lacks a fluorosulfate. PARP1cat bound to rucaparib (Fig. 4d, Protein Data Bank (PDB)
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
ID 4RV6), led us to posit that the inhibitory effects of 23 are medi- 1980 demonstration that the inhibition of PARP1 sensitized leukae-
ated primarily through non-covalent interactions with PARP1, in mia cells to cytotoxic alkylating agents41. This premise was built on
contrast with the considerable conjugation demonstrated by 2 and 5. by an abundance of preclinical evidence that supports the ability of
To test this hypothesis, we subjected 2, 5, 20 and 23 to reactions with PARP inhibitors to sensitize and potentiate radiation and cytotoxic
PARP1cat for 20minutes or 18hours, followed by a CuAAC reaction chemotherapy42. However, the pivotal breakthrough was made with
with TMR-N3. The results were quantified by fluorescent SDS-PAGE the observation that BRCA-mutant cells were up to 1,000 times more
(Fig. 4e,f). It is clear that 23 did not react with PARP1cat above back- sensitive to PARP inhibitors43,44. The enhanced sensitivity observed in
ground. We hypothesize that 23 adopts a binding orientation close BRCA-mutant cancer cells enabled the clinical validation of synthetic
to that of rucaparib in the NAD+-binding site of PARP1cat, which lethality in oncology, a concept first described in 1922, wherein simul-
prevents the SAF from engaging with Tyr907. However, SAFs 2 and taneous targeting of two genes or proteins can be lethal, even when
5 react substantially with PARP1cat, with some reaction occurring deletion and/or inhibition of each individually is itself tolerated45,46
after just 20minutes at 25°C. These results suggest that to simply
.
The treatment of cancers with existing PARP1 inhibitors is asso-
modify the existing structures of known protein ligands with SAF ciated with a high incidence of toxicity (such as neutropenia and
electrophiles does not generally result in a conjugation reaction, anaemia)47. New PARP1 inhibitors based on a covalent mechanism
which emphasizes the importance of a proper positioning of the SAF. of action may allow for the circumvention of side effects associated
with existing inhibitors. It was recently reported that PAR synthe-
SAF 2 inhibits PAR synthesis in HeLa cells. To investigate whether sis helps drive α-synuclein aggregation both in vitro and in vivo,
SAFs inhibit PARP1 activity in living cells, we treated HeLa cells which highlights PARP1 inhibition as a potential therapy for the
with 2, 5 or olaparib for 24hours prior to the addition of H2O2 for treatment of Parkinson’s disease48. Such a therapy will most prob-
15minutes to induce PARylation. No cell death was observed over ably require an extended duration of action, a convenient feature of
this period. After cell lysis, the extent of the PAR modification was SAF-based PARP1 inhibitors49. It is notable that our PARP1-reactive
measured by immunoblot using antibodies that recognize PAR and compounds bear little structural resemblance to the FDA-approved
PARP1. Cells treated with 2 exhibited a substantial reduction in inhibitors (for example, olaparib). Our compounds represent a fresh
both general PARylation and PARP1 automodification, unlike cells start for inhibitor design and synthesis. Covalent PARP1 inhibitors
treated with 5 or vehicle alone (Fig. 5a). As expected, treatment with may be optimized to achieve selective PARP1 or PARP2 inhibition,
olaparib (positive control) resulted in the total ablation of PAR syn- whereas non-covalent inhibitors are expected to bind to a wider
thesis. The ability of 2 to inhibit PARP1 activity in cells is notable, array of PARP family members38.
given that its structure has not been optimized for cellular perme-
ability nor PARP1 binding by medicinal chemistry efforts.
The apparent low reactivity of SAFs means that only a subset of
the human proteome is even capable of reacting with this functional
To scrutinize the cellular activity, we subjected HeLa cells to a group, which substantially reduces the off-target reactivity observed
treatment with either olaparib or 2 for 24 hours. Cells were then with more-reactive electrophiles. This feature, combined with their
washed and fresh media (free of inhibitor) was added for 6hours. chirality and straightforward synthesis, makes SAFs ideally suited
PARP1 activity was then induced via treatment with H2O2. This for the rapid generation of compound libraries, and so potentially
treatment regimen by 2 retained most of its PARP1 inhibitory activ- simplify the optimization of identified hits based on medicinal
ity, whereas the olaparib activity was dramatically reduced (Fig. 5b), chemistry. For IDD that involves arylfluorosulfates, the electrophilic
which indicates that conjugation by 2 inhibits PAR synthesis until warhead and affinity handle must be installed at separate sites via
the enzyme is turned over.
separate reactions or the judicious use of one-pot reaction schemes.
As a result, the applicability of the arylfluorosulfates in library con-
struction is more challenging. For these reasons, we recommend
Discussion
Past drug-discovery efforts, by and large, have not deliberately lev- that future IDD efforts take seriously the potential of SAFs.
eraged functional groups capable of protein conjugation. This may
be due to concerns about off-target reactivity and associated toxic- Online content
ity. However, the recent emergence of new functional groups that Any methods, additional references, Nature Research reporting
exhibit highly attenuated or latent protein reactivity, such as those summaries, source data, extended data, supplementary infor-
capable of undergoing a SuFEx reaction, suggest that drug discov- mation, acknowledgements, peer review information; details of
ery efforts that make deliberate use of these functionalities could author contributions and competing interests; and statements of
produce highly selective covalent drugs2,6,26. Our previous IDD data and code availability are available at https://doi.org/10.1038/
approach with arylfluorosulfates led to validated covalent probes for s41557-020-0530-4.
11 human proteins4. In this study, a new set of proteins closely asso-
Received: 25 April 2019; Accepted: 23 July 2020;
ciated with pathology (for example, BCAT1, EPHX2, PARP1 and
Published: xx xx xxxx
MIF) were matched with SAFs via the IDD strategy. Recombinant
BCAT1 and EPHX2 exhibited a low reactivity towards their cor-
References
responding SAF probes, which suggests that extensive medicinal
chemistry would be required to modulate function with SAFs.
Conversely, MIF and PARP1 exhibited a robust reactivity towards
SAFs from our first-generation library, which suggests that the dis-
covery of more reactive and selective SAF compounds towards these
proteins would probably require less effort. Notably, we demon-
strated that 2 inhibited PARP1 function in living cancer cells even
after compound washout, which implies that the SAF-based inhibi-
tion of PARP1 may represent a viable anticancer strategy.
1. Macarron, R. et al. Impact of high-throughput screening in biomedical
research. Nat. Rev. Drug Disc. 10, 188–195 (2011).
2. Singh, J., Petter, R. C., Baillie, T. A. & Whitty, A. Te resurgence of covalent
drugs. Nat. Rev. Drug Disc. 10, 307–317 (2011).
3. Bauer, R. A. Covalent inhibitors in drug discovery: from accidental
discoveries to avoided liabilities and designed therapies. Drug Disc. Today 20,
1061–1073 (2015).
4. Mortenson, D. E. et al. ‘Inverse drug discovery’ strategy to identify proteins
that are targeted by latent electrophiles as exemplifed by aryl fuorosulfates.
J. Am. Chem. Soc. 140, 200–210 (2018).
5. Dong, J., Krasnova, L., Finn, M. G. & Sharpless, K. B. Sulfur(vi) fuoride
exchange (SuFEx): another good reaction for click chemistry. Angew. Chem.
Int. Ed. 53, 9430–9448 (2014).
Acquired or inherited defects in the DNA system for damage
response and repair increase the lifetime risk of cancer. Mutations
in BRCA1 and BRCA2, both DNA repair proteins, were some of
the first mutations to be associated with familial breast and ovarian
cancers39,40. The targeting of PARP1 is appealing because of the
6. Chen, W. et al. Arylfuorosulfates inactivate intracellular lipid binding
protein(s) through chemoselective SuFEx reaction with a binding site Tyr
residue. J. Am. Chem. Soc. 138, 7353–7364 (2016).
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
7. Dong, J., Sharpless, K. B., Kwisnek, L., Oakdale, J. S. & Fokin, V. V. SuFEx-
based synthesis of polysulfates. Angew. Chem. Int. Ed. 53, 9466–9470 (2014).
8. Gao, B. et al. Bifuoride-catalysed sulfur(vi) fuoride exchange reaction for the
synthesis of polysulfates and polysulfonates. Nat. Chem. 9, 1083–1088 (2017).
9. Wang, H. et al. SuFEx-based polysulfonate formation from ethenesulfonyl
fuoride–amine adducts. Angew. Chem. Int. Ed. 56, 11203–11208 (2017).
10. Liu, Z. et al. SuFEx click chemistry enabled late-stage drug functionalization.
J. Am. Chem. Soc. 140, 2919–2925 (2018).
11. Li, S., Wu, P., Moses, J. E. & Sharpless, K. B. Multidimensional SuFEx
click chemistry: sequential sulfur(vi) fuoride exchange connections of
diverse modules launched from an SOF4 hub. Angew. Chem. Int. Ed. 56,
2903–2908 (2017).
12. Wang, Q. et al. Bioconjugation by copper(i)-catalyzed azide–alkyne [3+2]
cycloaddition. J. Am. Chem. Soc. 125, 3192–3193 (2003).
13. Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise
Huisgen cycloaddition process: copper(i)-catalyzed regioselective ‘ligation’ of
azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).
14. Verhelst, S. H., Fonovic, M. & Bogyo, M. A mild chemically cleavable linker
system for functional proteomic applications. Angew. Chem. Int. Ed. 46,
1284–1286 (2007).
29. Morisseau, C. & Hammock, B. D. Epoxide hydrolases: mechanisms, inhibitor
designs, and biological roles. Ann. Rev. Pharmacol. Toxicol. 45, 311–333 (2005).
30. Spencer, E. S. et al. Multiple binding modes of isothiocyanates that inhibit
macrophage migration inhibitory factor. Eur. J. Med. Chem. 93, 501–510 (2015).
31. Lee, K. S. et al. Optimized inhibitors of soluble epoxide hydrolase improve
in vitro target residence time and in vivo efcacy. J. Med. Chem. 57,
7016–7030 (2014).
32. Barbosa-Sicard, E. et al. Inhibition of the soluble epoxide hydrolase by
tyrosine nitration. J. Biol. Chem. 284, 28156–28163 (2009).
33. Fong, P. C. et al. Inhibition of poly(ADP-ribose) polymerase in tumors from
BRCA mutation carriers. N. Engl. J. Med. 361, 123–134 (2009).
34. Dawicki-McKenna, J. M. et al. PARP-1 activation requires local unfolding of
an autoinhibitory domain. Mol. Cell 60, 755–768 (2015).
35. Langelier, M.-F., Servent, K. M., Rogers, E. E. & Pascal, J. M. A third
zinc-binding domain of human poly(ADP-ribose) polymerase-1 coordinates
DNA-dependent enzyme activation. J. Biol. Chem. 283, 4105–4114 (2008).
36. Gibson, B. A. & Kraus, W. L. New insights into the molecular and cellular
functions of poly(ADP-ribose) and PARPs. Nat. Rev. Mol. Cell Biol. 13,
411–424 (2012).
37. Coleman, R. L. et al. Rucaparib maintenance treatment for recurrent
ovarian carcinoma afer response to platinum therapy (ARIEL3): a
randomised, double-blind, placebo-controlled, phase 3 trial. Te Lancet
390, 1949–1961 (2017).
15. Dayon, L. et al. Relative quantifcation of proteins in human cerebrospinal
fuids by MS/MS using 6-plex isobaric tags. Anal. Chem. 80, 2921–2931 (2008).
16. Konecny, G. E. & Kristeleit, R. S. PARP inhibitors for BRCA1/2-mutated and
sporadic ovarian cancer: current practice and future directions. Br J. Cancer
115, 1157–1173 (2016).
38. Torsell, A.-G. et al. Structural basis for potency and promiscuity in
poly(ADP-ribose) polymerase (PARP) and tankyrase inhibitors. J. Med.
Chem. 60, 1262–1271 (2017).
17. Lord, C. J. & Ashworth, A. BRCAness revisited. Nat. Rev. Cancer 16,
110–120 (2016).
39. Futreal, P. et al. BRCA1 mutations in primary breast and ovarian carcinomas.
Science 266, 120–122 (1994).
18. Lue, H., Kleemann, R., Calandra, T., Roger, T. & Bernhagen, J. Macrophage
migration inhibitory factor (MIF): mechanisms of action and role in disease.
Microbes Infect. 4, 449–460 (2002).
40. Wooster, R. et al. Identifcation of the breast cancer susceptibility gene
BRCA2. Nature 378, 789–792 (1995).
19. Calandra, T. & Roger, T. Macrophage migration inhibitory factor: a regulator
of innate immunity. Nat. Rev. Immunol. 3, 791–800 (2003).
41. Durkacz, B. W., Omidiji, O., Gray, D. A. & Shall, S. (ADP-ribose)n
participates in DNA excision repair. Nature 283, 593–596 (1980).
42. Tentori, L. & Graziani, G. Chemopotentiation by PARP inhibitors in cancer
therapy. Pharmacol. Res. 52, 25–33 (2005).
20. Imig, J. D. & Hammock, B. D. Soluble epoxide hydrolase as a therapeutic
target for cardiovascular diseases. Nat. Rev. Drug Disc. 8, 794–805 (2009).
21. Tonjes, M. et al. BCAT1 promotes cell proliferation through amino acid
catabolism in gliomas carrying wild-type IDH1. Nat. Med. 19, 901–908 (2013).
22. Xu, M. et al. BCAT1 promotes tumor cell migration and invasion in
hepatocellular carcinoma. Oncol. Lett. 12, 2648–2656 (2016).
43. Bryant, H. E. et al. Specifc killing of BRCA2-defcient tumours with
inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).
44. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a
therapeutic strategy. Nature 434, 917–921 (2005).
23. Zhang, L. & Han, J. Branched-chain amino acid transaminase 1 (BCAT1)
promotes the growth of breast cancer cells through improving
mTOR-mediated mitochondrial biogenesis and function. Biochem. Biophys.
Res. Commun. 486, 224–231 (2017).
45. Nijman, S. M. B. Synthetic lethality: general principles, utility and detection
using genetic screens in human cells. FEBS Lett. 585, 1–6 (2011).
46. Bridges, C. B. Te origin of variations in sexual and sex-limited characters.
Am. Nat. 56, 51–63 (1922).
47. Pujade-Lauraine, E. et al. Olaparib tablets as maintenance therapy in patients
with platinum-sensitive, relapsed ovarian cancer and a BRCA1/2 mutation
(SOLO2/ENGOT-Ov21): a double-blind, randomised, placebo-controlled,
phase 3 trial. Lancet Oncol. 18, 1274–1284 (2017).
48. Kam, T.-I. et al. Poly(ADP-ribose) drives pathologic α-synuclein
neurodegeneration in Parkinson’s disease. Science 362, eaat8407 (2018).
49. Berger, N. A. et al. Opportunities for the repurposing of PARP inhibitors for
the therapy of non-oncological diseases. Br. J. Pharmacol. 175, 192–222 (2018).
24. Zhao, Q. et al. Broad-spectrum kinase profling in live cells with lysine-
targeted sulfonyl fuoride probes. J. Am. Chem. Soc. 139, 680–685 (2017).
25. Zheng, Q. et al. SuFEx-enabled, agnostic discovery of covalent inhibitors of
human neutrophil elastase. Proc. Natl Acad. Sci. USA 116, 18808–18814 (2019).
26. Hett, E. C. et al. Rational targeting of active-site tyrosine residues using
sulfonyl fuoride probes. ACS Chem. Biol. 10, 1094–1098 (2015).
27. Hanoulle, X. et al. A new functional, chemical proteomics technology to
identify purine nucleotide binding sites in complex proteomes. J. Proteome
Res. 5, 3438–3445 (2006).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
28. Goto, M. et al. Structural determinants for branched-chain aminotransferase
isozyme-specifc inhibition by the anticonvulsant drug gabapentin.
J. Biol. Chem. 280, 37246–37256 (2005).
© The Author(s), under exclusive licence to Springer Nature Limited 2020
NatURE CHEMiStRy | www.nature.com/naturechemistry

NATure CHemisTry
Articles
curve. MIF (10µM) in PBS was reacted with 17 (100µM) for 24h at room
Methods
temperature (~25°C), after which time reaction completion was confirmed via
LC–MS analysis using a 1260 Infinity LC System (Agilent Technologies) and a
6130 Quadrupole LC/MS (Agilent Technologies). Protein m/z was calculated using
the Deconvolution Tool in the OpenLab Software Sutie (Agilent Technologies).
MIF was diluted to 3µM in PBS and subjected to CuAAC reaction conditions with
TMR-N3 (described above). This solution was then diluted threefold five times into
PBS that contained SDS loading buffer to make the standard curve.
Proteome labelling, fuorescence SDS–PAGE and SAF–protein conjugate
afnity purifcation. Conjugation reactions were carried out as follows: 5µl of
either DMSO or a 10mM solution of the SAF compound in DMSO were added
to 995µl of HEK293T lysate, followed by incubation at 25°C for 18h. Afer the
proteome reaction period, 50µl of each reaction was subjected to CuAAC reaction
conditions with TMR-N3 (TermoFisher Scientifc). Te CuAAC conditions
were: 2µl of 20µM CuSO4, 2µl of 40mM BTTAA (Click Chemistry Tools), 2.5µl
of sodium ascorbate (Sigma-Aldrich) and 1µl of 5mM TMR-N3 (in DMSO)
were added to 50µl of each proteome labelling reaction, and the reactions were
incubated at 30°C for 1h. Aferwards, proteins in the reaction mixture were
precipitated by the addition of MeOH/CHCl3 (3:1). Te proteins were resuspended
and subsequently pelleted twice in MeOH to facilitate the removal of excess
TMR-N3. Afer the fnal removal of MeOH by aspiration, the proteins were
dissolved in reducing SDS loading bufer. Proteins were resolved by SDS–PAGE,
and the fuorescently labelled proteins were visualized using a ChemiDoc XRS+
imager (Bio-Rad). Gels were then stained with a NOVEX Colloidal Blue Stain Kit
(Invitrogen) to assess equal protein loading across all lanes.
Fluorescence-based analysis of the reaction between MIF and 13 (with or
without BITC). MIF (5µM) in PBS was treated with 13 (50µM) along with either
50µM BITC or an equivalent volume of DMSO. The reactions were carried out at
room temperature for 24h. After this time, 50µl of each reaction was subjected to
CuAAC conditions with TMR-N3 and subsequently treated with 12µl of 6× SDS
loading buffer, followed by SDS–PAGE. The fluorescently labelled protein was
then visualized using a ChemiDoc XRS+ imager (Bio-Rad). The gels were then
stained with a NOVEX Colloidal Blue Stain Kit (Invitrogen) and imaged using a
ChemiDoc XRS+ imager (Bio-Rad).
Protein–SAF conjugates were purified, digested and TMT mass tagged in
accordance with a published experimental procedure4. Briefly, 950µl of each
proteome conjugation reaction described above were subjected to CuAAC
reaction conditions, with TMR-N3 substituted by diazo biotin-N3 (Click
Chemistry Tools, catalogue no. 1041). The reaction conditions were: 20µl of
20µM CuSO4, 20µl of 40mM BTTAA, 50µl of sodium ascorbate and 10µl
of 5mM biotin-N3 (DMSO) were added to 950µl of each proteome labelling
reaction, and the reactions were incubated at 30°C for 2h. After the CuAAC
reaction, the proteins subjected to the conjugation reaction were precipitated by
the addition of MeOH/CHCl3 (3:1) and subsequently pelleted by centrifugation.
The supernatant was removed by aspiration, and the pellet was washed with
MeOH three times to remove excess biotin-N3. After briefly drying the pellets
in air following the removal of MeOH, proteome samples were resuspended in
a 6M urea solution that contained 25mM (NH4)HCO3. SDS (as a 10% solution
w/v in water) was added to a final concentration of 2.2% to solubilize the protein
pellets. Next, proteins were reduced by the addition of 1M dithiothreitol to a
final concentration of 7.8mM and incubation for 15min at 65°C. After cooling
briefly on ice, the proteins were alkylated by the addition of 0.5M iodoacetamide
(Sigma-Aldrich) to a final concentration of 29mM and incubated in the dark
for 30min at 25°C. Proteome samples were each diluted into 6ml of PBS that
contained High-Capacity Streptavidin Agarose Resin (ThermoFisher Scientific)
and allowed to incubate for 16h with gentle agitation. The resin was then washed:
once with PBS+1% SDS, and then twice with PBS that contained no detergent.
The labelled proteins were then eluted from the resin by incubating for 1h at 25°C
with PBS that contained 1% SDS and 50mM Na2S2O4 (the solution was quickly
adjusted to pH ~7 after the dissolution of Na2S2O4). The elution procedure was
carried out twice and the elution volumes combined. The subsequently enriched
proteins were then precipitated once again with MeOH/CHCl3 (3:1), the pellets
were washed and centrifuged twice with MeOH to remove traces of SDS and were
then dried in air to remove residual MeOH. The proteins were then resuspended
in a solution that contained 0.2% Rapigest detergent (Waters) and 100mM HEPES
pH 8.0, and were digested overnight with trypsin protease (mass-spectrometric
grade; ThermoFisher Scientific). The digested samples were then reacted with
their designated TMT 6-plex reagent (ThermoFisher Scientific) for 1h (reactions
were conducted in 40% acetonitrile), and the reactions were quenched by the
addition of 10% (NH4)HCO3 (w/v) for 1h to a final concentration of 0.4% (w/v).
The quenched reactions were then combined and acidified to pH < 2 by the
addition of formic acid. The acetonitrile used in the TMT-labelling reaction
was removed via centrifugation under reduced pressure, and the samples were
incubated at 42°C for 1h to precipitate the remaining Rapigest. The samples were
then centrifuged and the supernatants collected and loaded into columns for
multidimensional protein identification technology experiments, as previously
described50. A heatmap was generated with R (version 3.2.4) using enrichment
ratios for hand-selected proteins identified by the multidimensional protein
identification technology experiments.
Fluorescence-based analysis of the reaction between EPHX2 and 9 (with or
without TPPU). EPHX2 (3µM) in PBS was treated with 9 (50µM) along with
either 10µM TPPU or an equivalent volume of DMSO. The reactions were
carried out at room temperature for 24h. After this time, 50µl of each reaction
was subjected to CuAAC conditions with TMR-N3 and subsequently treated with
12µl of 6× SDS loading buffer, followed by SDS–PAGE. The fluorescently labelled
protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). The gels
were then stained with a NOVEX Colloidal Blue Stain Kit (Invitrogen) and imaged
using a ChemiDoc XRS+ imager (Bio-Rad).
Fluorescence-based analysis of the reaction between PARP1cat and 2 (with or
without olaparib). PARP1cat (3µM) in PBS was treated with 2 (100µM) along
with either 10µM olaparib or an equivalent volume of DMSO. The reactions were
carried out at room temperature for 24h. After this time, 50µl of each reaction
was subjected to CuAAC conditions with TMR-N3 and subsequently treated with
12µl of 6× SDS loading buffer, followed by SDS–PAGE. The fluorescently labelled
protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). The gels
were then stained with a NOVEX Colloidal Blue Stain Kit (Invitrogen) and imaged
using a ChemiDoc XRS+ imager (Bio-Rad).
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
Additional methods and data are provided in the Supplementary Information. All
data generated or analysed during this study are included in this published article
(and its supplementary files). Source data are provided with this paper.
References
50. Ryno, L. M. et al. Characterizing the altered cellular proteome induced by
the stress-independent activation of heat shock factor 1. ACS Chem. Biol. 9,
1273–1283 (2014).
acknowledgements
This work was supported by the Skaggs Institute for Chemical Biology, the Lita
Annenberg Hazen Foundation and National Institutes of Health grants DK046335
(J.W.K.). R.C.B. was supported by a grant from the American Cancer Society. D.E.M. was
supported by a grant from the George E. Hewitt Foundation for Medical Research. This
work was supported in part by the National Institute of Environmental Health Sciences
(NIEHS) Grant R35 ES030443, and NIEHS Superfund Research Program P42 ES004699.
author contributions
G.J.B., R.C.B. and J.W.K. conceived and designed the experiments G.J.B., R.C.B., S.L.,
L.N., D.E.M., G.L., C.M. and H.W. carried out the experiments and performed the data
analysis G.J.B., R.C.B., S.L., C.M., B.D.H., K.B.S. and J.W.K. co-wrote the paper.
Fluorescence-based analysis of reactions between selected SAFs and selected
proteins. Reactions were carried out at room temperature in PBS (pH 7.4).
Protein (MIF, BCAT1, EPHX2 or PARP1cat; 3µM) were incubated with 30µM
of the indicated SAF compound for 24h at room temperature. Aliquots (50µl)
of each reaction were subjected to CuAAC reaction conditions with TMR-N3
(described above). The reactions were then treated with 12µl of 6× SDS loading
buffer, followed by SDS–PAGE. The fluorescently labelled protein was visualized
using a ChemiDoc XRS+ imager (Bio-Rad). Fluorescent bands were quantified
using Image Lab (BioRad) and plotted using Prism 6 (GraphPad). Error bars
represent s.e.m. for three independent experiments. Statistical significance was
calculated with the unpaired Student’s t-test: **P≤0.01, ***P≤0.001. The extent
of the SAF–protein reactions was quantified using a fluorescent MIF standard
Competing interests
The authors declare no competing interests.
additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41557-020-0530-4.
Correspondence and requests for materials should be addressed to K.B.S. or J.W.K.
Reprints and permissions information is available at www.nature.com/reprints.
NatURE CHEMiStRy | www.nature.com/naturechemistry

Corresponding author(s):
Last updated by author(s):
Jeffery W. Kelly
Apr 2, 2020
Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency
in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.
Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement
A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly
The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons
A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient)
AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated
Our web collection on statistics for biologists contains articles on many of the points above.
Software and code
Policy information about availability of computer code
Xcalibur Data Acquisition and Interpretation Software (Thermo Fisher Scientific) was used to collect MS1 and MS2 spectra from the Q
Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. Image Lab (version 6.0) was used to visualize fluorescence or coomassie
stained electrophoresis gels.
Data collection
Tandem mass spectra were extracted into MS1 and MS2 files from raw files using RawExtract 1.9.9. Protein identification and
quantification analysis were conducted with Integrated Proteomics Pipeline (IP2 ver. 6.5.5, Integrated Proteomics Applications, Inc. San
Diego, CA. (http://www.integratedproteomics.com) using ProLuCID/Sequest, DTASelect (version 2), and Census. Enrichment ratios and
statistics were conducted using Microsoft Excel (version 16), and heatmap was generated using RStudio (version 3.2.4). Image Lab
(version 6.0) was used to quantify band/lane fluorescence intensities of electrophoresis gels. Plots were generated using Microsoft Excel
(version 16) and Prism 6 (Graphpad)
Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers.
We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
Data
Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:
- Accession codes, unique identifiers, or web links for publicly available datasets
- A list of figures that have associated raw data
- A description of any restrictions on data availability
All data generated and analyzed during this study are included in this published article (and its supplementary information)
1

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf
Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample sizes were chosen to guarantee the reproducibility of the techniques used in the study. The techniques have low variability, therefore
2 - 3 replicates were considered sufficient.
Sample size
No data was excluded from this study.
Data exclusions
Replication
Quantitative proteomics experiments (e.g. MS/MS) were conducted as duplicates of each treatment condition (e.g. with SAF X or DMSO).
Reactions between recombinant proteins and SAF compounds were conducted three times in all cases. All attempts at replication were
successful.
Formal randomization was not relevant for this study. The techniques used to identify the targets of each compound are inherently unbiased
towards a specific protein(s) or family of proteins.
Randomization
Blinding
Formal blinding was not relevant for this study. The techniques used to identify the targets of each compound are inherently unbiased
towards a specific protein(s) or family of proteins.
Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,
system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Materials & experimental systems
n/a Involved in the study
Antibodies
Methods
n/a Involved in the study
ChIP-seq
Eukaryotic cell lines
Flow cytometry
Palaeontology
MRI-based neuroimaging
Animals and other organisms
Human research participants
Clinical data
Antibodies
Antibodies used
Antibodies for PARP1 (Cell Signaling, 9542), PAR (Trevigen, 4336-BPC-100), and GAPDH (Cell Signaling, 2118) were detected with
an anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074).
For the PARP1 antibody (Cell Signaling, 9542), the supplier (Cell Signaling) validated it via western blot under three different
conditions, including PARP1 knock-out conditions. Use of this antibody has been cited 1988 times, according to CiteAb.
Validation
For the GAPDH antibody (Cell Signaling, 2118), the supplier (Cell Signaling) validated it using western blot, immunohistochemical
staining, immunofluorescent staining, and flow cytometry. Use of this antibody has been cited 2988 times, according to CiteAb.
For the PAR antibody (Trevigen, 4336-BPC-100), the supplier (Trevigen) recommended applications are for Western and dot
blotting. Use of this antibody has been cited at least 15 times, according to the supplier. According to CiteAb, its use has been
cited 3 times.
Eukaryotic cell lines
Policy information about cell lines
Both HEK293T cells (ATCC® CRL-3216™, Lot# 62296864) and HeLa cells (ATCC® CCL-2™, Lot# 58930571) were purchased
from ATCC.
Cell line source(s)
2

None of the cell lines used in this study were authenticated by the authors
Cell lines were not tested for mycoplasma contamination
No commonly misidentified cell lines were used.
Authentication
Mycoplasma contamination
Commonly misidentified lines
(See ICLAC register)
3