Anticancer Pyrroloquinazoline LBL1 Targets Nuclear Lamins

Article abstract of DOI:10.1021/acschembio.8b00266

Target identification of bioactive compounds is critical for understanding their mechanism of action. We previously discovered a novel pyrroloquinazoline compound LBL1 with significant anticancer activity. However, its molecular targets remain to be estab

Full text of DOI:10.1021/acschembio.8b00266

Subscriber access provided by - Access paid by the | UCSB Libraries
Anticancer pyrroloquinazoline LBL1 targets nuclear lamins
Bingbing X Li, Jingjin Chen, Bo Chao, Larry L. David, and Xiangshu Xiao
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00266 • Publication Date (Web): 12 Apr 2018
Downloaded from http://pubs.acs.org on April 14, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 32
ACS Chemical Biology
1
2
3
4
5
1
Anticancer pyrroloquinazoline LBL1 targets nuclear lamins
2
6
7
8
9
3
Bingbing X. Li,#,* Jingjin Chen,# Bo Chao,# Larry L. David,& Xiangshu Xiao#,*
10
11
12
4
5
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
8
#Program in Chemical Biology, Department of Physiology and Pharmacology, &Department of
Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam
Jackson Park Rd, Portland, Oregon 97239, USA
9
10
11
12
13
14
15
16
*Correspondence and requests for materials should be addressed to one of the following coꢀ
corresponding authors:
B.X.L (lib@ohsu.edu) or X.X. (xiaoxi@ohsu.edu)
1
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 32
1
2
3
4
17
TOC graphics
5
6
7
8
9
NH2
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
O
N
N
H
N
N
colocalization
LBL1-P
CF3
LA
18
19
20
2
ACS Paragon Plus Environment

Page 3 of 32
ACS Chemical Biology
1
2
3
4
5
21
ABSTRACT
22
Target identification of bioactive compounds is critical for understanding their mechanism of
action. We previously discovered a novel pyrroloquinazoline compound LBL1 with significant
anticancer activity. However, its molecular targets remain to be established. Herein, we
developed a clickable photoaffinity probe based on LBL1. By extensive chemical, biochemical
and cellular studies with this probe and LBL1, we found that LBL1 targets nuclear lamins,
which are type V intermediate filament (IF) proteins. Further studies showed that LBL1 binds to
the coiledꢀcoil domain of lamin A. These results revealed that IF proteins can also be targeted
with appropriate small molecules besides two other cytoskeletal proteins actin filaments and
microtubules, providing a novel avenue to investigate lamin biology and a novel strategy to
develop distinct anticancer therapies.
6
7
8
9
23
24
25
26
27
28
29
30
31
32
33
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 32
1
2
3
4
5
6
7
8
9
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
INTRODUCTION
Identification of small molecule ligands to various biological macromolecules is critical
for drug discovery to provide hit compounds or chemical probes for target biology discovery.
Various sources including natural products and screening compound collections can contribute to
the identification of initial hits.1ꢀ2 Despite the rapid advances in recombinant technology and
genomic sciences, phenotypic screening still plays a pivotal role in hit discovery in today’s drug
discovery activities especially in disease areas with complex etiologies.3 In fact, an analysis of
FDAꢀapproved firstꢀinꢀclass drugs between 1999 and 2008 showed that while only 34% of the
drugs were originated from targetꢀbased strategies, phenotypic screens contributed to up to 56%
of the approved drugs.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Critical to a phenotypic screen is the availability of highꢀquality library compounds.
Along this line, derivatives from privileged scaffolds, initially coined by Evans,4 are fruitful
grounds for novel bioactive compounds and many such focused libraries have been prepared.5ꢀ6
We previously developed a unique suite of chemical methods to prepare a focused library of
privileged acylpyrroloquinazolines.7 From this library of unique compounds, we discovered
LBL1 with significant anticancer activities (Figures 1A and S1).7 While we excluded a few
potential targets,7 LBL1’s direct molecular target(s) remain unknown. In this article, we
employed an unbiased chemoproteomics strategy8 with a clickable photocrosslinking probe
LBL1-P to identify LBL1’s direct binding targets. Using this analysis as well as additional
biochemical and cellular supporting studies, we show that LBL1 targets nuclear lamins, which
are type V intermediate filament (IF) proteins and have not been successfully targeted by small
molecules.9
4
ACS Paragon Plus Environment

Page 5 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
56
57
Figure 1. Overview of a target identification approach using LBL1-P. (A) Chemical structures
of LBL1 and LBL1-P. (B) Schematic diagram of the chemoproteomics approach using a
clickable photocrosslinking probe LBL1-P.
58
59
60
61
62
63
64
65
66
67
68
69
RESULTS AND DISCUSSION
We envisioned that LBL1-P would covalently crosslink with its direct binding target(s)
in living cells upon UVꢀirradiation of LBL1-Pꢀtreated cells (Figure 1B). Then the covalently
crosslinked target(s) could be clicked8 with an appropriatelyꢀtagged azide for further downstream
applications (Figure 1B). The photocrosslinking probe LBL1-P was designed after investigating
the preliminary structureꢀactivity relationships of LBL1 in inhibiting breast cancer cell growth in
MDAꢀMBꢀ468 and MDAꢀMBꢀ231 cells (Table 1). Placing a clickable propargyl group at N-7 of
LBL1 resulted in compound 1, which showed only slightly decreased activity compared to
LBL1. We considered both benzophenone (2) and trifluoromethyldiazirine (3) as potential
5
ACS Paragon Plus Environment

ACS Chemical Biology
Page 6 of 32
1
2
3
4
5
6
7
8
9
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
photoaffinity labels. It was determined that 3 had more potent activity than 2 (Table 1). Based on
these results, we designed LBL1-P (Figure 1A). LBL1-P was synthesized as shown in Scheme 1
starting from previously made parent compound 4.7 Propargylation at N-7 of 4 with NaH and
propargyl bromide generated 5, whose N-1 amine was temporarily converted into a –OH group.
Amide formation of between 6 and NHS ester 8 provided 9. Final regeneration of N-1 amine7
was achieved by BOP/DBU followed by a SNAr reaction with NH3 to yield desired compound
LBL1-P. The synthetic details for compounds 1ꢀ3 are provided in the Supporting Information.
Gratifyingly, LBL1-P retained growth inhibitory activities in both of the cell lines albeit with a
slightly reduced potency compared to LBL1 (Table 1). It is interesting to note that LBL1-P was
slightly more potent than compound 3 in MDAꢀMBꢀ231 cells, but the opposite was true in
MDAꢀMBꢀ468 cells. The reason for this difference remains to be determined.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
ACS Paragon Plus Environment

Page 7 of 32
ACS Chemical Biology
1
2
3
4
5
93
94
6
7
8
9
95
96
Table 1. Growth inhibitory activities of LBL1 and its analogs in breast cancer cell lines.a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
GI50 (ꢀM)
compd
R1
R2
ꢀH
MDAꢀMBꢀ468
MDAꢀMBꢀ231
0.44 ± 0.14
1.60 ± 0.51
LBL1
1
2
1.19 ± 0.18
6.99 ± 2.98
3.27 ± 0.38
ꢀH
ꢀH
14.13 ± 4.99
0.97 ± 0.11
3.71 ± 0.07
9.24 ± 1.90
6.71 ± 1.21
3
LBL1-P
97
98
a The growth inhibitory activities of the compounds are presented as the mean GI50 ± SD
of at least two independent experiments in duplicates. The GI50 is the concentration needed to
inhibit the cell growth by 50% after incubating the cells with the compounds for 72 h.
99
100
7
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
101
102
Scheme 1. Synthesis of LBL1-P.
103
104
105
106
107
108
109
110
111
112
113
To investigate if LBL1-P could label its target(s), MDAꢀMBꢀ468 cells were treated with
LBL1-P in the absence or presence of LBL1 as a competitor. After UV (365 nm) irradiation, the
cell lysates were clicked with a biotinꢀN3.10 The crude cell lysates were then separated on a SDSꢀ
PAGE gel and the biotinylated proteins were directly detected by horseradish peroxidase (HRP)ꢀ
conjugated streptavidin blotting. The results in Figure 2A showed that three prominent bands
migrated between 60 and 75 kDa were specifically labeled by LBL1-P. Importantly, this
labeling was reduced with excess LBL1. Labeling of these three bands was fairly specific and no
other major bands were labeled even at 10 ꢀM of LBL1-P, which is well above its GI50 (Figure
2A and Table 1).
8
ACS Paragon Plus Environment

Page 9 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
114
36
37
115
38
Figure 2. Identification of lamins as the targets of LBL1. (A) LBL1ꢀP labeling in MDAꢀMBꢀ
468 cells. The cells were treated with LBL1-P with or without LBL1. Then the cells were UVꢀ
irradiated followed by cell lysis and click reaction with a biotinꢀN3. The lysates were then
separated on a SDSꢀPAGE gel for streptavidin blotting. (B) Mass spectrometry identification of
lamins as the targets of LBL1. The spectra counts of identified proteins in the presence and in
the absence of LBL1 were plotted on the xꢀ and yꢀaxis, respectively. (C) LBL1-Pꢀlabeled
proteins were localized in the nucleus. The cells were treated as in (A) except that the cells were
fractionated into cytosolic and nuclear fractions. Then the individual fractions were clicked with
39
116
117
118
119
120
121
122
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
ACS Paragon Plus Environment

ACS Chemical Biology
Page 10 of 32
1
2
3
4
5
6
7
8
9
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
a rhodamineꢀN3 for inꢀgel fluorescence imaging. The same gel was also used for western blotting
with indicated antibodies. C, cytosol; N, nucleus.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To identify the proteins that were labeled by LBL1-P, we enriched the photocrosslinked
and biotinꢀN3 clicked cell lysates with streptavidin beads for mass spectrometryꢀbased protein
identification. The relative abundance of the enriched proteins was quantified by labelꢀfree
spectra counting. This analysis revealed nuclear lamin A (LA), lamin B1 (LB1), lamin B2 (LB2)
and lamin C (LC) as the putative targets of LBL1 (Figure 2B and Table S1). Lamins are type V
intermediate filament (IF) proteins and are the major component of nuclear lamina, a meshwork
of proteins that lie underneath the inner nuclear membrane.11ꢀ12 In mammals, there are three
lamin genes (LA, LB1 and LB2) encoding four major proteins (LA, LB1, LB2 and LC). LA and
LC are alternative splicing products of LA gene.13ꢀ14 All the lamin proteins are highly
homologous in sequence and predicted structure and share the same domain organization. They
contain a long central, αꢀhelical coiledꢀcoil rod domain 15, which is flanked by a nonꢀαꢀhelical
Nꢀterminal head and a Cꢀterminal immunoglobulin (Ig)ꢀlike tail domain.14 Lamins are the major
scaffold proteins to maintain the mechanical stability of the nucleus and have been considered as
undruggable proteins. Therefore, our identification of lamins as the potential targets of LBL1
was unanticipated.
To determine the localization of the labeled proteins by LBL1-P, we separated the
cytosolic fraction from the nuclear fraction from the cells treated with LBL1-P. Each fraction
was individually clicked with a rhodamineꢀN3 for inꢀgel fluorescence imaging.10 The labeled
signals were found to be exclusively localized in the nuclear fraction (Figure 2C) and could be
10
ACS Paragon Plus Environment

Page 11 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
146
competed by LBL1, which is consistent with the finding that lamins are the targets of LBL1.
The purity of cytosolic and nuclear fractions was confirmed by the presence and absence of
cytosolic GAPDH and nuclear histone 3 (H3) in individual fractions through western blotting
(Figure 2C).
147
148
149
150
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
151
152
Figure 3. Validation of lamins as the targets of LBL1. (A) LBL1 bound to nuclear lamins. The
photocrosslinked and biotinꢀN3 clicked cell lysates were pulled down with streptavidin beads.
The precipitated proteins were separated on SDSꢀPAGE and probed with indicated antibodies.
(B) LBL1-P bound to LA and LB1 with higher efficiency than LB2. HEK293T cells were
transfected with indicated FLAGꢀtagged plasmid. Then the cells were treated as in (A) except
that antiꢀFLAG was used. SE, short exposure; LE, long exposure. IB: immunoblot. (C) LBL1
bound to LA in living cells. The cells were treated with LBL1-P with or without LBL1 followed
by UV irradiation and inꢀcell click reaction with a rhodamineꢀN3. The cells were then analyzed
by fluorescence microscopy after antiꢀLA staining. (D) Shown are the deconvoluted Zꢀstacks of
153
154
155
156
157
158
159
160
11
ACS Paragon Plus Environment

ACS Chemical Biology
Page 12 of 32
1
2
3
4
5
6
7
8
9
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
images of the cells treated with LBL1-P alone as shown in (C). “Colocalization” was presented
as a new channel to specifically label the colocalization between LBL1-P and LA.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We used western blot as an independent method to validate that lamins were truly and
specifically pulled down by LBL1-P. Therefore, the photocrosslinked and biotinꢀN3ꢀclicked cell
lysates were pulled down with streptavidin beads and probed with antiꢀLA/C, LB1 and LB2. The
data in Figure 3A showed that LA/C and LB1 were pulled down and again the efficiency was
decreased when LBL1 was included as a competitor. The labeling efficiency was doseꢀ
dependently inhibited by LBL1 (Figure S2). LB2 was not detected in the pulldown (Figure 3A).
To distinguish if lack of LB2 in the pulldown was due to lower binding affinity or inferior
antibody performance, we ectopically expressed FLAGꢀtagged LA, LB1 and LB2 in HEK293T
cells and performed the same photocrosslinking and streptavidin pulldown as shown in Figure
3A. Then the proteins being pulled down were detected by antiꢀFLAG antibody. It was found
that all three of them could be pulled down and detected by antiꢀFLAG antibody (Figure 3B) yet
LB2 clearly presented significantly lower labeling efficiency in comparison to LA and LB1,
suggesting that LBL1-P binds less avidly with LB2. Upon inspection of the sequence alignment
among LA, LB1 and LB2, we found that the Nꢀterminal sequence of LB2 is longer than that of
LA or LB1 (Figure S3), suggesting that this portion of sequence might be important for binding.
Lamins are abundant nuclear proteins. To exclude that LBL1-P simply pulled down abundant
cellular proteins, we investigated other highly abundant cellular proteins including Hsp90 and
GAPDH from the enrichment and we did not detect either of these (Figure 3A). Others have
attempted to inventorize the nonspecific proteins that could be enriched using clickable
12
ACS Paragon Plus Environment

Page 13 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
184
photoaffinity probes and indeed lamins were not found to be present in the background inventory
using a variety of photoreactive groups.16 These results suggest that LBL1 binds to lamins. This
is the first small molecule ligand known to bind lamins and thus the name of LBL1 for laminꢀ
binding ligand 1.
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To further confirm that LBL1 could target LA in cells, we investigated the localization of
LBL1-P in MDAꢀMBꢀ231 cells by inꢀcell click reaction with a rhodamineꢀN3 after
photocrosslinking. The cells were also stained with antiꢀLA. When the cells were treated with
LBL1-P only, a strong rhodamine signal was observed and this signal colocalized with LA
(Figure 3C, top panel). To quantitatively evaluate the colocalization between LBL1-P and LA,
we analyzed deconvoluted Zꢀstacks of cell images using Imaris® Coloc module and the
colocalization coefficient was found to be nearly perfect at 0.92 ± 0.022 (Figure 3D). Similar to
the SDSꢀPAGE analysis (Figures 2A and 3A), the signal labeled by LBL1-P was significantly
decreased in the presence of excess LBL1 (Figure 3C, middle panel), further supporting that
LBL1 targets LA in living cells. The specificity of the click reaction was confirmed by the lack
of rhodamine signal from the cells that were not treated with LBL1-P (Figure 3C, bottom panel).
Fullꢀlength LA is very challenging to be biochemically characterized due to its tendency
to aggregate in physiological buffers.17 To facilitate the biochemical characterization of the
binding interaction between LBL1 and LA, we employed photocrosslinking followed by click
reaction with biotinꢀN3 and streptavidin pulldown to identify the minimal fragment of LA to
mediate the binding interaction. We generated a series of FLAGꢀtagged LA fragments that
encompass different domains and expressed them in HEK293T cells. The transfected cells were
then treated and processed as Figure 3B for antiꢀFLAG blotting. As expected, the fullꢀlength LA
could be labeled by LBL1-P (Figures 4A and S4A). Surprisingly, we found that LA(1ꢀ427)
13
ACS Paragon Plus Environment

ACS Chemical Biology
Page 14 of 32
1
2
3
4
5
6
7
8
9
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
consisting of the coiledꢀcoil domain maintained robust labeling by LBL1-P while the Igꢀlike
domain LA(428ꢀ664) did not (Figures 4A and S3A), although the Igꢀlike domain mediates
interactions with many other proteins.18ꢀ19 LA(1ꢀ427) contains two coiledꢀcoils. We further
divided LA(1ꢀ427) into coil 1 [LA(1ꢀ218)] and coil 2 [LA(219ꢀ427)] to identify which coil might
mediate the interaction with LBL1. However, neither coil 1 nor coil 2 alone was sufficient to
crosslink with LBL1-P (Figures 4AꢀB), indicating that both coils are required for binding with
LBL1. Then a series of shorter fragments containing both coils were prepared. While LA(1ꢀ387)
mediated robust labeling, no labeling of LA(1ꢀ355) with LBL1-P was observed (Figures 4AꢀB),
suggesting the Cꢀterminal end of the nonꢀhelical coil is critical for binding. At the Nꢀterminal
end, a series of deletions of 10aa [i.e. LA(10ꢀ387), LA(20ꢀ387) and LA(30ꢀ387)] also presented
deleterious effect on binding (Figures 4A and S4B). Minimal labeling of LA(30ꢀ387) was
observed while no labeling was detected with LA(78ꢀ387). These results showed that the
minimal fragment required for binding with LBL1 was LA(1ꢀ387) consisting of the nonꢀhelical
head domain (36aa), both coil 1 and 2, and ~30aa Cꢀterminal to the end of the predicted coil 2
(Figure 4A). Together, these results suggest that there likely exists intermolecular interaction
between Nꢀ and Cꢀterminal ends in LA(1ꢀ387) fragment to maintain a functional assembly
required for optimal binding with LBL1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14
ACS Paragon Plus Environment

Page 15 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
224
20
21
22
23
24
225
Figure 4. LBL1 binds to LA(1ꢀ387). (A) The domain organization of LA and different
truncation constructs prepared. The LBL1-P labeling efficiency of these constructs is indicated
on the right. (B) Representative blots to show that LBL1-P labeled LA(1ꢀ387). HEK293T cells
were transfected with indicated FLAGꢀtagged LA fragments. Then the cells were treated as in
Figure 3B. (C-D) The indicated proteins were incubated with different concentrations of LBL1.
Then trypsin was added to initiate partial proteolysis. The residual proteins were analyzed by
SDSꢀPAGE followed by coomassie blue staining. (E-F) LBL1 binds to LA(1ꢀ387) as assessed
by TSA. LA(1ꢀ387) was incubated with different concentrations of LBL1. Then Sypro Orange
was added and the fluorescence intensity of the samples was measured during gradual heating.
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To assess the biochemical binding interaction between LBL1 and LA(1ꢀ387), we purified
LA(1ꢀ387) to homogeneity (Figure S5). As a negative control, LA(78ꢀ387) was also purified. We
first employed limited proteolysis assay20 of purified LA(1ꢀ387) by trypsin to assess its binding
with LBL1. Binding of a small molecule to a protein will protect the protein from partial
proteolysis.20 Incubation of increasing concentrations of LBL1 with LA(1ꢀ387) doseꢀ
15
ACS Paragon Plus Environment

ACS Chemical Biology
Page 16 of 32
1
2
3
4
5
6
7
8
9
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
dependently protected it from limited proteolysis by trypsin (Figure 4C), indicating direct
binding interaction between LBL1 and LA(1ꢀ387). As a negative control, partial proteolysis of
LA(78ꢀ387) was not affected by LBL1 (Figure 4D), indicating no direct binding with LA(78ꢀ
387). We further exploited a thermal shift assay (TSA)21 to determine the binding between LBL1
and LA(1ꢀ387). Ligands binding to a protein will increase its apparent melting point (Tm).21ꢀ23 As
shown in Figure 4E, LA(1ꢀ387) showed two apparent thermal transitions in the absence of LBL1
(Tm1 ~40 oC, Tm2 ~65 oC), likely reflecting unfolding of two different assembly states. Upon
titrating LBL1 into LA(1ꢀ387), Tm2 was unchanged while Tm1 was doseꢀdependently shifted to
higher Tm (Figure 4F) with an apparent Kd = 5.11 ꢀM, which is in line with the results from the
limited proteolysis assay (Figure 4C). Under the same conditions, no change in the Tm of
glutathione Sꢀtransferase (GST) was observed (Figure S6). Collectively, these results illustrate
that LBL1 binds to LA(1ꢀ387) both in vitro and in living cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
The current study details the identification of the direct molecular targets of anticancer
agent LBL1 with the assist of an unbiased chemoproteomics strategy. Unexpectedly, we found
that LBL1 targets nuclear lamins. The mammalian cytoskeleton is comprised of three protein
classes, i.e. actin filaments or microfilaments (~8 nm in diameter), microtubules (~25 nm in
diameter) and intermediate filaments (~10 nm in diameter).24 Although the former two classes
have been successfully targeted to develop cancer therapies and/or unique cell biology tools,25
small molecules for IF proteins have not been developed.9 Our present results demonstrate that
IF proteins can also be targeted with small molecules such as LBL1. While LBL1’s interaction
with LB1 remains to be characterized, its interaction with LA is mediated by the coiledꢀcoil
16
ACS Paragon Plus Environment

Page 17 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
264
domain. Given the known roles of LA in genome stability and DNA doubleꢀstrand break (DSB)
repair,26ꢀ27 we found that cells treated with LBL1 resulted in generation of DSBs as evidenced by
phosphorylation of a histone variant H2AX (referred to as γꢀH2AX)28 and induction of cellular
apoptosis (Figure S7). The unique targeting of LBL1 shall provide us ample opportunities to
further dissect its mechanism of action and also provide us an excellent chemical tool for
elucidating lamin biology and generating potential anticancer therapies. Further studies are
warranted and will be reported in due course.
265
266
267
268
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
MATERIALS AND METHODS
Chemistry. General. Glass Contout solvent purification system was used to purify all the
anhydrous solvents to be used for reactions. Melting points were determined in capillary tubes
1
13
using MelꢀTemp and are uncorrected. All H and C NMR spectra were obtained in a Bruker
Avance 400 MHz spectrometer using CDCl3 or DMSOꢀd6 as the solvent and the chemical shifts
of the residual CHCl3 (δ 7.24) or DMSO (δ 2.50) were taken as reference. Chemical shifts (δ) are
reported in parts per million (ppm), and the signals are described as brs (broad singlet), d
(doublet), dd (doublet of doublet), td (triplet of doublet), m (multiplet), q (quartet), s (singlet) and
t (triplet). Coupling constants (J values) are given in Hz. Silica gel flash chromatography was
performed using 230–400 mesh silica gel (EMD). All reactions were monitored using thinꢀlayer
chromatography (TLC) on silica gel plates (EMD). Yields were of purified compounds. The
mass spectra of the small molecules were obtained from a Thermo Electron LTQꢀOrbitrap
Discovery highꢀresolution mass spectrometer (Thermo Scientific) or Advion PlateExpress with
electrospray operated either in positive or negative mode. All the final compounds for biological
17
ACS Paragon Plus Environment

ACS Chemical Biology
Page 18 of 32
1
2
3
4
5
6
7
8
9
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
testing were confirmed to be >95% purity based on reverse phase HPLC (Waters, Milford, MA)
analysis using an XBridge C18 column (4.6 × 150 mm) and detected at 254 nm. The mobile
phases for HPLC are water and acetonitrile, both of which contained 0.1% formic acid.
7-(Prop-2-yn-1-yl)-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (5). To a stirred solution of 47
(400 mg, 2.0 mmol) in dry DMF (24 mL) was added NaH (83 mg, 2.2 mmol). The resulting
reaction mixture was stirred at room temperature for 1 h. Then propargyl bromide (169 µl, 2.2
mmol) was added. The reaction mixture was stirred at room temperature for another 2 h. The
solvent was removed under reduced pressure and the residue was purified by column
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
chromatography on silica gel, eluting with 15:1 DCM:MeOH containing 1% DIPEA to give the
desired compound 5 (404 mg, 85%) as a pale yellow solid: mp 224ꢀ226. 1H NMR (400 MHz,
DMSOꢀd6) δ 7.78 (d, J = 8.4 Hz, 1 H), 7.49 (d, J = 3.2 Hz, 1 H), 7.09 (d, J = 9.2 Hz, 1 H), 7.07
(d, J = 3.2 Hz, 1 H), 6.68 (brs, 2 H), 5.70 (s, 2 H), 5.17 (d, J = 2.4 Hz, 2 H), 3.44 (t, J = 2.4 Hz, 1
H); 13C NMR (100 MHz, DMSOꢀd6) δ 162.0, 159.3, 150.3, 129.9, 127.8, 121.0, 119.5, 117.3,
102.6, 101.9, 79.3, 75.8, 35.4; HRESIMS, calcd 238.10872, found 238.10915.
3-Amino-7-(prop-2-yn-1-yl)-7H-pyrrolo[3,2-f]quinazolin-1-ol (6). A mixture of 5 (194 mg,
0.82 mmol) in 3 N HCl solution (5 mL) was heated under reflux for 3 h. The white solid was
collected by filtration and further treated with 1 N NaOH (5 mL) for 30 min at room temperature.
The solid was collected by filtration and further purified by column chromatography on silica
gel, eluting with 10:1 DCM:MeOH containing 1% DIPEA to give the desired compound 6 (145
mg, 80%) as a white solid: mp >230 oC (dec). 1H NMR (400 MHz, DMSOꢀd6) δ 7.84 (d, J = 8.8
Hz, 1 H), 7.51 (d, J = 3.2 Hz, 1 H), 7.13 (d, J = 3.2 Hz, 1 H), 7.10 (d, J = 8.8 Hz, 1 H), 6.57 (brs,
13
2 H), 5.16 (d, J = 2.4 Hz, 2 H), 3.44 (t, J = 2.4 Hz, 1 H); C NMR (100 MHz, DMSOꢀd6) δ
18
ACS Paragon Plus Environment

Page 19 of 32
ACS Chemical Biology
1
2
3
4
5
309
163.1, 151.2, 143.5, 131.3, 129.9, 124.8, 117.7, 115.7, 107.9, 102.6, 79.2, 75.9, 35.4; HRESIMS,
calcd 239.09274, found 239.09330.
310
6
7
8
9
311
312
313
314
315
316
2,5-Dioxopyrrolidin-1-yl-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate (8). NHS (32.5
mg, 0.28 mmol) and EDCI.HCl (54.1 mg, 0.28 mmol) were added to a stirred solution of 7 (50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
mg, 0.22 mmol) in dry DMF (1 mL) at 0 C. The resulting mixture was warmed up to room
temperature and stirred for overnight. Then H2O (10 mL) was added to the reaction mixture. The
white solid was collected by filtration to give compound 8 (66 mg, 93%): mp 88ꢀ90 oC. 1H NMR
(400 MHz, CDCl3) δ 8.18ꢀ8.15 (m, 2 H), 7.32 (d, J = 8.4 Hz, 2 H), 2.92 (s, 4 H); 13C NMR (100
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
MHz, CDCl3) δ 169.0, 160.9, 135.8, 130.8, 126.7, 126.1, 121.6 (q, J = 273 Hz), 28.3 (q, J = 41
Hz), 25.6.
N-(1-Hydroxy-7-(prop-2-yn-1-yl)-7H-pyrrolo[3,2-f]quinazolin-3-yl)-4-(3-(trifluoromethyl)-
3H-diazirin-3-yl)benzamide (9). A mixture of 6 (145 mg, 0.61 mmol) and 8 (260 mg, 0.80
o
mmol) in dry DMF (4 mL) was stirred at 90 C for 4 h. Then the solvent was removed under
reduced pressure and the residue was purified by column chromatography on silica gel, eluting
with DCM to 20:1 DCM:THF containing 1% DIPEA to give compound 9 as a yellow solid (160
o
1
mg, 58%): mp >300 C. H NMR (400 MHz, DMSOꢀd6) δ 13.0ꢀ11.9 (m, 2 H), 8.22 (d, J = 7.2
Hz, 2 H), 8.03 (d, J = 9.2 Hz, 1 H), 7.66 (d, J = 2.8 Hz, 1 H), 7.44ꢀ7.40 (m, 3 H), 7.25 (d, J = 2.4
13
Hz, 1 H), 5.24 (d, J = 2.0, 2 H), 3.48 (s, 1 H); C NMR (100 MHz, DMSOꢀd6) δ 160.4, 132.4,
131.3, 130.9, 129.4, 126.3, 124.6, 121.8 (q, J = 274 Hz), 118.2, 102.8, 79.0, 76.1, 35.5, 28.1 (q, J
= 40 Hz); HRESIMS, calcd 451.11248, found 451.11291.
N-(1-Amino-7-(prop-2-yn-1-yl)-7H-pyrrolo[3,2-f]quinazolin-3-yl)-4-(3-(trifluoromethyl)-
3H-diazirin-3-yl)benzamide (LBL1-P). To a stirred solution of 9 (135 mg, 0.30 mmol) in dry
DMF (5 mL) was added BOP (199 mg, 0.45 mmol) and DBU (90 µl, 0.60 mmol). The resulting
19
ACS Paragon Plus Environment

ACS Chemical Biology
Page 20 of 32
1
2
3
4
5
6
7
8
9
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
reaction mixture was stirred for 7 h at room temperature. Then NH3 (7 N in MeOH, 1.7 mL, 12
mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solvent
was removed under reduced pressure and the residue was purified by column chromatography on
silica gel, eluting with 8:1 DCM:THF containing 1% TEA to give the desired compound LBL1-
P (13 mg, 10% yield) as a gray solid: mp >130 oC (dec). 1H NMR (400 MHz, DMSOꢀd6) δ 10.7
(s, 1 H), 8.08 (d, J = 8.0 Hz, 2 H), 8.04 (d, J = 8.4 Hz, 1 H), 7.67 (d, J = 2.4 Hz, 1 H), 7.40ꢀ7.26
(m, 6 H), 5.28 (d, J = 2.0, 2 H), 3.49 (t, J = 2.4 Hz, 1 H); 13C NMR (100 MHz, DMSOꢀd6) δ
164.5, 162.2, 152.6, 148.5, 136.5, 131.7, 130.6, 128.9, 128.8, 126.4, 121.8 (q, J = 273 Hz),
120.6, 120.4, 118.0, 105.5, 102.6, 79.1, 76.1, 35.6, 28.1 (q, J = 40 Hz); HRESIMS, calcd
450.12847, found 450.12875.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Plasmids. The LA, LB1 and LB2 expression plasmids were obtained from DNASU repository
and subcloned into pEGFPꢀN3 (Clontech). All the plasmids were sequenceꢀverified by Sanger
sequencing. The mammalian expression plasmids encoding different domains of LA were
subcloned into pEGFPꢀN3 (Clontech). The final constructs did not contain the original EGFP
sequence. The LA fragments for bacterial expression were subcloned into pETꢀ15b (Novagen).
The primers used for subcloning are available upon request.
Cell lines and culture. MDAꢀMBꢀ231 and MDAꢀMBꢀ468 were purchased from Developmental
Therapeutics Program at the National Cancer Institute. HEK 293T cells were purchased from
American Tissue Culture Collection (ATCC). The cells were tested for mycoplasma
contamination regularly by PCR. Cells were cultured in highꢀglucose Dulbecco’s Modified
Eagle’s Medium (DMEM, Life Technologies) supplemented with 10% FBS (Hyclone) and 10%
20
ACS Paragon Plus Environment

Page 21 of 32
ACS Chemical Biology
1
2
3
4
5
354
nonꢀessential amino acids (Life Technologies) at 37 °C with 5% CO2. All the cells were used
within 50 passages.
355
6
7
8
9
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
Cell growth inhibition assay. The cellular growth inhibition assay was carried out as described
before.29 The growth inhibition of different cell types was assessed by MTT assay using MTT
reagent (Sigma, St. Louis, MO). Briefly, the cells were plated into 96ꢀwell plates and the cells
were allowed to attach to the bottom of the plates for overnight. Then the cells were treated with
different concentrations of different drugs for 72 h. The media were removed and MTT reagent
in complete tissue culture media (0.5 mg/mL) was added to each well and incubated at 37 oC for
3 h. The incubation media were removed and 100 ꢀL of DMSO was added to each well. The
absorbance of the formed purple formazan solution was read at 570 nm using Packard Fusion
plate reader. The percent of growth is defined as 100% × (AtreatedꢀAinitial)/(AcontrolꢀAinitial), where
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Atreated represents absorbance in wells treated with a compound, Ainitial represents the absorbance
at time 0 and Acontrol denotes mediaꢀtreated cells. The GI50 was derived from nonꢀlinear
regression analysis of the percent of growthꢀconcentration curve in Prism 5.0.
Photocrosslinking and streptavidin enrichment. MDAꢀMBꢀ468 cells at ~80% confluency
were washed once with PBS and then the cells were covered with PBS. The cells were treated
with or without LBL1 for 10 min followed by treatment with LBL1-P for 30 min at 37 oC. At
the end of incubation, the cells were cooled to 4 oC and UVꢀirradiated at 365 nm for 5 min. Then
the cells were collected by scraping and pelleted by centrifugation at 1000x rpm for 5 min. The
cell pellets were washed once with iceꢀcold PBS. The washed cell pellets were lysed in 1%
SDS/PBS with brief sonication followed by centrifugation at room temperature at 14,000x rpm
for 10 min. The protein concentration of the supernatant was determined by BCA Protein Assay
Kit (Pierce). The crude lysates were adjusted with PBS to give a final concentration of ~1.0
21
ACS Paragon Plus Environment

ACS Chemical Biology
Page 22 of 32
1
2
3
4
377
mg/mL for click reaction with biotinꢀazide using the following condition: azide (50 ꢀM), TCEP
5
6
7
8
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
(1 mM), TBTA (100 ꢀM), CuSO4 (1 mM). The mixture was incubated with shaking at room
temperature for 1.0 h in the dark. For direct SDSꢀPAGE analysis, the reaction mixture was
separated by SDSꢀPAGE followed by blotting with HRPꢀstreptavidin (GEꢀHealthcare, 1:5000).
For streptavidin enrichment and protein identification by mass spectrometry, the reaction mixture
was further processed as follows. The reaction mixture was mixed with equal volume of MeOH
and 0.25 volume of CHCl3. The mixture was vigorously vortexed for 30 s and centrifuged at
8000x g for 10 min. The supernatant was removed and the protein solid was washed three times
with MeOH: CHCl3 (1:1). Then MeOH was added to the washed solid and briefly sonicated to
resuspend the solid. CHCl3 was added to a final mixture of MeOH:CHCl3 (4:1). The mixture was
vigorously vortexed for 30 s and centrifuged at 14000x rpm for 10 min to pellet the solid. The
resulting protein solid was redissolved in PBS with 8 M urea and 2% SDS with brief sonication.
The solution was centrifuged at 14000x rpm for 10 min to remove any insoluble material. The
proteins in the supernatant was reduced with DTT (10 mM) at 55 oC for 30 min followed by
alkylation with iodoacetamide (25 mM) for 30 min at room temperature in the dark. The solution
was diluted 10x with PBS and mixed with PBSꢀwashed streptavidinꢀagarose beads. The mixture
was tumbled at room temperature for 2 h. Then the beads were pelleted by centrifugation at 700x
g for 2 min. The beads were extensively washed 3 times in 1% SDS/PBS followed by 6 times in
PBS. The bound proteins were then eluted with 1%SDS/PBS containing 1 mM free biotin with
heating at 95 oC for 5 min. The supernatant was collected and the beads were further washed
with 2 x 200 ꢀL 100 mM NH4HCO3. The washes and the supernatant were combined and
lyophilized.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Protein LC-MS/MS. Affinity purified proteins from above were dissolved in 20 ꢀL of SDSꢀ
22
ACS Paragon Plus Environment

Page 23 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
400
PAGE sample buffer, briefly electrophoresed into a SDSꢀPAGE gel. The top of the gel was
excised and all proteins in each sample were subjected to an inꢀgel digestion with trypsin as
previously described30. The tryptic digests were then separated by capillary reverse phase
chromatography during a 110 min dataꢀdependent tandem mass spectrometric analysis as
previously described 30, except using a LTQ Velos Pro linear ion trap mass spectrometer. Data
collection used the dynamic exclusion feature of the instrument’s control software (repeat count
equal to 1, exclusion list size of 100, exclusion duration of 30 sec, and exclusion mass width of ꢀ
1 to +4 ) to obtain MS/MS spectra of the 5 most abundant parent ions following each survey scan
from m/z 350ꢀ2000. The tune file was configured with a 325°C capillary temperature, no
averaging of microscans, a maximum inject time of 200 msec and AGC target of 3 x 104 in MS
mode and maximum ion time of 100 msec and AGC target of 1 x 104 in MS2 mode. Sequest
(Thermo Scientific) was used to search MS2 spectra against a May 2013 version of the Sprot
human FASTA protein database, with concatenated sequenceꢀreversed entries to estimate error
thresholds. Results were then processed using Python scripts to map peptides to their parent
proteins and calculate peptide and protein false discovery as previously described31. A minimum
of 2 unique peptide sequence matches were required for the identification of proteins and the
resulting peptide and protein false discovery rates were approximately 0.4% and 1.5%,
respectively.
401
402
403
12
404
13
14
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Transient transfections. HEK293T cells in a well of a 6ꢀwell plate were transfected overnight
with 1 ꢀg of indicated FLAGꢀtagged plasmid using Lipofectamine2000 (Life Technologies)
following the manufacturer’s instructions. Then the cells were processed as above for
photocrosslinking and clicking with biotinꢀN3 and streptavidin pulldown, but with the following
exceptions: 1) no reduction or alkylation step was included; 2) the eluted proteins were analyzed
23
ACS Paragon Plus Environment

ACS Chemical Biology
Page 24 of 32
1
2
3
4
5
6
7
8
9
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
by Western blot using antiꢀFLAG antibody.
Western blot. For western blot analysis, the following primary antibodies were used: antiꢀLA/C
(mouse, Sigma, cat # SAB4200236, 1:2000), antiꢀFLAG M2 (mouse, Sigma, cat # F4049,
1:4000), antiꢀHsp90 (rabbit, Cell Signaling Technology, cat # 4874, 1:1000), antiꢀLB1 (rabbit,
Cell Signaling Technology, cat # 12586, 1:1000), antiꢀGAPDH (mouse, Santa Cruz
Biotechnology, cat # scꢀ32233, 1:4000), antiꢀphosphoꢀhistone H2AX (rabbit, Cell Signaling
Technology, cat # 9718, 1:1200), antiꢀLB2 (rabbit, Antibody Verify, cat # AAS57463C, 1:200),
antiꢀHistone 3 (H3) (mouse, Cell Signaling Technology, cat#14269, 1:1000). The HRPꢀ
conjugated secondary antibodies were from Cell Signaling Technology or Jackson
ImmunoResearch Laboratories.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Protein Purification. GST protein was purified as described previously.32 LA(1ꢀ387) and
LA(78ꢀ387) were expressed and purified from BL21(DE3)pLysS cells. The E. coli culture was
grown in Luria broth supplemented with ampicillin (50 ꢀg/mL) at 37 oC to an OD600 of ~0.6,
then IPTG (0.4 mM) was added and the cells were incubated for another 3 h at 37 oC. Then the
cells were lysed and the proteins were purified using HisTrap HP columns (GE Healthcare Life
Sciences). The binding buffer was buffer A (20 mM TrisHCl, 20% sucrose, pH 7.50)
supplemented with 6 M urea. The elution buffer was buffer A supplemented with 6 M urea and
10ꢀ300 mM imidazole. The purified proteins were snap frozen in liquid nitrogen and stored at ꢀ
80 oC. To prepare the proteins for binding experiments, the proteins were dialyzed into HBS (20
mM HEPES, 250 mM NaCl, pH 8.0) at 4 oC.
Limited proteolysis. The purified proteins (0.1 mg/mL) in HBS were incubated with different
concentrations of LBL1 in a PCR reaction plate at 20 oC for 30 min, when sequencingꢀgrade
24
ACS Paragon Plus Environment

Page 25 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
446
trypsin (Sigma) was added to give a ratio of trypsin/protein = 1/500. Proteolysis was allowed for
20 min at 20 oC. The reaction was quenched by adding SDSꢀPAGE buffer and the samples were
analyzed by SDSꢀPAGE followed by Coomassie blue staining.
447
448
449
450
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Thermal shift assay (TSA). Different concentrations of LBL1 were incubated with LA(1ꢀ387)
(0.3 mg/mL) or GST (2 ꢀM) for 30ꢀ60 min at room temperature. Then 5x Sypro Orange (Life
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
Technologies) was added. Twenty ꢀL/well was aliquoted into a qPCR plate (Life Technologies).
Then the TSA assay was performed in QuantStudio 7 Flex (Life Technologies) at a ramping rate
of 0.05 oC/s. The Tm was calculated from the peak of the first derivative of the fluorescence
intensityꢀtemperature plot.
Subcellular fractionation. For subcellular localization of the crosslinked proteins, the UVꢀ
irradiated cells were lysed in a hypotonic buffer (25 mM TrisHCl, 5 mM KCl, 0.5 mM DTT, pH
7.40) supplemented with protease inhibitor cocktail (Pierce) and 1 mM PMSF for 10 min on ice.
Then the cells were homogenized with a dounce homogenizer (15 strokes). The supernatant was
separated by centrifuging at 600x g for 10 min at 4 oC. The supernatant was further centrifuged
at 14000x rpm for 20 min at 4 oC and the supernatant was collected as cytosolic fraction. The
nuclear pellets obtained from initial 600x g centrifugation were washed 5 times with an isotonic
buffer (10 mM TrisHCl, 6 mM MgCl2, 0.5% Triton Xꢀ100, 10% glycerol, pH 7.40). The washed
nuclear pellets were then lysed in 1%SDS/PBS with brief sonication followed by centrifugation
at 14000x rpm for 10 min at room temperature. The supernatant was collected as nuclear
fractions. The protein concentrations of both cytosolic and nuclear fractions were determined by
BCA Protein Assay Kit. Equal amount of proteins were then clicked with rhodamineꢀN3 using
the same click reaction condition as above. The reaction mixture was then separated on a SDSꢀ
PAGE for inꢀgel fluorescence gel scanning with ChemiDocꢀMP (BioꢀRad).
25
ACS Paragon Plus Environment

ACS Chemical Biology
Page 26 of 32
1
2
3
4
5
6
7
8
9
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
In-cell click reaction. MDAꢀMBꢀ231 cells growing on the cover slips were washed once with
PBS and then the cells were covered with PBS. The cells were treated with DMSO or LBL1 for
20 min followed by LBL1-P for 20 min. After UVꢀirradiated for 5 min, the cells were fixed with
3.7% formaldehyde and permeabilized with 0.5% triton Xꢀ100. After the cells were washed with
3% BSA in PBS, the cells were incubated with ClickꢀiT® Cell reaction cocktail (Life
Technologies) supplemented with rhodamineꢀN3 for 30 min at room temperature. Then the cells
were washed with 3% BSA in PBS before being incubated with antiꢀLA (mouse, Sigma, cat#
SAB4200236, 1:1000) overnight. The cells were then incubated with Alexa Fluor 488 donkey
antiꢀmouse secondary antibody for one hour at room temperature and the coverslips were
mounted as above. The coverslips were then mounted in ProLong Gold AntiFade Reagent with
DAPI (Life Technologies) and the images were acquired with a fluorescence microscope
ApoTome (Zeiss). For colocalization analysis, the Zꢀstack of images were reconstructed and
colocalization Pearson correlation coefficients were determined using the Coloc module in the
Imaris software package (Bitplane).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Apoptosis analysis. MDAꢀMBꢀ231 cells were treated with indicated concentrations of LBL1 for
5 days. Then the cells were collected by trypsinization and washed twice with iceꢀcold PBS. The
cells were resuspended in 1x binding buffer (BD Pharmingen, Annexin VꢀFITC Apoptosis
Detection Kit I) and stained with Annexin VꢀFITC and PI at room temperature according to the
manufacturer’s protocol. The stained cells were analyzed by BD FACSCANTO II.
Supporting Information available. Synthesis of compounds 1-3, Table S1 and Figures S1ꢀS6
are available in the online version.
26
ACS Paragon Plus Environment

Page 27 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
491
ACKNOWLEDGEMENTS. We thank S. Kaech and A. Snyder for technical help on
fluorescence microscopy image acquisition and analysis. This work was supported by NIH
R01CA211866 (XX) and partially by R01GM122820 (XX). Oregon Health & Science
University School of Medicine, Office of the Technology Transfer and Business Development,
Oregon Clinical and Translational Research Institute, Medical Research Foundation of Oregon
and Lloyd Fund partially supported this research. Mass spectrometry was partially supported by
NIH grants P30EY010575 (LD), P30CA069533 (LD) and S10RR025571 (LD).
492
493
494
495
496
497
498
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27
ACS Paragon Plus Environment

ACS Chemical Biology
Page 28 of 32
1
2
3
4
5
6
7
8
9
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
REFERENCES:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(1) Neamati, N.; Barchi, J. J., Jr., New paradigms in drug design and discovery. Curr. Top. Med.
Chem. 2002, 2, 211ꢀ227.
(2) Shelat, A. A.; Guy, R. K., Scaffold composition and biological relevance of screening
libraries. Nat. Chem. Biol. 2007, 3, 442ꢀ446.
(3) Swinney, D. C.; Anthony, J., How were new medicines discovered? Nat. Rev. Drug Discov.
2011, 10, 507ꢀ519.
(4) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger, R. M.; Whitter, W. L.;
Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen,
T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J., Methods for drug discovery:
development of potent, selective, orally effective cholecystokinin antagonists. J. Med. Chem.
1988, 31, 2235ꢀ2246.
(5) Welsch, M. E.; Snyder, S. A.; Stockwell, B. R., Privileged scaffolds for library design and
drug discovery. Curr. Opin. Chem. Biol. 2010, 14, 347ꢀ361.
(6) Chao, B.; Li, B. X.; Xiao, X., The chemistry and pharmacology of privileged
pyrroloquinazolines. MedChemComm 2015, 6, 510ꢀ520.
(7) Chen, J.; Kassenbrock, A.; Li, B. X.; Xiao, X., Discovery of a Potent Antiꢀtumor Agent
through Regioselective Monoꢀꢀacylation of 7ꢀPyrrolo[3,2ꢀ]quinazolineꢀ1,3ꢀdiamine.
Medchemcomm 2013, 4, 1275ꢀ1282.
(8) Lee, J.; Bogyo, M., Target deconvolution techniques in modern phenotypic profiling. Curr.
Opin. Chem. Biol. 2013, 17, 118ꢀ126.
28
ACS Paragon Plus Environment

Page 29 of 32
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
522
(9) Lee, S. J.; Jung, Y. S.; Yoon, M. H.; Kang, S. M.; Oh, A. Y.; Lee, J. H.; Jun, S. Y.; Woo, T.
G.; Chun, H. Y.; Kim, S. K.; Chung, K. J.; Lee, H. Y.; Lee, K.; Jin, G.; Na, M. K.; Ha, N. C.;
Barcena, C.; Freije, J. M.; LopezꢀOtin, C.; Song, G. Y.; Park, B. J., Interruption of progerinꢀ
lamin A/C binding ameliorates HutchinsonꢀGilford progeria syndrome phenotype. J. Clin. Invest.
2016, 126, 3879ꢀ3893(This recent study identified compounds targeting a mutant lamin A).
(10) Speers, A. E.; Cravatt, B. F., Profiling enzyme activities in vivo using click chemistry
methods. Chem. Biol. 2004, 11, 535ꢀ546.
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(11) Burke, B.; Stewart, C. L., The nuclear lamins: flexibility in function. Nat. Rev. Mol. Cell
Biol. 2013, 14, 13ꢀ24.
(12) Aebi, U.; Cohn, J.; Buhle, L.; Gerace, L., The nuclear lamina is a meshwork of
intermediateꢀtype filaments. Nature 1986, 323, 560ꢀ564.
(13) Lin, F.; Worman, H. J., Structural organization of the human gene encoding nuclear lamin A
and nuclear lamin C. J. Biol. Chem. 1993, 268, 16321ꢀ16326.
(14) Dechat, T.; Adam, S. A.; Taimen, P.; Shimi, T.; Goldman, R. D., Nuclear lamins. Cold
Spring Harb. Perspect. Biol. 2010, 2, a000547.
(15) Strelkov, S. V.; Schumacher, J.; Burkhard, P.; Aebi, U.; Herrmann, H., Crystal structure of
the human lamin A coil 2B dimer: implications for the headꢀtoꢀtail association of nuclear lamins.
J. Mol. Biol. 2004, 343, 1067ꢀ1080.
(16) Kleiner, P.; Heydenreuter, W.; Stahl, M.; Korotkov, V. S.; Sieber, S. A., A Whole Proteome
Inventory of Background Photocrosslinker Binding. Angew. Chem. Int. Ed. Engl. 2017, 56,
1396ꢀ1401.
29
ACS Paragon Plus Environment