Structure-based design, docking and binding free energy calculations of a366 derivatives as spindlin1 inhibitors

Article abstract of DOI:10.3390/ijms22115910

The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure–activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.

Full text of DOI:10.3390/ijms22115910

International Journal of
Molecular Sciences
Article
Structure-Based Design, Docking and Binding Free Energy
Calculations of A366 Derivatives as Spindlin1 Inhibitors
Chiara Luise 1, Dina Robaa 1, Pierre Regenass 2, David Maurer 2 , Dmytro Ostrovskyi 2, Ludwig Seifert 3,
Johannes Bacher 3, Teresa Burgahn 3, Tobias Wagner 3, Johannes Seitz 3, Holger Greschik 4, Kwang-Su Park 5
,
Yan Xiong 5, Jian Jin 5 , Roland Schüle 4, Bernhard Breit 2, Manfred Jung 3,6 and Wolfgang Sippl 1,
*
1
Institute of Pharmacy, Martin Luther University of Halle-Wittenberg, Kurt-Mothes-Str. 3,
06120 Halle (Saale), Germany; luisechi@hotmail.it (C.L.); dina.robaa@pharmazie.uni-halle.de (D.R.)
Institute for Organic Chemistry, University of Freiburg, Albertstr. 21, 79104 Freiburg, Germany;
pm.regenass@gmail.com (P.R.); DM.Maurer@web.de (D.M.); dmytroostrovskyi@googlemail.com (D.O.);
bernhard.breit@chemie.uni-freiburg.de (B.B.)
Institute of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany;
ludwig.seifert@pharmazie.uni-freiburg.de (L.S.); johannes.bacher@pharmazie.uni-freiburg.de (J.B.);
teresa.burgahn@web.de (T.B.); dr.wagner.tobias@gmail.com (T.W.);
2
3
johannes.seifert@pharmazie.uni-freiburg.de (J.S.); manfred.jung@pharmazie.uni-freiburg.de (M.J.)
Department of Urology and Center for Clinical Research, University Freiburg Medical Center,
Breisacherstr. 66, 79106 Freiburg, Germany; holger.greschik@uniklinik-freiburg.de (H.G.);
roland.schuele@uniklinik-freiburg.de (R.S.)
Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological
Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA;
kwang-su.park@mssm.edu (K.-S.P.); yan.xiong@mssm.edu (Y.X.); jian.jin@mssm.edu (J.J.)
CIBSS—Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany
Correspondence: wolfgang.sippl@pharmazie.uni-halle.de
4
5
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
6
Citation: Luise, C.; Robaa, D.;
Regenass, P.; Maurer, D.;
*
Ostrovskyi, D.; Seifert, L.; Bacher, J.;
Burgahn, T.; Wagner, T.; Seitz, J.; et al.
Structure-Based Design, Docking and
Binding Free Energy Calculations of
A366 Derivatives as Spindlin1
Inhibitors. Int. J. Mol. Sci. 2021, 22,
5910. https://doi.org/10.3390/ijms
22115910
Abstract: The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation,
through which it has been linked to several types of malignant tumors. In the current work, we report
on the development of novel analogs of the previously published lead inhibitor A366. In an effort to
improve the activity and explore the structure–activity relationship (SAR), a series of 21 derivatives
was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking
studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the
structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light
on the important interactions. Our study highlighted the main structural features that are required
for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded
into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the
amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184.
Different protocols were tested to identify a fast in silico method that could help to discriminate
between active and inactive compounds within the A366 series. Rescoring the docking poses with
MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor,
the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the
selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds,
among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a
and GLP. Finally, future design hypotheses were suggested based on our findings.
Academic Editor: Giulio Poli
Received: 5 May 2021
Accepted: 26 May 2021
Published: 31 May 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: Spindlin1; structure–activity relationship (SAR); docking; molecular dynamics (MD) simula-
tions; MM-GBSA
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Spindlin1 is a chromatin reader protein that plays an important role in epigenetic
regulation through the recognition and interpretation of histone modifications. It con-
sists of three Tudor domains, with the second domain binding to the H3K4me3 (H3
Int. J. Mol. Sci. 2021, 22, 5910. https://doi.org/10.3390/ijms22115910
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 5910
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trimethylated at lysine 4) mark [
1
–3
]. More recently, its interaction with H4K20me3 (H4
trimethylated at lysine 20) was also described [4,5]. Since the binding of the latter histone
mark displayed a weaker affinity than that of H3K4me3, it has been hypothesized that the
H4K20me3 mark acts as a secondary binding partner for Spindlin1 [5]. Interestingly, the
additional binding of the first domain with asymmetrically dimethylated arginine residues
(Rme2a) has been shown to have different effects on the histone peptide affinity. Indeed,
the H3K4me3R8me2a methylation pattern increases the affinity of H3K4me3, whereas
H4K20me3R23me2a shows a lower binding affinity than H4K20me3 [3,5]. Very recently,
another bivalent methylation pattern has been reported, namely H3K4me3K9me3/2, which
has been described to enhance the histone binding affinity of H3K4me3 [6].
H3K4 is one of the most studied histone modifications, and it is well known to be
tightly associated with the promoters of active genes that lead to transcriptional acti-
vation [
the methylation states; H4K20me1 is associated with transcriptional activation, while
H4K20me3 is linked with repression of transcription when present at promoters [ ]. How-
7]. In contrast, H4K20 has been reported to have different functions based on
8
ever, further studies are needed to investigate the biological relevance of the H4K20me3
mark and its functions correlated with Spindlin1 interactions.
Spindlin1 has been linked to several types of malignant tumors including ovarian
cancer, breast cancer and triple negative breast cancer, non-small-cell lung cancers, liposar-
coma, and very recently, liver cancer [9–14]. Moreover, it was found to be associated with
drug-resistant breast cancer, as a study established that Spindlin1, which is negatively
regulated by the miR-148/152 family, enhances Adriamycin resistance [15]. The literature
also reports that Spindlin1 may play a role in tumorigenesis, and it was additionally sug-
gested that Spindlin1 is a proto-oncogene [16
functions have been related to diverse signaling pathways, such as Wnt (
TCF-4) [ 21], RET [13], PI3K/Akt [10], and uL18-MDM2-p53 [19] pathways and SREBP1c-
–20]. Some of the above-mentioned Spindlin1
β-catenin and
3
,
triggered FASN signaling [14]. In addition, Spindlin1 was found to control skeletal muscle
development in mice and to play an important role in the first meiotic division of mam-
malian oocytes, thus providing potential links of Spindlin1 to human skeletal muscle
diseases and human infertility, respectively [22,23]. Therefore, Spindlin1 inhibitors might
be useful chemical tools to further investigate the physiological and pathological roles of
this relatively new target.
To date, 15 crystal structures of Spindlin1 have been deposited in the RCSB Protein
Data Bank (PDB) [24], both in apo form [25] and in complex with histone peptides [
2,3,5],
bivalent inhibitors, and small molecule inhibitors [26 28]. The crystal structure analysis and
–
mutagenesis studies of some residues present in the first and second Tudor domains clearly
indicated, as mentioned above, that the second domain is responsible for the recognition of
the trimethylated lysine [2,3,5,13]. Specifically, four residues (Phe141, Trp151, Tyr170, and
Tyr177) form the so-called aromatic cage in which the trimethylated lysine is embedded
and stabilized by cation–pi interactions and hydrophobic van der Waals contacts.
In recent years, there has been growing interest in Spindlin1, which has led to the
identification of several inhibitors blocking the Spindlin–H3K4me3 interaction. Through in
silico studies coupled with in vitro testing, we identified novel Spindlin1 inhibitors active
in the low µM range; as an example, compound Robaa-1k is illustrated in Figure 1 [29].
Using a screening platform, diverse compounds were identified, leading to the discovery
of A366, a previously described G9a inhibitor (IC50: 3.3 nM, [30]), as a nanomolar Spindlin1
inhibitor (IC50: 182.6 nM, [31]) (Figure 1). Recently, applying the same approach to the
screening of an epigenetic compound library, another hit was discovered, which was
then simplified to a fragment-like inhibitor (MS31, Figure 1) [28]. Two different studies
have identified bivalent inhibitors of Spindlin1 that simultaneously interact with its Tudor
domain I and II (EML631 and VinSpinIn, Figure 1) [26,27].

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Figure 1. Structures of reported Spindlin1 inhibitors.
Herein we report on the development of novel analogs of the A366 inhibitor. In an
attempt to improve the activity of A366 and explore the structure–activity relationship
(SAR), a congeneric series containing 21 A366 derivatives was synthesized and tested
in vitro for their inhibitory activity against Spindlin1. This was accompanied by various
computational approaches in order to investigate the possible binding mode of the syn-
thesized derivatives, to explore the impact of the structural variations on the Spindlin1
inhibitory activity, and to identify a fast in silico method that could help to discriminate
between active and inactive compounds within the A366 series. Additionally, to verify the
selectivity of the new analogs, the most active compounds were also tested in vitro against
G9a and GLP (G9a like protein).
2. Results and Discussion
In order to explore the SAR of A366 and to develop modified analogs, a congeneric
series was synthesized and tested. This led to the generation of a dataset consisting of
21 compounds, 17 of which showed IC50 values in the range of 0.15–11.8 µM, two of
which were found to be weakly active (Table 1, 1a and 1b), and three of which displayed
a considerably decreased activity and were, thus, considered inactive (Table 1, 1c, 1d, 1e).
Apart from A366 [31], 1f, and 1g [27], the other compounds are reported here for the
first time as novel Spindlin1 inhibitors. The attained dataset was subsequently used for
computational studies aimed to investigate the structural differences responsible for the
activity of the compounds. Docking studies, molecular dynamics (MD) simulations, and
molecular mechanics/generalized born surface area (MM-GBSA) rescoring calculations
were performed using the Spindlin1 second domain as a binding site (PDB ID: 6I8Y).

Int. J. Mol. Sci. 2021, 22, 5910
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Table 1. Chemical structures and inhibitory activity of the A366 derivatives.
R4
R2
R4
R1O
R3O
NH2
N
Spin1
G9a
GLP
Compounds
R1
R2
R3
R4
IC50 ± SD (nM) [a]
182.6 ± 9.1 [31]
IC50 (nM) [b]
IC50 (nM) [b]
N
38 [c]
A366
1a
Me
Et
H
OEt
H
51
N
70% @ 25 µM [d]
81% @ 100 µM [d]
N
O
1b
Me
42% @ 50 µM [d]
Inactive
N
1c
1d
1e
Me
Me
Me
H
H
H
>20,000
>20,000
N
N
Inactive
N
1f
1g
1h
1i
Me
Et
H
H
169 ± 13
316 ± 26
431
243
334
N
2969
N
Me
Me
CN
Cl
3900 ± 700
1600 ± 200
N
N
N
N
>20,000
>20,000
1j
1k
1l
Me
Me
Et
OMe
Ac
H
2500 ± 200
11,800 ± 210
567 ± 35
N
1m
1n
2-Pr
Me
H
3300 ± 470
255 ± 35
N
H
779
527
N
1o
1p
Me
Me
H
H
1210 ± 90
157 ± 12
>20,000
759
>20,000
470
N
(R): 470 ± 27
(S): 478 ± 34
(rac): 584 ± 31
N
HO
1q
1r
Me
Me
H
H
(rac): 2321
3191
(rac): 4432
3185
N
408 ± 35
360 ± 20
467 ± 15
N
1s
1t
Me
Me
H
H
>20,000
>20,000
>20,000
>20,000
N

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Table 1. Cont.
R4
R2
R4
R1O
R3O
NH2
N
Spin1
G9a
GLP
Compounds
R1
R2
R3
R4
IC50 ± SD (nM) [a]
IC50 (nM) [b]
IC50 (nM) [b]
N
1u
Me
H
1250 ± 50
>20,000
>20,000
HO
[a]
[b]
Fluorescence polarization assay, based on competition to a Fluorescein-H3K4me3 peptide.
Fluorescence based coupled enzyme assay,
[c]
[d]
based on SAM-SAH conversion. For details, see experimental part. Reported IC50 G9a for A366: 3.3 nM [30].
If no IC50 was obtained,
inhibition is reported in percentage at specified concentration.
2.1. Analysis of Spindlin1-A366 Complex
The binding mode of A366 in the Spindlin1 second domain was first analyzed
(Figure 2a,c) (PDB ID: 6I8Y [27]). This crystal structure shows a different shape of the
binding pocket compared to the histones-bound crystal structures (e.g., PDB ID: 4H75 [2]).
Indeed, A366 induces a flip of Phe141 in the aromatic cage; this conformational change of
the phenylalanine side chain consequently opens the pocket and changes its shape and
size. The protonated pyrrolidine moiety is embedded in the aromatic cage and undergoes
cation–pi interactions with the surrounding amino acids. Notably, also the side chain of
Trp151 is slightly adapted to better interact with A366. The amidine group is involved in
salt bridge interactions with Asp184. The ligand binding mode is further stabilized by
the formation of an intramolecular hydrogen bond between the methoxy group and the
positively charged pyrrolidine-NH group. For comprehension and comparison, the crystal
structure of Spindlin1 bound to the H3K4me3 histone peptide with a focus on the second
domain is shown in Figure 2b,d (PDB ID: 4H75).
2.2. Docking Studies
Initially, our attention was focused on understanding the role of the spacer length
(R3 group), since biological assays revealed that changing the linker from a propyl chain
to ethyl/butyl chains results in a drop in activity (Table 1, compounds 1c, 1d, 1e). The
compounds were hence docked into the crystal structure of Spindlin1 (PDB ID: 6I8Y) using
Glide in the standard precision (SP) mode [32]. It is noteworthy that the used docking
setup could successfully reproduce the experimentally determined binding mode and the
interactions of A366 with an RMSD of 0.54 Å (Supplementary Materials, Figure S1).
The obtained docking poses and docking scores (DS) of the inactive compounds were
analyzed and compared to A366. Interestingly, the docking scores showed no substantial
differences between the active and inactive compounds (DS range of inactive ligands:
−
11.16/
−
11.28 kcal/mol; DS of A366
:
−
11.37 kcal/mol—Table S1). Docking of the inactive
derivative 1e (Figure 3a), which bears a shorter ethoxy spacer as compared with A366
,
resulted in the same binding mode and showed the same interactions as observed for
A366 (Figure 2c). On the other hand, the inactive derivative bearing a butoxy spacer,
compound 1c, displayed a slightly different binding mode in which the amidine moiety
is tilted towards Asp189 and establishes a salt bridge with it (Figure 3b). Nevertheless,
the pyrrolidine ring of 1c is still properly located in the aromatic cage where it undergoes
cation–pi interactions.
The main difference detected in the binding modes of all three inactive compounds
(1c, 1d, and 1e) lies in the absence of the intramolecular hydrogen bond observed in the
binding mode of A366. There is reason to believe that the formation of an intramolecular
hydrogen bond plays an important role in stabilizing the active conformation.

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Figure 2. Crystal structures of Spindlin1 in complex with (a,c) A366 (PDB ID: 6I8Y) and (b,d) H3K4me3 (PDB ID: 4H75).
(a,b) show the protein surfaces of the three Tudor domains, which are colored in cyan (domain I), white (domain II), and
green (domain III). A366 bound to the first Spindlin1 domain is represented as orange sticks. (c,d) focus on domain II and
the binding interactions of A366 (yellow sticks) and H3K4me3 histone peptide (pink sticks). Only the side chains of the
surrounding residues are shown for clarity, and they are displayed as sticks; for residues Gly93, Asp95, and Glu142, the
main chains are shown. Phe141 is also depicted by the surface mesh. Hydrogen bonds are represented as dashed yellow
lines, cation–pi interactions as dashed green lines, pi–pi stacking interactions as dashed cyan lines, and salt bridges as
dashed magenta lines.
2.3. Molecular Dynamic Simulations
Given the poor understanding of the compounds’ inactivity by docking scores and
predicted binding mode, the next step was the investigation of these ligands by means
of MD simulations. The obtained docking poses of A366 and two inactive compounds
(1e and 1c) were subjected to 200 ns MD simulations using Amber18 package [33] to gain
insight into the stability of the predicted binding modes and protein–ligand interactions.

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Figure 3. Docking poses of 1e (a) and 1c (b) in the Spindlin1 second domain (PDB ID: 6I8Y). Docking poses and side chains
of the surrounding residues are shown as sticks. Binding interactions are represented with dashed lines colored in green
(cation–pi), cyan (pi–pi stacking), and magenta (salt bridge).
The analysis of the MD simulation performed for A366 indicated a stable binding
mode, where the interactions observed in the crystal structure (Figure 2c) are preserved
throughout the simulation. It is noteworthy that the intramolecular hydrogen bond be-
tween the methoxy group and the pyrrolidine-N+H was also maintained throughout the
MD simulation. Meanwhile, for both inactive derivatives, 1c and 1e, this intramolecular
hydrogen bond was barely observed during the MD simulation. Figure 4a shows the
root-mean-square deviation (RMSD) plot, while the occupancy rates of the most important
interactions are reported in the heat map (Figure 5).
Compound 1c, despite its inactivity, was found to be relatively stable throughout the
MD simulation in terms of RMSD values, albeit less stable than A366 (Figure 4b). Although
the obtained docking pose of 1c shows an interaction between the amidine moiety of
the ligand and Asp189 (Figure 3b), this interaction is lost during the MD simulation
(occupancy 2%). Indeed, directly at the beginning of the MD simulation, the aminoindole
moiety moves so that its amidine group establishes a salt-bridge with the side chain
of Asp184, which is maintained for ca. 94% of the simulation time. Apart from the
aminoindole group, small fluctuations of the ligand are observed, which mainly affect
the pyrrolidine moiety and, consequently, the formation of cation–pi interactions. This
was highlighted by the decreased occupancy of these interactions during the simulation
(Figure 5). In fact, with the exception of the cation–pi interactions established with Trp151,
the occupancies of the other aromatic interactions are dramatically decreased, pointing out
the important role of these interactions for the activity.
On the other hand, the instability of the binding mode obtained for 1e could give a
plausible explanation for its inactivity (Figure 4c). Indeed, diverse binding conformations
were detected during the MD simulation (Supplementary Materials, Figure S2), all of which
seem to be unstable, as demonstrated also by the low occupancy rates of the observed
interactions, as detailed in Figure 5. The salt bridge between the amidine group and
Asp184 (occupancy rate 31%) is completely lost after 70 ns simulation time (Supplementary
Materials, Figure S3). Moreover, the analysis of the cation–pi interactions underlined that
even though the positively charged pyrrolidine moiety is located in the aromatic cage, its
position is not optimal for establishing aromatic interactions with all amino acids that form

Int. J. Mol. Sci. 2021, 22, 5910
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the cage. This can be seen in Figure 5, specifically, in the low occupancy rates obtained for
the interactions with Phe141 and Tyr177 (54.1% and 55.5%, respectively).
Figure 4. Analysis of 200 ns MD simulations of reference ligand A366 and inactive compounds 1c and 1e. Root mean square
deviation (RMSD) plots of the systems with (a) A366, (b) 1c, and (c) 1e.
In conclusion, based on the MD simulations analysis of these three compounds, we
could confirm the assumption that the spacer length is important for stabilizing the binding
pose and optimizing the binding interactions. The finding shows that the linker chain can
affect not only the presence of intramolecular hydrogen bond, but also the optimal location
of the pyrrolidine ring in the pocket and generally the binding conformation, which in
turn influences the formation of cation–pi interactions with the aromatic cage as well as
the interactions of the amidine moiety with Asp184.
2.4. Re-Scoring Using Prime MM-GBSA
Although the analysis of the MD simulations was able to rationalize the difference in
the compounds’ activity, this approach is time-consuming for a bigger dataset. Thus, the
synthesis of the new derivatives was coupled with docking and MM-GBSA rescoring to
find a fast approach that could help in discriminating active from inactive compounds of
the same congeneric series. The structural differences between the active and inactive com-
pounds lie in the linker chain, where the actives bear a propyl chain, whereas the inactive
compounds bear either an ethyl or a butyl chain (Table 1, compounds 1c, 1d, and 1e). Only
ligands with determined IC50 values were used as actives.

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Figure 5. Summary of the binding interactions identified during 200 ns MD simulations of the reference ligand A366 and
the inactive compounds 1c and 1e. The binding poses were obtained from docking into Spindlin1 second domain binding
site (PDB ID: 6I8Y). (a) Heat map showing the occupancy rates of the observed interactions. (b) Common structure of the
compounds highlighting the atoms involved in the interactions.
First, since from the above-described docking studies and MD simulations analy-
sis the key role of the intramolecular hydrogen bond was evident, its contribution was
incorporated into the docking scores. This was done by allowing the option “reward in-
tramolecular hydrogen bonds” in Glide [32]; in this way, the compounds that exhibit these
intramolecular interactions pay a smaller entropic penalty upon binding, which affects the
final docking scores (DS_Intra-H-bonds). For the analysis of the results, box-plots were
generated showing the distribution of docking scores without the “reward intramolecular
hydrogen bonds” option (DS) and DS_Intra-H-bonds attained for our active and inactive
ligands. As illustrated in Figure 6a, the DS values retrieved for active and inactive com-
pounds are distributed in the same range, which clearly demonstrates that this scoring
function is not able to discriminate between active and inactive compounds. On the other
hand, a small improvement is observed when DS_Intra-H-bonds values are evaluated
(Figure 6b). However, the difference between active and inactive ligands is still too small to
be considered reliable, as highlighted by the mean values ( 12.0 kcal/mol for the actives
−
and −11.2 kcal/mol for the inactive compounds).
Figure 6. Box-plots showing docking scores distribution obtained for active and inactive compounds using glide standard
precision (a) without intramolecular hydrogen bonds contribution and (b) rewarding intramolecular hydrogen bonds.
Owing to the low discriminatory power of the glide docking scores (DS and DS_Intra-
H-bonds), the docking poses were post-processed using more accurate binding free energy
(BFE) calculations with Prime MM-GBSA. Diverse settings were tested; here, we report the
results obtained when only the ligands were relaxed, and when also the protein residues
within 3 Å of the ligands were treated as flexible. The computed binding affinity values

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were visualized by box-plots, seen in Figure 7. Interestingly, the re-scoring methods
were able to successfully discriminate between active and inactive compounds. The
calculated BFE values retrieved for the active compounds were more favorable than those
of the inactive compounds, and good discrimination ranges were found, as emphasized
by the significant gaps between the mean values (MM-GBSA
−76.3 kcal/mol (actives) and 60.35 kcal/mol (inactives); MM-GBSA
values of −76.3 kcal/mol (actives) and −62.6 kcal/mol (inactives)).
∆
Gbind mean values of
−
∆
Gbind–3 Å mean
Figure 7. Box-plots showing ∆Gbind distribution obtained for active and inactive compounds using Prime MM-GBSA.
(a) Default setting, only ligands are relaxed (b) also protein residues around 3 Å from the ligands are treated as flexible.
The two re-scoring methods performed equally well from an efficiency perspective,
but differently for the computation time. The CPU time required for the calculation of
one inhibitor was 0.6 min when only the ligand was treated as flexible, but 1.7 min when
the protein residues were included (MM-GBSA-3 Å). Given the promising findings, this
developed protocol can be applied to guide further optimization steps in order to prioritize
compounds for synthesis and biological characterization.
2.5. Structure–Activity Relationships
Once the role of the spacer was clear, we were interested in exploring the effect of
other substitutions on the ligand’s inhibitory activity. The synthesized dataset of A366
derivatives encompassed compounds bearing different spiro and dialkyl substituents at
position-3 of the indole ring (R4), different substituents at the pyrrolidine ring, as well as
varying groups at the R1 and R2 positions of the indole ring.
To investigate the influence of these modifications on the Spindlin1 inhibitory activity,
the fluorescence polarization assay established earlier [31] was used to determine the
potency of the compounds. Furthermore, the most active Spindlin1 inhibitors were tested
in vitro against G9a and GLP to analyze their selectivity profile.
The biological assays revealed that the introduction of a substituent on R2 position
led to a significant decrease in the Spindlin1 inhibitory activity as observed for 1a
1j, and 1k (IC50 values: 1.6–11.8 M) when compared to the parent compound A366 (IC50
182.6 nM). Meanwhile, the replacement of the methoxy group at R1 with ethoxy (1g 1l
, 1h, 1i,
µ
,
)
and isopropoxy (1m) groups showed different impacts on the activity of the compounds.
The ethoxy derivatives showed only a two-fold decrease in the inhibitory activity, as
observed when comparing 1g and 1l with 1f and 1n, respectively. On the other hand, the
isopropoxy derivative 1m displayed a significant decrease in activity with an IC50 value in
the micromolar range.
Modifications of the spiro moiety (R4) had no major impact on the Spindlin1 in-
hibitory activity of the compounds; cyclobutyl, -pentyl, and -hexyl rings as well as the
dimethyl analogues (A366
,
1f
,
1n
,
1p) were all well tolerated. Meanwhile, the 3-ethyl-
3-methyl analogue 1r (IC50 408
±
35 nM) displayed only a two-fold decrease in activ-
ity as compared to A366. However, no selectivity was detected for these compounds.
Among the attempted R4-modifications, the major impact was observed with compound
1o bearing the geminal diethyl group, which exhibited a significantly decreased activity
for Spindlin1 (IC50: 1.21 ± 0.09 µM). Interestingly, this was coupled with an increase in

Int. J. Mol. Sci. 2021, 22, 5910
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selectivity for Spindlin1 over G9a and GLP (G9a IC50 >20
µM, GLP IC50 >20
µM). Note-
worthy, the analogous derivative with dimethyl groups, 1p, which showed an activity
comparable to A366 and was one of the most active Spindlin1 inhibitors of our series
(Spindlin1 IC50: 157 ± 12 nM), showed no selectivity over G9a and GLP (G9a IC50: 759 nM,
GLP IC50: 470 nM). The decrease in the Spindlin1 activity going from geminal dimethyl
(
1p) to 3-ethyl-3-methyl (1r) and ultimately to geminal diethyl (1o) is inversely related to
the number of rotatable bonds and can, thus, be associated with the loss of conformational
entropy of the compounds.
Given the important role of the pyrrolidine moiety in the binding pocket, it was not
surprising that its modifications could impact the activity of the compounds. Notably,
the replacement of the pyrrolidine ring in 1f by a 3-hydroxypyrrolidine moiety (1q) only
led to three-fold decrease in the Spindlin1 activity (IC50 values of 169 nM and 584 nM,
respectively). The docking studies of 1q predicted binding modes in which the hydroxy
group is engaged in a hydrogen bond interaction with Tyr177 (Figure 8a). On the other
hand, the replacement of the pyrrolidine ring by an isoindoline moiety led to compound 1s
which showed strong inhibition of Spindlin1 (IC50: 360 20 nM) combined with selectivity
(G9a IC50 >20 M, GLP IC50 >20 M). Our docking studies in the Spindlin1 second domain
,
±
µ
µ
binding site reveal that the aromatic ring of the isoindoline moiety can further stabilize the
ligand through the formation of additional pi–pi stacking interactions with Phe141 and
Trp151 in the aromatic cage (Figure 8b). To further explore the binding mode of compound
1s, the obtained docking pose was subjected to 100 ns MD simulation. The analysis showed
that the predicted binding mode is stable, and the key interactions (cation–pi, salt bridge,
and intramolecular hydrogen bond) are conserved during the simulation time. However,
the occupancy rates of the additional pi–pi stacking highlighted that such interactions are
less preserved throughout the MD simulation (Figure S4).
Figure 8. Predicted binding modes of some derivatives in the Spindlin1 second domain binding site (PDB ID: 6I8Y).
) Compound 1q (S, green; R, beige). ( ) Compound 1s (S, cyan; R, light pink). Docking poses and side chains of the
(
a
b
surrounding residues are shown as sticks. Binding interactions are represented with dashed lines colored in yellow
(hydrogen bond), green (cation–pi), cyan (pi–pi stacking), and magenta (salt bridge).
A good selectivity profile was also observed for compound 1t, which bears a 3-
methylpyrrolidine moiety and displays a distinct inhibition of Spindlin1 (IC50: 467
±
15 nM)
coupled with selectivity over G9a (G9a IC50 >20 M, GLP IC50 >20 M). In conclusion, the
µ
µ

Int. J. Mol. Sci. 2021, 22, 5910
12 of 19
obtained data underlined that specific substitutions on the pyrrolidine moiety by small
substituents like 3-methyl and 3-hydroxymethyl groups as well as replacement of the py-
rollidine ring by an isoindoline moiety led to a significant drop in the activity for G9a and
GLP whilst maintaining the potent inhibitory activity against Spindlin1. These substitution
patterns can hence be used to design selective compounds.
2.6. Synthesis
All compounds were synthesized from easily accessible starting materials such as gua-
iacol derivatives
first route, bromination of 2 was achieved in a regioselective manner at low temperatures
in order to obtain phenols , which were directly benzylated and conveniently purified
by recrystallization, resulting in excellent yields of intermediates . The organolithium
species resulting from subsequent bromine–lithium exchange were then added to a variety
of ketone derivatives resulting in benzyl alcohols . Mediated by a Lewis acid activation,
benzyl cyanides 10 were obtained by a substitution reaction. After benzylation of their
phenol moiety, the second route was initiated by the reduction of aldehydes with lithium
aluminum hydride. Benzyl alcohols were generally obtained in excellent yields and could
be transformed into benzyl cyanides via two successive substitution steps using first
2 (Scheme 1, Route A) or vanillin derivatives 3 (Scheme 1, Route B). In the
7
8
5
6
7
9
concentrated hydrochloric acid and then potassium cyanide as nucleophile sources. Double
alkylation in benzyl position under basic conditions led to either symmetrically dialkylated
or cycloalkylated benzyl cyanides 10, which were intermediates common to both synthetic
routes. Nitration was then performed at low temperatures and under mild conditions; the
lower yields of the desired regioisomers 11 resulted from poor regioselectivity for certain
derivatives. Deprotection of the phenol moiety was selectively achieved using aluminum
chloride. Typical hydrogenation conditions involving palladium on carbon as the catalyst
partially resulted in a reduction of the nitro group for several derivatives, leading to a
significant loss of desired materials 12. Alkylation of the unprotected phenol moiety was
performed either directly with 1-(3-chloropropyl)pyrrolidine under basic conditions or as a
two-step sequence including first a terminal dihaloalkane with the desired spacer length
and then the N-heterocyclic compound of choice. The final step consisted of reducing
compounds 13 using zinc dust in acetic acid, delivering the 2-aminoindole derivatives 1a-u
resulting from direct ring closure under the chosen conditions.
◦
◦
Scheme 1. Reagents and conditions: (a) Br2 (1.1 eq), DCM, −20 to 0 C, 1 h; (b) K2CO3 (1.2 eq), BnBr (1.2 eq), DMF, 90 C,

Int. J. Mol. Sci. 2021, 22, 5910
13 of 19
◦
2 h, 91–95% over two steps; (
c
) n-BuLi (1.1 eq); ketone (1.1 eq), THF,
−
78 C to rt, 1 h, 44–70%; (
d
) TMSCN (3.0 eq); SnCl4
) conc. HCl (15 eq), Et2O,
◦
0 C to rt, 30 min, 93–98%; (g) KCN (1.1 eq), NaI (0.5 eq), DMF, 80 C, 17 h, 74–79%; (h) KOH (7 eq), 1,n-dibromo-n-alkane
(1◦.0 eq), DCM,
−
78 ◦C to rt, 0.5 h, 39–90%; (
e
) LiAlH4 (1.5 eq), THF, 0 ◦C to rt, 4 h, 94–97%; (
f
(1.0 eq), TBAB (1 mol%), PhMe/H2O, reflux, 1.0 h, 56–63%; or NaH (2.5 eq); iodomethane (4 eq), THF, reflux, 16 h, 90%;
◦
◦
(i) HNO3 (1.5 eq), Ac2O/AcOH, 0 C, 1.0 h, 56–92%; (
j
) AlCl3 (3.0 eq), DCM, 0 C, 1 h, 80%-quant.; (
k
) K2CO3 (1.4 eq), NaI
◦
(0.2 eq), 1-(3-chloropropyl)pyrrolidine (1.2 eq), DMF, 80 C, 1.5 h, 67–92%; (
DMF, 0 to 100 C, 2 h, 2. N-heterocyclic compound (2 to 10 eq), DMF, 70 C, 2–3 h, 30–86% over two steps; (
l
) 1. NaH (1.2 eq); 1,n-dihalo-n-alkane (1.5 eq),
◦
◦
m) Zn (10–15 eq),
◦
HOAc, 85 C, 1 h, 61–99%.
3. Materials and Methods
3.1. Synthesis
3.1.1. General Methods and Materials
Reagents and solvents were obtained from commercial sources and used without
any further purification. Column chromatography was accomplished using MACHEREY-
NAGEL silica gel 60® (230–400 mesh). Thin layer chromatography was performed on
aluminum plates pre-coated with silica gel (MERCK, 60 F254) unless specified. TLCs were
visualized by UV fluorescence (λmax = 254/320 nm) and/or by staining.
NMR spectra were acquired on a BRUKER Avance 400 spectrometer (400 MHz and
100.6 MHz for 1H and 13C respectively) or a Bruker 500 DRX NMR spectrometer with
TBI probe head (499.6 and 125.6 MHz for 1H and 13C respectively) at a temperature of
303 K unless specified. Chemical shifts are reported in parts per million (ppm) relative to
residual solvent.
Data for 1H NMR are described as follows: chemical shift (δ in ppm), multiplicity
(s, singlet; d, doublet; t, triplet; q, quartet; quin, quintuplet; hept, heptuplet; m, multiplet;
br, broad signal), coupling constant (Hz), and integration. Data for 13C NMR are described
in terms of chemical shift (δ in ppm).
HR-MS were obtained on a THERMO SCIENTIFIC Advantage and a THERMO SCI-
ENTIFIC Exactive instrument (APCI/MeOH: spray voltage 4–5 kV, ion transfer tube:
250–300 ◦C, vaporizer: 300–400 ◦C).
Optical rotation of chiral compounds was determined on a PERKIN-ELMER PE
241 apparatus and transformed for a given temperature according to the following formula
(Equation (1)):
α × 100
[α]TD=
(1)
c × l
α
: measured value for optical rotation; c: concentration in g/100 mL; l: length of the
cuvette in dm; T: temperature in ◦C.
3.1.2. Synthesis and Analytical Data
Detailed synthetic route for each final derivative as well as syntheses and data for
intermediary compounds are given in the Supplementary Materials.
3.2. Biological Assays
3.2.1. Fluorescence Polarization Assay
Fluorescence polarization assays were carried out as described before [31]. The in-
hibitors were diluted in assay buffer (25 mM HEPES pH 7.5, NaCl 100 mM, 0.05% CHAPS,
1 mg/mL BSA) in a serial dilution of 12 inhibitor concentrations with equal DMSO con-
centrations, and added in a 384-well black non-binding microplate (Greiner Bio-One)
to mixtures of H3(1–14)K4me3-Fluorescein (Peptide Specialty Laboratories) and His-
Spindlin1(49–262), with final concentrations of 10 and 100 nM, respectively. After 30 min
incubation at room temperature, the fluorescence polarization (Ex480, Em535) was mea-
sured using an Envision Plate reader (PerkinElmer). For the controls, a buffer solution with
the same DMSO concentration as the inhibitor solutions was added to the plate instead
of the inhibitor solution, the negative control contained the H3(1–14)K4me3-Fluorescein

Int. J. Mol. Sci. 2021, 22, 5910
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without Spindlin1. The blank control contained buffer solution with DMSO. Every inhibitor
concentration and the controls were done in triplicates. Fluorescence polarization values
IS−G×IP
(mP) were determined after blank correction as
×
1000, where G is a device-specific
IS+G×IP
factor and IS and IP are the fluorescence intensities of the S and P plane, respectively. Inhi-
ꢀ
ꢀ
ꢁꢁ
PI −Pneg
bition values were then calculated as I = 100% × 1 −
, with PI, Pneg, and Ppos
Ppos−Pneg
being the fluorescence polarization results of the ligand solution, the negative control, and
the positive control, respectively. Curve fitting was carried out using GraphPad Prism 7,
applying a non-linear sigmoidal fit with a variable slope. Inhibition curves used for IC50
determination are reported in the Supplementary Materials (Figure S5).
3.2.2. Reagents for Histone Methyltransferase Assays
H3 peptide (1–25) is purchased from Anaspec (Cat. NO. AS-61703), thiol fluorescent
probe IV is purchased from Millipore-Sigma (Cat. NO. 595504), SAH (S-Adenosyl-L-
homocysteine) is purchased from NEB (Cat. NO. B9003S), and adenosine deaminase (ADA)
is purchased from Sigma (Cat. NO. 10102105001).
3.2.3. Expression and Purification of G9a and GLP and SAHH
Catalytic domains of Human G9a (913–1193) and GLP (982–1266) were cloned, ex-
pressed, and purified as previously described [34]. Full length of S. solfataricus S-
adenosylhomocysteine hydrolase (SAHH) was cloned, expressed, and purified using
the published protocol [35].
3.2.4. Coupled Enzyme Fluorescent Histone Methyltransferase Assay
The assay was performed using the previously published protocol [35]. Tested com-
pounds were pre-dissolved in assay buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 0.01%
Triton X-100, 3 mM MgCl2, 0.1 mg/mL BSA) with 5
µ
M of purified SAHH, 1 unit of ADA,
L in black 96-well plate. After 5 min,
M) in assay buffer was added. After incubation for 10 min at room
M, 50 L) was added. After 10 min, the
10
20
µ
M of H3, and 5 nM of either G9a or GLP as 30
µ
µ
L of SAM (25
µ
temperature, thiol fluorescent probe IV (20
µ
µ
fluorescence of probe at Ex400/Em465 was measured by Infinite M Plex (Tecan, San Jose,
CA, USA). Inhibition curves used for IC50 determination are reported in the Supplementary
Materials (Figures S6 and S7).
3.3. Computational Studies
3.3.1. Docking Studies
The crystal structure of the Spindlin1 in complex with A366 (PDB ID: 6I8Y) was re-
trieved from the Protein Data Bank (PDB; www.rcsb.org (accessed on 17 March 2019)) [24
]
and prepared with Schrödinger’s Protein Preparation Wizard tool [36]. Solvent molecules
and sodium ions were removed; meanwhile, hydrogen atoms and missing side chain
residues were added to the protein structure. Protonation states were assigned by PROPKA
at pH 7.0, and the hydrogen bonding network was consequently optimized. A restrained en-
ergy minimization step was then executed using the OPLS3 force field with default settings.
A Maestro 2D sketcher [37] was used to draw the ligand structures, which were
then converted into 3D structures and prepared with Schrödinger’s LigPrep tool [38]. All
possible tautomeric forms and stereoisomers were generated at pH 7.0
±
1.0 using Epik.
Afterwards, a multi-conformational dataset was generated with ConfGen, allowing a maxi-
mum of 50 conformers per ligand and energy minimization of the output conformations
using the default force field (OLPS_2005) [39,40].
Molecular docking studies were carried out by means of Glide using the standard
precision (SP) mode [32]. Prior, a grid box was generated employing the Receptor Grid
Generation tool, the co-crystallized A366 inhibitor was selected as the center of the grid,
and a cube of 15 Å was defined as the inner box. During docking, the option “sample ring
conformation” was switched on, and a maximum of three docking poses were output for
each ligand conformer; all other settings were kept as default. Moreover, the contribution

Int. J. Mol. Sci. 2021, 22, 5910
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of the intramolecular hydrogen bonds was evaluated and incorporated into the docking
scores. This was done by allowing the option “reward intramolecular hydrogen bonds”
in Glide [32]. In this way, the compounds that exhibit these intramolecular interactions
pay a smaller entropic penalty upon the binding, which affects the final scores—referred
to as DS_Intra-H-bonds in this article. Instead, DS refers to the default docking scores
without the intramolecular hydrogen bonds’ contribution. The above-mentioned protocol
was able to reproduce the binding mode of A366 as observed in the co-crystal structure
with Spindlin1 with a root-mean-square deviation (RMSD, heavy atoms) of 0.54 Å when
using the top-scored pose (Supplementary Materials, Figure S1).
Box-plots were generated using the R package to evaluate the distribution of the
docking scores (DS and DS_Intra-H-bonds) retrieved for the active and inactive ligands.
Only the ligands with determined IC50 values were used as actives, whereas compounds
1c, 1d, and 1e were treated as inactive.
3.3.2. Molecular Dynamic Simulations
The previously generated docking poses of A366, 1c, and 1e in complex with Spindlin1
(PDB ID: 6I8Y) were used as initial coordinates for the generation of the MD systems. The
simulations were performed using the Amber18 package, and the systems were prepared
by means of AmberTools18 tools [33]. First, topologies and force field parameters were
assigned to the ligands with the Antechamber package using General Amber Force Field
(GAFF) and AM1-BCC as atomic charges method (AM1-BBC, semi-empirical (AM1) with
bond charge correction (BCC)) [41,42]. The ligands were considered in the positively
charged state; thus, a net molecular charge of 2 was assigned to them. Then, each sys-
tem was prepared in TLEaP using Amber ff03 force field for the protein and GAFF for
the ligands. The systems were solvated in a TIP3P octahedral water box of 10 Å and
neutralized using Na+ and counterions. The prepared systems were then subjected to a pro-
tocol that encompassed diverse steps. Initially, two consecutive minimization steps using
constant-volume periodic boundaries were carried out. In each step, 4000 iterations (first
2000 steepest descent and then 2000 conjugate gradient) were applied to the system. In the
first minimization step, only solvent atoms were minimized, while the protein and ligand
−2
atoms were restrained to their initial coordinates with a force constant of 10 kcal mol−1 Å
.
In the second minimization step, the positional restraints were removed, and the whole
system was minimized. Next, the system was heated, starting from a temperature of 0 K up
to 300 K through 100 ps of MD, applying constant-volume periodic boundaries. To prevent
large structural deviations, the complex atoms (protein and ligand) were restrained with a
force constant of 10 kcal mol−1 Å−2. SHAKE was turned on and used to constrain bonds
involving hydrogen [43]. The Langevin dynamics was employed to control the temperature
using a collision frequency of 1 ps−1. Then, after a density step, the system was subjected to
an equilibration run of 2 ns with a time step of 2 fs per step. The temperature and pressure
were kept constant: 300 K and a constant-pressure periodic boundary maintained at an
average pressure of 1 atm by using isotropic position scaling with a relaxation time of 2 ps.
As for the heating step, SHAKE was turned, while the Langevin dynamics was employed
to control the temperature using a collision frequency of 2 ps−1
.
Finally, each system was subjected to a production run of 200 ns under the same
conditions used in the equilibration step (time step of 2 fs per step, constant temperature
of 300 K, Langevin dynamics with a collision frequency of 2 ps−1, constant-pressure
periodic boundary with an average pressure of 1 atm using isotropic position scaling with a
relaxation time of 2 ps). In all steps, a cutoff of 10 Å was used for non-bonded interactions,
and long-range electrostatic interactions were treated by means of the Particle Mesh Ewald
(PME) method [44,45].
The trajectories were analyzed using the CPPTRAJ module [46] from AmberTools18 [33
and visualized in VMD. Plots were generated by means of R package. For the analysis
of the protein–ligand hydrogen bonds, the default settings were used, which encompass
a distance cutoff of 3 Å between the donor and acceptor atoms and an angle cutoff of
]

Int. J. Mol. Sci. 2021, 22, 5910
16 of 19
135 degrees. Meanwhile, the distance cutoff was extended to 3.5 Å for the analysis of the
ligand intramolecular hydrogen bonds. Two criteria were applied to detect the presence of
cation–pi interactions between the ligand and the residues of the aromatic cage (Phe141,
Trp151, Tyr170, and Tyr177): (i) a distance cutoff of 6 Å between the center of mass of the
positively charged pyrrolidine nitrogen and the centroid of the aromatic ring and (ii) an
angle cutoff of 45 degrees; the angle was defined as the angle between the normal vector
of the aromatic ring plane and the vector connecting the positively charged pyrrolidine
nitrogen and the centroid of the aromatic ring. Salt bridge interactions were considered
to be formed if the distance between the negatively charged oxygen atom of Asp184 (or
Asp189) and the positively charged amidine nitrogen of the ligand were within the cutoff
distance of 4 Å.
All 100,000 frames retrieved from the 200 ns MDs were subjected to the analysis. The
occupancy rates of the interactions were calculated as the percent of the frames in which a
specific interaction was observed.
3.3.3. Prime MM-GBSA
The first docking poses were rescored with Prime MM-GBSA, a tool implemented in
Schrödinger suite that performs protein–ligand complex minimization and refinement [47].
The VSGB solvation model and OPLS3 force field were used [48]. Several settings were
tested, and we report to two different protocols: (i) only the ligands were relaxed, and
(ii) also the protein residues within 3 Å from the ligands were treated as flexible. For
the analysis of the results, for the docking studies, box-plots were generated using the
R package to show the distribution of the computed binding affinity values (MM-GBSA
∆
Gbind) obtained for the active and inactive ligands. Only the ligands with determined
IC50 values were used as actives, whereas compounds 1c, 1d, and 1e were treated as
inactive compounds.
4. Conclusions
In previous work, we reported the discovery of A366, an originally developed G9a
inhibitor [30], as a nanomolar Spindlin1 inhibitor (Figure 1) [31]. Later, the crystal structure
of Spindlin1 in complex with A366 was resolved (PDB ID: 6I8Y, [27]), and some modifica-
tions, as well as bivalent derivatives, were investigated [27]. In this study, we synthesized,
tested, and computationally analyzed a series of 21 A366 derivatives as Spindlin1 inhibitors
to further explore the SAR of this series. The main modification that had a drastic im-
pact on the activity was the length of the linker chain, where the actives bear a propyl
chain, while the inactive compounds either an ethyl or a butyl chain. Since the pyrrolidine
function mimics the trimethylated lysine moiety, it is crucial for the inhibitory activity.
Minor modification of the pyrrolidine ring did not lead to significant changes in the in-
hibitory activity (e.g., 1q), whereas its replacement with an isoindoline moiety yielded
1s, a good Spindlin1 binder, and the most selective compound of our series (Spindlin1
IC50: 360 ± 20 nM, G9a IC50 >20 µM, and GLP IC50 >20
µM). Likewise, a good selectiv-
ity profile was observed for compound 1t bearing a 3-methylpyrrolidine moiety and a
3-ethyl-3-methyl substitution on R4. Substituents on R2 position were not well tolerated,
as highlighted by a significant decrease in the inhibitory activity of several compounds
(
1a
,
1h
,
1i
,
,
1j, and 1k). Meanwhile, the replacement of the methoxy group at R1 displayed
1l) to more significant (1m) decreases in the activity based on the substitutions.
minor (1g
Finally, modifications of the spiro moiety showed no major impact on the inhibitory activity
of the compounds; more relevant effects were observed with the geminal dimethyl and
diethyl modifications. Notably, compound 1p was the most active derivative of the series
(Spindlin1 IC50: 157
exhibited a decreased activity for Spindlin1 coupled with an increase in selectivity over
G9a and GLP (Spindlin1 IC50: 1.21 0.09 M, G9a IC50 >20 M, GLP IC50 >20 M).
±
12 nM) but showed no selectivity over G9a; while compound 1o
±
µ
µ
µ
The synthesis and in vitro testing were coupled with various computational approaches.
Docking studies and MD simulations were carried out to rationalize the lack of activity

Int. J. Mol. Sci. 2021, 22, 5910
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of some derivatives. Interestingly, while from the predicted binding modes and docking
scores it was not possible to explain the differences in the activity, the analysis of the MD
simulations shed light on the important interactions and their contributions. Moreover,
rescoring the docking poses with MM-GBSA calculations was successful in discriminating
active from inactive compounds within our congeneric series of A366 inhibitors.
Considering the SAR of our series and the structural features of the binding site of
Spindlin1 and G9a, future work will concentrate on attempting more substitutions on
R2 and exploring other replacement for the pyrrolidine moiety. Specifically, longer and
hydrophilic substitutions will be tested on R2, which are postulated, on the one hand,
to be able to interact with the surrounding Spindlin1 residues (e.g., Asp95 and Glu142),
and on the other clash with the smaller G9a pocket and ensure selectivity. Additionally,
bisubstitutions on the pyrrolidine moiety as well as other bicyclic basic moieties will be
probed to achieve further potent and selective compounds.
It was reported that high levels of Spindlin1 have been observed in liposarcoma
and other types of tumors including ovarian cancer [13]. Thus, targeting the Spindlin1/
H3K4me3 pocket with small molecule inhibitors as described in the present work might be
an interesting therapeutic approach for such cancer treatment. The developed potent and
selective Spindlin1 inhibitors are drug-like molecules that can be used for further cellular
studies to achieve this goal.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22115910/s1.
Author Contributions: C.L. and D.R. carried out the docking, molecular dynamics simulations
and binding free energy calculations and wrote the manuscript. P.R., D.M. and D.O. synthesized
compounds and provided them for in vitro testing. L.S., J.S., J.B., T.B. and T.W. developed and
applied the Spindlin1 in vitro assays. H.G. and R.S. expressed and provided the proteins for testing.
K.-S.P. and Y.X. carried out the G9a testing. B.B. supervised the synthesis of the compounds and the
analytics. J.J. and M.J. supervised the in vitro testing and wrote the manuscript. W.S. designed and
supervised the computational studies and was in charge of revising and reviewing the manuscript.
All authors have read and agreed to the published version of the manuscript.
Funding: B.B., M.J. and R.S. received funding from the Deutsche Forschungsgemeinschaft (German
Research Foundation, DFG Project-ID 192904750—SFB 992) and M.J. and R.S. under Germany’s
Excellence Strategy—EXC-2189—Project ID 390939984). R.S. was further supported by grants from the
European Research Council (AdGrant 322844) and the DFG (CRC 850, CRC 746, and SCHU688/12-1).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We acknowledge the financial support of the Open Access Publication Fund of
the Martin-Luther-University Halle-Wittenberg.
Conflicts of Interest: The authors declare no conflict of interest.
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