A structurally guided dissection-then-evolution strategy for ligand optimization of smoothened receptor

Article abstract of DOI:10.1039/c7md00104e

We present herein a novel dissection-then-evolution strategy for ligand optimization. Using the co-crystal structure of the smoothened receptor (SMO) as a guide, we studied the modular contribution of LY2940680 by systematically silencing the specific interaction between the individual residue(s) and the fragment in the ligand. Following evolution by focusing on the benzoyl part finally yielded an improved ligand 21.

Full text of DOI:10.1039/c7md00104e

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Commun., 2017, DOI: 10.1039/C7MD00104E.
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A Structurally Guided Dissection-then-Evolution Strategy for
Ligand Optimization of Smoothened Receptor†
Received 00th January 20xx,
Accepted 00th January 20xx
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Lintao Ye, Kang Ding, Fei Zhao, Xiaoyan Liu, Yiran Wu, Yang Liu, Dongxiang Xue, Fang
abef
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Zhou, Xianjun Zhang, Raymond C. Stevens, Fei Xu, Suwen Zhao and Houchao Tao
DOI: 10.1039/x0xx00000x
www.rsc.org/
We present herein a novel dissection-then-evolution strategy for fundamental questions remain regarding understanding SMO’s
19
ligand optimization. Under the guidance of the co-crystal structure involvement
in
these
physiological
mechanisms.
of smoothened receptor (SMO), we digested the modular Development of a persistent and reliable ligand that would
contribution of LY2940680 by systematically “silencing” the address these issues is highly sought after.
specific interaction between individual residue(s) and the
The evolution of SMO ligands can now be guided by the
2
fragment in the ligand. Following evolution by focusing on the high resolution structures that were obtained recently. As
0
benzoyl part finally yielded an improved ligand 21.
part of the achievements from the intense and flourishing
research on SMO, there have emerged several atomic
resolution structures of SMO including those of the
The smoothened receptor (SMO) is a transmembrane protein
categorized as a member of Frizzled G protein-coupled
receptors (GPCRs). SMO plays an essential role in the
regulation of the evolutionarily conserved and physiologically
2
1, 22
transmembrane domain (TMD),
2
extracellular domain
25
and the very recent multi-domain structures.
3, 24
(ECD),
These structures clearly defined the molecular contacts of co-
crystallized SMO ligands and provided great insights for the
design of tools and probes for further biophysical studies. For
example, originating from the first co-crystallized SMO ligand
1
, 2
important Hedgehog (Hh) signaling pathway.
Disruption of
this pathway results in disorders of embryonic development
and tissue regeneration that are related to birth defects or
3, 4
2
1, 26
cancers.
Thus, SMO has been a well-pursued and now
LY2940680,
our groups designed a super-stabilizing tool
5
-8
validated target for anticancer agent developments. Many
SMO ligands have been discovered, including a steroidal
ligand TC114 (Fig. S1, ESI†) by the introducꢀon of a new
functional group. This modification established an extra
interaction that restricts motion to hold the receptor’s
conformation and facilitate crystallization for structural studies
9
-11
alkaloid cyclopamine and other synthetic compounds.
1
2
Among these, vismodegib (GDC-0449)
and sonidegib
1
LDE225), have been approved by the FDA in 2012 and 2015,
3
2
7
(
of multi-domain SMO. This success encouraged us to look for
an additional improved ligand with potential applications in
pharmacological development and probe design.
respectively, for the treatment of basal-cell carcinoma (BCC).
Nevertheless, their clinical use has been associated with many
1
4, 15
severe side reactions,
as well as several instances of
1
Under the guidance of known SMO structures, we present
6-18
mutation related drug resistance.
In addition, many
herein
a
dissection-then-evolution strategy for ligand
optimization. To this end, we first carried out a comprehensive
digestion on LY2940680, whose interactions between the
receptor residues and ligand fragments were pinpointed at a
2
1
high resolution (Fig. 1A). According to the key interactions as
indicated, the molecule was dissected into four variable parts
(I-IV) and one linker (Fig. 1B). Sequential “silencing”, namely
introducing a chemical mutation on LY2940680 to weaken or
eliminate a specific ligand-receptor interaction, generated a
series of compounds (1-4, Fig. 1B). All of the new compounds
were synthesized (Scheme S1, ESI†) and then tested for
activity at inhibiting the Hedgehog signaling pathway by a
luciferase reporter assay using light II cells, which were NIH-
3
T3 cells stably transfected with a Gli-responsive firefly
luciferase reporter. As clearly indicated (Fig. 1C), compounds
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2
with substituents removed on the benzoyl (1) or deletion of mentioned above. As shown (Fig. 1C and Table S1, ESI†),
8
the pyrazole (
activity. While the replacement of phthalazine with binding energy (ddG = 2.5 kcal/mol). In comparison, the
naphthalene ( ) only accounts for a slight reduction of the alteration of other fragments would result in large expense on
activity. This may indicate there is little difference between the the binding. For compound , the loss of pyrazole lead to a
4) almost completely abolished the receptor’s modification of the pyradiazine lead to the least loss of relative
3
4
electronic interaction of Arg400 with phthalazine and the huge loss of ddG (21.6 kcal/mol), which is mainly from van der
cation-π interaction with naphthalene. Surprisingly, the Waals energy (ddG VDW = 11.0 kcal/mol). Similarly, removal of
replacement of the amide by a tertiary amine in
moderate activity with an IC50 of about 70 nM, possibly kcal/mol). Interestingly, elimination of the carbonyl renders
because of compensation resulting from the missing H- compound with a unique basic tertiary amine that, under
bonding between the carbonyl and Asn219. Additionally, to physiological conditions, produces a protonated
2 maintained substituents in benzoyl also results in large loss of ddG (~ 10
2
+
2
-H which
exclude the potential interruption from the metabolism of can still maintain a similar binding free energy as the parent
these compounds, we checked their stability in cell media (Fig. compound LY2940680 because the solvation energy (ddG solv
S3, ESI†) which indicated that this group of compounds were = -28.1 kcal/mol) compensates for the loss of coulomb energy
+
-H .
stable and thus this dissecting effect is only dependent on the (ddG Coulomb = 28.6 kcal/mol) of
2
structural variation.
Overall, in this context the computational performance
). The key showed good agreement with the IC50 values from cell-based
functional assays. These results confirmed our hypothesis that
modification on part I or IV would be valuable, while that on
part II or III would be less sensitive. Furthermore, considering
the space limitations of the deeply buried pocket that interacts
with part IV, the more outward situated part I fragment seems
to be in a better position for modification. It may not be a
coincidence that our previous development of TC114 (Fig. S1,
ESI†) in which a nitro was introduced to the benzoyl yielded
successful co-crystallization. However, an aromatic nitro
group, like in TC114, was generally avoided by medicinal
chemists since its reductive metabolite causes undesired
toxicity. Thus, our aim here is to search for a better and more
drug-like surrogate of the nitro group (Fig. 1D). To simplify the
synthesis and characterization, all the compounds were
initially designed without the 6-trifluoromethyl group on the
benzoic fragment. For this reason, all the compounds then
Fig. 1 Digestion study of SMO ligand LY2940680. (
A
were compared to the reference
from TC114 by removal of the trifluoromethyl group.
A series of compounds ( 20) were then synthesized
Scheme S2, ESI†). Following our established procedure in
5, a 4-NO compound derived
2
5-
(
search for stabilizing molecules on GPCRs, compounds were
first screened via a thermal stability assay on purified SMO
(
Table 1 and Fig. S2, ESI†). This assay records the fluorescence
365/460 nm) increase during the thermal denaturation upon
(
reaction of exposed cysteines with
interactions observed in the co-crystal structure of SMO with fluorochrome, CPM dye (N-[4-(7-diethylamino-4-methyl-3-
LY2940680. ( ). Design of LY2940680 analogs to digest the coumarinyl)phen-yl]maleimide). The thermal stabilization
a
thiol-specific
B
contribution from each part. LY2940680 was dissected into capability of test compounds was denoted as the midpoint
four parts (I-IV) and one linker. Analogs were designed by temperature (Tm) at which 50% of fluorescence increase was
partially “silencing” the interaction by the removal or observed. The CPM assay has been widely used in the high
substitution of the functional groups, as highlighted in coral in throughput screening of tool compounds on membrane
2
9, 30
every compound. (
calculated and the potency was evaluated by Gli assay. IC50
values represent the mean ± SEM of at least three separate able to stabilize SMO to some extent when compared to the
experiments carried out in duplicate. All compounds were APO sample (Tm = 56.3 °C). Among of them,
remained the
evaluated following 24 h incubation. ( ). New analogs were best stabilizer (Tm = 61.0 °C). Surprisingly, little improvement
designed by focusing on the variation on the benzoyl unit.
was observed on the negatively charged compounds 12 and
which, according to the design with gradient acidity and
C
). The relative binding energy was proteins.
As indicated in Table 1, almost all the compounds were
5
D
8,
1
5
Glide XP docking followed by Prime MM-GBSA was used to linker length, are expected to form an ionic pair with the
estimate the relative binding affinities of the compounds positive charged ε-amine of Lys395. This may be due to a high
2
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desolvation penalty on the highly solvated ligand anion that 20
a
OCH
2
C
6
H
5
58.4
-113.2
1799 ± 443
weakens the contribution from the ionic pair. Simple
IC50 values represent the mean ± SEM of at least three independent
substitutes, like fluorine (
stabilization (Tm = 59.1 °C). Other neutral compounds without following 24 h incubation.
a negatively induced dipole, such as and 10, exhibited even
To further elucidate the structure activity relationships
6), showed a slight benefit on experiments carried out in duplicate. All analogues were evaluated
7
less favorable interactions (Tm ≈ 58 °C), while compounds with (SAR), we performed MM-GBSA calculations and measured
possible negative dipoles to mimic the nitro group, like sulfone IC s via a Gli-luciferase reporter assay. As indicated (Table 1
5
0
11
and ester 14, could indeed promote the expected and Fig. S6, ESI†), the relaꢀve binding free energy and Tm
interaction. The corresponding sulfonamide 13 and amide 16
,
values correlate well, in a linear manner with only a few
however showed less capability to stabilize. This may be due to exceptions. Compounds that deviated from the correlation
an attenuated π-π interaction between the ligand and Phe484 include the highly acidic phenyl sulfonic acid 12 and phenyl
by a relatively weaker electron-withdrawing group than the attached 19 and 20. For the latter two compounds,
nitro in compound
covalent Schiff base with amine. However,
significantly enhance the stability of SMO. 18 with an extended from VDW and lipophilicity (Table S1, ESI†) when compared
linker is a better ligand in this regard as it may have a higher with nitro embedded
. The low Tm value reflected that the
5. Aldehyde is proposed to form reversible calculations provided an extremely low binding free energy
9
did not (dG bind < -110). The major difference in the formula came
5
tendency to form a covalent bond. Boronic acids, though not loading of such a large rigid and hydrophobic group is not
often, have been found in marketed drugs. Functionally, favored for stabilizing SMO in micelles. Nevertheless, the
phenyl boronic acid usually has a pKa of 9~10 that makes it correlations indicated that the calculation method can be a
protonated under physiological conditions. It has been rough alternative for prediction of a ligand’s stabilizing effect.
3
1
reported to behave as the bioisostere of phenol, and it was
On the other hand, the Gli-luciferase reporter assay
proposed to form a hydrogen bond or boron-nitrogen bond at showed a slight consistency with the CPM assay or MM-GBSA
times. Thus, it could be an alternative to explore the calculation. Roughly, compounds with lower binding energy
interaction with Lys395. However, compound 17 did not behave relatively well with a few exceptions. 14 has a high Tm
provide encouraging results (Tm = 59.4 °C). Finally, two phenyl value and a low binding energy but exhibited no activity in the
substituted compounds 19 and 20 gave moderately enhanced Gli assay. This may be ascribed to its in vivo hydrolysis to acid
stabilization on SMO.
15 which, in general in this assay, lost activity like
the nitro compound remained the best with an IC50 of 235
Table 1. Exploration of structure activity relationships (SAR) on nM. Excitingly, 19 ranked second with a close IC , though not
8. Overall,
5
5
0
potential analogs.
as potent as reflected in the MM-GBSA calculation. Other
molecules were generally much less effective, in a micro molar
range of IC50 values in this assay.
R
Me
O
N
5-20
Compds.
R
CPM
assay
Tm (°C)
MM-GBSA Gli-Luc
dG Bind reporter
(kcal/mol) IC50 (nM)
a
APO
NA
NO
56.3
NA
NA
5
2
61.0
-100.6
235.6
±
8
8.5
6
7
8
9
F
59.1
58.2
56.8
59.0
58.5
56.3
59.4
57.1
60.3
56.8
59.7
59.4
60.2
59.6
-93.6
-92.3
-88.8
-97.9
-96.3
-87.3
-86.6
-98.1
-100.6
-89.5
-97.5
-101.1
-99.0
-111.8
1575 ± 506
2018 ± 556
>10,000
NH
2
NHCH
CHO
2 2
CO H
2891 ± 407
1415 ± 338
>10,000
Fig. 2 Compound 21 exhibited enhanced potency. (A).
10
11
12
13
14
15
16
17
18
19
NMe
2
Evolution of 19 by the addition of a trifluoromethyl group on
the benzoyl unit generated compound 21 with IC50 at 1.0 nM.
SO
SO
SO
2
3
2
Me
H
2682 ± 898
3242 ± 712
>10,000
(B). Docking study of 21 in the two-domain SMO showed that
NH
2
all the original key interactions were retained. Besides, the
embedded benzene ring at the outer end of molecule, 21
established two additional interactions with Lys395 and
Phe484, respectively.
CO
CO
2
Me
H
2
>10,000
CONH
2
2
3018 ± 223
870 ± 358
>10,000
B(OH)
We then further modified 19 to 21 by adding a
CH
2
CHO
trifluomethyl group on the benzoyl unit (Fig. 2A). As observed
OC
6
H
5
269.7
9.1
±
2
and TC114, this evolution
7
in the transformation of
5
5
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dramatically enhanced the activity in the Gli assay.
Trifluomethyl groups play multiple roles in pharmaceutical
development. Likely, this strong electron-withdrawing group
enhanced the πꢀπ interaction with Phe484. In comparison to
TC114, 21 could be a promising drug candidate for further
evolution since 21 maintained comparable potency while
replacing the nitro with a pharmacologically safer group. We
carefully determined the docking of compound 21 in our
7
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The authors (FX, RCS, SZ, and HT) are thankful to Shanghai
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support and Shanghai PuJiang Talent Program (16PJ1407300;
SZ) and Natural Science Foundation of Shanghai
2
(16ZR1448500; SZ). We thank J. Yang and W. Tan for
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