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J Mol Model
(2019) 25:193
was coupled to the respective C-terminally extended com-
pound. The program MODELLER 9v4 [28] was used accord-
ing to a procedure that was successfully applied in a previous
work [7], We created 100 models of each ligand–receptor
complex, which mainly showed conformational differences
at the C-terminally extended residues. We manually selected
three representative conformations of the test compound 1a at
NTS1 (models 1–3) that (1) showed a comparable conforma-
tion to the parent peptide NT(8-13) (as indicated by our com-
plementary experimental results) and (2) showed a different
conformation of the C-terminal extension. Subsequently,
models 1–3 were investigated to check the stability of their
initial conformations using MD simulations. According to the
conformation observed within model 1 of 1a, we manually
selected one model each of compounds 3a and 4a at NTS1.
The ligand–receptor complexes were submitted to energy
minimization using the SANDER module of AMBER10, as
previously described [29]. The all-atom force field ff99SB
was transiently transfected into human embryonic kidney
(HEK 293) cells. To investigate the intrinsic activities of some
of the described elongated peptides in comparison with NT(8-
13), we used an inositol phosphate (IP) accumulation assay in
which the Gα -promoted modulation of IP production in cells
q
expressing NTS1 was recorded (Tables 1 and 2).
The obtained binding data support the existence of the pos-
tulated allosteric binding pocket below the C-terminus of
bound NT(8-13), and point to interesting structure–activity
relationships (SAR) for this cavity (Table 1). The K values
i
of the glycine derivatives 1a–c, which bear a carboxylic acid
(1a), an amide (1b), or a hydroxamic acid (1c) as a C-terminal
functional group, are all comparable for both subtypes, where-
as deletion of the carbonyl group, as implemented in the alco-
hol 1d, leads to a fiftyfold or fivefold loss of affinity at NTS1
or NTS2, respectively, indicating the importance of the pres-
ence of a carbonyl function in combination with a group
which is able to form hydrogen bonds (but not necessarily
an ionic interaction) for receptor recognition. These results
are supported by data obtained for the alanine derivatives 2a
and 2c as well as the serine derivatives 3a, 3c, and 3d. With
the introduction of more complex amino acids, we were able
to enhance the affinity as well as the selectivity compared to
our starting compound NT(8-13)-glycine 1a. Introducing both
a methyl group (2a) and an aromatic system (4a) at the Cα
atom increased NTS1 affinity (1 nM and 0.9 nM, respective-
ly), whereas the additional insertion of a hydroxyl function
(3a) led to enhanced selectivity (> 18-fold) for NTS1 over
NTS2, in combination with a good single-digit nanomolar
NTS1 affinity (3.3 nM). Modifying the aromatic moiety of
4a by inserting the recently described 5-substituted azaindolyl
alanine (peptide 5) did not result in a significant improvement.
In contrast, introducing a tyrosine at this position (peptide 6)
led us to the very promising NTS1-selective compound 6,
offering NTS1 binding of 1.3 nM and a 26-fold selectivity
for NTS1 over NTS2. Further modifications of the parent
compound 6 that led to compounds 7a–7g did not result in
any further gain in affinity or selectivity (Table 1). The same is
true of the insertion of other functional amino acid side chains,
such as basic or acidic residues, or homologation of the back-
bone via the insertion of β-amino acids (see Supporting
Information S2 in the ESM).
[30] was used. Minimization was carried out as reported in
the literature [7]. The AMBER parameter topology and coor-
dinate files for the minimized complexes were converted into
GROMACS [31, 32] input files and applied to a lipidic bilayer
of DOPC residues as previously described [33]. The charges
on the simulation systems were neutralized by adding 12 chlo-
rine atoms each. The simulation systems were submitted to
molecular dynamics simulation runs as previously described,
using the GROMACS simulation package [34]. An overview
of the simulation systems and their simulation times is provid-
ed in the ESM. Trajectory analysis was performed using
PTRAJ of the AMBER package, and figures were prepared
using PyMOL [35] and Chimera [36]. Representative confor-
mations for compound 8d were obtained by the homology
modeling procedure described above.
Results and discussion
Our synthesis of C-terminally extended NT(8-13)-derivatives
of type NT(8-13)-Xaa (Fig. 1b) involved microwave-assisted
solid-phase supported peptide synthesis (SPPS), starting from
chlorotrityl chloride resin, Rink amide resin, or Wang resin
(
preloaded with a suitable Fmoc-amino acid in some cases),
which were reacted as described in the BMethods^ section. A
more detailed description of the syntheses and analytical data
of the test compounds explored in this study is provided in
Supporting Information S1 of the ESM.
Radioligand binding studies were conducted to evaluate
the NTS1 and NTS2 affinities of all the synthesized com-
pounds (Tables 1–3, Supporting Information S2 of the
Interestingly, the addition ofL-amino acids increased NTS1
selectivity compared to the parent peptide NT(8-13), whereas
the addition of the corresponding D-amino acids yielded a
decrease in NTS1 selectivity or even a preference for NTS2
binding (Table 1, Supporting Information S2 in the ESM).
Remarkably, all of the investigated peptides showed ago-
nist behavior and were able to activate NTS1 at levels of 92–
100% compared to NT(8-13); see Table 1.
ESM). Binding data were determined utilizing the radioligand
3
[
H]neurotensin and Chinese hamster ovary (CHO) cells that
To learn more about the possible binding modes of our
bitopic ligands, we investigated representative test com-
pounds exhibiting glycine (1a), serine (3a), or phenylalanine
3
stably expressed human NTS1. [ H]NT(8-13) was used for
binding assays carried out to investigate human NTS2, which
Kling, Ralf Christian
