ACS Chemical Biology
Letters
(3) Ghosh, E., Kumari, P., Jaiman, D., and Shukla, A. K. (2015)
Methodological advances: the unsung heroes of the GPCR structural
revolution. Nat. Rev. Mol. Cell Biol. 16, 69−81.
pharmacophores with micromolar Ki values as we have shown
for disulfide-functionalized analogs of biogenic amines.22 For
such examples, a careful design of the linker providing
additional binding energy is helpful. The knowledge about
this particular covalent bond was used to facilitate the
prediction of reliable binding modes for the covalent ligand 1
and SR142948A by long-term MD simulations. Molecular
interactions between the neurotensin receptors and non-
peptidic ligands have been investigated.28,33 This work presents
the first model of an antagonist-bound human NTS1 that
combines an experimental with a computational approach.
Taken together, our strategy has provided us with a plausible
structural basis for the binding mode of SR142948A-derived
antagonists at NTS1, which may facilitate a rational, structure-
based development of subtype selective antagonists, non-
peptidic agonists, as well as tracers for PET and radioactive
tumor therapeutics.
(4) Shonberg, J., Kling, R. C., Gmeiner, P., and Lober, S. (2015)
̈
GPCR crystal structures: Medicinal chemistry in the pocket. Bioorg.
Med. Chem. 23, 3880−3906.
(5) White, J. F., Noinaj, N., Shibata, Y., Love, J., Kloss, B., Xu, F.,
Gvozdenovic-Jeremic, J., Shah, P., Shiloach, J., Tate, C. G., and
Grisshammer, R. (2012) Structure of the agonist-bound neurotensin
receptor. Nature 490, 508−513.
(6) Egloff, P., Hillenbrand, M., Klenk, C., Batyuk, A., Heine, P.,
Balada, S., Schlinkmann, K. M., Scott, D. J., Schutz, M., and Pluckthun,
A. (2014) Structure of signaling-competent neurotensin receptor 1
obtained by directed evolution in Escherichia coli. Proc. Natl. Acad. Sci.
U. S. A. 111, 655−662.
(7) Krumm, B. E., White, J. F., Shah, P., and Grisshammer, R. (2015)
Structural prerequisites for G-protein activation by the neurotensin
receptor. Nat. Commun. 6, 7895.
(8) Kinkead, B., Binder, E. B., and Nemeroff, C. B. (1999) Does
neurotensin mediate the effects of antipsychotic drugs? Biol. Psychiatry
46, 340−351.
(9) Binder, E. B., Kinkead, B., Owens, M. J., and Nemeroff, C. B.
(2001) Neurotensin and dopamine interactions. Pharmacol Rev. 53,
453−486.
(10) Koschatzky, S., Tschammer, N., and Gmeiner, P. (2011) Cross-
receptor interactions between dopamine D2L and neurotensin NTS1
receptors modulate binding affinities of dopaminergics. ACS Chem.
Neurosci. 2, 308−316.
(11) Gully, D., Canton, M., Boigegrain, R., Jeanjean, F., Molimard, J.
C., Poncelet, M., Gueudet, C., Heaulme, M., Leyris, R., Brouard, A.,
et al. (1993) Biochemical and pharmacological profile of a potent and
selective nonpeptide antagonist of the neurotensin receptor. Proc. Natl.
Acad. Sci. U. S. A. 90, 65−69.
METHODS
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Full details for all experimental methods (site-directed mutagenesis,
saturation-binding experiments and receptor binding studies, radio-
ligand depletion assay, luciferase reporter-gene assay, synthesis
protocols, and computational methods) are provided in the
Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
(12) Lang, C., Maschauer, S., Hubner, H., Gmeiner, P., and Prante,
O. (2013) Synthesis and evaluation of a (18)F-labeled diarylpyrazole
glycoconjugate for the imaging of NTS1-positive tumors. J. Med. Chem.
56, 9361−9365.
(13) Maschauer, S., Ruckdeschel, T., Tripal, P., Haubner, R.,
Einsiedel, J., Hubner, H., Gmeiner, P., Kuwert, T., and Prante, O.
(2014) In vivo monitoring of the antiangiogenic effect of neurotensin
receptor-mediated radiotherapy by small-animal positron emission
tomography: a pilot study. Pharmaceuticals 7, 464−481.
(14) Leach, A. R., Shoichet, B. K., and Peishoff, C. E. (2006)
Prediction of protein-ligand interactions. Docking and scoring:
successes and gaps. J. Med. Chem. 49, 5851−5855.
Figures S1−S6, Tables S1 and S2, complete experimental
procedures for the biological characterization of the test
compounds and NTS1 receptor mutants (site-directed
mutagenesis, saturation-binding experiments and recep-
tor binding studies, radioligand depletion assay, and
luciferase reporter-gene assay), synthesis protocols, and a
detailed description of computational methods (PDF)
Coordinates of representative, energy-minimized snap-
shots of compound 1 and SR142948A in complex with
Coordinates of representative, energy-minimized snap-
shots of compound 1 and SR142948A in complex with
(15) Shoichet, B. K., and Kobilka, B. K. (2012) Structure-based drug
screening for G-protein-coupled receptors. Trends Pharmacol. Sci. 33,
268−272.
(16) Dror, R. O., Pan, A. C., Arlow, D. H., Borhani, D. W., Maragakis,
P., Shan, Y., Xu, H., and Shaw, D. E. (2011) Pathway and mechanism
of drug binding to G-protein-coupled receptors. Proc. Natl. Acad. Sci.
U. S. A. 108, 13118−13123.
(17) Dror, R. O., Green, H. F., Valant, C., Borhani, D. W., Valcourt, J.
R., Pan, A. C., Arlow, D. H., Canals, M., Lane, J. R., Rahmani, R., Baell,
J. B., Sexton, P. M., Christopoulos, A., and Shaw, D. E. (2013)
Structural basis for modulation of a G-protein-coupled receptor by
allosteric drugs. Nature 503, 295−299.
(18) Erlanson, D. A., Wells, J. A., and Braisted, A. C. (2004)
Tethering: fragment-based drug discovery. Annu. Rev. Biophys. Biomol.
Struct. 33, 199−223.
(19) Buck, E., and Wells, J. A. (2005) Disulfide trapping to localize
small-molecule agonists and antagonists for a G protein-coupled
receptor. Proc. Natl. Acad. Sci. U. S. A. 102, 2719−2724.
(20) Weichert, D., and Gmeiner, P. (2015) Covalent Molecular
Probes for Class A G Protein-Coupled Receptors: Advances and
Applications. ACS Chem. Biol. 10, 1376−1386.
(21) Rosenbaum, D. M., Zhang, C., Lyons, J. A., Holl, R., Aragao, D.,
Arlow, D. H., Rasmussen, S. G., Choi, H. J., Devree, B. T., Sunahara, R.
K., Chae, P. S., Gellman, S. H., Dror, R. O., Shaw, D. E., Weis, W. I.,
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: +49 9131 85-29383. Fax: +49 9131 85-22585. E-mail:
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the German Research Foundation (DFG grant Gm-
13/10).
REFERENCES
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(1) Wise, A., Gearing, K., and Rees, S. (2002) Target validation of G-
protein coupled receptors. Drug Discovery Today 7, 235−246.
(2) Kobilka, B. (2013) The structural basis of g-protein-coupled
receptor signaling (nobel lecture). Angew. Chem., Int. Ed. 52, 6380−
6388.
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