Development of Covalent Ligand–Receptor Pairs to Study the Binding Properties of Nonpeptidic Neurotensin Receptor 1 Antagonists

Abstract

The neurotensin receptor NTS1 has been suggested to be of pharmaceutical relevance, as it was found to exert modulatory effects on dopaminergic signal transduction and to be involved in tumor progression. Rational drug design of NTS1 receptor ligands requires molecular insights into the binding behavior of a particular lead compound. Although crystal structures of NTS1 have revealed the molecular determinants of peptide-agonist interactions, the binding mode of small-molecule antagonists remains largely unknown. Employing a disulfide-based tethering approach, we developed covalently binding molecular probes. The ligands 1 and 2 are based on the pharmacophore of the nonpeptidic NTS1 antagonist SR142948A, allowing the formation of a disulfide bond to an engineered cysteine residue of NTS1. The position of the covalent bond between Cys127(2.65) and the ligand was used to predict the binding mode of the covalent antagonist 1 and its parent compound SR142948A by molecular dynamics simulations.

En route towards the peptide γ‐helix: X‐ray diffraction analyses and conformational energy calculations of Adm‐rich short peptides

“…The ϕ values are in the range − 57° ÷ −61°, a common observation for helical C α ‐tetrasubstituted α ‐amino acid residues , whereas the ψ values, from −51° to −66°, are larger in magnitude than those typical for 3 10 ‐helices (−30°) or α ‐helices (−42°) . These findings compare well with the few occurrences of N‐acylated Adm derivatives of the R‐CO‐Adm‐OH or R‐CO‐Adm‐OR′ type reported in the literature , from which the average values of ϕ , ψ = −54°, −55° can be calculated for a helical Adm residue.…”

Computational investigation of the conformational dynamics in Tom20‐mitochondrial presequence tethered complexes

“…In the Tom20‐presequence complex, we found the common major and minor distributions of the conformational ensemble, independent of the choice of Ala or Tyr in the spacer region of the linker, but absence of a certain minor distribution due to the bulky side chain of the Tyr residue. In general, the cross‐linking via a disulfide‐bond is widely used as a biochemical technique to fix a certain conformation among multiple conformations of protein molecules or to stabilize the bound state of a ligand . The primary goal of cross‐linking is to trap one particular state/conformation for which preservation of dynamics in the cross‐linked complexes is generally not considered.…”

“…In general, the cross-linking via a disulfide-bond is widely used as a biochemical technique to fix a certain conformation among multiple conformations of protein molecules [17][18][19] or to stabilize the bound state of a ligand. [20][21][22][23] The primary goal of cross-linking is to trap one particular state/conformation for which preservation of dynamics in the cross-linked complexes is generally not considered. However, as demonstrated by our simulations, disulfide-bond tethering is also useful for the study of the dynamics in weakly binding complexes.…”

“…Disulfide‐bond formation has been successfully used for co‐crystallization of protein‐peptide complexes with low affinity, fixing a certain state of a biological complex among many conformations and studies on the binding properties of ligands . In these experiments, a disulfide‐bond was introduced to trap one particular state/conformation, without considering residual dynamics in the stabilized state/conformation.…”

“…Disulfide-bond formation has been successfully used for cocrystallization of protein-peptide complexes with low affinity, 16 fixing a certain state of a biological complex among many conformations [17][18][19] and studies on the binding properties of ligands. [20][21][22][23] In these experiments, a disulfide-bond was introduced to trap one particular state/ conformation, without considering residual dynamics in the stabilized state/conformation. However, it is pertinent to consider the effects of the disulfide-bond tethering on the conformational dynamics, since the NMR relaxation analysis was performed with covalent bond tethering of the presequence to Tom20.…”