In Search of the Optimal Macrocyclization Site for Neurotensin

Neurotensin exerts potent analgesic effects following activation of its cognate GPCRs. In this study, we describe a systematic exploration, using structure-based design, of conformationally constraining neurotensin (8–13) with the help of macrocyclization and the resulting impacts on binding affinity, signaling, and proteolytic stability. This exploratory study led to a macrocyclic scaffold with submicromolar binding affinity, agonist activity, and greatly improved plasma stability

Use of Molecular Modeling to Design Selective NTS2 Neurotensin Analogues

Neurotensin exerts potent analgesia by acting at both NTS1 and NTS2 receptors, whereas NTS1 activation also results in other physiological effects such as hypotension and hypothermia. Here, we used molecular modeling approach to design highly selective NTS2 ligands by investigating the docking of novel NT­[8-13] compounds at both NTS1 and NTS2 sites. Molecular dynamics simulations revealed an interaction of the Tyr¹¹ residue of NT­[8-13] with an acidic residue (Glu¹⁷⁹) located in the ECL2 of hNTS2 or with a basic residue (Arg²¹²) at the same position in hNTS1. The importance of the residue at position 11 for NTS1/NTS2 selectivity was further demonstrated by the design of new NT analogues bearing basic (Lys, Orn) or acid (Asp or Glu) function. As predicted by the molecular dynamics simulations, binding of NT­[8-13] analogues harboring a Lys¹¹ exhibited higher affinity toward the hNTS1-R212E mutant receptor, in which Arg212 was substituted by the negatively charged Glu residue

Use of Molecular Modeling to Design Selective NTS2 Neurotensin Analogues

Neurotensin exerts potent analgesia by acting at both NTS1 and NTS2 receptors, whereas NTS1 activation also results in other physiological effects such as hypotension and hypothermia. Here, we used molecular modeling approach to design highly selective NTS2 ligands by investigating the docking of novel NT­[8-13] compounds at both NTS1 and NTS2 sites. Molecular dynamics simulations revealed an interaction of the Tyr¹¹ residue of NT­[8-13] with an acidic residue (Glu¹⁷⁹) located in the ECL2 of hNTS2 or with a basic residue (Arg²¹²) at the same position in hNTS1. The importance of the residue at position 11 for NTS1/NTS2 selectivity was further demonstrated by the design of new NT analogues bearing basic (Lys, Orn) or acid (Asp or Glu) function. As predicted by the molecular dynamics simulations, binding of NT­[8-13] analogues harboring a Lys¹¹ exhibited higher affinity toward the hNTS1-R212E mutant receptor, in which Arg212 was substituted by the negatively charged Glu residue

C‑Terminal Modifications of Apelin-13 Significantly Change Ligand Binding, Receptor Signaling, and Hypotensive Action

Apelin is the endogenous ligand of the APJ receptor, a member of the G protein-coupled receptor family. This system plays an important role in the regulation of blood pressure and cardiovascular functions. To better understand the role of its C-terminal Phe¹³ residue on ligand binding, receptor signaling, and hypotension, we report a series of modified analogues in which Phe¹³ was substituted by unnatural amino acids. These modifications delivered new compounds exhibiting higher affinity and potency to inhibit cAMP accumulation compared to apelin-13. In particular, analogues Bpa¹³ or (α-Me)­Phe¹³ were 30-fold more potent to inhibit cAMP accumulation than apelin-13. Tyr­(OBn)¹³ substitution led to a 60-fold improvement in binding affinity and induced stronger and more sustained drop in blood pressure compared to apelin-13. Our study identified new potent analogues of apelin-13, which represent valuable probes to better understand its structure–function relationship

Synthesis and Characterization in Vitro and in Vivo of (l)‑(Trimethylsilyl)alanine Containing Neurotensin Analogues

The silylated amino acid (l)-(trimethylsilyl)­alanine (TMSAla) was incorporated at the C-terminal end of the minimal biologically active neurotensin (NT) fragment, leading to the synthesis of new hexapeptide NT[8–13] analogues. Here, we assessed the ability of these new silylated NT compounds to bind to NTS1 and NTS2 receptors, promote regulation of multiple signaling pathways, induce inhibition of the ileal smooth muscle contractions, and affect distinct physiological variables, including blood pressure and pain sensation. Among the C-terminal modified analogues, compound <b>6</b> (JMV2007) carrying a TMSAla residue in position 13 exhibits a higher affinity toward NT receptors than the NT native peptide. We also found that compound <b>6</b> is effective in reversing carbachol-induced contraction in the isolated strip preparation assay and at inducing a drop in blood pressure. Finally, compound <b>6</b> produces potent analgesia in experimental models of acute and persistent pain

Identification of 1‑({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)‑1<i>H</i>‑pyrazol-3-yl]carbonyl}amino)cyclohexane Carboxylic Acid as a Selective Nonpeptide Neurotensin Receptor Type 2 Compound

Compounds active at neurotensin receptors (NTS1 and NTS2) exert analgesic effects on different types of nociceptive modalities, including thermal, mechanical, and chemical stimuli. The NTS2 preferring peptide JMV-431 (<b>2</b>) and the NTS2 selective nonpeptide compound levocabastine (<b>6</b>) have been shown to be effective in relieving the pain associated with peripheral neuropathies. With the aim of identifying novel nonpeptide compounds selective for NTS2, we examined analogues of SR48692 (<b>5a</b>) using a FLIPR calcium assay in CHO cells stably expressing rat NTS2. This led to the discovery of the NTS2 selective nonpeptide compound 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1<i>H</i>-pyrazol-3-yl]­carbonyl}­amino)­cyclohexane carboxylic acid (NTRC-739, <b>7b</b>) starting from the nonselective compound <b>5a</b>

A Systematic Exploration of Macrocyclization in Apelin-13: Impact on Binding, Signaling, Stability, and Cardiovascular Effects

The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue <b>15</b>, <i>K</i>_i 0.15 nM and <i>t</i>_1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues <b>13</b> and <b>15</b>, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system

Identification of 2‑({[1-(4-Fluorophenyl)-5-(2-meth­oxy­phen­yl)‑1<i>H</i>‑pyr­azol-3-yl]­carb­onyl}ami­no)tri­cyclo[3.3.1.13,7]­dec­ane-2-carb­oxy­lic Acid (NTRC-844) as a Selective Antagonist for the Rat Neurotensin Receptor Type 2

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (<b>8</b>) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound <b>9</b> in the NTS2 binding assay

A Systematic Exploration of Macrocyclization in Apelin-13: Impact on Binding, Signaling, Stability, and Cardiovascular Effects

The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue <b>15</b>, <i>K</i>_i 0.15 nM and <i>t</i>_1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues <b>13</b> and <b>15</b>, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system

χ‑Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the <i>N</i>-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH₂) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH₂ was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the <i>C</i>-terminus of the peptide sequences, (partial) (ant)­agonism at MOP and weak (ant)­agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism