Neurotensin Analogues Containing Cyclic Surrogates of Tyrosine at Position 11 Improve NTS2 Selectivity Leading to Analgesia without Hypotension and Hypothermia

Abstract

Neurotensin (NT) exerts its analgesic effects through activation of the G protein-coupled receptors NTS1 and NTS2. This opioid-independent antinociception represents a potential alternative for pain management. While activation of NTS1 also induces a drop in blood pressure and body temperature, NTS2 appears to be an analgesic target free of these adverse effects. Here, we report modifications of NT at Tyr11 to increase selectivity toward NTS2, complemented by modifications at the N-terminus to impair proteolytic degradation of the biologically active NT(8–13) sequence. Replacement of Tyr11 by either 6-OH-Tic or 7-OH-Tic resulted in a significant loss of binding affinity to NTS1 and subsequent NTS2 selectivity. Incorporation of the unnatural amino acid β3hLys at position 8 increased the half-life to over 24 h in plasma. Simultaneous integration of both β3hLys8 and 6-OH-Tic11 into NT(8–13) produced a potent and NTS2-selective analogue with strong analgesic action after intrathecal delivery in the rat formalin-induced pain model with an ED50 of 1.4 nmol. Additionally, intravenous administration of this NT analogue did not produce persistent hypotension or hypothermia. These results demonstrate that NT analogues harboring unnatural amino acids at positions 8 and 11 can enhance crucial pharmacokinetic and pharmacodynamic features for NT(8–13) analogues, i.e., proteolytic stability, NTS2 selectivity, and improved analgesic/adverse effect ratio.

Insightful Backbone Modifications Preventing Proteolytic Degradation of Neurotensin Analogs Improve NTS1-Induced Protective Hypothermia

“…Then, the presence of the reduced amine bond combined with D-Trp 11 and TMSAla 13 (14) favored the binding to NTS1 by 65-fold without modifying binding at NTS2 (Table 2). Finally, as shown previously (Lugrin et al, 1991;Fanelli et al, 2017;Eiselt et al, 2019), NT(8-13) analogs harboring a Tyr mimic at position 11 (i.e., Dmt 11 ) and a Tle residue at position 12, coupled or not to a CH 2 NH reduced amine bond (15 and 16) showed lower binding affinity at NTS1 with no significant influence on NTS2 binding.…”

“…This substitution was combined with the incorporation of TMSAla at position 13 to afford compounds 13 (H-Lys-Lys-Pro-D-Trp-Ile-TMSAla-OH) and 14 (H-Lysψ[CH 2 NH]Lys-Pro-D-Trp-Ile-TMSAla-OH). Finally, we evaluated the plasma stability and its ability to regulate body temperature for 15 and 16, which carry the unnatural amino acids 2 ′ ,6 ′ -di-methyl-tyrosine (Dmt) and tert-leucine (Tle) in positions 11 and 12, respectively (15: H-Lys-Lys-Pro-Dmt-Tle-Leu-OH, 16: H-Lysψ[CH 2 NH]Lys-Pro-Dmt-Tle-Leu-OH) (Eiselt et al, 2019).…”

“…The synthesis of all NT(8-13) analogs are detailed in the Supporting Information. Please note that the synthesis of some of these NT(8-13) derivatives have already been published elsewhere: compounds 1, 9, and 11 in Lugrin et al (1991) and Fanelli et al (2017); compound 2 in Fanelli et al (2017) and Lugrin et al (1991); compounds 3 and 5 in Doulut et al (1992), Vivet et al (2000), René et al (2013), and Fanelli et al (2015); and compounds 15 and 16 in Fanelli et al (2015) and Eiselt et al (2019). Briefly, the designed hexapeptides were synthesized in solution starting from Boc-Leu-OMe commercially available, or Boc-TMSAla-OMe, which was previously described following the standard Boc strategy (Fanelli et al, 2015).…”

“…The results are summarized in Table 2 and Figure 1. As indicated in Table 2, the radioligand binding studies on some NT(8-13) analogs, already reported in previous studies (Lugrin et al, 1991;Fanelli et al, 2015Fanelli et al, , 2017Eiselt et al, 2019), were repeated in a same set of experiments for comparison purposes.…”

“…Data are expressed ± SEM of at least three separate experiments. For compounds 3, 5, 15, and 16, K i values were taken from the literature (Fanelli et al, 2015;Eiselt et al, 2019).…”