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

Abstract

Therapeutic hypothermia represents a brain-protective strategy for multiple emergency situations, such as stroke or traumatic injury. Neurotensin (NT), which exerts its effects through activation of two G protein-coupled receptors, namely NTS1 and NTS2, induces a strong and long-lasting decrease in core body temperature after its central administration. Growing evidence demonstrates that NTS1 is the receptor subtype mediating the hypothermic action of NT. As such, potent NTS1 agonists designed on the basis of the minimal C-terminal NT(8-13) bioactive fragment have been shown to produce mild hypothermia and exert neuroprotective effects under various clinically relevant conditions. The high susceptibility of NT(8-13) to protease degradation (half-life <2 min) represents, however, a serious limitation for its use in pharmacological therapy. In light of this, we report here a structure-activity relationship study in which pairs of NT(8-13) analogs have been developed, based on the incorporation of a reduced Lys 8-Lys 9 bond. To further stabilize the peptide bonds, a panel of backbone modifications was also inserted along the peptide sequence, including Sip 10 , D-Trp 11 , Dmt 11 , Tle 12 , and TMSAla 13. Our results revealed that the combination of appropriate chemical modifications leads to compounds exhibiting improved resistance to proteolytic cleavages (>24 h; 16). Among them, the NT(8-13) analogs harboring the reduced amine bond combined with the unnatural amino acids TMSAla 13 (4) and Sip 10 (6) or the di-substitution Lys 11-TMSAla 13 (12), D-Trp 11-TMSAla 13 (14), and Dmt 11-Tle 12 (16) produced sustained hypothermic effects (−3 • C for at least 1 h). Importantly, we observed that hypothermia was mainly driven by the increased stability of the NT(8-13) derivatives, instead of the high binding-affinity at NTS1. Altogether, these results reveal the importance of the reduced amine bond in optimizing the metabolic properties of the NT(8-13) peptide and support the development of stable NTS1 agonists as first drug candidate in neuroprotective hypothermia.

Metabolically stable neurotensin analogs exert potent and long-acting analgesia without hypothermia

“…Our results showed that NT(8-13) and JMV449 had no effect on body temperature, compared to saline-treated animals while JMV5170 and JMV5206 induced a strong and sustained decrease of body temperature of more than 2°C for up to 1 hour. These results are consistent with previous findings showing that the stability of NT(8-13) analogs is a key factor driving hypothermic response and that the increased stability can even compensate for a reduction in binding affinity at NTS1 [39,71]. Indeed, NT(8-13) and JMV449, which exhibit a plasma half-life of less than 10 min did not induce hypothermia.…”

“…Bold indicates synthetic changes from native peptide NT (8)(9)(10)(11)(12)(13). Data are expressed as mean ± SEM [39].…”

“…Full synthetic procedures and characterization of the compounds presented in this study are reported in [39].…”

“…Therefore, the subsequent chemical modifications of the NT(8-13) sequence were based on the JMV438 derivative. (JMV449), this N-terminal protection conferring resistance against proteolytic degradation, with minor impact on NT receptor binding [39,51,52]. On JMV5170, Pro 10 was then substituted by the silylated proline analog silaproline (Sip) [57].…”

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis

“…Among all the possible approaches, the introduction of unnatural amino acids and peptide backbone modifications has led to ligands with improved selectivity toward the target [36][37][38][39][40]. While the incorporation of an unnatural amino acid could be limited to a substitution, an extensive variety of backbone modifications could be inserted [41,42].…”