Metabolically stable neurotensin analogs exert potent and long-acting analgesia without hypothermia
Graphical abstract
Introduction
Chronic pain is an important public health problem affecting more than 20 % of the worldwide population [1]. Opioid drugs are extensively used in the treatment of chronic pain despite a long list of undesired effects and their limited long-term efficacy to relieve pain for many patients [2,3]. Even with the growing awareness of the risks associated with opioid misuse, overdose and addiction, opioid use is still rising, thus leading to the current opioid crisis in North America [4,5]. Thus, the societal and economic burden, healthcare costs and high prevalence of chronic pain encourage researchers to seek for new pain medications with an increased benefit/risk ratio [6,7]. Among the current development strategies, drugs targeting non-opioid G protein-coupled receptors (GPCR) represent a promising therapeutic avenue in pain research [7].
The development of peptide-based therapeutics is undergoing an exciting revival in the last decade, when compared to small molecule drugs [8,9]. Peptides often offer high target selectivity and specificity as well as enhanced efficacy, safety and tolerability profiles. However, naturally occurring peptides are often not directly suitable for clinical use due to low oral bioavailability, poor blood-brain barrier (BBB) penetration, and short half-life in physiological fluids related to their poor resistance to proteolytic degradation [8,10].
Among the promising alternatives to opioid pain medications, neurotensin (NT) receptors emerge as attractive targets for the treatment of pain [7,11]. Neurotensin (NT) is a small neuropeptide of 13 amino acids (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) [12] known to mediate its physiological effects through its binding to two receptors that belongs to 7TMRs superfamily, namely NTS1 and NTS2 [13]. Peripherally, NT acts as a hormone in the cardiovascular system where it induces a drop in blood pressure [14,15], controls appetite [16] and regulates gastrointestinal motility [17]. When administered directly into the central nervous system (CNS), NT is known to play a role in the regulation of anxiety [18,19] as well as in dopaminergic (DAergic)-associated diseases, such as schizophrenia, drug abuse, and Parkinson’s disease [[20], [21], [22]]. NT and its analogs also produce persistent hypothermia [20,23] and analgesia [[24], [25], [26]]. Both NTS1 and NTS2 receptors are present in CNS regions involved in nociceptive transmission and pain modulation, such as the spinal dorsal horn, periaqueductal gray (PAG), rostroventral medulla (RVM), dorsal raphe nucleus, raphe magnus and pallidus [25,[27], [28], [29]]. Moreover, the neurotensinergic system is gaining further interests for pain relief as NT-induced analgesia is not altered by the administration of the opioid antagonists’ naloxone and naltrexone, thereby supporting that NT receptor activation mediates its antinociception action independently of the opioid system [30].
Amino acids forming the C-terminal moiety of the native NT peptide, at position 8–13 (Arg-Arg-Pro-Tyr-Ile-Leu), were identified as the minimal sequence for NT receptor binding and biological activity [31,32]. Therefore, compound synthesis and structure-activity relationship studies have allowed the development of new NT(8−13) selective analogs targeting either NTS1 or NTS2 receptors and permitted the discrimination of their respective physiological effects [33,34]. While analgesia in acute, tonic, and chronic pain paradigms was demonstrated to be mediated by the activation of both receptors [24,25,28,[34], [35], [36]], only NTS1 activation was associated with hypotension [14,37] and hypothermia [38].
These adverse effects (hypothermia and hypotension) combined with the low metabolic stability of the native NT peptide preclude its therapeutic use as non-opioid pain-relieving medications. Therefore, chemical modifications of the backbone and the incorporation of unnatural amino acids are necessary to optimize the pharmacological properties of newly synthesized NT(8−13) drug candidates. In the present study, we evaluate the impact of site-selective chemical modifications of NT(8−13) to improve the peptide metabolic stability and its analgesic efficacy in different experimental pain models as well as to reduce the unwanted effects triggered by NTS1 activation.
Section snippets
Peptide chemistry and characterization
Full synthetic procedures and characterization of the compounds presented in this study are reported in [39].
Competitive radioligand binding assay
CHO-K1 cells stably expressing hNTS1 (ES-690-C from PerkinElmer, Montréal, Canada) or 1321N1 cells stably expressing hNTS2 (ES-691-C from PerkinElmer) were cultured respectively in DMEM/F12 or DMEM. Culture media were supplemented with 10 % FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 20 mM HEPES and 0.4 mg/mL G418, and cells were incubated at 37 °C in a humidified chamber at 5% CO2
Rational design of NT(8−13) analogs
Both Arg residues in positions 8 and 9 were replaced by two Lys for ease synthesis (JMV438). It was previously demonstrated that this amino acid di-substitution had no impact on NTS1 and NTS2 receptor binding or activation [[51], [52], [53]]. Therefore, the subsequent chemical modifications of the NT(8−13) sequence were based on the JMV438 derivative.
First, the chemical modifications performed on the NT(8−13) backbone were focused on increasing the metabolic stability of NT(8−13) analogs to
Conclusion and perspectives
The present study reports the characterization of a series of NT(8−13) analogs harboring a reduced amine bond and additional substitutions with unnatural silylated amino acids to give rise to metabolically stable and powerful analgesic pseudopeptide compounds. Incorporation of a reduced amine bond between Lys8-Lys9, Sip in position 10 and a TMSAla in position 13 of NT(8−13) resulted in the generation of JMV5296. These modifications produced a fairly NTS2-selective analog with extended
Funding sources
This research was supported by a grant from the Canadian Institutes for Health Research [grant number FDN-148413].
CRediT authorship contribution statement
Mélanie Vivancos: Methodology, Investigation, Formal analysis, Writing - original draft, Visualization. Roberto Fanelli: Resources. Élie Besserer-Offroy: Validation, Writing - review & editing. Sabrina Beaulieu: Methodology, Investigation. Magali Chartier: Methodology, Investigation. Martin Resua-Rojas: Investigation. Christine E. Mona: Resources. Santo Previti: Resources. Emmanuelle Rémond: Resources. Jean-Michel Longpré: Validation, Writing - review & editing. Florine Cavelier: Validation,
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors thank Professors Éric Marsault and Pedro D’Orléan-Juste (Dept. Pharmacology-Physiology, Université de Sherbrooke) for allowing them to use, the UPLC/MS instrument for plasma stability assays and the use of Micro-Med transducer for the blood pressure measurements, respectively.
MV was supported by a PhD scholarship from the Institut de Pharmacologie de Sherbrooke (IPS) and Centre d′Excellence en Neurosciences de l′Université de Sherbrooke (CNS). Funding from theUniversité de
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- 1
Present address: Ahmanson Translational Theranostics Division, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA
- 2
Present address: Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Vial Annunziata, Messina, Italy.
- 3
Lead Author.