Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Magafa, Vassiliki; Matsoukas, Minos‐Timotheos; Karageorgos, Vlasios; Dermitzaki, Eirini; Exarchakou, Revekka; Stylos, Evgenios Κ.; Pardalos, Michail; Margioris, Andrew N.; Varvounis, George; Tzakos, Andreas G.; Spyroulias, Georgios A.; Liapakis, George

doi:10.1007/s00726-019-02741-2

Cited by 8 publications

(10 citation statements)

References 52 publications

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“…Addition of the reduced amine bond to this double substitution to produce analog 8 seems to be detrimental for NTS1, with a 400-fold decrease in affinity and no apparent change in the binding to NTS2. We and others have previously demonstrated that Tyr 11 is a critical position for NTS1/NTS2 affinity and selectivity (Richelson et al, 2008;Einsiedel et al, 2011;Fanelli et al, 2017;Magafa et al, 2019). As, expected, insertion of Lys at position 11 (9) resulted in an important loss of affinity on NTS1 and NTS2, of more than 5,000-fold and 110-fold, respectively, compared to NT(8-13).…”

Section: Impact Of the Peptide Backbone Modifications On Receptor Binsupporting

confidence: 62%

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

et al. 2020

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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.

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Section: Impact Of the Peptide Backbone Modifications On Receptor Binsupporting

confidence: 62%

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

et al. 2020

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“…The constrained amino acids have, as anticipated, a remarkable effect: a decreased affinity with a substantially more prominent influence on NTS1, therefore increasing selectivity toward NTS2. Modifications at Tyr 11 with other unnatural amino acids, such as d -Tyr(Et) and d -1-Nal, were also previously reported to dramatically decrease NTS1 binding affinity . This was particularly true for compound 10 , containing a 6-OH-Tic residue at this position, achieving 50-fold selectivity toward NTS2.…”

Section: Resultsmentioning

confidence: 90%

“…Modifications at Tyr 11 with other unnatural amino acids, such as D-Tyr(Et) and D-1-Nal, were also previously reported to dramatically decrease NTS1 binding affinity. 45 This was particularly true for compound 10, containing a 6-OH-Tic residue at this position, achieving 50-fold selectivity toward NTS2. This enhanced NTS2 selectivity seems to be related to both the hydroxyl group position and the presence of a conformational constraint.…”

Section: ■ Results and Discussionmentioning

confidence: 98%

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

Eiselt

González

Martin

et al. 2019

ACS Chem. Neurosci.

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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.

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“…As an alternative, researchers can use N-terminal acetylation and/or C-terminal amidation, or otherwise blocking a peptide terminus, or “stapling” (i.e., linking two positions in the peptide with a hydrocarbon or other chain) . Glycosilation , and N -methylation of some of the backbone nitrogens or arginine residues is another such strategy, , as is “capping” of some side-chain hydroxyl groups . Backbone N -methylation also favors membrane permeation .…”

Section: Challengesmentioning

confidence: 99%

“… 458 Glycosilation 459 , 460 and N -methylation of some of the backbone nitrogens or arginine residues is another such strategy, 461 , 462 as is “capping” of some side-chain hydroxyl groups. 463 Backbone N -methylation also favors membrane permeation. 464 Once the protease-sensitive sites in the peptide have been identified, they can be “reinforced” by substituting some of the amino acids so as to make the cut less likely.…”

Section: Challengesmentioning

confidence: 99%

Peptides as Pharmacological Carriers to the Brain: Promises, Shortcomings and Challenges

et al. 2022

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Central nervous system (CNS) diseases are among the most difficult to treat, mainly because the vast majority of the drugs fail to cross the blood-brain barrier (BBB) or to reach the brain at concentrations adequate to exert a pharmacological activity. The obstacle posed by the BBB has led to the in-depth study of strategies allowing the brain delivery of CNS-active drugs. Among the most promising strategies is the use of peptides addressed to the BBB. Peptides are versatile molecules that can be used to decorate nanoparticles or can be conjugated to drugs, with either a stable link or as pro-drugs. They have been used to deliver to the brain both small molecules and proteins, with applications in diverse therapeutic areas such as brain cancers, neurodegenerative diseases and imaging. Peptides can be generally classified as receptor-targeted, recognizing membrane proteins expressed by the BBB microvessels (e.g., Angiopep2, CDX, and iRGD), "cell-penetrating peptides" (CPPs; e.g. TAT 47−57 , SynB1/3, and Penetratin), undergoing transcytosis through unspecific mechanisms, or those exploiting a mixed approach. The advantages of peptides have been extensively pointed out, but so far few studies have focused on the potential negative aspects. Indeed, despite having a generally good safety profile, some peptide conjugates may display toxicological characteristics distinct from those of the peptide itself, causing for instance antigenicity, cardiovascular alterations or hemolysis. Other shortcomings are the often brief lifetime in vivo, caused by the presence of peptidases, the vulnerability to endosomal/lysosomal degradation, and the frequently still insufficient attainable increase of brain drug levels, which remain below the therapeutically useful concentrations. The aim of this review is to analyze not only the successful and promising aspects of the use of peptides in brain targeting but also the problems posed by this strategy for drug delivery.

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Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Cited by 8 publications

References 52 publications

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

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

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

Peptides as Pharmacological Carriers to the Brain: Promises, Shortcomings and Challenges

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