Preparation and Evaluation at the Delta Opioid Receptor of a Series of Linear Leu-Enkephalin Analogues Obtained by Systematic Replacement of the Amides

Abstract

Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.

Systematic replacement of amides by 1,4-disubstituted[1,2,3]triazoles in Leu-enkephalin and the impact on the delta opioid receptor activity

“…Among all compounds tested, analog 17 showed the highest affinity (K i = 89 nM) for DOPr (Table 1). At this fourth amide position, we previously showed that introducing an E-alkene induced an even larger decrease in the affinity, 11 while the replacement by the hydrogen bond acceptors ester 12 and N-Methyl amide 12 generated peptidomimetics almost as potent as Leu-enkephalin itself (K i = 6.3 nM). 11 The analog 17 also displayed a weak potency to inhibit electrical field-induced mouse vas deferens contraction (Table 1) and to induce ERK1/2 phosphorylation ( Figure S1).…”

“…In previous studies, we have shown that the systematic replacement of amides in Leuenkephalin by E-alkenes 11 , esters 12 and N-Methyl amides 12 can reveal important information regarding the biological role of each amide in relevant analogs of Leuenkephalin and can therefore increase our understanding of the non-covalent interactions between DOPr and its endogenous ligands. To our knowledge the use of a triazole as a dipeptide isostere has never been applied to the field of opioid peptidomimetics, although some tetrazoles mimicking cis-amides have already been reported 5d .…”

The delta opioid receptor tool box

“…There are some reports that DPDPE and deltorphins can cross the blood-brain barrier (Williams et al, 1996; Fiori et al, 1997); and there has also been some effort to make more stable peptidomimetics (Rochon et al, 2013). In addition, considerable effort has been expended in developing small molecule (non-peptide) agonists to study the delta opioid receptor.…”

Synthesis and Opioid Activity of Tyr1‐ψ[(Z)CF=CH]‐Gly2 and Tyr1‐ψ[(S)/(R)‐CF3CH‐NH]‐Gly2 Leu‐enkephalin Fluorinated Peptidomimetics

“…However, clinical administration of Leu‐enkephalin is limited by a poor pharmacokinetic (PK) profile, including rapid proteolysis of Tyr 1 ‐Gly 2 by aminopeptidase N in human plasma ( t 1/2 =0.69 min; 0.50 U mL −1 purified enzyme) and of Gly 3 ‐Phe 4 by angiotensin‐converting enzyme at the blood–brain barrier ( t 1/2 =130 min; 0.12 U mL −1 purified enzyme), both of which inhibit penetration into the central nervous system . To improve potency and selectivity, many cyclic and linear analogues of enkephalin have been developed that possess excellent affinities and selectivities for the DOPR, and in some cases, the replacement of hydrolyzable amide bonds at various positions with peptidomimetics (e.g., trans ‐alkene, thioamide, ester or N ‐methyl amide dipeptide isostere) have modulated stability and PK properties of enkephalins, without major loss of agonist activity at the DOPR. However, many of these analogues still lack appropriate physicochemical and biophysical properties for in vivo use.…”