Design, Synthesis And Biological Evaluation Of Endomorphin Analogues And Synthesis Of Drug-Like Libraries

Opioid receptors are members of the superfamily of G-protein coupled receptors (GPCRs), and they are consist of three typical subtypes (μ,δ and κ) and ORL1. μ. opioid receptor mediates potent analgesia, but also followed by side effects such as tolerance, dependence and drug abuse;8opioid receptor has less effect on relieving pain, but can decrease the potential addition, and is less prone to develop tolerance; κ opioid receptor can mediate the peripheral antinociception, and is also related to neuroendocrine and immune regulation; ORL1opioid receptor is relevant with body self-stabilization, neuroendocrine and autonomic nervous system regulation. As a common drug for analgesia, morphine alkaloid is well-known for its side effects such as serious tolerance, addiction and respiratory depression. Endomorphin-1and endomorphin-2exhibit high affinity and selectivity for μ opioid receptor, while inhibiting pain without some undesired side effects like morphine. However, they have several deleterious properties that make them currently unsuitable for clinical use, such as unwanted side effects, metabolic enzyme instability and limited ability to cross blood-brain barrier. It’s reported that μ and δ receptors can form dimers, and8agonists can enhance the analgesic potency and efficacy of μ agonists, while δ antagonists can prevent or diminish the development of tolerance and physical dependence by μ agonists. Based on these observations, peptides and peptidomimetics possessing mixed μ/δ profiles may maintain potent antinociception while developing less side effects.This paper is based on endomorphins to synthesize β-peptidomimetics by incorporating a-hydroxyl-β-amino acids or a-hydrazino acids, aimed at developing endomorphins based opioid ligands to improve metabolic stability and maintain potent analgesic activity while decreasing the generation of tolerance like morphine.Initially, this paper took a-hydroxyl-β-peptidomimetics3as a lead compound, aided by computer molecular flexible docking,49endomorphins analogues were designed and synthesized by the solution-phase synthesis of α-hydroxyl-β-amino acids and a positional screening strategy. In the meanwhile, a series of in vitro and in vivo biological screening were conducted for these analogs:the opioid receptor binding assay revealed that some of these analogues and endomorphins had a similar affinity range for the μ receptor, while incorporating Dmt’-Tic2at the same time, some of them had high affinities for both μ and δ receptors, which could be potential peptidomimetics possessing with mixed μ/δ profiles; the metabolic stability experiment showed that half-lives of a-hydroxyl-β-peptidomimetics had been improved to some extend; the functional response indicated that compound34was a partial δ agonist; in the mouse tail-withdrawal test, compound34and morphine exhibited statistically equivalent antinociceptive potency; in different mouse models, compound34was confirmed as a mixed opioid receptor agonist and8antagonist; further liability evaluation showed that compound34was without significant locomotor effect at any time tested with a maximal-therapeutic dose (100nmol), while in the acute tolerance model, compound34didn’t demonstrate acute antinociceptive tolerance like morphine. Finally, the flexible docking between μ receptor and compounds with high affinity and selectivity for μ receptor (compound19and48) revealed that the binding modes of these compounds were similar to ((3-FNA); as a mixed μ agonist and8antagonist, compound34was docked into the inactive form of8receptor, and the result showed that its binding mode resembled Naltrindole, and DIPP-NH2which was also a mixed μ agonist and8antagonist.Similar to a-hydroxyl-β-amino acids, a-hydrazino acids were also introduced into endomorphins, and a-hydrazino-peptidomimetics were directly synthesized on solid-phase, which were then screened for in vitro biological assays. Some of these analogues showed high inhibition rates for μ receptor (>50%), and some exhibited high inhibition rates for both μ and8receptors (>50%). Furthermore, the affinity data from the opioid receptor binding assay revealed the importance of the substitution types and position of a-hydrazino acids in endomorphins. Most of these analogues displayed substantially decreased affinity for μ receptor, but compound M-29and M-51had equivalent affinity to endomorphins. On the basis of designing and synthesizing peptidomimetics with mixed μ/δ activity, while incorporating Dmt1-Tic2at the same time, most of these analogues retained the high affinity for8receptor, but with varying decreased μ affinity. However, for compound M-55, the μ affinity was as high as DIPP-NH2and the8affinity was four times higher than DIPP-NH2. Beyond all that, in the metabolic stability experiments, these a-hydrazino-peptidomimetics were much more stable than the endomorphins. Finally, when compound M-55was docked into the inactive form of8receptor, the result showed that its binding mode resembled Naltrindole, and DIPP-NH2. Compound M-55is likely to be a mixed μ agonist and δ antagonist. Further biological screening is still undergoing.In conclusion, our results reveal that whether a-hydroxyl-β-peptidomimetics or a-hydrazino-peptidomimetics, they can enhance the metabolic stability of endomorphins while maintaining the antinociceptive potency, and moreover, they are likely to improve the bioavailability. The subsequent biological test is in progress. Besides, glycosylation and lipidation can be used in improving BBB penetration and bioavailablity. The free hydroxyl functionality and amine functionality from a-hydroxyl-β-peptidomimetics and a-hydrazino-peptidomimetics respectively, can be a potential glycosylation or lipidation site, which could be modified to make endomorphins suitable for clinical use in the future. Overall, it is still challenging in the process of research and development of peptides as analgesic drugs. We are looking forward to develop novel peptides as analgesic drugs suitable for clinical use, which are effective but not producing side effects such as tolerance at the same time in the near future. This will definitely bring gospel to the majority of patients.In the drug discovery process, high through put screening is still one of the main tools. I have developed8structurally diverse drug-like libraries with519compounds during the Ph. D. Program in Zhejiang University. Further extensive biological evaluation is undergoing.