Andrzej W. Lipkowski

Topographical requirements for delta opioid ligands: presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity.

To investigate the role of the carboxyl group in deltorphin molecules, we have synthesized three new analogues in which the acidic amino acid residues in position 4 of the deltorphins were replaced by non-acidic but hydrophilic amino acids residues. The three analogues, [Ser4]-, [Gln4]-, and [Cys4]-deltorphin, all are as potent or more potent than either deltorphin I or II at delta opioid receptors and possess good delta selectivities. The excellent correlation between their in vitro delta receptor potencies and their intrathecal antinociception activity forms a strong argument for involvement of those receptors in spinal nociceptive modulation in the rats.

Endomorphins interact with tachykinin receptors.

Soon after the discovery of endomorphins several studies indicated differences between pharmacological effects of endomorphins and other MOR selective ligands, as well as differences between the effects of endomorphin I and endomorphin II. We now propose that these differences are the result of an additional non-opioid property of endomorphins, namely, their weak antagonist properties with respect to tachykinin NK1 and NK1 receptors.

Biological properties of a new fluorescent biphalin fragment analogue.

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.

[125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes

Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125I-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr1]biphalin to the delta and mu opioid receptors are not independent. [125I-Tyr1]Biphalin binds to delta receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to delta receptors in rat brain membranes was hardly evident and mu receptor binding predominated or at least was much more readily detectable in this preparation.

Topographical requirements for delta opioid ligands: The synthesis and biological properties of a cyclic analogue of deltorphin I

Abstract A cyclic constrained analogue of deltorphin, has been proposed on the basis of an energetically favored model of a delta-selective conformation for deltorphin. The biological properties of this synthetic analogue demonstrate that incorporation of a disulfide bridge into Deltorphin I does not affect its high affinity for delta opioid receptors. The analogue shows low receptor selectivity as a result of a large increase in its affinity for mu receptors when compared to the parent deltorphin.

Amino acids with amphiphilic side chains: Deltorphin analogues with Phe3 replaced by all β-hydroxyphenylalanine diastereoisomers

Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I containing each of the four stereoisomers of the unusual amphiphilic amino acid β-hydroxyphenylalanine in position 3. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays and by bioassay in MVD and GPI. The results show that introducing a hydrophilic group into the β-carbon of Phe3 decreases the affinity and biological activity of δ-opioid receptors, which strongly depend on the chirality of the α-carbon, but not on that of the β-carbon.

Biological properties of Phe°-opioid peptide analogues

Biological properties of new analogues, which represent Phe(o)-propeptides of a variety of opioid peptides, are described. All Phe(o)-opioid analogues expressed both receptor binding affinities and in vitro biological activities at least at the level of the primary opioid peptides. Surprisingly, some of the propeptides expressed slightly higher activity than the primary opioid peptides. Nevertheless, no significant shift in receptor selectivity was observed, which indicate that these Phe(o)-analogues undoubtedly are propeptides. The possible role of membrane proteolytic enzymes associated with opioid receptors in transformation of propeptides is discussed.

The synthesis and opioid receptor binding affinities of analogues of dermorphin and its N-terminal tetrapeptide fragment with dibasic acids in position 2

Analysis of possible mu opioid receptor active conformations for dermorphin suggested that the topographical location of the tyramine moiety of the N-terminal tyrosine can be simulated with the phenol of tyrosine or desamino-tyrosine (4-hydroxyphenylpropionic acid) and a basic group located on the side chain of a dibasic acid residue located in position 2. The biological properties of respective analogs with D- or L-arginine, and D- or L-lysine in the position 2 of dermorphin or desamino-dermorphin and their N-terminal tetrapeptide fragments, has provided evidence in support of this prediction, and questions the dogma that an N-terminal tyrosine is a necessary element for opioid agonist peptides.

Topographic model of delta opioid selective ligands; DPLPE-deltorphin hybrid analogues

They are attempts to correlate the topography of the bioactive structures of DPDPE and deltorphins, two delta active peptide families. One of the methods utilized by us is the hybridization of necesary elements for bioactivity of these opioid peptide groups. We have found that a major point determining high activity of hybrid analogues is also the chirality of the amino acid residue in position 5. Replacing D-Pen(5) with L-Pen(5) resulted in dramatic increases in affinity for opioid delta receptors