Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N 2 A cells (EC 50 in nM range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca 2؉ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.

Strategies for the design of bi-or multifunctional drugs are reviewed. A distinction is made between bifunctional drugs interacting in a monovalent fashion with two targets and ligands containing two distinct pharmacophores binding in a bivalent mode to the two binding sites in a receptor heterodimer. Arguments are presented to indicate that some of the so-called "bivalent" ligands reported in the literature are unlikely to simultaneously interact with two binding sites. Aspects related to the development of bi-or multifunctional drugs are illustrated with examples from the field of opioid analgesics. The drug-like properties of the tetrapeptide Dmt 1 [DALDA] with triple action as a μ opioid agonist, norepinephrine uptake inhibitor and releaser of endogenous opioid peptides to produce potent spinal analgesia are reviewed. Rationales for the development of opioid peptides with mixed agonist/antagonist profiles as analgesics with reduced side effects are presented. Progress in the development of mixed μ opioid agonist/δ opioid antagonists with low propensity to produce tolerance and physical dependence is reviewed. Efforts to develop bifunctional peptides containing a μ opioid agonist and a cholecystokinin antagonist or an NK1 receptor antagonist as analgesics expected to produce less tolerance and dependence are also reviewed. A strategy to improve the drug-like properties of bifunctional opioid peptide analgesics is presented.

Opioid compounds with mixed mu agonist/delta antagonist properties are expected to be analgesics with low propensity to produce tolerance and dependence. In an effort to strengthen the mu agonist component of the mixed mu agonist/delta antagonist H-Tyr-Tic-Phe-Phe-NH(2) (TIPP-NH(2)), analogues containing structurally modified tyrosine residues in place of Tyr(1) were synthesized. Among the prepared compounds, H-Dmt-Tic-Phe-Phe-NH(2) (DIPP-NH(2); Dmt = 2',6'-dimethyltyrosine) and H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2) (DIPP-NH(2)[Psi]) retained a mixed mu agonist/delta antagonist profile, as determined in the guinea pig ileum and mouse vas deferens assays, whereas H-Tmt-Tic-Phe-Phe-NH(2) (Tmt = N,2',6'-trimethyltyrosine) was a partial mu agonist/delta antagonist and H-Tmt-TicPsi[CH(2)NH]Phe-Phe-NH(2) was a mu antagonist/delta antagonist. DIPP-NH(2)[Psi] showed binding affinities in the subnanomolar range for both mu and delta receptors in the rat brain membrane binding assays, thus representing the first example of a balanced mu agonist/delta antagonist with high potency. In the rat tail flick test, DIPP-NH(2)[Psi] given icv produced a potent analgesic effect (ED(50) = 0.04 microg), being about 3 times more potent than morphine (ED(50) = 0.11 microg). It produced less acute tolerance than morphine but still a certain level of chronic tolerance. Unlike morphine, DIPP-NH(2)[Psi] produced no physical dependence whatsoever upon chronic administration at high doses (up to 4.5 microg/h) over a 7-day period. In conclusion, DIPP-NH(2)[Psi] fulfills to a large extent the expectations based on the mixed mu agonist/delta antagonist concept with regard to analgesic activity and the development of tolerance and dependence.

Opioid peptide analogs consisting entirely of aromatic amino acid residues and contining conformationally restricted phenylalanine derivatives in position 2 of the peptide sequence were synthesized and pharmacologically characterized in vitro. Whereas the existence of at least three major opioid receptor classes (u, 6, and K) is now well-established, the development ofpotent opioid agonists and antagonists with high specificity for each receptor type and of ligands with receptor-specific agonist/antagonist properties continues to be an important goal in opioid pharmacology. The fact that A and 8 opioid receptors differ from one another in their conformational requirements for peptide ligands was first established through comparison of the receptor binding profiles of a cyclic enkephalin analog and its linear correlate (1). This observation led to the realization that conformational restriction of peptides either locally through incorporation of backbone or side-chain conformational constraints at a specific amino acid residue or more globally through peptide cyclizations may often result in improved receptor selectivity. The use of this strategy resulted in a number of conformationally restricted opioid peptide analogs with agonist properties that showed high preference for either 1L or 6 receptors (for a review, see ref.2). It has often been speculated but never demonstrated unambiguously that conformational restriction of peptides in some cases might also reduce or even totally abolish their intrinsic activity ("efficacy") and, thus, may produce partial agonists or antagonists. No examples of opioid peptide analogs with significant antagonist properties as a consequence of conformational restriction have been reported to date. The only opioid-peptide-derived antagonists with reasonable potency described so far were obtained through diallylation of the N-terminal amino group. An enkephalin analog of this type, NN-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864; Aib = aminoisobutyric acid) (3), has been useful as a 6-selective antagonist.In this paper we report that the tetrapeptide amide H-Tyr-D-Phe-Phe-Phe-NH2 (la) is a potent p-selective opioid agonist. This compound consists entirely of aromatic amino acids that can be conformationally restricted in a number of interesting ways. We show that substitution of the D and L isomers of the conformationally restricted phenylalanine analogs Na-methylphenylalanine (NMePhe) and tetrahydro-3-isoquinoline carboxylic acid (Tic) (Fig. 1) for D-Phe2 in peptide la produced astonishing changes in receptor affinities and intrinsic activities. Most importantly, these structureactivity studies defined a class of potent and selective 6 antagonists, characterized by the N-terminal sequence H-Tyr-Tic-Phe-. MATERIALS AND METHODSPeptide Synthesis. Peptide analogs 1-7 were synthesized by the usual solid-phase technique with N"-t-butyloxycarbonylprotected amino acids and with benzotriazol-1-yl-oxytris-(dimethylamino)phosphonium hexafluorophosphate as coupling agent as described el...

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