Structure-based design, synthesis, and pharmacologic evaluation of peptide RGS4 inhibitors.

Regulators of G-protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G-protein Galpha-subunit and acting as GTPase-activating proteins (GAPs), thereby rapidly terminating G protein-coupled receptor (GPCR) signaling. Using the published RGS4-Gialpha1 X-ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Gialpha Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.

Tetrapeptide derivatives of [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) lacking an N-terminal tyrosine residue are agonists at the mu-opioid receptor.

The Phe(1) cyclic tetrapeptide Phe-c[D-Cys-Phe-D-Pen]NH(2) (Et) (JH-54) has been shown previously to exhibit high affinity and selectivity for the mu-opioid receptor. To examine the role of the Phe(1) residue in the unexpected high affinity of this peptide, 11 analogs of JH-54 have been synthesized and evaluated for opioid ligand binding and for efficacy using the [(35)S]GTPgammaS assay. Alteration of the bridging groups between the D-Cys(2) and D-Pen(4) residues of JH-54 from dithioether to disulfide revealed the importance of the relative position of the aromatic rings of the first and third residues in determining mu- and delta-affinities. The one carbon distance between the alpha carbon and phenyl ring in the N-terminal residue was critical. Additional steric bulk in the N-terminal Phe(1) residue was accommodated without large reductions in affinity in two naphthyl analogs, but not with 3, 3-(diphenyl)alanine. Conformational restriction of the Calpha-Cbeta and/or Cbeta-Cgamma bonds had little effect on affinities in two peptides with 2-amino-2-carboxytetralin in position 1, but it abolished activity in an isoquinoline analog and differentially altered activity in four phenylproline(1)-containing peptides. Most surprisingly, replacement of the Phe(1) aromatic ring with cyclohexyl resulted in a peptide of moderate affinity (K(i) = 32.5 nM) and potency (EC(50) = 58.8 nM). Thus, the tyrosyl para-hydroxyl substituent and even aromaticity in the N-terminal amino acid of these tetrapeptides are shown to be important, but not critical, features for mu-opioid receptor affinity, agonist potency, and efficacy.

Development of a model for the delta opioid receptor pharmacophore. 2. Conformationally restricted Phe3 replacements in the cyclic delta receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13).

The in vitro pharmacological properties and conformational features of analogs of the delta opioid receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) in which the Phe3 residue was replaced by each of the four stereoisomers of beta-methylphenylalanine (beta-MePhe) were investigated. Both analogs in which the alpha carbon of the Phe3 replacement has L-stereochemistry display high affinity for delta receptors with the (2S,3S)-MePhe3 analog exhibiting approximately 8-fold higher affinity than the (2S,3R)-MePhe3 diastereomer. Surprisingly, one analog with D-stereochemistry in residue 3, the (2R,3R)-MePhe3 analog, also displays high affinity for the delta receptor and is extraordinarily selective for this receptor. All analogs were agonists in the mouse vas deferens (MVD) and guinea pig ileum (GPI) smooth muscle bioassays, displaying MVD and GPI potencies consistent with their delta and mu opioid receptor affinities, respectively. The use of beta-MePhe as a replacement for Phe3 was based upon the desire to reduce the conformational flexibility of the Phe3 side chain by imposing a steric rotational constraint in the form of the beta-methyl substituent and to thus deduce the residue 3 side chain orientation in the delta receptor-bound conformation from the correlation between delta receptor binding affinities and conformational preferences. Molecular mechanics computations revealed, however, that the conformational constraints imposed by the beta-methyl group in the (2S,3S)-MePhe3 and (2S,3R)-MePhe3 analogs were too modest to allow unequivocal determination of delta receptor-bound residue 3 side chain conformation. However, analysis of the high-affinity (2R,3R)-MePhe3 analog revealed a strong preference for a single side chain conformer (chi 1 approximately 60 degrees). Low-energy conformers of this analog could only be effectively superimposed with low-energy conformers of the parent peptide in which the Phe3 side chain conformation was limited to chi 1 approximately -60 degrees. This observation eliminates the last remaining uncertainty regarding conformational features of the pharmacophore elements in the delta receptor-bound state, allowing the proposal of a complete model.

Opioid receptor affinity and selectivity effects of second residue and carboxy terminal residue variation in a cyclic disulfide-containing opioid tetrapeptide.

The previously described cyclic, delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH, where Pen, penicillamine, is beta-beta-dimethylcysteine, was modified at residues 2 and 4 by varying combinations of D- and L-Cys and D- and L-Pen, and effects on mu and delta opioid receptor binding affinities and on potency in the mouse vas deferens (MVD) smooth muscle assay were evaluated. A comparison was drawn between consequences of alterations in this series of analogs and those of analogous modifications in the related cyclic pentapeptide series which includes the highly delta receptor-selective [D-Pen2,D-Pen5]enkephalin, DPDPE. Unlike effects observed in the cyclic pentapeptide series, the mu receptor binding affinities of the cyclic tetrapeptides are not dramatically influenced by substitution of Pen for Cys at residue 2. Conversely, while binding of the pentapeptides is only slightly affected by alteration of the chirality of the carboxy-terminal residue, modification of stereochemistry at the carboxy terminus in the tetrapeptides critically alters binding behavior at both mu and delta sites. In contrast with the pentapeptide series, the tetrapeptides appear to be highly dependent upon primary sequence for binding and activity, as only the lead compound binds with high affinity to the delta site. Results suggest that the less flexible cyclic tetrapeptides, lacking the Gly3 residue, display more stringent structural requirements for binding and activity than do the corresponding cyclic pentapeptides.

Pharmacological characterization of [D-Ala2,Leu5,Ser6]enkephalin (DALES): antinociceptive actions at the delta non-complexed-opioid receptor.

Substantial evidence has been accumulated which suggests that opioid delta receptors may be distinguished on the basis of their involvement in the modulation (i.e., increase or decrease in potency) of mu-mediated antinociception. On this basis, it has been hypothesized that some opioid delta receptors exist within a functional complex with mu receptors (delta complexed (delta cx) receptors) while other delta sites do not (delta non-complexed (delta ncx) receptors). Recent work with [D-Ala2,Leu5,Cys6]enkephalin (DALCE) has demonstrated that this compound produces initial antinociceptive actions, does not modulate morphine antinociception and appears to bind irreversibly to the delta ncx site, presumably by means of thiol-disulfide exchange between the receptor and the cysteine sulfhydryl group. To determine if a structural basis exists for actions at the hypothesized delta ncx receptor, in the present study we report the synthesis and pharmacological characterization of [D-Ala2,Leu5,Ser6] enkephalin (DALES), a close structural analogue of DALCE. If a structural basis for action at the delta ncx site exists, then DALES would be predicted to produce antinociception, fail to modulate morphine antinociception and, since it lacks the free sulfhydryl group present in DALCE, fail to exhibit irreversible antagonistic actions; these predictions were supported. Additionally, pretreatment with DALCE at -24 h, but not with DALES, blocked DALES-induced antinociception. These observations in vivo support the concept of a structural basis for activity at the hypothesized delta ncx site and suggest that DALES, like DALCE, may be a useful probe for pharmacological characterization of putative delta receptor subtypes.

Cyclic dermorphin-like tetrapeptides with delta-opioid receptor selectivity. 3. Effect of residue 3 modification on in vitro opioid activity.

A series of residue 3-modified analogs of the cyclic, delta-opioid receptor-selective, dermorphin-like tetrapeptide Tyr-D-Cys-Phe-D-Pen and the corresponding residue 4-modified analog of the related delta receptor-selective cyclic pentapeptide [D-Pen2,D-Pen5] enkephalin were synthesized and evaluated in opioid receptor binding assays and in the in vitro mouse vas deferens (MVD) bioassay. In both series, substitutions that would be expected to alter the orientation of the phenylalanine-substituted aromatic side chain relative to the rest of the peptide, due to changes in the conformation of the peptide backbone, had deleterious effects on binding affinity and MVD potency. In general, these adverse effects were more pronounced in the pentapeptide series, owing, most likely, to the greater rigidity and, therefore, reduced susceptibility to conformational perturbation of the tetrapeptides. Substitution of phenylalanine by p-fluorophenylalanine enhances binding affinity in the pentapeptide series, consistent with previous observations in the enkephalins, but is without effect on binding in the tetrapeptide series. Substitution of phenylalanine by homophenylalanine, which alters the relationship of the aromatic phenyl ring to the remainder of the peptide by inserting an additional methylene group between the aromatic moiety and the backbone, greatly reduces binding affinity and MVD potency in the pentapeptide. The corresponding modification in the tetrapeptide series has little effect on delta receptor binding affinity and MVD potency and enhances binding to mu opioid receptors. Several possible interpretations of these results are discussed.

Direct dependence studies in rats with agents selective for different types of opioid receptor.

The objective of this study was to describe, quantitate and compare naloxone-induced abstinence syndromes in rats infused centrally (Sylvian aqueduct) with agonists that are currently the most selective for mu [( D-Ala2, MePhe4, Gly-ol5]enkephalin), delta [( D-Pen2, D-Pen5]enkephalin) and kappa (3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeneacetamide) (U-50,488H) opioid receptors, respectively. Morphine, ethylketazocine and dynorphin A served as reference compounds. After 70 hr of infusion from s.c. implanted osmotic minipumps, three levels of abstinence were associated with the injection of naloxone (3 mg/kg s.c.): 1) negligible syndromes (scores of less than 21) were obtained in rats on water or the kappa-directed ligands, U-50,488H and dynorphin A; 2) a low-to-moderate abstinence score (37-38) was recorded with rats receiving [D-Pen2, D-Pen5]enkephalin and ethylketazocine; and 3) a high abstinence score (64-73) was obtained with rats on morphine and DAGO. These results reinforce the concept of developing selective, nonbenzomorphan kappa agonists as clinically useful analgesics and emphasize that, when evaluating new analgesics, high selectivity for delta receptors does not, in itself, guarantee freedom from physical dependence.

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: development of a ligand with high delta opioid receptor selectivity and affinity.

Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.

Structural requirements for delta opioid receptor binding.

Structural features influencing opioid activity of enkephalin analogs were investigated through the synthesis and evaluation of opioid receptor binding affinities of a series of cyclic dithioether-containing analogs and structurally related linear analogs of the cyclic, disulfide-containing peptides, [D-Pen2, D-Pen5]enkephalin and [D-Pen2, L-Pen5]enkephalin, where Pen (penicillamine) is beta, beta-dimethylcysteine. The major effect of increasing the ring size of the cyclic moiety from disulfide to dithioether analogs was a large decrease in delta opioid receptor binding affinity which suggests that relatively compact conformations of the peptide ligand are necessary for optimal binding to this receptor. The effect of bulky, hydrophobic residues at position 2 in the peptide chain was evaluated by preparing the linear analogs, [D-t-Leu2, D-t-Leu5]enkephalin (t-Leu, 2-amino-3,3-dimethylbutanoic acid) and [D-Abu2, D-t-Leu5]enkephalin (Abu, 2-aminobutanoic acid). The former analog was found to be 36- and 450-fold less potent at delta and mu receptor sites, respectively, than was the latter, suggesting that bulky side chain substituents in position 2 of enkephalin analogs lead to a deleterious steric interaction at delta and particularly at mu receptors.

Role of mu and delta receptors in the supraspinal and spinal analgesic effects of [D-Pen2, D-Pen5]enkephalin in the mouse.

The opioid receptors involved in the supraspinal and spinal actions of [D-Pen2, D-Pen5]enkephalin (DPDPE) for production and/or modulation of analgesia were investigated in two thermal analgesic tests, the mouse warm water (55 degrees C) tail-withdrawal assay and the radiant heat tail-flick test. Two approaches were used at supraspinal and spinal sites: determination of possible cross-tolerance between morphine and a variety of receptor selective/nonselective agonists (DPDPE, [D-Pen2, L-Pen5]enkephalin (DPLPE), [D-Ala2, MePhe4, Gly-ol]enkephalin, [D-Ala2, Met5]enkephalin amide, [D-Ser2, Leu5, Thr6]enkephalin and [D-Thr2 Leu, Thr6]enkephalin) and possible potentiation of morphine (mu) analgesia by proposed delta agonists (DPDPE, DPLPE and [D-Ala2, D-Leu5]enkephalin) in naive and morphine-tolerant mice. Additionally, proposed mu (morphine) and delta (DPDPE) agonists were evaluated for their i.c.v. analgesic effectiveness in the absence, and in the presence, of the proposed delta antagonist ICI 174,864. The present communication now reports that after i.c.v. administration analgesic cross-tolerance could be demonstrated between morphine and a variety of relatively selective or nonselective opioids but not to the highly delta selective DPDPE and DPLPE. This result was consistent with direct antagonism of i.c.v. DPDPE, but not morphine analgesia, by ICI 174,864. Furthermore, i.c.v. DPDPE and DPLPE were able to potentiate morphine analgesia in either naive or morphine-tolerant mice. In contrast, after intrathecal administration, cross-tolerance could be demonstrated between DPDPE or DPLPE and morphine, and no potentiation of morphine by DPDPE could be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Supraspinal and spinal potency of selective opioid agonists in the mouse writhing test.

Three agonists with the highest degree of selectivity available for mu ([D-Ala2,NMePhe4,Gly-ol]enkephalin, DAGO), delta ([ D-Pen2,D-Pen5]enkephalin, DPDPE) and kappa (U-50,488H, U50) opioid receptors were compared for their activity in inhibiting acetic acid-induced writhing in mice. Additionally, three reference agonists for mu (morphine), delta ([ D-Ala2,D-Leu5]enkephalin, DADLE) and kappa (ketocyclazocine, KC) receptors were also studied in this test. The agonists were given directly into the lateral cerebral ventricle (i.c.v.) or into the lumbar spinal subarachnoid space (intrathecal), and the potency of each compound was compared across injection sites and with data previously obtained in a thermal analgesic test (mouse hot-plate test). The rank order of potency for inhibition of writhing after i.c.v. administration was DAGO greater than DADLE greater than morphine greater than DPDPE; KC and U50 showed no significant activity by this route. After intrathecal administration, the compounds inhibited writhing with a rank potency order of DAGO greater than KC greater than morphine = DADLE greater than DPDPE greater than U50. All compounds were more potent in inhibiting writhing at spinal sites than at supraspinal sites; DPDPE and DAGO were 15 and 24 times more potent after intrathecal than after i.c.v. administration, respectively. The proposed delta agonists DPDPE and DADLE inhibited writhing at both spinal and supraspinal sites. Further, although the proposed kappa-acting compounds KC and U50 were effective at relatively low doses at spinal levels, these compounds lacked activity at supraspinal sites at doses not causing sedation.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent structure of biodegradative threonine dehydratase of Escherichia coli: homology with other dehydratases.

The 987-base-pair coding region of the tdc gene of Escherichia coli K-12 encoding biodegradative threonine dehydratase [Tdc; L-threonine hydro-lyase (deaminating), EC 4.2.1.16], previously cloned in this laboratory, was sequenced. The deduced polypeptide consists of 329 amino acid residues with a calculated Mr of 35,238. Although the purified enzyme was shown to contain tryptophan, no tryptophan codon was found in the tdc reading frame. Incubation of purified Tdc with [14C]tryptophan revealed apparent "covalent" binding of tryptophan, indicating posttranslational modification of the enzyme. A heptapeptide, 54Thr-55Gly-56Ser-57Phe-58Lys-59Ile- 60Arg, was found to contain Lys-58, which binds pyridoxal phosphate coenzyme. A comparison of amino acid sequences between the Tdc polypeptide and the biosynthetic threonine dehydratases of yeast (encoded by ILV1) and E. coli (encoded by ilvA) and the E. coli D-serine dehydratase (DsdA, encoded by dsdA) revealed various extents of homology: five domains of the Tdc polypeptide were 63-93% homologous with the yeast enzyme, and three of these same regions were 80% homologous with the biosynthetic E. coli dehydratase; two different domains showed 67% and 83% homology with DsdA. In addition, two other sequences were highly conserved in all four proteins, one of which was shown to contain the conserved lysine residue that binds pyridoxal phosphate in the Tdc and DsdA polypeptides. These observations suggest that, despite their diverse origin and metabolic significance, these enzymes may have evolved from a common ancestral protein.