Preparation and properties of novel gramicidin S analogs possessing a tri-, tetra- or pentamethylene bridge between ornithine side chains.

Gramicidin S (GS) analogs in which the Ndelta atoms of the two Orn side chains are linked by an oligomethylene bridge [-(CH2)n-; n=3-5] were prepared via the bis(p-nitrobenzenesulfonyl) derivative [Orn(NBS)2,2']GS. For comparison the nonbridged secondary amino group-containing analog [Orn(Me)2,2']GS was also prepared. 1H NMR and CD spectral analysis indicated that these analogs adopt the same beta-sheet conformation as GS. The antimicrobial activities of these analogs were very similar, but were slightly dependent on the bridge chain length, the trimethylene-bridged analog being the most potent.     

Oxytocin binding sites in lactating rabbit mammary gland.

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 X 10(-9) M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 X 10(-9) M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 X g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.     

Synthesis and biological activities of oxytocin and lysine vasopressin analogs containing glutamic acid gamma-hydrazide in position 4

Solution methods, using N-hydroxysuccinimide esters, were used to synthesize [Glu(NHNH2)4] oxytocin and [Glu(NHNH2)4, Lys8] vasopressin. In these analogs of neurohypophyseal hormones, the side-chain carboxamide function of a glutamine residue is formally replaced by a hydrazide group at position 4. The hormone analogs were assayed for uterototonic activity, milk ejection activity, antidiuretic activity, and rat pressor activity. The specific biological activities of the oxytocin and vasopressin analogs were decreased compared to the respective parent hormones in all assay systems.     

Photoaffinity labeling of the angiotensin II receptor; pharmacology of the labeling peptides in the dark.

The biological activities of photoaffinity labeling analogs of angiotensin II (ATII) and their precursors were measured in rabbit aorta strips in the dark. Most of the analogs behave as reversible, specific agonists, one as a competitive inhibitor. The activities are discussed in line with the current view of structural requirements. The modifications consisted of substitutions on the aromatic nuclei of Tyr4 and Phe8 in [Sar1]ATII with (4'-NO2) Phe, (4'-NH2)Phe, (4'N3)Phe, (4'-N2 +)Phe, and (4'-NH2-3', 5'-I2)Phe. It is shown that the affinity of the ATII analogs modified in position 4 depends on the electronegativity and not on space-filling properties of the aromatic residue; rising electronegativity lowers the affinity, i.e. [sar1, (4'-NO2)Phe4]ATII has no more measurable activity. Substituting the aromatic side chain in position 8 of [Sar1]ATII gives well-binding analogs with intrinsic activities from 0 to 100% and activity seems to depend only on stereochemical requirements. Agonists and partial agonists bear rather small groups like -NH2, -N3, -NO2, and -N2 +. The only antagonist [Sar1, (4'-NH2-3',5'-I2)Phe8]ATII resembles the antagonist E1Sar1, Leu8]ATII in competitivity and binding.     

Fluorescent, photoaffinity, and biotinyl analogs of oxytocin

This study reports the solid phase synthesis and biological activities of two oxytocin analogs, [1-desamino, 4-lysine,7-(L-3,4,-dehydroproline)]oxytocin and [1-desamino, 4-threonine,7-(L-3,4-dehydroproline),8-lysine]oxytocin, and several fluorescent, photoaffinity, or biotinylated derivatives of these analogs and of oxytocin. The activities (in IU/mg) of the lysine-containing parent compounds, respectively, were as follows: uterus (without Mg++) 4.8 and 54; uterus (with Mg++) 19 and 440; milk ejection 65 and 414. The above analogs were coupled through the chemically reactive epsilon-amino group of lysine in position 4 or 8 or, in the case of oxytocin, through the N-terminal amino group of fluoresceine, photoaffinity, or biotinyl ligands. Fluoresceine coupled in position 1 of oxytocin gave an analog of low to moderate uterine (3.8 without Mg+ and 1.9 with Mg++) and milk ejection (7.9) activities. Analogs with biotin or fluoresceine coupled to lysine in position 4 had moderate uterine (11 and 23 without Mg++; 38 and 11 with Mg++) and milk ejection (33 and 13) activities. Analogs with fluoresceine, photoaffinity, or biotinyl labels coupled to lysine in position 8 retained good uterine (106, 62, and 147 without Mg++; 79, 78, and 509 with Mg++) and milk ejection (101, 181, and 247) activities and represent potentially useful experimental tools for studying hormone-receptor interactions and for receptor localization and isolation.     

Importance of the character and configuration of residues B24, B25, and B26 in insulin-receptor interactions

By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.     

Design of neurohypophyseal peptides that exhibit selective agonistic and antagonistic properties.

Within the spectrum of the characteristic pharmacological activities (oxytocic (O), milk-ejecting (ME), antidiuretic (A), pressor (P) associated with the known natural and synthetic analogs of oxytocin and vasopressin it is possible to discern patterns of selectivity of these types: 1) interpeptide-like (a) O/A, (b) O/P; 2) intraoxytocin-like (a) O/ME; (b) ME/O; 3) intravasopressinlike (a) A/P, (b) P/A. Consideration of structural modifications of oxytocin or vasopressin, which individually or in combination give rise to peptides possessing enhanced selectivity of a given type, can in some cases provide a rational basis for the design of peptides with even greater selectivity. [1-Deamino-4-valine-8-D-arginine]vasopressin, the most highly specific antidiuretic peptide known to date, was designed in this fashion. By contrast, intraoxytocin-like selectivity, is manifested to only a minor degree in all peptides studied to date. Enhanced interpeptide-like selectivity of the type 1a (O/A; O/P) is readily attainable by specific single substitutions at positions 4 or 7 in oxtocin. Substitution of threonine in the 4 position of the oxytocic antagonist [1-deaminopenicillamine]oxytocin brought about a threefold enhancement in oxytocic inhibitory activity. Thus [1-deaminopenicillamine-4-threonine]oxytocin (dPTOT) is the most potent antagonist of the in vitro oxytocic response to oxytocin known to date. Thus analysis of the pharmacological data from over 300 analogs of oxytocin and vasopressin allows the delineation of those structural modifications that can optimize selectivities. The potential and limitations of this approach for the design of peptides possessing desired agonistic or antagonistic selectivity for potential clinical use and for studies on oxytocin and vasopressin receptors is discussed.     

1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1alpha,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the previously well established main side chain modification pathway, is initiated by hydroxylation at C-24 of the side chain. The C-3 epimerization pathway, the newly discovered A-ring modification pathway, is initiated by epimerization of the hydroxyl group at C-3 of the A-ring. The end products of the metabolism of 1alpha,25(OH)2D3 through the C-24 oxidation and the C-3 epimerization pathways are calcitroic acid and 1alpha,25-dihydroxy-3-epi-vitamin-D3 respectively. During the past two decades, numerous noncalcemic analogs of 1alpha,25(OH)2D3 were synthesized. Several of the analogs have altered side chain structures and as a result some of these analogs have been shown to resist their metabolism through side chain modifications. For example, two of the analogs, namely, 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-D3] and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-D3], have been shown to resist their metabolism through the C-24 oxidation pathway. However, the possibility of the metabolism of these two analogs through the C-3 epimerization pathway has not been studied. Therefore, in our present study, we investigated the metabolism of these two analogs in rat osteosarcoma cells (UMR 106) which are known to express the C-3 epimerization pathway. The results of our study indicate that both analogs [1alpha,25(OH)2-16-ene-23-yne-D3 and 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3] are metabolized through the C-3 epimerization pathway in UMR 106 cells. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-D3 [1alpha,25(OH)2-16-ene-23-yne-3-epi-D3] was confirmed by GC/MS analysis and its comigration with synthetic 1alpha,25(OH)2-16-ene-23-yne-3-epi-D3 on both straight and reverse-phase HPLC systems. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-3-epi-D3] was confirmed by GC/MS and 1H NMR analysis. Thus, we indicate that vitamin D analogs which resist their metabolism through the C-24 oxidation pathway, have the potential to be metabolized through the C-3 epimerization pathway. In our present study, we also noted that the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 is about 10 times greater than the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-D3. Thus, we indicate for the first time that certain structural modifications of the side chain such as 20-epi modification can alter significantly the rate of C-3 epimerization of vitamin D compounds.     

Synthesis and some pharmacological properties of (2-tryptophan)-oxytocin.

Previous studies have suggested that an aromatic amino acid residue at position 2 in oxytocin facilitates the expression of the hormone's biolgocial activities. [2-Tryptophan]-oxytocin, in which a residue of tryptophan has replaced that of tyrosine in oxytocin, has been synthesized by the method of azide coupling of the N-terminal dipeptide and C-terminal heptapeptide amide. It was found to have approximately 0.1% of the potency of oxytocin in milk ejection and uterotonic biological activities.     

Conformational studies of oxytocin analogues with different ring sizes. I. Circular dichroism.

Circular dichroic spectra were measured for three analogues of deamino-oxytocin of different ring sizes where the disulfide group of oxytocin is replaced by the (CH2)n group. Their backbone rings are composed of different numbers of atoms, i.e., they are nineteen, twenty and twenty-one for [1,6-aminopimelic acid]oxytocin (n = 1), [1,6-aminosuberic acid]oxytocin (n = 2) and [1,6-aminoazelaic acid]oxytocin (n = 3), respectively. The pH dependence of the circular dichroism spectra indicates that the conformation of [1,6-aminoazelaic acid]oxytocin is different from those of others and the temperature dependency reveals that the conformation of [1,6-aminopimelic acid]oxytocin is most rigid. [1,6-Aminosuberic acid]oxytocin is biologically most active among three derivatives and their biological activities are related to the conformation and internal motions of the peptide hormone analogues.     

Vitamin D: structure-function analyses and the design of analogs.

There is continuing and emerging new interest in the development of vitamin D analogs resulting from the recognition that analogs of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] may be therapeutically useful. Side chain analogs of this steroid hormone are of particular interest because a family of lead structures have recently emerged for possible use in the treatment of certain types of cancers and skin diseases. Because of the chaotic array of side chain structures which exhibit useful therapeutic indices for these purposes, a more systematic approach towards developing intelligible structure-function information needs development. Accordingly, a method has been devised to analyze analogs as to their side chain topology based on identifying specific occupancy volumes through conformational analysis. Dot maps have been constructed as an indication of the volume in space which the side chain of 1 alpha,25-(OH)2-D3 or analogs is permitted to occupy. Volume exclusion analyses based on comparison of structural and biological data for 1 alpha,25-(OH)2-D3 and analogs are anticipated to lead to a more cogent model for drug design. A cautionary note on the limitations of this approach is discussed.     

Effects of conformational constraint in 2- and 8-cycloleucine analogues of oxytocin and [1-penicillamine] oxytocin examined by circular dichroism and biosassay

The analogues of oxytocin and [1-penicillamine]oxytocin, containing a cycloleucine (Cle) residue in position 2 or 8, were investigated by means of circular dichroism measurements in different solvents, and the results examined in terms of their biological activities. A cycloleucine residue in position 2 substantially reduces the free conformational space of the hormone 20-membered ring moiety (including the disulfide group), and stabilizes a conformation which is close to one of the possible conformations of oxytocin and involves a -turn. In position 8, the Cle residue affects the conformation of the Tyr² side chain, apparently forcing it away from the space above the 20-membered disulfide ring. However, it does not appear that the Cle residue has any significant effect on the overall backbone conformation of the hormone. The steric effect of the penicillamine residue in position 1 on the conformation of the disulfide group and Tyr² side chain from previous investigations is further confirmed. The synthesis and biological potency of [1-penicillamine, 8-cycloleucine]oxytocin is described. This analogue exhibits a strong inhibitory effect on the uterotonic activity of oxytocinin vitro. It also inhibited the vasopressor response to vasopressin.     

Photoaffinity labelling of the oxytocin receptor in plasma membranes from rat mammary gland

Plasma membranes from rat mammary gland containing a high concentration of [3H]oxytocin binding sites (2.8 pmol/mg protein) were used for photoaffinity labelling experiments. Competitive binding experiments show that these receptors bind with high affinity the specific oxytocin agonist [Thr4, Sar7]oxytocin and the analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidobenzoyl group (Abz) at the side chain of lysine. The tritium-labelled (50 Ci/mol) photoreactive analogue incorporated into a membrane protein with an apparent relative molecular mass of 65,000 +/- 3000 Da (n = 16). The labelling of this protein was completely suppressed by an excess of oxytocin.     

Benzophenone-containing cholesterol surrogates: synthesis and biological evaluation

Eight analogs of cholesterol (1) containing a benzophenone group have been synthesized as prospective photoaffinity labels for studies in cellular sterol efflux and HDL formation. Six of these compounds (4-9) have the photophore replacing different portions of the cholesterol alkyl side chain, and two (10 and 11) have it attached via nitrogen at carbon 3. The suitability of these analogs as cholesterol surrogates was determined by examining their ability to replace [3H]1 in fibroblasts preequilibrated with [3H]1. All eight analogs were effective in replacing natural 1 in competition with [3H]1 for apolipoprotein A-I-induced efflux. These are the first compounds shown to replace cholesterol successfully in a complex pathway of multiple intracellular steps. The results suggest an unexpected tolerance of biological membranes regarding the incorporation of sterols of differing chemical structure.     

Binding of [3H]oxytocin to cells isolated from the mammary gland of the lactating rat.

More than 90 percent of the cells isolated from the mammary gland of lactating rats with 0.1 percent collagenase were viable by dye exclusion. Myoepithelial cells comprised about one-third of the mammary cells and appeared to be morphologically intact in electron micrographs. [(3)H]Oxytocin-binding activity was localized in an enriched myoepitheial cell fraction obtained by density gradient centrifugation of the isolated cells. The amount of [(3)H] oxytocin bound at 20 degree C and pH 7.6 was proportional to the concentration of oxytocin and the number of cells, reaching a steady state by 40 min. About 0.45 fmol of oxytocin were bound per 10(6) cells. There was a single class of independent binding sites with an apparent K(d), estimated from equilibrium conditions, of 5 nM. This value agrees within experimental error with the value calculated from the ratio of reverse to forward rate constants (5.8 x 10(-4)s(-1) and 2.2 x 10(5) M(-1)s(-1), respectively), consistent with a single-step model for the interaction of oxytocin with binding sites on the cells. Erythrocytes bound only 3.5 percent of the amount of oxytocin bound by an equal number of mammary cells. Oxytocin analogues competed with [(3)H]oxytocin for binding sites in the following order: [deamino]oxytocin > [4-threonine]oxytocin > oxytocin > [O- methyltyrosine]oxytocin > [8-lysine]vasopressin; [lysine]-bradykinin and [4-proline]oxytocin were not inhibitory in the dose ranges tested. These results demonstrate that isolated mammary cells possess oxytocin receptors with properties comparable to those found in broken mammary cell preparations.     

Peptide N-alkylamides by solid phase synthesis

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Synthesis and bioactivity of fatty acid-conjugated GnRH derivatives.

Transdermal delivery of peptidic drugs is usually inefficient, notably due to their hydrophilic character which makes it difficult to cross the hydrophobic layers of the skin. In order to obtain transdermally deliverable analogs of gonadotropin releasing hormone (GnRH), we have synthesized related hydrophobic derivatives by attaching various aliphatic acids to the N(epsilon)-amino side chain of [D-Lys]6GnRH, a superactive GnRH agonist. It was found that the affinity toward the GnRH receptor gradually decrease with increased hydrophobicity, i.e. increase in chain length of the attached aliphatic acid. Nevertheless, analogs with 12-carbon or shorter aliphatic acids were shown to be GnRH superagonists, with in vitro and in vivo potencies similar to that of [D-Lys]6GnRH. [D-Lys-lauryl]6GnRH was shown to have a longer duration of action in vivo, as compared to [D-Lys]6GnRH. The transdermal penetration of the peptides was evaluated by in vivo functional experiments in rats. According to these studies the efficiency of penetration is gradually lowered in increasingly hydrophobic analogs. These results are discussed with respect to the circular dichroism spectra of the peptides in trifluoroethanol. The spectra of the aliphatic acid-conjugated superagonists examined do not express a significant tendency towards a beta-turn conformation, typical of GnRH and its agonists. This finding contradict previous publications which suggested a correlation between the conformations of GnRH analogs in trifluoroethanol and their biological activities.     

Oxytocin receptors in the mammary gland and reproductive tract of a marsupial, the brushtail possum (Trichosurus vulpecula).

Previous studies of marsupial lactation have shown that the milk-ejection reflex changes in sensitivity, being greater in small mammary glands sucked by small pouch young and lesser in larger glands supplying milk to larger young. The involvement of oxytocin receptors in these changes was examined in the brushtail possum Trichosurus vulpecula. Oxytocin receptors were measured in the mammary glands, uterus, and medial vaginal sacs by radioreceptor assay, using [3H]oxytocin as radioligand. In the mammary gland, a single oxytocin binding site was found with an affinity and receptor concentration of 0.81 +/- 0.41 l/nmol and 10.2 +/- 4.8 pmol/g tissue respectively (SD, 10 possums). Competitive displacement curves with related peptides and analogs showed the following order of specificity: d(CH2)5[Tyr(Me)2,Thr4,Tyr9-NH2]-vasotocin much greater than vasotocin greater than oxytocin = Arg-vasopressin greater than mesotocin greater than [Thr4,Gly7]-oxytocin = Lys-vasopressin greater than [deamino-Pen1, O-methyl-Tyr2, Arg8]-vasopressin greater than isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. [3H]Oxytocin did not bind to vasopressin receptors in the thoracic aorta. The concentration of oxytocin receptors was very high in small mammary glands (18.6 pmol/g tissue in a 2-g gland) and decreased logarithmically as the size of the mammary gland increased. It is suggested that the changes in the sensitivity of milk ejection to oxytocin is related to the concentration of mammary oxytocin receptors. The presence of oxytocin receptors in both uterus and median vaginal sacs extends previous observations and supports the hypothesis that in marsupial parturition, the uterus and medial vaginal sacs respond as a single functional unit to oxytocin.     

Conformationally biased analogs of oxytocin.

Four diastereomeric analogs of oxytocin containing substituted phenylalanine in position 2 were synthesized. This modified phenylalanine side chain contained one methyl group attached to the beta-carbon and the second one at the 2' position of the aromatic ring. All analogs were found to be inhibitors of uterotonic activity of oxytocin with pA2 values ranging from 6.0 to 8.3; the most potent one (pA2 = 8.3) contained dimethylphenylalanine of the D-erythro configuration.     

Oxytocin receptors coupled to uterine contraction in estrogen-dominated rabbits

The present study investigated whether specific [3H]oxytocin binding sites previously demonstrated in estrogen-dominated rabbit uterus have properties expected of physiologic receptors coupled to uterine contraction. Microsomal membranes from estrogen-dominated rabbit uterus were found to contain high-affinity specific oxytocin binding sites with Kd = 2-3 nM. These sites were predominantly myometrial in locus. Specific oxytocin binding exhibited a pH optimum between 7.5 and 8.0. Mg2+ or Mn2+ was necessary for maximal specific [3H]oxytocin binding; in contrast, Ca2+ at submillimolar concentrations inhibited specific binding. Oxytocin binding sites were not detectable in microsomal membranes isolated from progesterone-dominated rabbit uterus. Relative binding and uterotonic activities of 10 synthetic neurohypophyseal hormone analogues were determined in estrogen-dominated rabbit uterus. A qualitative correlation was observed between binding and uterotonic responses. Angiotensin II and insulin did not compete with [3H]oxytocin for uterine binding sites. It is concluded that the specific high affinity [3H]oxytocin binding sites demonstrated in estrogen-dominated rabbit uterus have the selectivity for neurohypophyseal hormone analogues expected for physiologic receptors coupled to uterine contraction.