Novel cyclic analogs of angiotensin II with cyclization between positions 5 and 7: conformational and biological implications

To study the conformational features of molecular recognition of angiotensin II (Asp1-Arg2-Val3-Tyr4-Val/IIe5-His6-Pro7-Phe8, AII), the synthesis and biological testing of several cyclic analogs of AII cyclized between positions 5 and 7 have been performed. The synthesized analogs were Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Pen7)-Phe8 (3), Sar1-Arg2-Val3-Tyr4-cyclo(Asp5-His6-Apt7)-Phe8 (4), Sar1-Arg2-Val3-Tyr4-cyclo(Glu5-His6-Apt7)-Phe8 (5), Sar1-Arg2-Val3-Tyr4-cyclo-(Cys5-His6-Mpt7)-Phe8 (6), Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Mpc7)-Phe8 (7), Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpt7)-Phe8 (8), and Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpc7)-Phe8 (9), where Apt stands for 4-amino-trans-proline, and Mpt and Mpc for 4-mercapto-trans- and -cis-prolines, respectively. Compound (9) showed good affinity at AT-1 receptors, namely a KD = 20 nM. In functional assays, it showed the characteristics of a weak partial agonist with a relative affinity of 0.26% of that for AII and an intrinsic efficacy, alpha E, of 0.42. Molecular modeling suggested a possible explanation for this finding: the relatively strong binding and the weak partial agonistic activity of compound 9 are due to interaction with AT-1 receptor of only two functionally important groups, namely, the side chains of the His6 and Phe8 residues.     

Human eosinophils produce biologically active IL-12: implications for control of T cell responses

The present study assessed the capacity of eosinophils (EOS) to synthesize the cytokine IL-12. Blood-derived, highly purified human EOS from six atopic patients and two nonatopic individuals were treated in culture with IL-4, IL-5, granulocyte-macrophage CSF, IFN-gamma, TNF-alpha, IL-1alpha, RANTES, and complement 5a, respectively. The expression of both IL-12 protein and mRNAs for the p35 and p40 IL-12 subunits was strongly induced in all donors by the Th2-like cytokines IL-4 and granulocyte-macrophage CSF and was also moderately induced by TNF-alpha and IL-1alpha. IL-5 treatment resulted in IL-12 synthesis in four atopic donors and one nonatopic donor, whereas IFN-gamma induced IL-12 synthesis in only two atopic donors. In contrast, RANTES exclusively induced mRNA for the p40 subunit without detectable protein release, and complement 5a had no effect on IL-12 mRNA or protein expression. EOS-derived IL-12 was biologically active, because supernatants derived from IL-4-treated EOS superinduced the Con A-induced expression of IFN-gamma by a human Th1-like T cell line. This activity was neutralized by anti-IL-12 Abs. In conclusion, EOS secrete biologically active IL-12 after treatment with selected cytokines, which mainly represent the Th2-like type. Consequently, EOS may promote a switch from Th2-like to Th1-like immune responses in atopic and parasitic diseases.     

Improved method for the purification of biologically active transcobalamin II

Transcobalamin II (TC II) was purified about 300,000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709-7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two column chromatographic steps originally reported by the above workers. The final preparation had 26.7 mug of vitamin B12 (B12) bound per mg of protein and an A280/A361 ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B12 into HeLa cells in tissue culture.     

Dimensional analysis of osteoclastic bone resorption and the measurement of biologically active calcitonin

Calcitonin inhibits bone resorption through a direct action on the osteoclast. We report a quantitative analysis of bone resorption by disaggregated rat osteoclasts. We then used our findings to develop a formal bioassay for calcitonin. Osteoclasts were mechanically disaggregated from neonatal rat long bones and dispersed at low densities on slices of devitalized bovine cortical bone. The resulting areas of bone excavation were quantified to micrometric precision by scanning electron microscopy together with computer-assisted image analysis. These findings were correlated with the volumes of bone resorption in the same slices measured by confocal scanning microscopy for the first time. The total planar areas of bone resorption per slice correlated linearly (r = 0.78) with the confocal microscopic measurements of total volume resorbed, provided that volume was expressed to its two-thirds power. The latter transformation resulted in representations of the determined areas ([length]2) and volumes ([length]3) which were dimensionally consistent. These findings thus demonstrate that osteoclastic bone excavations show a consistent relationship between area and volume and that assessments of the area of excavations accordingly provide an empirical representation of the volume of bone resorbed. Furthermore, in view of the skewed nature of the distributions of area measurements, we assessed the effect of transforming the response variable to derive a metameter, (planar area of resorption)1/2. Such transformed data points, which expressed the data in the dimensions of [length], were more normally distributed than the raw data points and had more stable variances over a wider concentration range. We accordingly determined relative potencies using parallel line analyses on the transformed data. The latter offered a consistent correlation to the volume measurements when these were also converted to dimensions of [length] (r = 0.805). It was confirmed that the inhibition of bone resorption by calcitonins from various species, namely, pig, salmon and eel, was quantitatively dependent upon concentration of the respective peptides. The resulting assay was also found to be sufficiently sensitive to measure picomolar peptide concentrations with a precision, lambda (standard deviation/slope), ranging between 0.3 and 0.8. Finally, we identified factors affecting assay precision and sensitivity.     

Amino acid side chain conformation in angiotensin II and analogs: correlated results of circular dichroism and 1H nuclear magnetic resonance

[1-Sarcosine,8-isoleucine]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent antagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue at position 4 (tyrosine) or position 5 (isoleucine) was replaced with the corresponding N-methylated residue. This change drastically reduced the antagonistic properties of this analog. The present work was therefore undertaken to investigate the effect of N-methylation on overall conformation of these peptides and to determine the conformational requirements for maximum agonistic or antagonistic properties. Conformation studies were carried out by circular dichroism and proton nuclear magnetic resonance spectroscopy in aqueous solution as a function of pH. The results indicated that: (i) angiotensin II and [1-sarcosine,8-isoleucine]angiotensin II gave practically identical spectroscopic data; and (ii) N-methylation in either position 4 or position 5 resulted in remarkable changes in the peptide backbone and a severe limitation in rotational freedom of side chains in tyrosine, isoleucine, and histidine residues. However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyrosine,8-isoleucine]angiotensin II than in [1-sarcosine,5-N-methylisoleucine,8-isoleucine]angiotensin II. Thus, these results suggest that: (i) the backbone and side chain structure of a potent angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing and binding with the receptor on the cell membrane; and (ii) greater impact of N-methylation in position 5 on the overall conformation of these peptides points to the controlling influence of position 5 (isoleucine) in aligning the residues in the central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly extended structure. Any change in this arrangement may lead to reduced biological activity.     

Capillary electrophoresis: a powerful microanalytical technique for biologically active molecules

Capillary electrophoresis (CE) is a versatile microanalytical technique that has gained much attention, particularly from those working with biologically active molecules. Its appealing characteristics include unprecedented sensitivity and the ability for automating the rapid electrophoretic separation of a number of low-volume samples in a reproducible manner, with relatively short analysis times. The picomole-femtomole (10(-12)-10(-15) mol) sensitivity of UV-CE has been enhanced tremendously by the interfacing of detection systems such as laser-induced fluorescence, which has extended the sensitivity into the attomole-zeptomole (10(-18)-10(-21) mol) range. Fluorescence detection has shown great potential for the CE analysis of a wide range of biomolecules including peptides, proteins and DNA. CE research and development has taken on directions focused primarily on improving detection, understanding and exploiting the basic chemistry of CE and devising new applications.     

Novel biologically active nonpeptidic inhibitors of myristoylCoA:protein N-myristoyltransferase

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds 16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition site of the enzyme. Moreover, 16-18 have an affinity comparable to that of 2 even though they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal activity.     

Conformational analysis and hemolytic activity of immunoglobulin G fragment 285-292 and its analogs

Theoretical conformational analysis was carried out for the 285-292 fragment of human immunoglobulin G (His-Asn-Ala-Lys-Thr-Lys-Pro-Arg) and its analogues containing Arg, Glu, Gly, Lys, or Trp residue instead of the His residue in position 1. Spectropolarimetic investigation of these peptides showed the analogues to have different activities in the C1q-mediated erythrocytes hemolysis assay. Comparison of the low-energy structures sets of the compounds tested allowed to suggest a model of the "biological active" conformation for the peptide molecule in the course of the C1q complement component binding.     

Synthesis of two biologically active insulin analogues with modifications at the N-terminal and N- and C-terminal amino acid residues

The synthesis and isolation in purified form of two analogues of insulin is described. [21-Isoasparagine-A] ([Iasn21-A]) insulin differs from the parent molecule in that the amino acid residue, asparagine, found at the C terminus of the A chain (A21) has been replaced by isoasparagine. [Sar1, Iasn21-A] insulin differs from insulin in that both the A1 and A21 amino acid residues, glycine and asparagine, have been substituted by sarcosine and isoasparagine, respectively. The synthesis of these analogues followed the pattern employed in this laboratory for the synthesis of insulin and its analogues. The S-sulfonated derivatives of the A chain analogues were chemically synthesized, converted to their sulfhydryl forms, and then combined with the S-sulfonated B chain to produce the respective insulin analogues. Isolation of the insulin analogues from the combination mixtures was effected by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. By the mouse convulsion assay method [Iasn21-A]insulin possessed a potency of 21 IU/mg and [Sar1, Iasn21-A] insulin 15 IU/mg. The radioimmunoassay method gave values of 16 IU/mg for the former and 7IU/mg for the altter analogue. The natural hormone has a potency of 23-25 IU/mg by both assay methods. These data indicate that the alpha- and beta-carboxyl groups of the A21 amino acid residue are nearly equivalent in terms of their contribution to the expression of the biological activity of insulin. Furthermore, these data strengthen the speculation (Cosmatos, A., Johnson, S., Breier, B., and Katsoyannis, P. G. (1975), J. Chem. Soc. Perkin Trans. 1, 2157) that the change in the relative positive charge at the N-terminal amino acid residue of the A chain is responsible for the considerable decrease in the immunoreactivity observed in such modified insulins.     

Fibrinogen coating density affects the conformation of immobilized fibrinogen: implications for platelet adhesion and spreading

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.     

Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16-24 carbons of which 16-18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Delta9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.     

Probing the importance of spacial and conformational domains in captopril analogs for angiotensin converting enzyme activity

A new synthesis of 4,5-methano-L-prolines and the enzymatic activity of the corresponding N-(3-mercapto-2-R-methyl-propionyl) analogs as inhibitors of angiotensin converting enzyme are described.     

Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.     

Synthesis of a cyclic peptide containing norlanthionine: effect of the thioether bridge on peptide conformation

Two diastereomeric analogues of ring C of nisin incorporating a novel norlanthionine residue have been synthesized via a triply orthogonal protecting group strategy. A full structural study was carried out by NMR, which elucidated the conformational properties of the two peptides and enabled the identity of each diastereoisomer to be proposed.     

Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: implications for PET dopamine studies

Affinities of dopamine (DA) analogs to both granular and plasma membrane uptake transporters were measured in vitro by inhibition of [3H]DA uptake in bovine chromaffin granule ghosts and C6 glial cells transfected with cDNA for the rat presynaptic dopamine transporter, respectively. Five amines were studied: DA, 6-fluorodopamine (6FDA), m-tyramine (MTA), 6-fluoro-m-tyramine (6FMTA), and beta-fluoromethylene-m-tyramine (FMMTA). Direct uptake of 18F labeled 6FDA and 6FMTA was also measured in the chromaffin granule system and compared with [3H]DA uptake. Results show that the transporter affinities of 6FDA and MTA were similar to that of DA in both transport systems while affinities of 6FMTA and FMMTA were lower. Furthermore while the direct uptake of DA and FDA in chromaffin granules were essentially identical and significantly reserpine-inhibitable, the direct uptake of 6FMTA was about 15-fold less and only minimally sensitive to reserpine pretreatment. Thus, although vesicular protection and reuptake may influence the turnover of FDA in 6-fluoroDOPA studies, they are unlikely to be important determinants of the kinetics of the slowly clearing components in studies with either 6-fluoro-m-tyrosine (6FMT) or 6-fluoro-beta-fluoro-methylene-m-tyrosine (6FFMMT), the bioprecursors of 6FMTA and 6-fluoro-FMMTA, respectively. These results are consistent with the finding that the longterm component in 6FMT PET studies is 6-fluoro-hydroxyphenylacetic acid (6FHPAC), which can be explained by the lack of vesicular protection of 6FMTA from MAO oxidation.     

Treatment of destructive acute pancreatitis with the new biologically active substance in experiments

An acute destructive pancreatitis (ADP) was simulated according to the original method with subsequent conduction of the conventional conservative therapy and pancreatic resection in 25 mongrel dogs. The mortality due to ADP lessening was promoted by the new biologically-active substance application.     

Comparative genomic hybridization and telomerase activity analysis identify two biologically different groups of 4s neuroblastomas

Chromosomal aberrations of 20 stage 4s neuroblastomas were analysed by comparative genomic hybridization (CGH). In a subset of 13/20 tumours, telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP). The CGH data were compared with the CGH results of ten stage 1 and 2 (stage 1/2) and 22 stage 3 and 4 (stage 3/4) neuroblastomas. A total of 17/20 stage 4s neuroblastomas did not progress clinically, whereas tumour progression with lethal outcome occurred in 3/20 cases. The CGH data of clinically non-progressing stage 4s tumours revealed a high rate of whole-chromosome aberrations (73.4%) with an overrepresentation of mainly chromosomes 2, 6, 7, 12, 13, 17, 18 and an underrepresentation of mainly chromosomes 3, 4, 11, 14. MYCN amplification or 1p deletion was observed in only 1/27 or 2/17 clinically non-progressing stage 4s tumours respectively, whereas all three progressive stage 4s neuroblastomas showed MYCN amplification, 1p deletion and, in 2/3 cases, distal 17q gains. Except for one case, telomerase activity was not observed in non-progressing stage 4s neuroblastomas. In contrast, 4s tumours with lethal outcome revealed elevated telomerase activity levels. Our data suggest that stage 4s neuroblastomas belong to two biologically different groups, one of which displays the genetic features of localized stage 1/2 tumours, whereas the other mimics advanced stage 3/4 neuroblastomas.