Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.     

Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis

Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50 μM), collagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A-23187 (20 μM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6 μM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists—collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.     

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.     

Effect of melanin concentrating hormone on pigment and adrenal cells in vitro

Highly purified synthetic salmonid melanin concentrating hormone (MCH) and some analogs were investigated for their ability to concentrate the pigment in scale melanophores of the Chinese grass carp, Ctenopharyngodon idellus, to produce melanin dispersion in frog or lizard melanophores and to inhibit alpha-MSH in its action on mouse melanoma and rat adrenal glomerulosa cells in vitro. In the grass carp, MCH produced half-maximal pigment aggregation at 6 X 10(-11) M and its oxidized form at 7 X 10(-11) M. Replacement of the two methionines at position 3 and 6 with norvaline lowered the potency by a factor of 2.7 and with propargylglycine by a factor of about 7. Linear, Cys5,14-Acm-protected MCH was a full agonist of MCH but with a 345-fold lower potency. Iodinated MCH showed similar, low activity. In tetrapods, salmonid MCH and its analogs displayed only marginal pigment dispersion at concentrations greater than 10(-5) M. Alkali-treatment of MCH increased the pigment-dispersing potency by a factor of about 30 whereas the activity for pigment aggregation in the grass carp was destroyed. At high concentrations (10(-6), 10(-5) M) MCH also stimulated tyrosinase activity in B-16 mouse melanoma cells but did not modify the effects of alpha-MSH in this system. By contrast, when tested on rat adrenal glomerulosa cells, salmonid MCH had no effect alone but at a concentration of greater than 10(-10) M it slightly reduced corticosterone production by an alpha-MSH concentration of 10(-7) M. Aldosterone production was not affected and MCH did not influence the response to ACTH.     

Hepoxilins, potential endogenous mediators of insulin release

Evidence is presented to show that pancreatic islets of Langerhans are capable of producing hepoxilins A3 and B3 from endogenous substrates as well as 14C-labeled 12-HPETE. Both hepoxilins are active in stimulating the release of insulin from these cells in the presence of 10 mM glucose. These experiments suggest that the hepoxilins may participate as potential endogenous mediators of insulin release in islets of Langerhans.     

The hepoxilin analog PBT-3 inhibits heparin-activated platelet aggregation evoked by ADP

We have previously shown that PBT-3, a stable synthetic analog of hepoxilins, inhibits the aggregation of human platelets in vitro evoked by collagen through inhibition of thromboxane A(2) formation and action on the TP receptor. We now show that PBT-3 is capable of potently inhibiting the second phase of aggregation evoked by ADP in both washed human platelets and platelet-rich plasma (PRP), a phase associated with thromboxane formation. Aspirin blocks this second phase as well; so does the thromboxane receptor antagonist SQ 29,548. When ADP-evoked aggregation in PRP is activated by heparin through an enhancement of thromboxane formation, PBT-3, aspirin as well as SQ 29,548 block this activation through different mechanisms. These data confirm the inhibitory action of PBT-3 on aggregation of human platelets through inhibition of both thromboxane formation and blockade of thromboxane receptor action and suggest that this family of compounds may be useful in the treatment of thrombotic disorders in combination with heparin.     

Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct

Alzheimer disease is characterized by the abnormal aggregation of amyloid beta peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid beta oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid beta aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid beta monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms.     

Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability

Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).     

Competitive inhibition of tritium-labeled platelet-activating factor binding to rabbit platelet membranes by amiloride and amiloride analogs

Amiloride and its structural analogs, ethylisopropyl amiloride, benzamil, and dichlorobenzamil, inhibit both the specific [3H]C18-PAF binding to rabbit platelet membranes and PAF-induced aggregation of gel-filtered rabbit platelets. Detailed analysis of binding inhibitions demonstrate that ethylisopropyl amiloride is a competitive inhibitor with an equilibrium dissociation constant (KB) of 23 microM. The concentration of amiloride and its analogs needed to inhibit the PAF-induced aggregation is high and there exists no correlation between their inhibitory activities of platelet aggregation and those of Na+/H+ antiporter. However, the inhibitory effects on the PAF-induced aggregation are parallel to those on the specific [3H]C18-PAF binding. The inhibitory effects of amiloride and its analogs on the activation of platelets are at the PAF-receptor binding step, rather than at the Na+/H+ antiporter.     

Potent and prolonged melanotropic activities of the alpha-MSH fragment analog, Ac-[Nle4,D-Phe7]-alpha-MSH4-9-NH2

Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 and Ac-[Nle4]-alpha-MSH4-9-NH2, fragment analogs of the tridecapeptide, alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to alpha-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than alpha-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than alpha-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 was considerably prolonged compared to alpha-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence.     

Structural features of prostanoid analogues involved in hepatocytes protection against CCl4-induced injury

The ability of natural prostaglandins (PGs) and synthetic prostanoids of B, H and E(1)-types to prevent CCl(4)-induced liver injury in vitro was examined. Seven analogs of PGB, 3 derivatives of PGH and two 11-deoxy-analogs of PGE(1) decreased cytotoxic index of CCl(4) by 2-fold and were more effective then such well-known hepatoprotectors as silymarin, curcumin and PGI(2). These prostanoids reduced trien conjugates formation by 25-95% and strongly prevented LDH leakage through plasma membrane. The results from the structure-activity relationship (SAR) analysis can be summarized as follows: (1) the presence of 2-pyridyl at the 15 position of ω-chain plays the main role in cytoprotection for synthetic analogs of PGH; (2) among 11-deoxy-analogs of PGE(1) the most active substances possess the phenyl residue at C-15 connected with C-13 isoxazolin or C-13 isoxazol; (3) C-13 cyclohexylamine or C-15 phenyl groups in ω-chain and metoxy-group in α-chain are important for protective activity of PGB analogs; (4) synthetic analogs of PGB demonstrate more pronounced cytoprotective properties than prostanoids of H and E-types. This information suggests paths for further exploration in the area of hepatoprotection and the design of novel therapeutic agents.     

Conformationally constrained analogs of protein kinase inhibitor (6-22)amide: effect of turn structures in the center of the peptide on inhibition of cAMP-dependent protein kinase.

The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide(Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly- Arg-Arg-Asn- Ala-Ile22-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr6 to Ile11 sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg15 to Ile22. Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J 264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a beta-turn structure might be located in the -Ala12-Ser-Gly-Arg15-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26:7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala12-Ser-Gly14-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D-alanine14 PKI analog was as potent as the parent peptide, whereas the beta-alanine14 and the sarcosine14 analogs were only 10-fold less active. Several peptides that promoted a beta-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a beta-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal beta-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).     

Synthesis and anti-platelet evaluation of 2-benzoylaminobenzoate analogs

Fifty-two 2-benzoylaminobenzoate analogs were synthesized and subjected to anti-platelet aggregation assay using arachidonic acid (AA), collagen (Col), thrombin (Thr), and U46619 as inducers. The results revealed that most of 2-benzoylaminobenzoic acid derivatives showed a selectively inhibitory effect on AA-induced platelet aggregation. As a result of the 2-benzoylaminobenzoic acid derivatives (18, 44, and 46), there were no inhibitory effects on platelet aggregation induced by U46619, but these elicited an inhibitory effect on thromboxane B(2) formation at 1.0microM. These 2-benzoylaminobenzoate analogs were therefore proposed as cyclooxygenase inhibitors.     

Structure–activity relationships in aminosterol antibiotics: The effect of stereochemistry at the 7-OH group

Squalamine and three aminosterol analogs have been shown to inhibit bacterial cell growth and induce lysis of large unilamellar phospholipid vesicles. The analogs differ in the identity of the polyamine attached at C3 of the sterol, and the stereochemistry of a hydroxyl substituent at C7. Analogs with a tetraammonium spermine polyamine are somewhat more active than analogs with a shorter trisammonium spermidine polyamine, and analogs with an axial (α) hydroxyl substituent at C7 are more active than analogs with the corresponding equatorial (β) hydroxyl group. There is some variability noted; the 7β-OH spermine analog is the most active compound against Escherichia coli, but the least effective against Pseudomonas aeruginosa. Lytic activity correlates well with antimicrobial activity of the compounds, but the lytic activity varies with the phospholipid composition of the vesicles.     

Structure-activity relationships of 19-norvitamin D analogs having a fluoroethylidene group at the C-2 position

We have synthesized four new geometric isomers of 1alpha,25-dihydroxy-2-(2'-fluoroethylidene)-19-norvitamin D analogs 1 and 2 having a 20R- and 20S-configuration, whose structures are correlated with 2MD possessing high potencies in stimulating bone formation in vitro and in vivo. The E-isomers of (20R)- and (20S)-2-fluoroethylidene analogs 1a and 1b were comparable with the natural hormone 1alpha,25-(OH)(2)D(3) in binding to the vitamin D receptor (VDR), while two Z-isomers 2a and 2b were about 15-20 times less active than the hormone. In inducing expression of the vitamin D responsive element-based luciferase reporter gene, the E-isomers 1a and 1b were 1.2- and 8.6-fold more potent than the hormone, respectively, while the Z-isomers 2a and 2b had 27-55% of the potency. On the basis of the biological activities and a docking simulation based on X-ray crystallographic analysis of the VDR ligand-binding pocket, the structure-activity relationships of the fluorinated 19-norvitamin D analogs are discussed.     

Conformationally restricted homotryptamines. Part 6: Indole-5-cycloalkyl methylamines as selective serotonin reuptake inhibitors

Racemic 5-(trans-2-aminomethylcyclopropyl)indoles, 5-(trans-2-aminomethylcyclopentyl) indoles, and 5-(cis-2-aminomethylcyclopentyl)indoles were synthesized and evaluated as selective serotonin reuptake inhibitors. These analogs followed SAR trends similar to those previously reported for 3-cycloalkyl substituted indoles. The most potent analogs exhibited single digit nanomolar inhibition at the human serotonin transporter but were 10-fold less active than the previously reported compounds.     

Vasopressin and oxytocin reduce plasma prolactin levels of conscious rats in basal and stress conditions. Study of the characteristics of the receptor involved

Subcutaneous administration of arginine vasopressin (AVP) to conscious rats induced a dose-dependent increase of plasma ACTH and beta-endorphin levels and decrease of plasma prolactin (PRL) levels 30 min later. AVP similarly reduced PRL increase induced by exposure to a novel environment stress. Oxytocin (OT) was also active but 5-fold less potent than AVP. The study of several analogs with specific agonistic and antagonistic activity on the oxytocic, vasopressor and antidiuretic receptors of OT and AVP suggests that the receptor involved in this effect does not fit into this classification.