Synthetic branched-chain analogues of distearoylphosphatidylcholine: structure-activity relationship in inhibiting and activating protein kinase C

A series of distearoylphosphatidylcholine (DSPC) analogues having various branched alkyl chains were synthesized and tested for their abilities to regulate protein kinase C (PKC). The greatest improvement (about 3-fold) in the PKC inhibitory activity over that seen for the parental lipid (i.e., DSPC) was accomplished by substitution of 8-methylstearate at sn-2 and 16-methylstearate at both sn-1 and sn-2 positions of glycerol; substitutions at both sn-1 and sn-2 with 8-methylstearate, on the other hand, caused a decrease (about 4-fold) in its inhibitory activity. Introduction of butyl, phenyl, or keto functions to various positions in the fatty alkyl chain substituted at both sn-1- and sn-2 positions imparted upon the DSPC analogues an ability to potently stimulate PKC to an extent comparable to those attainable by diacylglycerol or phorbol ester; the analogues having substitution only at the sn-2 position, in comparison, had no or reduced stimulatory activity. The butyl, phenyl, and keto analogues of DSPC, as with DSPC itself and its methyl analogues, inhibited PKC at high concentrations. Kinetic analysis indicated that the methyl DSPC analogues inhibited the enzyme competitively with respect to phosphatidylserine (PS; a phospholipid cofactor) and Ca2+. The butyl analogues activated the enzyme without affecting its affinity for PS or Ca2+, indicating a mechanism different from that seen for diacylglycerol or phorbol ester. The inhibitory activity of the methyl DSPC analogues and the stimulatory activity of the butyl DSPC analogues were reduced when PKC was activated by phorbol ester. Both classes of the analogues were unable to compete for the binding of [3H]phorbol dibutyrate to PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural and synthetic analogues of actinomycin D as Grb2-SH2 domain blockers

Natural analogues (D, C2, and VII) of actinomycin inhibit Grb2 SH2 domain binding with phosphopeptide-derived from Shc in vitro and in intracellular system. To study structure-activity relationships, 13 actinomycin analogues were synthesized and we found that the inhibition activity depended on the substituents of cyclic peptide groups in actinomycin and two analogues with Tyr residue are the most potent inhibitors with IC50 value of 0.5 and 0.8 microM, respectively.     

3-Deazaadenosine analogues of p5'A2'p5'A2'p5'A: synthesis, stereochemistry, and the roles of adenine ring nitrogen-3 in the interaction with RNase L

Sequence-specific 3-deazaadenosine (c(3)A)-substituted analogues of trimeric 2',5'-oligoadenylate, p5'A2'p5'A2'p5'A, were synthesized and evaluated for their ability to activate human RNase L (EC 3.1.2.6) aiming at the elucidation of the nitrogen-3 role in this biochemical process. Substitution of either 5'-terminal or 2'-terminal adenosine with c(3)A afforded the respective analogues p5'(c(3)A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(c(3)A) that were as effective as the natural tetramer itself as activators of RNase L (EC(50)=1nM). In contrast, p5'A2'p5'(c(3)A)2'p5'A showed diminished RNase L activation ability (EC(50)=10nM). The extensive conformational analysis of the c(3)A-substituted core trimers versus the parent natural core trimer by the (1)H and (13)C NMR, and CD spectroscopy displayed close stereochemical similarity between the natural core trimer and (c(3)A)2'p5'A2'p5'A and A2'p5'A2'p5'(c(3)A) analogues, thereby strong evidences for the syn base orientation about the glycosyl bond of the c(3)A residue of the latter were found. On the contrary, an analogue A2'p5'(c(3)A)2'p5'A displayed rather essential deviations from the spatial arrangement of the parent natural core trimer.     

Synthesis of derivatives of (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) modified at the 1-aromatic moiety as novel NMDA receptor antagonists: the aromatic group is essential for the activity

(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, 4a), which is a conformationally restricted analogue of antidepressant milnacipran [(±)-1], is a new class of potent noncompetitive NMDA receptor antagonists. A series of PPDC analogues modified at the 1-phenyl moiety, that is, the analogue 6 lacking 1-phenyl group, the 1-(fluorophenyl) analogues 4b,c,d, the 1-(methylphenyl) analogues 4e–g and the 1-(naphthyl) analogues 4h,i were synthesized. Analogue 6, lacking the 1-phenyl group, was completely inactive showing that the aromatic moiety is essential for the NMDA receptor binding. Among the analogues synthesized, the 1-o-fluorophenyl and 1-m-fluorophenyl analogues 4b and 4c showed potent affinities for the NMDA receptor [IC₅₀=0.16±0.001 μM (4b), 0.15±0.02 μM (4c)], which were improved to some extent compared to those of the parent compound PPDC (IC₅₀=0.20±0.02 μM). On the other hand, compounds 4b and 4c showed none of the 5-HT-uptake inhibitory effect, while PPDC turned out to be a weak 5-HT-uptake inhibitor.     

Study in vitro and in vivo of nociceptin/orphanin FQ(1-13)NH2 analogues substituting N-Me-Gly for Gly2 or Gly3

In the present study, two analogues containing N-Me-Gly (Sarcosine, Sar) were synthesized to further investigate the structural-activity relationships of orphanin FQ/nociceptin (OFQ/NC, NC). The replacement of Gly(2) or Gly(3) with Sar increased the flexibility and decreased the hydrophobicity of the N-terminal tetrapeptide. The activity of the analogues was investigated in a series of assays in vivo and in vitro. [Sar(2)]NC(1-13)NH(2) was found to (1) produce dose-dependent inhibition of the electrically induced contraction in MVD assay (pEC(50) = 6.14); (2) produce significant hyperalgesia effects in a dose-dependent manner when intracerebroventricularly (i.c.v.) injected in mice. The inhibitive effects of [Sar(2)]NC(1-13)NH(2) in MVD assay could be significantly antagonized by [Nphe(1)]NC(1-13)NH(2), and partially antagonized by naloxone; the hyperalgesic effect of [Sar(2)]NC(1-13)NH(2) could be significantly antagonized by naloxone, and partially antagonized by [Nphe(1)]NC(1-13)NH(2). On the contrary, [Sar(3)]NC(1-13)NH(2) showed no effects in these assays. All the findings suggest that the flexibility of the peptide bond between Phe(1) and Gly(2) and between Gly(2) and Gly(3) play an important role in NC-OP(4) receptor interaction, and the hydrophobicity of the N-terminal tetrapeptide showed no significant effect on this interaction. The present work also helps to provide a novel method to elucidate structural and conformational requirements of the opioid peptide-receptor interaction.     

Design and synthesis of new water-soluble tetrazolide derivatives of celecoxib and rofecoxib as selective cyclooxygenase-2 (COX-2) inhibitors

In an attempt to prepare a new water-soluble, parenteral COX-2 inhibitor, rofecoxib (9) and celecoxib (13) analogues were designed and synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors with in vivo anti-inflammatory activity. In this experiment, respective SO(2)Me and SO(2)NH(2) hydrogen-bonding pharmacophores were replaced by a tetrazole ring. Molecular modeling (docking) studies showed that the tetrazole ring of these two analogues (9 and 13) was inserted deep into the secondary pocket of the human COX-2 binding site where it undergoes electrostatic interaction with Arg(513). The rofecoxib (9) and celecoxib (13) analogues exhibited a high in vitro selectivity (9, COX-1 IC(50) = 3.8 nM; COX-2 IC(50) = 1.8 nM; SI = 2.11; 13, COX-1 IC(50) = 4.1 nM; COX-2 IC(50) = 1.9 nM; SI = 2.16) relative to the reference drug celecoxib (COX-1 IC(50) = 3.7 nM; COX-2 IC(50) = .2 nM; SI=1.68) and also showed high aqueous solubility at pH higher than 7 and good anti-inflammatory activity in a carrageenan-induced rat paw edema assay. However, 9 and 13 had no significant damage on gastric mucosa.     

Superoxide dismutase mimetics: synthesis and structure-activity relationship study of MnTBAP analogues

Carboxylic ester and amide-substituted analogues of [5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrinato]manganese(III) chloride (MnTBAP) were synthesized and assayed as potential superoxide dismutase (SOD) mimetics. The tetraester analogues 4a and 4b were found to have comparable SOD activity to the known SOD mimetic MnTBAP, while amides 4c-4e exhibited reduced SOD activity. In the substituted methyl benzoate/acid and disubstituted porphyrin series, analogues 12c, 12f, and 12m were found to have comparable to improved SOD activity relative to MnTBAP and analogues 12j, 13a, and 13d exhibited improved activity in both the SOD and thiobarbituric acid reactive species (TBARS) assays relative to MnTBAP.     

Fluoroanalogues of anti-cytomegalovirus agent cyclopropavir: synthesis and antiviral activity of (E)- and (Z)-9-{[2,2-bis(hydroxymethyl)-3-fluorocyclopropylidene]methyl}-adenines and guanines

Synthesis of fluorinated cyclopropavir analogues 13a, 13b, 14a, and 14b is described starting from alkene 15. Addition of carbene derived from dibromofluoromethane gave bromofluoro cyclopropane 16. Reduction (compound 17) followed by desilylation gave intermediate 18, which was transformed to 2-nitrophenylselenenyl derivative 19. Oxidation to selenoxide 20 was followed by beta-elimination to afford methylenecyclopropane 21. Addition of bromine provided compound 22 for alkylation-elimination of adenine and 2-amino-6-chloropurine. The resultant E,Z isomeric mixtures of methylenecyclopropanes 23a + 24a and 23c + 24c were resolved and the individual isomers were deprotected to give adenine analogues 13a and 14a as well as compounds 13c and 14c. Hydrolytic dechlorination of 13c and 14c furnished guanine analogues 13b and 14b. The only significant antiviral effects were observed with analogue 13a against HCMV and 14a against VZV in cytopathic inhibition assays.     

Synthesis, antiviral, and antitumor activity of 2-substituted purine methylenecyclopropane analogues of nucleosides

The Z- and E-2-fluoro- and 2-chloropurine methylenecyclopropanes 9a,b and 10a,b as well as enantiomeric Z-isoguanine methylenecyclopropanes 11a,b and their phenyl phosphoralaninate pronucleotides 11c,d were synthesized and their antiviral activity against several viruses was evaluated. Fluoro analogues 9a and 10a were active against human cytomegalovirus but they were cytotoxic at approximately the same concentrations. Chloro derivatives 9b and 10b were non-cytotoxic and effective against Epstein-Barr virus in Daudi cells. Isoguanine analogues 11a-d lacked antiviral activity but pronucleotides 11c,d were substrates for porcine liver esterase. From the group of 9a,b and 10a,b, the fluoro analogues 9a and 10a exhibited antitumor activity but only the Z-isomer 9a had a selective effect.     

A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages

Superoxide production in alveolar macrophages is stimulated by agonists which act through Ca2+-mediated (concanavalin A) and/or protein kinase C (phorbol ester or diacylglycerol analogues) -mediated events. Simultaneous addition of saturating concentrations of concanavalin A and a protein kinase C activator (either phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol) caused a supra-additive enhancement of the initial rate of O2-. production. This synergism closely correlated with the known time-course of Ca2+ mobilization induced by concanavalin A; however, it occurred under conditions in which protein kinase C activation is reportedly not Ca2+ dependent. Phorbol ester-induced O2-. production was partially inhibited by the Ca2+ ionophore, A23187. Although phorbol ester-stimulated O2-. production initially was enhanced by concanavalin A, the duration of this O2-. production was reduced in comparison to that induced by phorbol ester alone. These results suggest a dual role for intracellular Ca2+ in both stimulatory and inhibitory regulation of O2-. production.     

Synthesis and Biological Activities of Sugar-Modified 2-(p-n-Butylanilino)-2′-deoxyadenosine Analogues

Abstract

Several sugar-modified 2-(p-n-butylanilino)-2′-deoxyadenosine analogues, including arabino and 2′(R)-azido-2′-deoxy analogues and their 5′-triphosphates were synthesized. These nucleosides thus obtained exhibited moderate cytotoxicity against P-388 leukemic cells in culture (IC₅₀ = 13–24 μ). In contrast to above results, the 5′-triphosphates have been shown to exert strong and selective inhibitory effects on mammalian DNA polymerase α (Ki= 0.02–0.04 μ).     

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH2 with aminophosphonate moiety

New series of N-modified analogues of the N/OFQ(1-13)NH(2) with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH(2) analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH(2), but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH(2) with aminophosphonate moiety was investigated for the first time.     

Short pseudopeptides containing turn scaffolds with high AT2 receptor affinity

Two pentapeptides, Ac-Tyr-Ile-His-Pro-Phe/Ile, were synthesized and shown to have angiotensin II AT2 receptor affinity and agonistic activity. Based on these peptides, a new series of 13 pseudopeptides was synthesized via introduction of five different turn scaffolds replacing the Tyr-Ile amino acid residues. Pharmacological evaluation disclosed subnanomolar affinities for some of these compounds at the AT2 receptor. Substitution of Phe by Ile in this series of ligands enhanced the AT2 receptor affinity of all compounds. These results suggest that the C-terminal amino acid residues can be elaborated on to enhance the AT2 receptor affinity in truncated Ang II analogues.     

Identification and optimisation of a series of substituted 5-pyridin-2-yl-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors

Further investigation of a series of thienyl-based hydroxamic acids that included ADS100380 and ADS102550 led to the identification of the 5-pyridin-2-yl-thiophene-2-hydroxamic acid 3c, which possessed modest HDAC inhibitory activity. Substitution at the 5- and 6-positions of the pyridyl ring of compound 3c provided compounds 5a-g, 7a, b, 9, and 13a. Compound 5b demonstrated improved potency, in vitro DMPK profile, and rat oral bioavailability, compared to ADS102550. Functionalisation of the pendent phenyl group of compounds 5b, 5e and 13a provided analogues that possessed excellent enzyme inhibition and anti-proliferative activity.     

Activation of a keratinocyte phospholipase A2 by bradykinin and 4 beta-phorbol 12-myristate 13-acetate. Evidence for a receptor-GTP-binding protein versus a protein-kinase-C mediated mechanism.

The release of arachidonic acid from cellular phospholipids and its subsequent conversion to eicosanoids is the common early response of skin keratinocytes to a wide variety of exogenous or endogenous agonists including irritant skin mitogens such as the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA) or the inflammatory peptide bradykinin. In mouse keratinocytes labeled with [14C]arachidonic acid, both PMA and bradykinin induced the release of the fatty acid in a dose-dependent and time-dependent manner. Three lines of evidence indicate phospholipase A2 activity to be involved in arachidonic acid release: (a) its inhibition by mepacrine, (b) the concomitant generation of lysophosphatidylcholine from [3H]choline-labeled cells and (c) an increase in arachidonic acid release from 14C-labeled phosphatidylcholine in particulate fractions from PMA-treated and bradykinin-treated keratinocytes. Inhibition or down regulation of protein kinase C (PKC) led to a suppression of PMA-induced but not bradykinin-induced arachidonic acid release, indicating a critical involvement of this kinase in phorbol-ester-induced activation of epidermal phospholipase A2 activity. Bradykinin-induced activation of phospholipase A2 was however, shown to be mediated by specific B2 receptors coupled to GTP-binding proteins (G protein). In support of this mechanism it was demonstrated that the bradykinin-induced phospholipase A2 activity was increased in the presence of non-hydrolysable GTP but decreased upon addition of non-hydrolysable GDP analogues. Moreover, cholera toxin stimulated both basal and bradykinin-induced phospholipase A2 activity in a cAMP-independent manner, whereas pertussis toxin was found to be inactive in this respect. The data suggest that epidermal phospholipase A2 activity can be stimulated by bradykinin via a B2 receptor-G-protein-dependent pathway, which is independent of PKC and a PKC-dependent pathway which is activated by phorbol esters such as PMA.     

Adenosine 3',5'-cyclic monophosphate (cAMP) inhibits phorbol ester-induced growth of an IL-2-dependent T cell line

We previously established a human T cell line, TPA-Mat, which can proliferate in response to not only interleukin-2 (IL-2), but also phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu). The present study demonstrated that the PDBu-dependent growth of TPA-Mat cells was inhibited up to 90% by adenosine 3',5'-cyclic monophosphate (cAMP] raising agents such as forskolin, cholera toxin and 1-methyl-3-isobutyl-xanthine, and cAMP analogues, whereas the IL-2-stimulated TPA-Mat growth was slightly inhibited. These findings suggest that the signal transduction pathway of PDBu-induced growth, which should involve activation of protein kinase C, is sensitive to cAMP, and that it cannot be exactly identical to the signal transduction pathway of Il-2-induced growth in TPA-Mat cells.     

Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints

ADAM17, also known as tumor necrosis factor α-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) → Glu(708) mutation and the Arg(708)Arg(710) → Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.     

Acute effects of tumor-promoting phorbol esters on hepatic intermediary metabolism

In hepatocytes isolated from meal-fed rats, phorbol 12-myristate 13-acetate as well as phorbol 12,13-didecanoate stimulated de novo fatty acid synthesis in a dose-dependent manner. Moreover, phorbol 12-myristate 13-acetate inhibited ketogenesis from exogenous oleate, but slightly enhanced oleate esterification. The stimulation of esterification was more pronounced with endogenously synthesized fatty acids. In hepatocytes from 24h-starved rats a moderate stimulation of gluconeogenesis and ureogenesis was observed with glutamine as substrate. It is concluded that tumor-promoting phorbol esters mimick the short-term effects of insulin on hepatic fatty acid metabolism.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.