Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

Comparative analysis of two different amide-to-ester bond mutations in the beta-sheet of 4-oxalocrotonate tautomerase

Here we describe the total chemical synthesis and biophysical characterization of two backbone-modified, ester bond-containing analogues of the homohexameric enzyme 4-oxalocrotonate tautomerase (4OT). The amide-to-ester bond mutations in the two analogues in this study, (OI2)4OT and (OI7)4OT, were designed to effectively delete specific backbone-backbone hydrogen bonds in the beta-sheet region of the native 4OT hexamer. The (OI2)4OT and (OI7)4OT analogues each contained one ester bond per monomer that effectively deleted 12 backbone-backbone hydrogen bonds per hexamer. The structural properties of each analogue were characterized by size-exclusion chromatography (SEC), far-UV CD spectroscopy, and catalytic activity measurements, and they were found to be very similar to the structural properties of the wild-type enzyme. The results of equilibrium unfolding studies revealed that the (OI2)4OT and (OI7)4OT analogues were stabilized by 47.7 +/- 2.5 and 45.0 +/- 2.5 kcal/mol, respectively, under standard state conditions (1 M hexamer) as compared to a value of 69.6 +/- 3.3 kcal/mol for the wild-type control. Our results suggest that the two different, but structurally similar, backbone-backbone hydrogen bonds deleted in (OI2)4OT and (OI7)4OT make nearly equivalent contributions to the thermodynamic stability of the 4OT hexamer.     

Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation

A wide range of piperine analogues has been synthesised in order to undertake a structure-activity study of their ability to stimulate melanocyte proliferation. Results demonstrate that an aromatic ring containing at least one ether function and a carbonyl group containing side chain is essential for this activity. A number of highly active piperine analogues have been identified, for instance 1-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienoic acid methyl ester (5a), 1-E,E-piperinoyl-isobutylamine (4f) and 1-(3,4-methylenedioxyphenyl)-pentanoic acid cyclohexyl amide (20). A selection of analogues has also been evaluated for their effect on melanocyte morphology and melanogenesis. The piperine analogues altered cell morphology by increasing dendrite formation leading to bi-, tri- and quadripolar cells. These same analogues were found to increase total melanin in cell cultures, although melanin content per cell was not significantly altered from control in the presence of these compounds.     

Synthesis and solution conformation studies of the modified nucleoside N(4),2'-O-dimethylcytidine (m(4)Cm) and its analogues

The dimethylated ribosomal nucleoside m(4)Cm and its monomethylated analogues Cm and m(4)C were synthesized. The conformations (syn vs anti) of the three modified nucleosides and cytidine were determined by CD and 1D NOE difference spectroscopy. The ribose sugar puckers were determined by the use of proton coupling constants. The position of modification (e.g., O vs N methylation) was found to have an effect on the sugar pucker of cytidine.     

Synthesis and anti-HIV activities of symmetrical dicarboxylate esters of dinucleoside reverse transcriptase inhibitors

Three nucleoside analogues, 3'-fluoro-2',3'-dideoxythymidine (FLT), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-dideoxy-3'-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC(50) values of 0.8-1.0nM and 3-4nM against HIV-1(US/92/727) and HIV-1(IIIB) cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC(50)=3-60nM) was improved by 1.5-66 fold when compared to 3TC (EC(50)=90-200nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile.     

Synthesis of (+/-)-4'-ethynyl and 4'-cyano carbocyclic analogues of stavudine (d4T)

The synthesis of (+/-)-4'-ethynyl (8) and 4'-cyano (9) carbocyclic analogues of the anti-HIV agent stavudine (5, d4T) is reported. The carbocyclic unit (16) was constructed from readily available beta-keto ester 10. The ethynyl or cyano group of 8 and 9 were prepared, after the introduction of thymine base to 16, by manipulation of the ester function. Evaluation of the anti-HIV activity of 8 and 9 was also carried out.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

Thiosugars. XII. Synthesis of new 3'-O-substituted 2',5'-anhydro-2'-thio-alpha-D-pentofuranosyl nucleoside analogues

Methyl 2,5-anhydro-3-O-(2-methoxyethyl)-2-thio-beta-D-arabinofuranoside and methyl 2,5-anhydro-3-O-(2-fluorobenzyl)-2-thio-alpha-D-lyxofuranoside were transformed into the corresponding uridine, thymidine, cytidine and adenosine analogues, which exclusively exhibited the alpha-configuration irrespective of the anomeric configuration of the donor. The structure, configuration, and conformation of the products was elucidated by X-ray structure analyses. The nucleoside analogues were tested for antiviral activities.     

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)     

Acyclic nucleoside/nucleotide analogues with an imidazole ring skeleton

Syntheses of a few acyclic nucleoside and acyclic nucleoside phosphonate analogues containing an imidazole ring have been reported. These analogues include methyl 1-(2-hydroxyethoxymethyl)imidazole-4, 5-dicarbo-xylate (1), 4,5-dicarbamoyl-1-(2-hydroxyethoxymethyl)imidazole (2), 4,5-dicyano-1-(2-hydroxyethoxymethyl)imidazole (4), Methyl 1-(2-bromoethoxymethyl)imidazole-4,5-dicarboxylate (7), 4,5-dicyano-(2-bromoethoxymethyl)imidazole (8), and Methyl 1-(2-phosphonomethoxyethyl)imidazole (10). Also reported are a few potential prodrugs of the above compounds, including the acetyl derivatives 5 and 6 (of 1 and 4, respectively), and the diethyl phosphonate ester 9 (of 10). In addition, the corresponding benzyl-protected precursors 11 and 12 (of 1 and 4, respectively), along with their common hydrolysis product, 1-(2-benzyloxy-ethoxymethyl)-4,5-imidazoledicarboxylic acid (3), are reported. Another potential prodrug included in the list is 1-(2-acetoxyethyl)-4,5-dicyanoimidazole (15). The compounds were screened for in vitro antiviral activity against a wide variety of herpes and respiratory viruses. The most active compound was the phosphonate analogue 9 which exhibited an anti-measles virus activity with an EC50 of <2.5 microg/mL and an SI value of > 176.     

Effects of 5-azacytidine and 5-aza-2-deoxycytidine on alphafetoprotein levels in mice.

1. Production of alphafetoprotein in adult C3H mice was monitored by radial immunodiffusion both in controls, and in animals treated with carbon tetrachloride, 5-azacytidine, or 5-aza-2-deoxycytidine, either alone or in combination. 2. Carbon tetrachloride routinely induced alphafetoprotein synthesis in our experiments, but neither of the cytidine analogues showed any effects on the serum levels of this protein when administered alone. 3. Treatment of mice with either cytidine analogue prior to carbon tetrachloride injection markedly reduced the consequent production of alphafetoprotein, whereas if carbon tetrachloride injection was followed by a subsequent injection with either cytidine analogue, a markedly enhanced level of serum alphafetoprotein was detected. 4. It is suggested that carbon tetrachloride induces alphafetoprotein production in adult mice by inducing liver damage, followed by synthesis of the protein in the dividing and differentiating cells during recovery. We also propose that the cytidine analogues ablate this response by a cytotoxic effect on the liver cells when they are administered prior to the CCl4, but enhance the alphafetoprotein levels when administered after the CCl4 because they inhibit the methylation of cytidine residues in the recovery cell population in the liver and thus prevent early cessation of synthesis of the protein.     

Structure-activity relationship of synthetic truncated analogues of vasoactive intestinal peptide (VIP): an enhancement in the activity by a substitution with arginine

In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.     

Selective antiproliferative activity of caffeic acid phenethyl ester analogues on highly liver-metastatic murine colon 26-L5 carcinoma cell line

Caffeic acid phenethyl ester (CAPE, 2) and its twenty analogues (1, 3-21) were prepared. These esters were tested by MTT assay on growth of murine colon 26-L5 carcinoma, murine B16-BL6 malonoma, murine Lewis lung carcinoma, human HT-1080 fibrosarcoma, human lung A549 adenocarcinoma, and human cervix HeLa adenocarcinoma cell lines. It was found that CAPE analogues possessed selective antiproliferative activity toward highly liver-metastatic murine colon 26-L5 carcinoma cell line. Among them, 4-phenylbutyl caffeate (4), (Z)-8-phenyl-7-octenyl (10a) and (E)-8-phenyl-7-octenyl (10b) caffeate showed the most potent antiproliferative activity (EC50 value, 0.02 microM). In addition, CAPE (2) induced DNA fragmentation at concentrations of 1 to 10 microg/mL towards murine colon 26-L5 carcinoma cells.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

RNA degradation in perfused rat liver as determined from the release of [14C]cytidine

The degradation of RNA in the cyclically perfused rat liver was determined from the release of labeled cytidine from RNA that had been previously labeled with [6-14C]orotic acid in vivo. Because cytidine is not appreciably degraded in rat liver (its deamination to uridine is virtually nil) or produced in significant amounts from free 5'-nucleotides, its release will directly reflect net RNA breakdown. This conclusion was substantiated by the fact that the specific radioactivity of released cytidine equaled that of CMP in RNA and remained unchanged for 180 min of perfusion. The initial rate of [14C]cytidine accumulation was slow, but after 10-20 min it increased abruptly by more than 4-fold and remained virtually constant. The addition of 0.5 mM unlabeled cytidine effectively prevented the reutilization of label and increased the rate of labeled cytidine release by an amount representing 13% of the maximal rate of cytidine accumulation. Rates of RNA degradation, measured between 20 and 60 min in the presence of 0.5 mM unlabeled cytidine, averaged 1.00 +/- 0.05 mg h-1 liver-1 (100-g rat), the equivalent of 65% of total RNA per day. This accelerated value, which was about 4-fold larger than the initial rate, is believed to be the direct consequence of amino acid deprivation since, in separate experiments, the increase was completely suppressed by the addition of plasma amino acids (Lardeux, B. R., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14514-14519). These findings demonstrate the potential value of cytidine as a marker for following moment-to-moment regulatory alterations in RNA degradation in the isolated liver or hepatocyte preparation.     

Functional Studies with Substance P Analogues: Effects of N-Terminal, C-Terminal, and C-Terminus-Extended Analogues of Substance P on Nicotine-Induced Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10^-5M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr¹²-NH₂, 65% by (Ala³)SP-NH₂, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 ×; 10^-5M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr¹²-NH₂ > (Ala³)SP-NH₂ > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala³)SP-NH₂ protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 ×; 10^-5M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr¹²-NH₂ at 3 × 10^-5M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH₂, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine-evoked secretion and protection against nicotinic desensitization, are regulated independently.     

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

Synthesis and antimycobacterial activities of ring-substituted quinolinecarboxylic acid/ester analogues. Part 1

Structural optimization of recently discovered new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC= 6.25 microg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of ring-substituted quinolinecarboxylic acids/esters constituting 45 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against M. tuberculosis H37Rv. Certain ring-substituted-2-quinolinecarboxylic acid ester and ring-substituted-2-quinoline acetic acid ester analogues described herein showed moderate to good inhibitory activity. In particular, three analogues methyl 4,5-dicyclopentyl-2-quinolinecarboxylate (3b), methyl 4,8-dicyclopentyl-2-quinolinecarboxylate (3c) and ethyl 2-(2,8-dicyclopentyl-4-quinolyl)acetate (14g) exhibited excellent MIC values of 1.00, 2.00 and 4.00microg/mL, respectively. Results obtained indicate that substitution of the quinoline ring with dicyclopentyl substituent presumably enhances the antimycobacterial activities in the quinoline analogues described herein.