Diversity of Cyclic Ureide Compound-, Dihydropyrimidine-, and Hydantoin-hydrolyzing Enzymes in Blastobacter sp. A17p-4

Two cyclic ureide compound-hydrolyzing enzymes were found in Blastohacter sp. A17p-4, and partially purified. One hydrolyzed 5-substituted hydantoins D-stereospecifically and had dihydropyrimidinase activity. The other was a novel enzyme which should be called an imidase. The imidase preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds, and produced monoamidated dicarboxylates.     

Evaluation of apoptotic effect of cyclic imide derivatives on murine B16F10 melanoma cells

Cyclic imides are a large class of compounds obtained by organic synthesis including several sub-classes (succinimides maleimide, glutarimide, phthalimides naphtalimides, and its derivatives). Recently, some cyclic imide derivatives have shown important results as potential antitumor agents, as a Mitonafide and Amonafide. Based on this fact, we have studied antitumoral properties of nine cyclic imide derivatives, four of which are unpublished compounds, against Murine Melanoma Cells (B16F10). Initially, the MTT assay was used to select the compound with the best cytotoxic potential. After this selection, the compound 2-benzyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (4), which showed the best cytotoxic effects, was evaluated by flow cytometry, and a significant increase was observed in the proportion of cells in the subG0/G1, S and G2/M phases accompanied by a significant decrease in the G0/G1 phases. Then the mechanism involved on the death route (necrosis or apoptosis) was evaluated the by bromide and acridine orange method and by an Annexin V-FITC Apoptosis Detection kit. These results confirm that the percentage of B16F10 cells observed in the sub G0/G1 phase were undergoing apoptosis. The biological effects observed in the current study for the cyclic imide derivatives suggested promising applications, especially for the prototype compound 4.     

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.     

A novel amidase (half-amidase) for half-amide hydrolysis involved in the bacterial metabolism of cyclic imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K(m) = 6.2 mM; k(cat) = 5.76 s(-1)) and glutaramic acid (K(m) = 2.8 mM; k(cat) = 2.23 s(-1)). However, the substrates of known amidases such as short-chain (C(2) to C(4)) aliphatic amides, long-chain (above C(16)) aliphatic amides, amino acid amides, aliphatic diamides, alpha-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M(r) of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Structure-intrinsic activity relationship studies in the group of 1-imido/amido substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives; new, potent 5-HT1A receptor agents with anxiolytic- like activity

Introduction of 1,4-disubstituted cyclohexane ring in the structure of flexible long chain arylpiperazines resulted in linearly constrained, potent serotonin (5-HT)(1A) ligands. In order to trace structure-intrinsic activity relationships in this group, a new series of 1-substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives with different cyclic imide/amide termini, and their flexible, tetramethylene analogues were synthesized and pharmacologically evaluated for 5-HT(1A) receptors. In vitro binding experiments revealed that all the compounds were potent 5-HT(1A) receptor agents (K(i) = 1.9-74 nM). Some derivatives tested additionally showed also high affinity for alpha(1)-adrenergic receptors (K(i) = 2.9-101 nM) and for 5-HT(7) receptors. Functional in vivo examination revealed that rigid ligands with o-OCH(3) group in the aryl moiety and cyclic imide system in the opposite terminal behaved like postsynaptic 5-HT(1A) receptor antagonists. On the other hand, unsubstituted, m-Cl, or m-CF(3) substituted derivatives as well as those with cyclic amide group in the terminal fragment exhibited agonistic or partial agonistic activity. Three out of four derivatives tested, that is, postsynaptic 5-HT(1A) antagonists 9 and 10, and partial agonist 16, showed anxiolytic-like activity in the conflict drinking (Vogel) test in rats.     

Design and synthesis of 6-amino-1,4-oxazepane-3,5-dione derivatives as novel broad spectrum anticonvulsants

Based on the structural estimations of the typical anticonvulsant drugs, a series of 6-amino-1,4-oxazepane-3,5-dione derivatives, novel structures of 7-membered heterocyclic imides, which were hybridized with pharmacophores such as cyclic imide and N-CO-C-N group in their molecule were designed and synthesized. Their anticonvulsant activities were evaluated by the maximal electroshock induced seizure (MES) and subcutaneous pentylenetetrazole (PTZ) tests. Almost all the designed compounds except 1c and 1f showed comparable anticonvulsant activities in at least one of the anticonvulsant tests. Moreover, some of the tested compounds exhibited moderate anticonvulsant activities in both MES and PTZ tests. From these results, 6-amino-1,4-oxazepane-3,5-dione derivatives could be recommended as novel structures of broad spectrum anticonvulsants.     

Selective deamidation and enzymatic methylation of seminal ribonuclease

Isoenzymatic forms alpha 2, alpha beta, and beta 2 of bovine seminal ribonuclease are generated by the transformation of beta-type into alpha-type subunit through deamidation of a single amide group [Di Donato, A., & D'Alessio, G. (1981) Biochemistry 20, 7232-7237]. The residue involved in this selective deamidation has been identified as Asn67. Deamidation occurs by formation of a cyclic imide intermediate involving the Gly at position 68. Opening of the cyclic imide may occur on either side of the nitrogen, generating both the normal alpha-aspartyl and an isoaspartyl residue at position 67. The alpha-carboxyl of the isoaspartyl residue is effectively methylated by bovine brain protein carboxylmethyltransferase.     

Synthesis and antidepressant-like activity evaluation of sulphonamides and sulphonyl-hydrazones

In this study a series of sulphonamides and sulphonyl hydrazones of maleimide, naphthalimide and phthalimide derivatives was synthesized. The antidepressant effect of these compounds was evaluated by the forced-swimming test in mice. The behavioural parameter observed in this test is a reduction in the immobility time, which is indicative of antidepressant activity. All compounds, except 8, 11 and 24, were active as antidepressants. The most active compound was the sulphonyl-hydrazone 10 which showed an activity of around 72.02% at 60 mg/kg, it thus being more active than imipramine (10mg/kg, ip), a commercial antidepressant. Other important results were obtained for the benzylnaphthalimide derivatives, the sulphonamides 21 and 22 showing activity of 64% at 10mg/kg, also being more active than imipramine. These results indicate that the sulphonamides and sulphonyl-hydrazone cyclic imide derivatives are potential compounds for use in the designing of new candidates for the treatment of depression.     

突变型环酰亚胺水解酶的底物专一性

摘要 目的：研究环酰亚胺水解酶（CIH293）C-末端区残基对其底物专一性的影响。方法：通过缺失或替代获得了环酰亚胺水解酶C-末端剔除2个或3个氨基酸残基及C-末端两个Lys替代为两个Glu的突变型酶CIH291、CIH290以及KK292,293EE,用比色法与高效液相色谱法分析了重组野生型酶与突变型酶的底物专一性和动力学参数。结果：突变型酶与野生型酶相比,底物专一性未发生显著改变,最适底物仍为琥珀酰亚胺,然突变型酶对最适底物的亲和力略有降低,导致反应速度减小。结论：环酰亚胺水解酶（CIH293）C-末端区残基的改变对其底物专一性的影响不大,但影响了酶对底物的亲和力。     

Design, synthesis, and evaluation of cyclic amide/imide-bearing hydroxamic acid derivatives as class-selective histone deacetylase (HDAC) inhibitors

A series of hydroxamic acid derivatives bearing a cyclic amide/imide group as a linker and/or cap structure, prepared during our structural development studies based on thalidomide, showed class-selective potent histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that the steric character of the substituent introduced at the cyclic amide/imide nitrogen atom, the presence of the amide/imide carbonyl group, the hydroxamic acid structure, the shape of the linking group, and the distance between the zinc-binding hydroxamic acid group and the cap structure are all important for HDAC-inhibitory activity and class selectivity. A representative compound (30w) showed potent p21 promoter activity, comparable with that of trichostatin A (TSA), and its cytostatic activity against cells of the human prostate cell line LNCaP was more potent than that of the well-known HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA).     

Carbonic anhydrase inhibitory activity of sulfonamides and carboxylic acids incorporating cyclic imide scaffolds

A series of sulfonamides incorporating cyclic imide moieties were investigated as inhibitors of several human α-carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. Several carboxylic acids possessing the same scaffolds as the sulfonamides were also included in the study, since the sulfonamidate and the carboxylate are among the frequently used zinc-binding groups (ZBGs) for obtaining zinc enzymes inhibitors. The cytosolic isoform hCA I was moderately inhibited by most of the 30 investigated derivatives; many low nanomolar hCA II inhibitors were detected, whereas some of these compounds were low nanomolar/subnanomolar inhibitors of the transmembrane, tumor-associated isoforms hCA IX and XII. In this series of compounds the SO₂NH⁻ and the COO⁻ ZBGs showed similar efficacy for obtaining potent inhibitors, although some carboxylates had isoform-selective inhibition profiles for the transmembrane CAs.     

Distribution of cyclic imide-transforming activity in microorganisms

Cyclic imide-transforming activity was found to be widely distributed in bacteria, yeast and molds. This activity was not correlated with cyclic ureide-transforming activity in bacteria, but there was some correlation in yeast and molds. These two activities are probably catalyzed by different enzymes in bacteria. Besides the well-known cyclic ureide transformation, cyclic imide transformation by microorganisms was common.     

Synthesis of α-aspartyl-containing cyclic peptides

Summary α-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.     

Novel Metabolic Transformation Pathway for Cyclic Imides in Blastobacter sp. Strain A17p-4

The metabolic transformation pathway for cyclic imides in microorganisms was studied in Blastobacter sp. strain A17p-4. This novel pathway involves, in turn, hydrolytic ring opening of a cyclic imide to yield a monoamidated dicarboxylate, hydrolytic deamidation of the monoamidated dicarboxylate to yield a dicarboxylate, and dicarboxylate transformation similar to that in the tricarboxylic acid cycle. The initial step is catalyzed by a novel enzyme, imidase. Imidase and subsequent enzymes involved in this metabolic pathway are induced by some cyclic imides, such as succinimide and glutarimide. Induced cells metabolize various cyclic imides.     

Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover

At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure.     

A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K_m = 6.2 mM; k_cat = 5.76 s⁻¹) and glutaramic acid (K_m = 2.8 mM; k_cat = 2.23 s⁻¹). However, the substrates of known amidases such as short-chain (C₂ to C₄) aliphatic amides, long-chain (above C₁₆) aliphatic amides, amino acid amides, aliphatic diamides, α-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M_r of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Cyclic-imide-hydrolyzing activity of D-hydantoinase from Blastobacter sp. strain A17p-4.

The cyclic-imide-hydrolyzing activity of a prokaryotic cyclic-ureide-hydrolyzing enzyme, D-hydantoinase, was investigated. The enzyme hydrolyzed cyclic imides with bulky substituents such as 2-methylsuccinimide, 2-phenylsuccinimide, phthalimide, and 3,4-pyridine dicarboximide to the corresponding half-amides. However, simple cyclic imides without substituents, which are substrates of imidase (ie.g., succinimide, glutarimide, and sulfur-containing cyclic imides such as 2,4-thiazolidinedione and rhodanine), were not hydrolyzed. The combined catalytic actions of bacterial D-hydantoinase and imidase can cover the function of a single mammalian enzyme, dihydropyrimidinase. Prokaryotic D-hydantoinase also catalyzed the dehyrative cyclization of the half-amide phthalamidic acid to the corresponding cyclic imide, phthalimide. The reversible hydrolysis of cyclic imides shown by prokaryotic D-hydantoinase suggested that, in addition to pyrimidine metabolism, it may also function in cyclic-imide metabolism.     

Synthesis and biological evaluation of rebeccamycin analogues modified at the imide moiety

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of antitumour drugs. In the course of our structure-activity relationship studies, new rebeccamycin analogues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4 (small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the A2780 xenograft model was also investigated.     

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.