Effect of the orthoquinone moiety in 9,10-phenanthrenequinone on its ability to induce apoptosis in HCT-116 and HL-60 cells.

9,10-Phenanthrenequinone (9,10-PQ) is one of the most abundant quinones among diesel exhaust particulates. Recent data have suggested that quinones induce apoptosis in immune, epithelial and tumor cells, leading to respirator illness; however, the mechanisms by which quinones induce apoptosis and the structure required for this remain unknown. We studied the antitumor activity of 9,10-PQ analogs against two human tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. The loss of the cis-orthoquinone unit in 9,10-PQ abrogated its ability to induce apoptosis in the two tumor cell lines, and the LC50 values of these analogs were indicated over 10 µM. An analog of 9,10-PQ in which the biaryl unit had been deleted displayed a reduced ability to induce tumor cell apoptosis, while the analogs 1,10-phenanthroline-5,6-dione (9) and pyrene-4,5-dione (10), which also had modified biaryl units, exhibited increased tumor cell apoptotic activity. The cis-orthoquinone unit in 9,10-PQ was identified as essential for its ability to induce apoptosis in tumor cells, and its biaryl unit is also considered to influence orthoquinone-mediated apoptotic activity.

Identification of a novel carbohydrate-mimicking octapeptide from chemical peptide library and characterization as selectin inhibitor.

We found a novel octapeptide (H-YRNWFGRW-NH2) mimicking sialyl Lewis X (sLe(X)) carbohydrate from a chemical peptide library with anti-sLe(X) monoclonal antibody (MAb) 2H5. The peptide libraries were constructed by Fmoc-based solid-phase methodology using the mix-split method. The octapeptide sequence was determined by the iterative deconvolution method using anti-sLe(X) MAb 2H5. To define the important residues for interaction with anti-sLe(X) MAb 2H5, alanine-scanning analogues of H-YRNWFGRW-NH2 were synthesized. Substitution of Tyr¹, Trp4, Arg7 and Trp8 to Ala resulted in a marked drop in affinity. This result indicates that aromatic and cationic amino residues have a key role in interacting with anti-sLe(X) MAb 2H5. The binding property of the octapeptide was evaluated with anti-sLe(X) MAb 2H5 and human E-selectin. The octapeptide showed high inhibitory potency (IC50=17.8 nM) for sLe(X) and competitively inhibited the binding of anti-sLe(X) MAb 2H5 in a dose-dependent manner. The octapeptide had high affinity (K(d)=0.168 µM) for E-selectin and this binding was inhibited by sLe(X). These results suggest that octapeptide binds to anti-sLe(X) MAb 2H5 or E-selectin at the sLe(X) binding site and sterically interferes with the recognition of anti-sLe(X) MAb 2H5 or E-selectin with sLe(X). This peptide may be a useful lead compound for an anti-inflammatory agent targeting selectin.

Structural basis for the design of novel Schiff base metal chelate inhibitors of trypsin.

The crystal structures of the complexes of bovine trypsin with m-guanidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 1), [N,N'-bis(m-guanidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 2), and [N,N'-bis(m-amidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 4) have been determined. The guanidine-containing trypsin-inhibitors (1 and 2) bind to the trypsin active site in a manner similar to that previously reported for amidine-containing inhibitors, for example, m-amidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 3). However, the binding mode of the guanidino groups of inhibitors 1 and 2 to Asp189 in the S1 pocket of trypsin was found to be markedly different from that of the amidino group of inhibitor 3. The present X-ray analyses revealed that the interactions of the metal ion of the inhibitors with the active site residues of trypsin play a crucial role in the binding affinity to the trypsin molecule. These structural information and inhibitory activity data for amidine- and guanidine-containing Schiff base metal chelate inhibitors provide new avenues for designing novel inhibitors against physiologically important trypsin-like serine proteases.

Atlantic cod trypsin-catalyzed peptide synthesis with inverse substrates as acyl donor components.

Atlantic cod trypsin-catalyzed peptide synthesis has been studied by using p-amidino- and p-guanidinophenyl esters of N-(tert-butyloxycarbonyl)amino acid as acyl donor components. The reaction temperature was optimized at 0 degrees C. The method was shown to be successful as effectively for synthesizing the peptide and useful for preparing dipeptide between D-amino acid with D-amino acid and beta-amino acid with beta-amino acid, respectively. The enzymatic hydrolysis of the resulting products was negligible.

A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta).

Three anionic salmon trypsin isoforms (CST-1, CST-2 and CST-3) were isolated from the pyloric caeca of chum salmon (Oncorhynchus keta). The order of catalytic efficiency (K(m)/k(cat)) of the isoforms during BAPA hydrolysis was CST-2 > CST-1 > CST-3. In order to find a structural rationalization for the observed difference in catalytic efficiency, the X-ray crystallographic structures of the three isoforms were compared in detail. Some structural differences were observed in the C-terminal alpha-helix, interdomain loop and active-site region. From the results of the detailed comparison, it appears that the structural flexibility of the C-terminal alpha-helix, which interacts with the N-terminal domain, and the substrate-binding pocket in CST-3 are lower than those in CST-1 and CST-2. In addition, the conformation of the catalytic triad (His57, Asp102 and Ser195) differs among the three isoforms. The imidazole N atom of His57 in CST-1 and CST-2 forms a hydrogen bond to the hydroxyl O atom of Ser195, but the distance between the imidazole N atom of His57 and the hydroxyl O atom of Ser195 in CST-3 is too great (3.8 A) for the formation of a hydrogen bond. Thus, the nucleophilicity of the hydroxyl group of Ser195 in CST-3 is weaker than that in CST-1 or CST-2. Furthermore, the electrostatic potential of the substrate-binding pocket in CST-2 is markedly lower than those in CST-1 and CST-3 owing to the negative charges of Asp150, Asp153 and Glu221B that arise from the long-range effect. These results may explain the higher catalytic efficiency of CST-2 compared with CST-1 and CST-3.

Cloning, expression, purification and preliminary crystallographic studies of the adenylate/uridylate-rich element-binding protein HuR complexed with its target RNA.

Adenylate/uridylate-rich elements (AREs), which are found in the 3'-untranslated region (UTR) of many mRNAs, influence the stability of cytoplasmic mRNA. HuR (human antigen R) binds to AREs and regulates various genes. In order to reveal the RNA-recognition mechanism of HuR protein, an RNA-binding region of human HuR containing two N-terminal RNA-recognition motif domains bound to an 11-base RNA fragment has been crystallized. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 42.4, b = 44.9, c = 91.1 A. X-ray diffraction data were collected to 1.8 A resolution.

Crystallization and preliminary x-ray studies of the unliganded wild-type bovine thrombin.

Wild type of bovine thrombin has been crystallized in a ligand-free form by the hanging drop vapor diffusion method with polyethylene glycol 4000 and 2-propanol. The crystals belong to space group P4 3 2 12 with unit cell parameters of a = b = 87.7 A, c = 195.9 A. X-ray diffraction data were collected to 2.8 A resolution.

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin.

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.

Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/µg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

Trypsin-catalyzed synthesis of dipeptide containing alpha-aminoisobutyric acid using p- and m-(amidinomethyl)phenyl esters as acyl donor.

Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An alpha-aminoisobutyric acid containing dipeptide was especially obtained in satisfactory yield. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. The optimum condition for the coupling reaction was studied by changing the organic solvent, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

Kinetic properties of three isoforms of trypsin isolated from the pyloric caeca of chum salmon (Oncorhynchus keta).

Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.8, 5.4, and 5.6, respectively. The apparent K(m) values of two isoforms (ST-1 and ST-2) for BAPA (benzoyl-L-arginine-p-nitroanilide) hydrolysis at 5, 15, 25 and 35 degrees C were slightly higher than that of the main isoform ST-3, depending on temperature. The turnover numbers, k(cat), of ST-1 and ST-2 were about twice as high as that of ST-3. Consequently, the catalytic efficiencies (k(cat)/K(m)) of ST-1 and ST-2 were more efficient than ST-3. There were marked differences in both apparent K(m) and k(cat) values of three anionic chum salmon trypsins as compared to bovine cationic trypsin. K(m) values of all chum salmon trypsins were approximately 10 times lower than those of bovine trypsin, depending on the temperature. The k(cat) values of all chum salmon trypsins were about 2- to 5-fold higher than those of bovine trypsin; therefore, the catalytic efficiencies (k(cat)/K(m)) of chum salmon trypsin were 20- to 40-fold more efficient than those of bovine trypsin. On the other hand, k(cat)/K(m) values of ST-1 for TAME (tosyl-L-arginine methyl ester) hydrolysis were lower than those of bovine trypsin, whereas k(cat)/K(m) values of ST-2 and ST-3 were comparable to those of bovine trypsin, depending on the temperature.

A facile synthesis of p- and m-(amidinomethyl)phenyl esters derived from amino acid and tryptic hydrolysis of these synthetic inverse substrates.

A facile synthetic method for p- and m-(amidinomethyl)phenyl esters derived from a variety of amino acids is presented. We analyzed the kinetic behavior of trypsin towards these synthetic esters, which are inverse substrates. The substituent (meta- and para-isomers) and isosteric effects of (amidinomethyl)phenyl esters are discussed.

Amidino-containing Schiff base copper(II) and iron(III) chelates as a thrombin inhibitor.

Four series of Schiff base copper(II) and iron(III) chelates were synthesized from 4-formyl-3-hydroxybenzamidine or 3-formyl-4-hydroxybenzamidine and various L- or D-amino acids. Their inhibitory activities for bovine alpha-thrombin (abbreviated as thrombin) were determined. The most potent thrombin inhibitor in this series is copper(II) chelate (1g') derived from 4-formyl-3-hydroxybenzamidine and D-Trp. Its Ki value, 2.7x10(-8) M, is comparable to that of Argatroban (MD-805), which is a clinically used compound. The iron(III) chelates derived from 4-formyl-3-hydroxybenzamidine and hydrophobic L-amino acids (Val, Ile, Leu, Phe, Trp, Met) also exhibited higher inhibitory potency. It appears that coordination geometry composed of metal ion, amidino group, amino acid side chain is well accommodated to the thrombin active site. From the Ki values of Schiff base metal chelates for thrombin, the structure-activity relationships between the chelates and active site of thrombin were discussed.

Trypsin-catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, Streptomyces griseus and chum salmon).

A procedure has been developed for the synthesis of oligopeptide amide using inverse substrates as acyl donors with amino acid amide instead of p-nitroanilide as acyl acceptor and trypsins of different origin (bovine, Streptomyces griseus and chum salmon trypsins) as the catalyst. The effectiveness of this procedure was demonstrated by the synthesis of a pentapeptide, Boc-[Leu5]-enkephalin amide, as a model compound. The method was the first enzymatic method shown to be successful at each successive coupling step for the synthesis of the oligopeptide. Bovine and chum salmon trypsins were superior to Streptomyces griseus trypsin as the catalyst.

Synthesis and evaluation of guanidine-containing Schiff base copper(II), zinc(II), and iron(III) chelates as trypsin inhibitors.

3-Formyl-4-hydroxyphenylguanidine hydrochloride and its Schiff base copper(II), zinc(II), and iron(III) chelates were synthesized and their inhibitory activity against bovine beta-trypsin were determined. Syntheses of Schiff base metal chelates were carried out from 3-formyl-4-hydroxyphenylguanidine, various L-amino acids, and divalent metal acetate. Their structures were established on the basis of spectroscopic evidence and elemental analysis. The inhibitory activity of these chelates against bovine beta-trypsin was determined. The guanidine-containing copper(II) and zinc(II) chelates behaved as potent competitive inhibitors of trypsin. However, similar inhibitory activity was not observed for guanidine-containing iron(III) chelates. The inhibition constants (K(i) values, ca. 10(-5) M) of guanidine-containing Schiff base copper(II) and zinc(II) chelates were slightly lower than those (ca. 10(-6) M) of the corresponding amidine-containing Schiff base chelates with regard to bovine trypsin.

Photoregulation of deacylation rate of acyl trypsin derived from photoresponsive inverse substrate.

The acyl trypsin was prepared by use of an inverse substrate, which is comprise of a photoresponsive 4-phenylazobenzoyl moiety. The acyl group in acyl trypsin has been shown to isomerize from trans-form (4t-trypsin) to cis-form (4c-trypsin)/from cis-form to trans-form by irradiation of UV-vis light. The deacylation rate of the cis-form (4c-trypsin) has been shown to be 18.6 times faster than that of the trans-form (4t-trypsin).