Seiichi Imajo

Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli.

Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed beta-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to beta-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. The sequence of the gene for Toho-1 was determined, and the open reading frame of the gene consisted of 873 or 876 bases (initial sequence, ATGATG). The nucleotide sequence of the gene (DDBJ accession number D37830) was found to be about 73% homologous to the sequence of the gene encoding a class A beta-lactamase produced by Klebsiella oxytoca E23004. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 290 or 291 amino acid residues, which contained amino acid motifs common to class A beta-lactamases (70SXXK, 130SDN, and 234KTG). Toho-1 was about 83% homologous to the beta-lactamase mediated by the chromosome of K. oxytoca D488 and the beta-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated beta-lactamase Toho-1 produced by E. coli TUH12191 is similar to beta-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A beta-lactamase. Detailed comparison of Toho-1 with other beta-lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity.

Synthesis and biological evaluation of new 4-arylpiperidines and 4-aryl-4-piperidinols: dual Na+ and Ca2+ channel blockers with reduced affinity for dopamine D2 receptors

Abstract A series of novel 4-arylpiperidines and 4-aryl-4-piperidinols ( 2a – f , 3a – f and 4a – f ) was synthesized and evaluated for blocking effects on both neuronal Na + and T-type Ca 2+ channels and binding affinity for dopamine D 2 receptors. Most of the compounds blockaded both ion channels with potency greater than or equal to flunarizine 1a which was adopted as a reference standard. In addition, these compounds had significantly reduced affinity for dopamine D 2 receptors which is common in this class of structure. Compounds 2a – f , 3a – f and 4a – f exhibited potent anticonvulsant effects following systemic (ip) administration on audiogenic seizures in DBA/2 mice, indicating their excellent brain permeability. The neuroprotective activity of 2a , 3a and 4a was also assessed in a transient middle cerebral artery occlusion (MCAO) model. These compounds significantly reduced neuronal damage without affecting ischemic hyperthemia, while flunarizine 1a produced only minor reductions. In particular, 4a had 1.7-fold the potency in this MCAO model but only 1/20 the affinity for dopamine D 2 receptors of 1a . The superposition of 2a , 3a and 4a on the basis of analyses of systematic conformation and similar structure has revealed that the cinnamyl, phenacyl and phenoxypropanol groups are likely to be structurally and biologically equivalent. Moreover, the superposition of 2a and 2f shows that diphenyl ether and biphenyl groups occupy a similar space, suggesting that both groups act as a bioisostere for the blockade of ion channels; however, this is not the case for dopamine D 2 receptors since only biphenyl compounds such as 2f had high affinity similar to flunarizine 1a . Compound 4a (SUN N5030) has a good pharmacological profile and may be useful in the alleviation and treatment of ischemic diseases.

ABSOLUTE CONFIGURATION OF C-1027 CHROMOPHORE

Abstract The absolute configuration of C-1027 chromophore was determined as 8 R , 9 R , 13 R , and 18 S using the modified Moshers, two-dimensional NMR, and molecular modeling methods.

Solution structure of the antitumour antibiotic neocarzinostatin, a chromophore–protein complex

The chromophore-binding structure of the neocarzinostatin complex is determined for the first time by two-dimensional 1H NMR spectroscopy, and reveals the elements of the specific binding and the stabilization interactions of the unstable dienediyne chromophore by the apoprotein.

Proton NMR studies on the chromophore binding structure in neocarzinostatin complex

Abstract NMR studies indicatethe hydrophobic pocket formedby the internalfour-stand β-sheet of apoprotein bindsNCS-chr with the conformation in wich its naphthoate moiety sits on the bottom of the pocket and the aminosugar and the carbonate group face outwards.

A facile conversion of the phenylthio group to acetoxy by copper reagents for a practical synthesis of 4-acetoxyazetidin-2-one derivatives from (R)-butane-1,3-diol

(3S,4R,1′R)-4-Acetoxy-3-(1′-tert-butyldimethylsilyloxyethyl)-azetidin-2-one 8, an important intermediate for the synthesis of penem and carbapenem antibiotics, was synthesized from (R)-butane-1,3-diol 1, using chlorosulphonyl isocyanate for the formation of β-lactam ring in which a significant solvent effect on the ratio of diastereoisomers 6a and 6b was observed; copper(II) acetate rather than the poisonous mercury(II) acetate was used to convert the phenylthio group of compound 6a to acetoxy.

On the conformation of Phe78 of a chromoprotein antibiotic, neocarzinostatin

A structure of neocarzinostatin, an antitumor chromoprotein antibiotic, has been built using X-ray crystallographic data and NMR data, particularly NOE data observed between the apoprotein and the chromophore. Chemical shift changes of protons of the chromophore upon binding to the apoprotein indicated that the aromatic plane of Phe52 has the conformation almost perpendicular to the C-2-C-3 triple bond of the core of the chromophore while Phe78 takes multiple conformations in solution although one of the stable conformations has been assigned for Phe78 in a crystal structure.

A survey of a functional amino acid of class Cβ-lactamase corresponding to Glu166 of class A β-lactamases

The class C β‐lactamase of Cltrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids. The aspartic acid at position 217 and glutamic acid at position 219 in this β‐lactamase were, respectively, previously shown not to be the counterpart of Glu166 (ABL166) in class A β‐lactamases, even though sequence alignment of class A and C enzymes strongly suggested this possibility [(1990) FEBS Lett. 264, 211‐214; (1990) J. Bacteriol. 172, 4348‐4351]. We tried again to assign candidates for the counterpart of Glu166 through sequence alignment based on other criteria, the glutamic acids at positions 195 and 205 in the class C β‐lactamase being selected. To investigate this possibility, these two glutamic acids were changed to glutamine, lysine or alanine, respectively. All the mutant enzymes showed more than 50% of the activity of the wild‐type enzyme, indicating that the possibility was ruled out. These results strongly suggested the possibility that the class C β‐lactamase lacks a functional acidic residue corresponding to Glu166 in class A enzymes.

Discovery and structure–activity relationship study of 1,3,6-trisubstituted 1,4-diazepane-7-ones as novel human kallikrein 7 inhibitors

Compound 1, composed of a 1,3,6-trisubstituted 1,4-diazepane-7-one, was discovered as a novel human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme, SCCE) inhibitor, and its derivatives were synthesized and evaluated. Structure-activity relationship studies of the amidoxime unit and benzoic acid part of this new scaffold led to the identification of 25 and 34, which were more potent than the hit compound, 1. The X-ray co-crystal structure of compound 25 and human KLK7 revealed the characteristic interactions and enabled explanations of the structure-activity relationship.

Discovery of novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as human chymase inhibitors using structure-based drug design.

A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones were explored as human chymase inhibitors using structure-based drug design according to the X-ray cocrystal structure of chymase and compound 1. The optimization focused on the prime site led to the attainment of compounds that showed potent inhibitory activity, and among them, 18R shows a novel interaction mode.