Kazunori Hanada

Novel epoxysuccinyl peptides Selective inhibitors of cathepsin B, in vitro

A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N‐(L‐3‐trans‐ethoxycarbonyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐030) and N‐(L‐3‐trans‐propylcarbamoyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or n‐propylamide group in the oxirane ring were necessary, the ethyl ester group or the n‐propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.

Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo

New derivatives of E‐64 (compound CA‐030 and CA‐074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K 1 values for cathepsin B were about 2–5 nM, like that of E‐64‐c, whereas their initial K 1 values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA‐030 or CA‐074 into rats, compound CA‐074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA‐030 does not show selectivity for cathepsin B, although both compounds CA‐030 and CA‐074 show complete selectivity for cathepsin B in vitro.

Transformation of vitamin D3 to 1α,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains

To enzymatically synthesize active metabolites of vitamin D3, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D3 to 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] via 25-hyroxyvitamin D3 [25(OH)D3]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D3 to 25(OH)D3 and 1α,25(OH)2D3 in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D3/l culture and 0.17 mg 1α,25(OH)2D3/l culture. A related compound, vitamin D2, could be also converted to 25-hydroxyvitamin D2 and 1α,25-dihydroxyvitamin D2 using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D3 in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D3 and at C-1 of 25(OH)D3 originates from cytochrome P-450.

The susceptibility of Campylobacter pylori to antiulcer agents and antibiotics

The antibacterial activities of antiulcer agents and antibiotics against Campylobacter pylori were studied. The MC90 values of three kinds of antibiotics—macrolides, β-lactams, and metronidazole—were 0.05–0.78, 0.39–1.56, and 12.5 μg/ml, respectively. They were more active than antiulcer agents such as H2blockers and cetraxate with MIC90 values of ≥1,600 and >1,600 μg/ml, respectively. Especially, clarithromycin, a new derivative of erythromycin, showed an MIC90 of 0.05 μg/ml. However, the other antiulcer agents such as sofalcone and tripotassium dicitrate bismuthate (TDB) also had MIC90 values of 50 and 6.25 μg/ml, respectively, Clarithromycin, sofalcone, and TDB showed bactericidal activity against C. pylori CLO2. The bactericidal actions of these drugs could be observed under electron microscopy.

A Novel Bioactive δ lactone FD-211

During our screening program for natural product drugs effective against multidrug-resistant mammalian cells, we have discovered a new delta lactone FD-211 from the fermantation broth of Myceliophthora lutea TF-0409. FD-211 had a broad spectrum activity against cultured tumor cell lines, including adriamycin-resistant HL-60 cells.

Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase

The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.

The importance of Val-157 hydrophobia interaction for papain inhibitory activity of an epoxysuccinyl amino acid derivative: A structure-activity relationship based on the crystal structure of the papain-E-64-c complex

Based on the crystal structure of the papain-E-64-c complex, 3-dimensional binding modes of a series of epoxysuccinyl amino acid derivatives to the papain active site have been constructed and the structure-inhibitory activity relationship has been analyzed using the accessible surface area and nonbonded energy parameters. The result indicates the importance of the hydrophobic interaction between the amino acid side chain of the inhibitor and the papain Val-157 residue for revealing the potent inhibitory activity.Based on the crystal structure of the papain‐E‐64‐c complex, 3‐dimensional binding modes of a series of epoxysuccinyl amino acid derivatives to the papain active site have been constructed and the structure‐inhibitory activity relationship has been analyzed using the accessible surface area and nonbonded energy parameters. The result indicates the importance of the hydrophobic interaction between the amino acid side chain of the inhibitor and the papain Val‐157 residue for revealing the potent inhibitory activity.

Effect of E-64, Thiol Protease Inhibitor, on the Secondary Anti-SRBC Response In Vitro

E‐64, L‐trans‐epoxysuccinyl‐leucylamido (4‐guanidino) butane, a specific inhibitor of thiol proteases originally isolated from the culture of a fungus, was examined in connection with the immune responses to the splenocytes of mice.

Chemoenzymatic synthesis of optically active 1,4-dihydropyridine derivatives via enantioselective hydrolysis and transesterification

Abstract (4 R )-(−)-2-(Nicotinoylamino)ethyl 3-nitrooxypropyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydro-3,5-pyridinedicarboxylate ( 1 ), a new calcium antagonist, was synthesized via both enantioselective hydrolysis and transesterification of prochiral bis[2-(nicotinoylamino)ethyl]ester · 2HCl ( 5 ) by using enzymes. Hydrolysis of 5 by proteases originated from Aspergillus melleus and Bacillus licheniformis etc. in aqueous media afforded (4 R )-(−)-3-[2-(nicotinoylamino)-ethoxycarbonyl]-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-5-carboxylic acid ( 6 ) in more than 99% e.e., which was converted to 1 by esterification with 3-nitrooxypropyl bromide. Enzymatic transesterification of 5 with 3-nitrooxypropanol gave 1 in more than 99.5% e.e. directly.

In vitro and in vivo Antibacterial Activity and Pharmacokinetics of SC-002 and Its Derivative, SC-004: New Oral Cephalosporins

SC-002 is a novel oral cephalosporin possessing a unique thiadiazolylethenyl moiety at the 3 position. In the present study, it was the most active against gram-positive bacteria among oral cephalosporins such as cefdinir (CFDN), cefpodoxime, cefditoren and cefaclor (CCL). It was equal to or 16 times more active than CFDN against standard and clinical strains. In particular, against clinical isolates of Morganella morganii and Haemophilus influenzae, SC-002 was 8–64 times more active than CFDN. The antibacterial activity of SC-002 against some β-lactam-resistant strains was superior to that of CFDN. The in vivo antibacterial activity of SC-004, a pivaloyloxymethyl ester of SC-002, was 1.2–8 times more protective against systemic infections due to Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae than that of CFDN. The therapeutic effects of SC-004 on experimental respiratory tract infections caused by S. pneumoniae or H. influenzae were superior to those of CFDN and CCL. SC-004 showed higher and longer-lasting blood levels and higher urinary excretion in pharmacokinetics in mice.