Hironobu Iinuma

Synthesis and Activity of Tyropeptin A Derivatives as Potent and Selective Inhibitors of Mammalian 20S Proteasome

Tyropeptin A, a potent proteasome inhibitor, was isolated from the culture broth of Kitasatospora sp. MK993-dF2. We synthesized the derivatives of tyropeptin A to enhance its inhibitory potency. Among the synthesized derivatives, the most potent compound, TP-104, exhibited a 20-fold inhibitory potency enhancement for chymotrypsin-like activity of 20S proteasome compared to tyropeptin A. Additionally, TP-110 specifically inhibited the chymotrypsin-like activity, but did not inhibit the post-glutamyl-peptide hydrolyzing (PGPH) and the trypsin-like activities of 20S proteasome. In vitro TP-110 strongly inhibited the growth of various cell lines.

Inhibition of glucocerebrosidase and induction of neural abnormality by cyclophellitol in mice

Cyclophellitol, a cyclitol with an epoxide, is a novel microbial secondary metabolite that inhibits beta-glucosidase and beta-glucocerebrosidase. Daily administration of cyclophellitol induces a severe abnormality of the nervous system in mice while it has no toxicity in various cultured cells. It was shown to inhibit glucocerebrosidase in vivo significantly in mice and the content of glucocerebroside in liver, spleen, and brain was increased markedly. The enzyme activity was completely suppressed in brain, liver, spleen, kidney, and muscle. On the other hand hexosaminidase activity was not affected in all tissues. After a single administration of cyclophellitol the maximal inhibition of glucocerebrosidase was observed within 30 min in brain and liver, and the inhibition lasted for 2-4 days. A single administration of cyclophellitol also induced a severe abnormality of the nervous system known as Gauchers-like disease in mice. Conduritol B epoxide is also known to inhibit glucocerebrosidase and induce Gauchers like-disease in mice by repetitive injection. Cyclophellitol was shown to be more potent than conduritol B epoxide in inhibition of glucocerebrosidase and in induction of the neural abnormality.

Inhibition of proteasome activity by tyropeptin A in PC12 cells.

Tyropeptin A, a potent proteasome inhibitor not reported before, was produced by Kitasatospora sp. MK993-dF2. In this study, we investigated the effects of tyropeptin A on proteasome activity in PC12 cells. Tyropeptin A inhibited the intracellular proteasome activity in a dose-dependent way and seemed to cause neurite outgrowth. As expected, ubiquitinated proteins that should be substrates for the proteasome accumulated in cells treated with tyropeptin A. Hence, it appears that tyropeptin A can permeate into cells and there inhibit the intracellular proteasome activity.

Bispolides, novel 20-membered ring macrodiolide antibiotics from microbispora.

Seven new related compounds named bispolides A1, A2, A3, B1, B2a, B2b and B3, have been found in a culture of Microbispora sp. A34030, isolated from a Malaysian soil sample. The planar structures were elucidated to be new 20-membered ring macrodiolide antibiotics on the basis of MS and NMR spectroscopic analyses. These antibiotics showed a good anti-MRSA activity in vitro.

A novel cyclic lipoundecapeptide, pholipeptin, isolated from Pseudomonas sp.

An inhibitor of phosphatidylinositol-specific phospholipase C, pholipeptin, was purified from the culture broth of Pseudomonas sp. Structural determination by 2D NMR spectroscopy with addition of a small amount of trifluoroacetic acid revealed that it was a novel cyclic lipodepsipeptide (1) consisting of 11 amino acids and a 3-hydroxydecanoic acid moiety.

In vitro and in vivo antiprotozoal activities of bispolides and their derivatives

During the course of our screening program to discover new antiprotozoal (antimalarial and antitrypanosomal) chemicals, we have evaluated isolates from soil microorganisms, as well as compounds from the antibiotic libraries of the Kitasato Institute for Life Sciences and Nimura Genetic Solutions. We have previously reported various microbial metabolites exhibiting potent antimalarial1–4 and antitrypanosomal properties.5–7 We have recently found that the known 20-membered ring macrodiolide antibiotics, the bispolides,8 together with new derivatives, exhibit selective antitrypanosomal and potent antimalarial activities, both in vitro and in vivo. Here, we report the antitrypanosomal and antimalarial profiles of bispolides, their derivatives (Figure 1) and the related 16-membered ring macrodiolide antibiotic as elaiophylin (azalomycin B)9,10 (Figure 1) in comparison with those of clinically used antitrypanosomal drugs, such as suramin and eflornithine, and two clinically used antimalarial drugs, artemisinin and chloroquine. We also present some conclusions on structure–activity relationships. Bispolides A1, A3, B1 and B3 were purified from the culture broth of Microbispora sp. A34030.8 Derivatives of bispolide A1 (derivatives I and II) were prepared as follows. The 13,13¢-dimethoxy compound, bispolide A3, prepared from bispolide A1 by reaction with 0.04%-HCl in MeOH, was reduced by NaBH3CN in EtOH to give the 13,13¢dideoxy compound (derivative I (ESI-MS m/z 1123.69114; C62 H100 Na1 O16)). Partial hydrolysis of derivative I in the presence of p-toluenesulfonic acid in aqueous acetonitrile gave derivative II (FAB-MS m/z 841 (M+)). Elaiophylin was obtained from the antibiotic library of the Kitasato Institute for Life Sciences. In vitro antiprotozoal activities against Trypanosoma brucei brucei strain GUTat 3.1, Plasmodium falciparum strains K1 (drugresistant) and FCR3 (drug-sensitive), and cytotoxicity against human diploid embryonic cell line MRC-5 were measured as described previously.1,5 In vivo antitrypanosomal activity for T. b. brucei strain S427 was measured as described previously.6 Test compounds were solubilized in an aqueous mixture of 10% DMSO-Tween 80 and EtOH (7:3) and administered i.p. to mice on the next day (day 1) following infection with parasites (day 0). Subsequently, the compounds were successively administered (i.p.) to the infected mice once a day for 3 days (days 2–4). Efficacies of compounds were determined by the parasitemia levels and the mean of survival days (MSD), compared with that of the untreated control mice. Table 1 shows the in vitro antiprotozoal activities of bispolides, their derivatives, elaiophylin and some standard antiprotozoal drugs. Bispolides, derivative I (13,13¢-dideoxybispolide A1) and elaiophylin showed the more potent antimalarial activity against the drug-resistant K1 strain of P. falciparum, in the 260–620 ng ml 1 range. The half-maximal inhibitory concentration (IC50) values against the drug-sensitive FCR3 strain of P. falciparum were similar to those against the K1 strain of P. falciparum (data not shown). The antimalarial activity was similar to that of chloroquine, but 43–103-fold less than that of artemisinin. However, the antimalarial activity of derivative II was 14-fold less than that of bispolide A3. With respect to antitrypanosomal activity, bispolides and derivative I showed the most potency against the GUTat 3.1 strain of T. b. brucei, in the 57–150 ng ml 1 range. The antitrypanosomal activities were 10–40-fold more potent than that of the standard drugs, suramin and eflornithine. However, the antitrypanosomal activities of derivative II and elaiophylin were 3–6-fold less than that of bispolide A3. The in vitro cytotoxicities of bispolides, their derivatives, elaiophylin and some standard antiprotozoal drugs are presented in Table 1. Among them, the IC50 values of bispolide B1 and elaiophylin were 0.96 and 0.87mg ml 1, respectively. The IC50 values of bispolide A1, A3 and B3, and derivative I were in the range of 1.4–3.9mg ml 1, whereas that of derivative II was 15.6mg ml 1. To compare the antiprotozoal activities and cytotoxicities, we introduced selectivity indexes (SIs: cytotoxicity (IC50 for the MRC-5 cells)/antimalarial or antitrypanosomal activity (IC50 for the K1 strain or the GUTat 3.1 strain), as presented in Table 1. In the case of the MRC-5 cells/K1 strain, bispolide A1 showed a medium SI, with a ratio of 15. Other bispolides and elaiophylin showed a low SI, with ratios of 2–9. In the case of the MRC-5 cells/GUTat 3.1 strain, bispolides A1 and B3 showed a moderate SI, with ratios of 57–62. Other bispolides and elaiophylin showed a medium or low SI (ratios of 2–26). Among the bispolides, we were interested in bispolides A3 and B3, which had an SI of 26 and 62, respectively. Among the tested bispolides, B3 showed the more potent antitrypanosomal activity and highest SI, whereas bispolide A3 exhibited potent antitrypanosomal activity but with medium SI. The preliminary in vivo antitrypanosomal activities of bispolides A3 and B3 were measured in the T. b. brucei S-427 acute mouse model. At a dose of 25 mg kg 1, bispolide B3 did not achieve cure but did extend the MSD to 9.5 days, representing a 1.4-fold increase over control MSD (7.0 days). The same dose of bispolide A3 did not achieve cure but also extended the MSD. Under the same conditions, suramin showed a curative effect (MSD: 430 days) at a dose of 1 mg kg 1. The bispolide B3 data suggest that it is possibly a new candidate compound for discovering new antitrypanosomal drugs with more potent activity. These efficacy tests, including in vivo antimalarial activity, are being investigated further. The Journal of Antibiotics (2010) 63, 275–277 & 2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10

Synthesis of anti-rheumatic agent epoxyquinomicin B.

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Migracins A and B, new inhibitors of cancer cell migration, produced by Streptomyces sp.

Anti-rheumatic agent (+/-)-epoxyquinomicin B was synthesized for a 22% overall yield in eight steps from commercially available 3-hydroxy-4-nitrobenzaldehyde via the intermediate quinone 6 prepared by selective phenol oxidation of 5 by use of Fremys salt as the key step.

3,4-dihydroxystyrene, a novel microbial inhibitor for phenylalanine hydroxylase and other pteridine-dependent monooxygenases

In the course of screening for breast cancer cell migration inhibitors, we isolated two novel compounds, migracins A and B from the culture broth of Streptomyces sp. MI264-NF2. Their structures are related to those of luminacins previously isolated from Streptomyces. Migracins A and B inhibited breast cancer cell migration, monitored by wound healing assay with IC50 values of 1.31 and 1.99 μg ml−1, respectively, in human breast carcinoma MDA-MB-231 cells without showing any cytotoxicity. Migracins also inhibited the migration of human lung adenocarcinoma A549 cells and human fibrosarcoma HT-1080 cells. Therefore, migracins may become new cancer metastasis inhibitors.

Isolation of a novel paxilline analog pyrapaxilline from fungus that inhibits LPS-induced NO production

A new microbial inhibitor for rat-liver phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine: oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) was isolated from a culture medium of Fomes tasmanicus, and its structure was determined as 3,4-dihydroxystyrene. This compound inhibited the enzyme by 50% at a concentration of 5 X 10(-6) M and 5 X 10(-7) M, respectively, without or with preincubation at 25 degrees C for 15 min. Without preincubation, dihydroxystyrene inhibited phenylalanine hydroxylase noncompetitively with phenylalanine and a pteridine cofactor, 6,7-dimethyltetrahydropterin, and uncompetitively with oxygen. A change in kinetic properties of the inhibition was observed when the enzyme was preincubated with dihydroxystyrene; the degree of inhibition was increased, and the purely noncompetitive-type inhibition with phenylalanine changed to a mixed-type inhibition. A study concerning the structure-inhibitory activity relationship using several compounds similar to dihydroxystyrene, indicated that the catechol structure is essential and that the structure of the aliphatic side-chain affects the inhibitory potency. A similar degree of inhibition was observed using 6,7-dimethyl- or 6-methyltetrahydropterin or tetrahydrobiopterin as a cofactor. Dihydroxystyrene also inhibited other pteridine-dependent monooxygenases, tyrosine hydroxylase (EC 1.14.16.2) and tryptophan hydroxylase (EC 1.14.16.4), indicating that dihydroxystyrene is a general inhibitor for pteridine-dependent monooxygenases.