Identification of ten KB425796-A congeners from Paenibacillus sp. 530603 using an antifungal assay against Aspergillus fumigatus in combination with micafungin.

The discovery and characterization of natural congeners is one approach for understanding the relationship between chemical structure and biological function. We recently isolated the novel antifungal metabolite KB425796-A produced by the recently isolated bacterium Paenibacillus sp. 530603. On the basis of morphological changes of Aspergillus fumigatus induced by KB425796-A in combination with micafungin, we developed a highly sensitive screening method for the specific detection of KB425796-A congeners. Using this method, we isolated ten congeners of KB425796-A, named KB425796-B, -C, -D, -E, -F, -G, -H, -I, -J and -K, which exhibited diverse antifungal potencies against A. fumigatus. One of the most potent congeners, KB425796-C, had antifungal activities against several micafungin-resistant infectious fungi. KB425796-C can be a potential drug candidate for treating micafungin-resistant fungal infections.

KB425796-A, a novel antifungal antibiotic produced by Paenibacillus sp. 530603.

The novel antifungal macrocyclic lipopeptidolactone, KB425796-A (1), was isolated from the fermentation broth of bacterial strain 530603, which was identified as a new Paenibacillus species based on morphological and physiological characteristics, and 16S rRNA sequences. KB425796-A (1) was isolated as white powder by solvent extraction, HP-20 and ODS-B column chromatography, and lyophilization, and was determined to have the molecular formula C79H115N19O18. KB425796-A (1) showed antifungal activities against Aspergillus fumigatus and the micafungin-resistant infectious fungi Trichosporon asahii, Rhizopus oryzae, Pseudallescheria boydii and Cryptococcus neoformans.

Synergistic antifungal activity of KB425796-C in combination with micafungin against Aspergillus fumigatus and its efficacy in murine infection models.

KB425796-C is a novel antifungal metabolite produced by the newly isolated bacterial strain Paenibacillus sp. No. 530603. This compound is a 40-membered macrocyclic lipopeptidolactone consisting of 12 amino acids and a 3-hydroxy-15-methylpalmitoyl moiety. KB425796-C displayed antifungal activity against micafungin-resistant fungi and was fungicidal to Trichosporon asahii in vitro. In a murine systemic infection model of T. asahii, KB425796-C showed excellent efficacy upon i.p. administration at 32 mg kg(-1). In addition, KB425796-C induced morphological changes in the hyphae of Aspergillus fumigatus and had fungicidal effects in combination with micafungin. In a mouse model of septic A. fumigatus infection, although non-treated mice survived for a maximum of only 6 days, the survival rate of micafungin-treated mice (0.1 mg kg(-1)) increased to 20%, while the survival rate of mice treated with a combination of micafungin (0.1 mg kg(-1)) and KB425796-C (32 mg kg(-1)) increased to 100% during the 31-day post-infection period. Our findings suggest that KB425796-C is a good candidate for the treatment of aspergillosis in combination with micafungin.

Histone deacetylase inhibitors from microorganisms: the Astellas experience.

Histone deacetylase (HDAC) inhibitors, such as trichostatin A and trapoxin, which were first found in microorganisms, potently and selectively inhibit HDAC enzymes. They have made a strong contribution to research on HDACs, chromatin control, abnormal epigenetic control in various diseases and the significance of acetylation in posttranslational modification. Recently, HDAC inhibitors have been focused on as potential drugs for the treatment of several diseases, including cancer, although trichostatin A and trapoxin show no effects in animal models because of their metabolic instability in vivo. Chemical modification has been conducted in order to overcome this drawback. We discovered the microbial metabolites FK228 (also known as FR901228, romidepsin, depsipeptide, NSC-630176 and NSC-630176D) and YM753 (spiruchostatin A). Both compounds have bicyclic structures and represent a novel structural class of HDAC inhibitor. The enzyme and tumor cell growth inhibitory activities of FK228 were found to be very potent. It also showed potent HDAC inhibitory activity in vivo. FK228 is the first potent HDAC inhibitor to undergo clinical development as a potential treatment for solid and hematological cancers. Due to its dramatic effect in patients with refractory cutaneous T-cell lymphoma (CTCL), in October 2004 the US Food & Drug Administration (FDA) granted fast-track status to FK228 as monotherapy for the treatment of CTCL in patients who have relapsed following, or become refractory to, another systemic therapy. Thus HDAC inhibitors such as FK228 and YM753 have potential as tools for life science studies and also as therapeutic agents for various intractable diseases.

Identification of a selective ERK inhibitor and structural determination of the inhibitor-ERK2 complex.

Selective inhibition of extracellular signal-regulated kinase (ERK) represents a potential approach for the treatment of cancer and other diseases; however, no selective inhibitors are currently available. Here, we describe an ERK-selective inhibitor, FR180204, and determine the structural basis of its selectivity. FR180204 inhibited the kinase activity of ERK1 and ERK2, with K(i) values 0.31 and 0.14microM, respectively. Lineweaver-Burk analysis of the binding interaction revealed that FR180204 acted as competitive inhibitor of ATP. In mink lung epithelial Mv1Lu cells, FR180204 inhibited TGFbeta-induced luciferase-expression. X-ray crystal structure analysis of the human ERK2/FR180204 complex revealed that Q105, D106, L156, and C166, which form the ATP-binding pocket on ERK, play important roles in the drug/protein interaction. These results suggest that FR180204 is an ERK-selective and cell-permeable inhibitor, and could be useful for elucidating the roles of ERK as well as for drug development.

Discovery of the first non-peptide full agonists for the human bradykinin B(2) receptor incorporating 4-(2-picolyloxy)quinoline and 1-(2-picolyl)benzimidazole frameworks.

In the course of our studies on non-peptide bradykinin (BK) B(2) receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B(2) receptors, as well as agonist/antagonist properties. We carried out an extensive investigation to elucidate the structure-activity relationships (SAR) for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist. This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster ovary cells expressing the cloned human B(2) receptor at concentrations greater than 10 nM and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective for the B(2) receptor and was inhibited by selective peptide and non-peptide B(2) antagonists. On the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human B(2) receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-peptide B(2) agonist, 19a. Successive optimization of the acyl side chain afforded 38, which exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimidazole derivative 47c increased PGE(2) production at a 1 microM concentration to the same level as the maximum effect of BK. Thus, we have established the medicinal chemistry modifications required to convert our highly potent non-peptide B(2) antagonists to agonists with potent efficacy.

A new class of nonpeptide bradykinin B(2) receptor ligand, incorporating a 4-aminoquinoline framework. Identification of a key pharmacophore to determine species difference and agonist/antagonist profile.

Introduction of various aliphatic amino groups at the 4-position of the quinoline moiety of our nonpeptide bradykinin (BK) B(2) receptor antagonists afforded highly potent ligands for human B(2) receptor with various affinities for guinea pig B(2) receptor, indicating remarkable species difference. A representative 4-dimethyamino derivative 40a exhibited subnanomolar and nanomolar binding affinities for human and guinea pig B(2) receptors, respectively, and significantly inhibited BK-induced bronchoconstriction in guinea pigs at 10 microg/kg by intravenous administration. Further chemical modification led us to discover unique partial agonists for the human B(2) receptor that increase inositol phosphates (IPs) production by themselves in Chinese hamster ovary (CHO) cells expressing the cloned human B(2) receptor. Although their potency and efficacy were much lower than those of BK, we identified them as screening leads for nonpeptide B(2) agonists. In these studies it was revealed the 4-substituent of the quinoline moiety is the key pharmacophore to determine species difference and agonist/antagonist profiles.

A new series of highly potent non-peptide bradykinin B2 receptor antagonists incorporating the 4-heteroarylquinoline framework. Improvement of aqueous solubility and new insights into species difference.

Introduction of nitrogen-containing heteroaromatic groups at the 4-position of the quinoline moiety of our non-peptide B(2) receptor antagonists resulted in enhancing binding affinities for the human B(2) receptor and reducing binding affinities for the guinea pig one, providing new structural insights into species difference. A CoMFA study focused on the diversity of the quinoline moiety afforded correlative and predictive QSAR models of binding for the human B(2) receptor but not for the guinea pig one. A series of 4-(1-imidazolyl)quinoline derivatives could be dissolved in a 5% aqueous solution of citric acid up to a concentration of 10 mg/mL. A representative compound 48a inhibited the specific binding of [(3)H]BK to the cloned human B(2) receptor expressed in Chinese hamster ovary cells with an IC(50) value of 0.26 nM and significantly inhibited BK-induced bronchoconstriction in guinea pigs even at 1 microg/kg by intravenous administration.