Pseudomonas bohemica strain ins3 eliminates antibacterial hinokitiol from its culture broth.

A bacterial strain, Pseudomonas bohemica strain ins3 was newly isolated as a resistant strain against high concentrations of hinokitiol. This strain was revealed not only to show resistance but also completely remove this compound from its culture broth. In addition, its mechanism was revealed to be independent of conventional aromatic dioxygenases, ie catechol-1,2- or 2,3-dioxygenases.

Effects of hemicelluloses on dehydrogenative polymerization of monolignols with cationic cell wall-bound peroxidase.

To elucidate the influence of polysaccharides on hardwood lignification, dehydrogenative polymerization of monolignols, coniferyl alcohol (CA) and sinapyl alcohol (SA), was attempted with recombinant cationic cell wall-bound peroxidase (rCWPO-C) and horseradish peroxidase (HRP) in measurement cells of a quartz crystal microbalance with dissipation (QCM-D). Hardwood cellulose nanofibers were anchored; hemicelluloses, xylan, partially acetylated xylan (AcXY), galactoglucomannan, and xyloglucan, and the enzymes were subsequently adsorbed onto the QCM-D sensor surface, enabling fabrication of artificial polysaccharide matrices. The largest amount of rCWPO-C is found to be adsorbed onto AcXY among all the polysaccharides, which affords the largest amount and size of spherical dehydrogenation polymers (DHPs) from both CA and SA. In contrast, no DHP and a small amount of DHPs are formed from SA and CA, respectively, by HRP catalysis in all of the polysaccharide matrices. This study demonstrates important functions of a real tree-derived peroxidase, rCWPO-C, and AcXY for hardwood lignification.

Direct deuteration of hinokitiol and its mechanistic study.

Hinokitiol has a broad antibacterial activity against bacteria and fungi. While its biosynthetic pathway has been intensively studied, its dynamics in natural environments, such as biodegradation pathway, remain unclear. In this study, the authors report a direct deuterium labeling of hinokitiol as a traceable molecular probe to serve those studies. Hinokitiol was subjected to the H2-Pd/C-D2O conditions and deuterated hinokitiol was obtained with excellent deuteration efficiencies and in moderate yield. The 1H and 2H NMR spectra indicated that all ring- and aliphatic hydrogens except that on C-6 were substituted by deuterium. According to the substrate scope and computational chemistry, deuteration on tropolone ring was suggested to proceed via D+-mediated process, and which was supported by the results of the experiment with trifluoroacetic acid and Pd(TPP)4. On the other hand, the deuteration on aliphatic group was predicted to be catalyzed by Pd(II) species.

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

We have found cyclophane-type adenosine derivatives having p-quinone amide moieties (1 and 2) as weak inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) from the Hokkaido University Chemical Library via the luciferase-based high-throughput screening. To obtain more potent inhibitors, we synthesized four new derivatives free from cyclophane rings (3-6). The N-H derivatives 3 and 5 showed more potent activities (24.4 and 11.1 µM, respectively) in the presence of dithiothreitol (DTT), whereas the N-methyl derivative 4 indicated more potent activity (2.1 µM) without DTT. Conformational analysis of compounds 3 and 4 suggested that N-H amide 3 binds to IMP-binding site in the DTT mediated manner.

Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.

Novel autophagy modulators: Design and synthesis of (+)-epogymnolactam analogues and structure-activity relationship.

(+)-Epogymnolactam (1) was discovered as a novel autophagy inducer from a culture of Gymnopus sp. in our laboratory. To determine structure-activity relationships among (+)-epogymnolactam analogues comparing with cerulenin (2), we synthesized 5 analogues including (-)-epogymnolactam (3) having each different functional group, and 3 analogues with different side-chain lengths. Five analogues, 3, 4, 5, 6, and 7 did not significantly increase the ratio of LC3-II to LC3-I as an autophagy marker in NIH3T3 cells. These results suggest that presence and stereochemistry of (2R,3S)-epoxy group and cyclic syn-form (1b) of 1 are important for the activity as autophagy inducer. Hexyl analogue (8) as well as 1 having butyl side-chain dose-dependently increased the ratio of LC3-II to LC3-I, whereas octyl analogue (9) and 2 rather decreased the ratio. Decyl analogue (10) did not give a change in the ratio. Although 8 seemed to be an excellent autophagy inducer, it dose-dependently increased SQSTM1 (p62) as in the case of 2, whereas 1 showed a slight dose-dependent decrease of p62 as an index of autophagic protein degradation. These observations suggest that 8 is an autophagy modulator with different molecular target from 1 or 2.

Identification and characterization of guanosine 5'-monophosphate reductase of Trypanosoma congolense as a drug target.

Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000_5_1940, as a guanosine 5'-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.

Zincmethylphyrins and coproporphyrins, novel growth factors released by Sphingopyxis sp., enable laboratory cultivation of previously uncultured Leucobacter sp. through interspecies mutualism.

We have identified coproporphyrins including structurally new zincmethylphyrins I and III as growth factors A-F for the previously uncultured bacterial strain, Leucobacter sp. ASN212, from a supernatant of 210 l of Sphingopyxis sp. GF9 culture. Growth factors A-F induced significant growth of strain ASN212 at the concentrations of picomolar to nanomolar which would otherwise be unculturable in liquid medium or on agar plate. More interestingly, we found that the growth factors functioned as self-toxic compounds for the growth-factor producing strain GF9 at the picomolar to nanomolar levels. As a variety of bacteria could potentially produce coproporphyrins, our findings suggest that these compounds function as a novel class of signal molecules across a boundary at phylum level in the complex bacterial communities.

First total synthesis of (+)-epogymnolactam, a novel autophagy inducer.

A novel autophagy inducer, (+)-epogymnolactam (1), was first synthesized from cis-4-benzyloxy-2-butene-1-ol (2) in eight steps. A reliable preparation of optically pure epoxy alcohol (+)-3 from monobenzyl derivative (2) was established by a tandem strategy, Sharpless epoxidation/lipase kinetic resolution.

(+)-Epogymnolactam, a novel autophagy inducer from mycelial culture of Gymnopus sp.

Mushrooms, including Ganoderma lucidum, have been used as a potential source of therapeutic compounds, and an autophagy inducer would be useful for treatment of diverse diseases in human. Reported here is a full account of screening, isolation, structural determination, and biological activity of an autophagy inducer, (+)-epogymnolactam (1) from a mycelial culture of a Gymnopus sp. strain. Its structure was elucidated by HR-ESI-MS, NMR, and its plus sign by specific rotation. It exists as a tautomeric mixture of 1a and 1b in MeOH. The major tautomer of 1 is (1R,5S)-4-butyl-4-hydroxy-3-aza-6-oxa-bicyclo[3.1.0]hexan-2-one (1a), and the minor tautomeric form is (2R,3S)-3-pentanoyloxirane-2-carboxamide (1b).

Maillard reaction inhibitors produced by Paecilomyces sp.

Maillard reaction inhibitors could be useful therapeutics for diabetes and other age-related diseases. We isolated for the first time 4-O-demethylsilvaticol (1) and (-)-mitorubrin (2) as Maillard reaction inhibitors from Paecilomyces sp. 3193B. Among the isolated inhibitors, 2 showed most potent inhibitory effect by an SDS-PAGE assay on cross-linked protein formation and by a fluorescent assay on AGE formation.

MurA as a primary target of tulipalin B and 6-tuliposide B.

(-)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.

Discovery of N-(2,3,5-triazoyl)mycophenolic amide and mycophenolic epoxyketone as novel inhibitors of human IMPDH.

Syntheses of ten derivatives of mycophenolic acid (MPA) at C-6' position, and structure-activity relationship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of N-(2,3,5-triazolyl)mycophenolic amide 4, (7'S) mycophenolic epoxyketone 9 and (7'R) mycophenolic epoxyketone 10 having potent inhibitory activity against human inosine-5'-monophosphate dehydrogenase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds 4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and 10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 µM. These observations suggest that the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of HDAC.

Cleavage of α-dicarbonyl compounds by terpene hydroperoxide.

The highly reactive α-dicarbonyl compounds, glyoxal, methylglyoxal (MGO), and 3-deoxyglucosone, react with the amino groups of proteins to form advanced glycation end-products (AGEs) which have been implicated in diabetic complications, aging, and Alzheimer's disease. We found that a test sample of terpinen-4-ol (T4) containing hydroperoxides showed cleaving activity toward an α-dicarbonyl compound, but that the freshly isolated pure sample did not. Prepared terpinen-4-ol hydroperoxide (T4-H) also efficiently cleaved the C-C bond of the α-dicarbonyl compounds via Baeyer-Villiger-like rearrangement and subsequent hydrolysis of an acid anhydride moiety in the rearranged product to give carboxylic acids. Other terpene hydroperoxides, as well as T4-H, showed significant cleaving activities, and all these hydroperoxides protected RNase A from the lowering of enzyme activity induced by MGO. The cleaving mechanism via Baeyer-Villiger-like rearrangement was confirmed by time-interval NMR measurements of the reaction mixture of the symmetrical α-dicarbonyl compound, diacetyl with T4-H.

Asymmetric total synthesis of 6-Tuliposide B and its biological activities against tulip pathogenic fungi.

The structure-activity relationship was investigated to evaluate the antifungal activities of tuliposides and tulipalins against tulip pathogenic fungi. 6-Tuliposide B was effectively synthesized via the asymmetric Baylis-Hillman reaction. Tuliposides and tulipalins showed antifungal activities against most of the strains tested at high concentrations (2.5 mM), while Botrytis tulipae was resistant to tuliposides. Tulipalin formation was involved in the antifungal activity, tulipalin A showed higher inhibitory activity than 6-tuliposide B and tulipalin B. Both the tuliposides and tulipalins showed pigment-inducing activity against Gibberella zeae and inhibitory activity against Fusarium oxysporum f. sp tulipae. These activities were induced at a much lower concentration (0.05 mM) than the antifungal MIC values.

The antibacterial properties of 6-tuliposide B. Synthesis of 6-tuliposide B analogues and structure-activity relationship.

6-Tuliposide B is a secondary metabolite occurring specifically in tulip anthers. Recently, a potent antibacterial activity of 6-tuliposide B has been reported. However, its molecular target has not yet been established, nor its action mechanism. To shed light on such issues, 6-tuliposide B and tulipalin B analogues were synthesized and a structure-activity relationship (SAR) was examined using a broad panel of bacterial strains. As the results of SAR among a total of 25 compounds, only tulipalin B and the compounds having 3',4'-dihydroxy-2'-methylenebutanoate (DHMB) moieties showed any significant antibacterial activity. Moreover, the 3'R analogues of these compounds displayed essentially the same activities as 6-tuliposide B and the structure of the 3'R-DMBA moiety was the same as that of the proposed active moiety of cnicin. These results suggest that 6-tuliposide B has the same action mechanism as proposed for cnicin and bacterial MurA is one of the major molecular targets of 6-tuliposide B.

Purification and characterization of a tuliposide-converting enzyme from bulbs of Tulipa gesneriana.

An enzyme that catalyzes the stoichiometric conversion of 6-tuliposide into tulipalin was purified and characterized from bulbs of Tulipa gesneriana. The enzyme appeared to be a dimer, the relative molecular mass (Mr) of each subunit being 34,900; it had maximum activity and stability at neutral pH and moderate temperature. The enzyme preferentially acted on such glucose esters as 6-tuliposides, and to a lesser extent on p-nitrophenylacetate.