Chemical biology research in RIKEN NPDepo aimed at agricultural applications

Abstract

This review outlines research on chemical biology using mainly microbial metabolites for agricultural applications. We established the RIKEN Natural Products Depository (NPDepo), housing many microbial metabolites, to support academic researchers who focus on drug discovery. We studied methods to stimulate secondary metabolism in microorganisms to collect various microbial products. The switch of secondary metabolism in microorganisms changes depending on the culture conditions. We discovered compounds that activate biosynthetic gene clusters in actinomycetes and filamentous fungi. Using these compounds, we succeeded in inducing the production of active compounds. Two approaches for screening bioactive compounds are described. One is phenotypic screening to explore antifungal compounds assisted by artificial intelligence (AI). AI can distinguish the morphological changes induced by antifungal compounds in filamentous fungi. The other is the chemical array method for detecting interactions between compounds and target proteins. Our chemical biology approach yielded many new compounds as fungicide candidates.

1. Introduction

Since the beginning of the 20th century, the world’s population has grown significantly, from approximately 2.5 billion in 1950 to more than 6 billion in 2000, and is expected to exceed 8.1 billion by 2024 (World Population Prospects 2024).¹⁾ Efforts to expand agricultural yields are necessary to secure food to support the increasing population, and using pesticides and fertilizers is inevitable.

Chemical pesticides except inorganic compounds emerged in Europe and the United States in the 1930s. The use of organochlorine pesticides, such as benzene hexachloride (mainly γBHC), in Japan began after World War II (in the 1940s), and agricultural production increased dramatically. However, organochlorine pesticides became a serious problem due to environmental pollution and their harmful effects on the human body, and they are no longer used nowadays.^2),3) Then, in the 1960s, screening of microbial metabolites that can be used for agriculture began. In general, microbial products were thought to be safer than synthetic compounds because most microbial metabolites are synthesized and degraded by microorganisms in nature and showed selective toxicity against fungi and parasites. Current pesticides, both microbial metabolites and synthetic compounds, are superior to previously developed pesticides in terms of safety and environmental issues. However, the development of new pesticides is still required because of the emergence of drug-resistant pathogens and insects.

The development of new pesticides needs to understand the interactions between hosts (farm animals and plants) and pathogenic microorganisms (fungi, bacteria, and viruses). Agricultural chemistry is an academic field that elucidates agricultural phenomena using chemistry or chemical methods; similarly, chemical biology elucidates complex biological phenomena using the power of chemistry, especially bioactive compounds.⁴⁾ This review will outline our chemical biology research aiming at agricultural applications (Fig. 1).

Fig. 1

Outline of drug discovery from microbial metabolites. (Left) Actinomycetes and filamentous fungi are isolated from soil samples from around the country, and each microorganism is cultured in various fermentation media. Culture extracts are stored as broth libraries, and partially purified culture extracts are stored as fraction libraries. When an interesting compound is found, the biosynthetic gene is cloned from the producer strain. New compounds are obtained by genetic modification to activate biosynthetic genes or by adding small molecules (biomediators) that activate gene expression. (Center) RIKEN Natural Products Depository was established with the isolated microbial products as the core. A Natural Product Plot (NPPlot) was constructed to display HPLC analysis data of the fraction library. (Right) A chemical array method was established to find compounds that bind to target proteins for finding candidate pesticide compounds. New compounds were isolated using phenotypic screening methods (such as morphological observation of filamentous fungi incorporating artificial intelligence).

2. Development of chemical resources as a foundation for chemical biology

The starting point of chemical biology research is the identification of useful chemical compounds, namely inhibitors, against a specific biological activity. This describes how to construct and effectively use microbial broth and fraction libraries, as well as compound libraries consisting of natural and synthetic compounds, as the basic platform for chemical biology research.

2.1. Chemical compound depository centers.

Since the discovery of penicillin, many antibiotics have been discovered from microorganisms and developed into therapeutic drugs against infectious diseases.⁵⁾ In particular, Japan was the first country to adapt antibiotics for agricultural use.⁶⁾ Blasticidin S,⁷⁾ polyoxin,⁸⁾ and kasugamycin⁹⁾ were discovered by researchers in non-profit organizations in Japan and agrochemical companies developed those compounds as fungicides. Furthermore, Japanese pharmaceutical companies discovered pharmacologically active compounds such as the cholesterol synthesis inhibitor ML-236B (mevastatin)^10),11) and the immunosuppressant FK-506 (tacrolimus)^12),13) from microbial cultures. The 1950s-1970s are referred to as the golden age for microbial drug discovery. Pharmaceutical companies introduced high-throughput screening (HTS) using automated robots, and the biological activities of many synthetic compounds have been evaluated since the 1990s using HTS. Then, drug discovery from microorganisms declined because it was thought to be inefficient.¹⁴⁾ However, with recent progress in the analysis of the genome sequences and gene engineering of microorganisms, the usefulness of microbial metabolites has been reconsidered.¹⁵⁾

Pharmaceutical and agrochemical companies have large-scale chemical libraries of compounds synthesized by combinatorial chemistry, but the chemical libraries available to academic researchers are limited.¹⁶⁾ The NIH Molecular Library Initiative, led by the National Institutes of Health in the United States and involving many universities and research institutes, synthesized and distributed chemical compounds to screening centers across the United States. Moreover, the National Cancer Institute (USA) has many natural products, including purified and semi-purified compounds, and is searching for natural products that are effective against cancer and drug-resistant microorganisms.¹⁴⁾ In Japan, RIKEN Natural Products Depository (NPDepo), which collects natural products derived from microorganisms, plants, and chemically synthesized compounds was established to accelerate drug discovery in academia.^16),17) The University of Tokyo’s Drug Discovery Initiative¹⁸⁾ has a chemical library centered on synthetic compounds, and the Japan Biological Informatics Consortium (JBIC),¹⁹⁾ compiled by the National Institute of Advanced Industrial Science and Technology, has a chemical library that includes many natural products derived from microorganisms. Osaka University recently established a drug discovery science research support center and is also distributing a chemical library for researchers (https://www.phs.osaka-u.ac.jp/souyaku_kyoten/about/clsc.html).

2.2. Fraction library.

Microorganisms, such as actinomycetes and filamentous fungi, produce metabolites with diverse biological activities and chemical structures, but conventional methods to find these bioactive compounds have not been advantageous.^20),21) Microbial culture broths were screened for specific biological activities, and the target compounds were purified using the limited activities as indicators. Therefore, even if thousands of strains of microorganisms were cultured, only a few compounds showing the desired activity remained, and other microbial metabolites that may be active if subjected to different assay systems were discarded. To compensate for this shortcoming, we stored microbial culture broths as broth libraries and prepared fraction libraries,²²⁾ yielded by systemic fractionation of microbial culture broths using medium-pressure liquid chromatography (MPLC) and high performance liquid chromatography (HPLC) (Fig. 2). The fractions obtained can be used immediately when a new bioassay system is established and can also be used for HTS. Furthermore, to prepare the fraction library, we used an HPLC equipped with a photodiode array (PDA) and mass spectrometer (MS), which made it possible to determine the approximate number and properties of the compounds contained in each fraction.

Fig. 2

Fraction library. A fraction library was prepared by partially purifying culture extracts of each strain. UV absorption and molecular weight data were obtained for each fraction using an HPLC equipped with a photodiode array and mass spectrometer.

2.3. Natural Product Plot (NPPlot).

The spectral information [ultraviolet-visible absorption (UV) and MS] and polarity (retention time of HPLC) obtained during the preparation of the fraction library were compiled into a database. The NPPlot was created with the HPLC retention time on the horizontal axis and the m/z value (molecular weight) on the vertical axis, with the UV information included for each spot, to simplify viewing the data. By comparing the distribution patterns of NPPlots created from various strains, it was possible to identify compounds that are commonly present in actinomycetes and filamentous fungi. Furthermore, compounds can be identified from the NPPlot pattern that are unique to the strain. These compounds are likely to be novel.

Using the above method, we discovered novel compounds, such as verticilactam,²³⁾ spirotoamide,²⁴⁾ and pyrrolizilactone,²⁵⁾ from the fraction libraries of various microorganisms. Verticilactam is the first natural compound with a β-ketoamide structure in the macrolactam skeleton (Fig. 3). Spirotoamides A and B are characteristic compounds with a 6,6-spiro ring structure and a terminal carboxamide group. Pyrrolizilactone has a characteristic structure in which the pyrrolizidinone and decalin moieties are connected via a ketone. Furthermore, this section present an example of the discovery of two novel cyclic depsipeptides, octaminomycins A and B,²⁶⁾ using NPPlots created from the fraction library of the actinomycete strain Streptomyces sp. RK85-270.

Fig. 3

Discovery of new compounds from the fraction library. Fraction libraries were prepared from the culture media of Streptomyces griseochromogenes, a tautomycetin-producing strain; Streptomyces spiroverticillatus, a tautomycin-producing strain; and an uncharacterized fungus isolated from a soil sample. Each fraction library contained the UV absorption profile and mass spectrometry data. Characteristic fractions with unique UV and mass profiles were purified to obtain new compounds. Verticilactam, spirotoamide A, pyrrolizilactone were also detected.

The fraction library of Streptomyces sp. RK85-270 was analyzed using LC-PDA-MS, and approximately 180 data sets were selected to create an NPPlot (Fig. 4).²⁷⁾ In the region with a retention time of approximately 25 min and molecular weight of approximately 1000 Da in the NPPlot, spots with similar UV absorption spectra (similar chromophore) and a difference of 14 Da in the m/z values (987 and 1001), were detected in adjacent fractions. Because these spots were not found in the NPPlots of other strains, these compounds were considered metabolites unique to this strain. Furthermore, a database search suggested that these compounds were novel, because compounds with these UV and molecular weight features were not found among known compounds. Compounds A (50 mg) and B (14 mg) were purified from 30 L of Streptomyces sp. RK85-270 fermentation broth to isolate them and confirm their structures. First, the structure of compound A was analyzed and its molecular formula was determined to be C₅₃H₇₆N₈O₁₁ using a high resolution electrospray ionization mass spectrometer (HR-ESI-MS). Based on the correlation of various data points from two-dimensional nuclear magnetic resonance (2D-NMR) spectrometry, compound A was identified as a depsipeptide comprising eight amino acids: two leucines, two prolines, valine, phenylalanine, threonine, and N-methyltyrosine, with the threonine hydroxyl group and the proline carboxyl group ester-bonded.

Fig. 4

Natural Product Plot (NPPlot) and chemical structures of octaminomycin A and B. An NPPlot was prepared, in which the parameters obtained for each strain are displayed as the HPLC elution time (min) on the X-axis, molecular weight (m/z) on the Y-axis, and ultraviolet absorption spectrum on the Z-axis. The NPP lot of the ascamycin-producing strain Streptomyces sp. The RK85-270 strain contained characteristic spots with molecular weights and UV absorption that differed from those of the other strains. Therefore, the components were isolated and named as new compounds octaminomycins A and B.

Next, the molecular formula of compound B was determined to be C₅₂H₇₄N₈O₁₁ by HR-ESI-MS. By comparing the ¹H and ¹³C NMR spectra with those of compound A, it was determined that threonine was propionylated in A and acetylated in B (Fig. 4). Because it is a novel compound consisting of eight amino acids, it was named octaminomycins A and B.²⁶⁾

The biological activities of octaminomycins A and B were evaluated. Both compounds showed no cytotoxicity at a concentration of 30 μM and antimalarial activity against the wild-type malarial parasite Plasmodium falciparum 3D7, with a 50% growth-inhibitory concentration (IC₅₀) of 1.5 μM. Furthermore, both compounds were effective against chloroquine-resistant Plasmodium falciparum Dd2 and K1 strains, with IC₅₀ values of approximately 1.5 μM. Taken together, it has been demonstrated that the NPPlot is useful for discovering novel compounds in the microbial fermentation broth.

3. From genetic resource to chemical resource

Microorganisms change the variety of secondary metabolites they produce when culture conditions are changed; however, the mechanisms by which this occurs are not fully understood. Recently, however, many microbial genomes have been decoded, and genetic information on microbial secondary metabolites has accumulated. Previously, it was a common process to isolate a compound and identify its biosynthetic gene cluster; however, a technique called genome mining, which predicts the compound that will be produced from genetic information to obtain the target compound, has become available.^28),29)

This section will introduce how various metabolic products can be produced by activating switches in biosynthetic genes.

3.1. Activation switch for biosynthetic genes of microbial secondary metabolites.

In collaboration with the Ōmura group at Kitasato University, we investigated the metabolome of the actinomycete Streptomyces avermitilis,³⁰⁾ the first actinomycete genome that was sequenced in Japan.³¹⁾ Although the genome of S. avermitilis contained more than 30 biosynthetic gene clusters, we detected the production of less than 10 compounds (Fig. 5). We focused on nocardamin, which is only produced in nutrient-poor synthetic media and not in nutrient-rich media.³²⁾ Nocardamin is a siderophore that chelates iron. We found that the iron-responsive regulator IdeR is switched on and the transcription of the nocardamin biosynthetic genes is suppressed when sufficient iron is present in the medium.³³⁾ Nocardamin production is maintained even in the presence of iron ions in the ideR-disrupted strain.³²⁾

Fig. 5

Whole genome sequence and secondary metabolite gene clusters in Streptomyces avermitilis. More than 30 biosynthetic gene clusters are shown; however, the production of eight compounds was confirmed at the time of this study. The production of nocardamin was also confirmed.

3.2. Activation of the secondary metabolite genes in actinomycete.

Reveromycin A (RM-A) was isolated from Streptomyces sp. SN-593 during screening for signal transduction inhibitors in cancer cells. Later, we found that RM-A inhibited eukaryotic isoleucine tRNA synthetase activity³⁴⁾ and killed animal osteoclasts³⁵⁾ and plant pathogenic fungi.³⁶⁾ When RM-A was discovered, the producing strain was classified as a member of the Streptomyces genus.³⁷⁾ However, owing to recent advances in taxonomy, this strain was re-classified into as a new genus and species, Actinacidiphila reveromycinica sp. nov.³⁸⁾

To improve the production of RM-A, we examined the medium in International Streptomyces Project (ISP) media.³⁹⁾ We found that RM-A production increased when ISP-8 medium containing V8 juice was used for fermentation.⁴⁰⁾ We assumed that there was a factor in the tomato extract that activated the RM-A biosynthetic gene cluster and attempted to purify the active substance in tomato extract. However, the active substance was difficult to isolate and identify from tomato extract. Then, we searched for small-molecule compounds related to enhancing RM-A production in the NPDepo library.⁴¹⁾ While screening compounds with RM-A production-inducing activity, we found a compound with a β-carboline skeleton. We then synthesized β-carboline related compounds to examine their structure-activity relationships. As a result, we created a biomediator for reveromycin (named BR-1) with an optimized chemical structure (Fig. 6).⁴⁰⁾ BR-1 binds to the transcription factor RevU (one of the LuxR⁴²⁾ family proteins of actinomycetes) and promotes transcription of the RM-A biosynthetic gene cluster.⁴³⁾

Fig. 6

HPLC analyses of reveromycin A production. When tomato juice was added to the culture medium of the reveromycin-producing strain, the amount of reveromycin A produced increased (top chart). The β-carboline derivative BR-1 (1 μg/mL) increased the amount of reveromycin produced to the same level as that by tomato juice (middle chart). A control sample was prepared by adding DMSO (0.5%) containing dissolved BR-1 to the normal medium (bottom chart).

Using the genes activated by tomato extract as clues, we succeeded in cloning the RM-A biosynthetic gene cluster, consisting of approximately 91 kb, including the polyketide synthase (PKS) genes (revA, revB, revC, revD) that form the carbon skeleton and 21 genes involved in expression control and structural modification (Fig. 7).⁴⁴⁾

Fig. 7

Biosynthetic gene cluster of reveromycin A. Polyketide synthases (PKS) are shown in blue, regulatory genes in purple, genes for synthesizing side chain 2-alkylmalonyl-CoA in pink, and genes related to post-PKS modification in yellow.

Many natural products containing a spiroacetal ring in their structure exhibit remarkable biological activity, leading to numerous researchers attempting to elucidate the formation of the spiroacetal ring and its biosynthetic mechanisms. Previous studies have suggested two main biosynthetic pathways (Fig. 8). An epoxide/ketone intermediate was assumed in the biosynthesis of monensin A,^45),46) whereas a dihydroxyketone intermediate was proposed in the biosynthesis of tautomycin.^47),48) When analyzing the biosynthetic mechanism of RM-A, the discovery of a cyclization precursor (RM-A1a) in the metabolic products of the producing strain provided a major clue. Dehydrogenase (RevG) uses RM-A1a as a substrate and performs a dehydrogenation reaction in the presence of the coenzyme, NAD^＋, to produce a dihydroxyketone. The RevG reaction product underwent non-enzymatic cyclization to produce spiroacetal compounds with different stereochemistries, i.e., 15S and 15R, at a ratio of 3:2, in the absence of RevJ. Previous predictions have suggested that the formation of a spiroacetal ring by dehydration cyclization, following the production of a dihydroxyketone, would non-enzymatically form a stable three-dimensional structure. However, contrary to expectations, we found that RevJ, the gene product of revJ present in the RM-A biosynthetic gene cluster, acts as a spiroacetal cyclase to control stereochemistry, converting all reaction products to the 15S configuration.⁴⁴⁾

Fig. 8

Pathway for spiroacetal formation of microbial metabolites. a) Monensin forms a spiroacetal ring via an epoxide intermediate. b) Tautomycin forms a spiroacetal ring via a dihydroxyketone intermediate. c) Reveromycin A produces a spiroacetal ring via a dihydroxyketone intermediate, in which two enzymes, RevG and RevJ, are involved.

Furthermore, we investigated the succinic acid at the C-18 position on the spiroacetal ring. RM-A is an acidic compound with three carboxylic acids; therefore, it is generally difficult to permeate cell membranes, but it is selectively taken up by osteoclasts³⁵⁾ and multiple myeloma cells,⁴⁹⁾ which secrete acid and create an acidic environment.⁵⁰⁾ In other words, without succinic acid at the C-18 position, the 6,6-membered spiroacetal structure is expected to be stabilized and easily taken up by cancer cells. We successfully analyzed the cocrystal structure of a P450 enzyme (RevI) that introduces a hydroxyl group at the C-18 position of the spiroacetal ring and its substrate, reveromycin T (RM-T) (Fig. 9).⁵¹⁾ We made a deletion mutant of RevI that can accumulate RM-T and evaluated its biological activity. Because RM-T has one less carboxylic acid than RM-A, its uptake into osteoclasts is reduced but its membrane permeability for plant pathogenic fungi was improved.

Fig. 9

Cocrystal structure of reveromycin T and RevI (P450). The P450 enzyme, RevI, adds a hydroxyl group to the C-18 position of reveromycin T, the biosynthetic precursor of reveromycin A.

3.3. Activation of the secondary metabolite genes in fungi.

Tenuazonic acid (TA) is a secondary metabolite of filamentous fungi known as a mycotoxin isolated from plant pathogenic filamentous fungi, such as Alternaria and Pyricularia genera.^52)-55) It was thought that TA may be involved in plant infection or lesion formation by plant pathogenic fungi, but there has been no conclusive evidence.⁵⁴⁾ Therefore, we investigated the relationship between TA productivity and pathogenicity. First, we confirmed the productivity of TA in the rice blast fungus (Pyricularia oryzae Kita1 strain) in our laboratory; however, it was produced at very low levels. Therefore, we searched for the conditions for TA production by generating gene-disrupted strains and screening compounds that induce TA production.

P. oryzae has a two-component signaling system (sensor system) consisting of two proteins: histidine kinase and response regulator proteins that respond to stresses and stimuli from the environment (e.g., light, heat, oxygen, and nutritional status).^56)-58) MAP kinase OSM1 works downstream of the two-component system in P. oryzae Kita1. We found that the OSM1 gene-disrupted strain induced the production of TA.⁵⁹⁾

We also screened the NPDepo library for compounds that induced TA production in P. oryzae Kita1. Surprisingly, most compounds in the NPDepo library were used for screening induced TA production. When we investigated the cause, we found that dimethyl sulfoxide (DMSO), which was used as a solvent for compounds, induced TA production in P. oryzae Kita1.⁵⁹⁾

Using DNA microarray analysis to search for genes activated in the above-mentioned TA production conditions, the TA biosynthetic gene TAS1 (tenuazonic acid synthetase) was identified.⁶⁰⁾ Furthermore, TAS2, which encodes a Zn(II)₂Cys₆-type pathway-specific transcription factor,⁶¹⁾ was found to be induced by DMSO and OSM1 disruption.⁵⁹⁾ It was revealed that upstream of this, LAE1,⁶²⁾ which is a global regulator of secondary metabolism in filamentous fungi, positively controls TA production (Fig. 10).⁶³⁾

Fig. 10

Regulation of biosynthetic genes of tenuazonic acid. Expression of the tenuazonic acid synthetase gene (TAS1) is regulated by the pathway-specific transcription factor TAS2, and the global regulator LAE1 is upstream of this gene. DMSO activates LAE1, and OSM1 is presumed to be a negative regulator of LAE1.

TAS1 is a hybrid enzyme consisting of a non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS), and its domain structure is unique and was first discovered in filamentous fungi.⁶⁰⁾ The C-A-T domain of NRPS links isoleucine and acetoacetyl-CoA, and the KS domain of PKS synthesizes TA via cyclization (Fig. 11). Because the KS domain of TAS1 performs the cyclization reaction, it was speculated that the KS domain of TAS1 might be similar to the KS domain of Type III PKS; however, when the X-ray crystal structure was compared, the KS domain of TAS1 was similar to that of Type I PKS (Fig. 12).^64)-66)

Fig. 11

Gene organization of TAS1 gene and enzymatic functions of the gene product. Although the domain structure of NRPS-PKS has been reported in bacteria, this is the first example in fungi. It consists of condensation (C), adenylation (A), and thioesterase (T) domains. The unique KS (keto synthetase) domain of the TAS1 PKS was found to undergo cyclization.

Fig. 12

X-ray crystal structures of the ketosynthase domain of PKS. The KS domain of TAS1 has a three-dimensional structure similar to that of the Type 1 KS domain in actinomycetes.^64),65)

After identifying TAS1, we investigated the relationship between plant pathogenicity and TA. Before our study, it was thought that TA might be involved in the pathogenicity of P. oryzae, but the pathogenicity of the P. oryzae TAS1-disrupted strain did not differ from that of its parent strain. We then constructed a TAS1-overexpressing strain (TAS1-OE strain) that constantly produced TA, it resulted in an unexpected decrease in pathogenicity compared with the parent strain (Fig. 13).⁶⁷⁾ When we investigated TAS1 gene expression during rice infection, we found that in the wild-type strain, TAS1 expression was suppressed immediately after infection, and no TA was produced. However, TAS1 was expressed and TA was produced from the late infection stage (72 hours after inoculation), whereas the overexpression strain continued to express TAS1 from the early infection stage, producing TA constitutively. Therefore, gene expression of the rice jasmonic acid signaling pathway was induced 48 hours after inoculation, and 72 hours after inoculation, the rice defense mechanism was observed to confine P. oryzae hyphae within the leaf cells. This confinement of P. oryzae was similar to that reported to be caused by the accumulation of jasmonic acid,⁶⁸⁾ suggesting that jasmonic acid production was induced by fungal infection.

Fig. 13

Effect of overexpression of TAS1 gene in Pyricularia oryzae upon infection of rice leaves. Rice leaves were sprayed with spore solutions of the wild type (wild type), tas1 (TAS1 deletion), and TAS1-OE (TAS1 overexpression), and lesions were counted 96 hours later (top and middle views). TAS1-OE injected hyphae into rice leaves but were immediately confined within rice cells (arrowheads in the bottom view). Bars, 50 μm.

These observations suggested that TA is produced in the late infection stage when P. oryzae switches from a biotrophic mode to a saprophytic infection mode and induces host cell death via the jasmonic acid signaling pathway. We speculated that the constitutive and induced production of TA reflects the difference in the infection strategies of necrotrophic Alternaria arborescens, which kills the host plant body and deprives it of nutrients, and hemibiotrophic P. oryzae,⁶⁹⁾ which infects a living host, ingests nutrients, and then transitions to the necrotrophic stage.

4. The one-strain-many-compounds (OSMAC) method

Isolating as many metabolites as possible from a specific strain is called the OSMAC method. The Zeeck group at the University of Gӧttingen, Germany has used this method to isolate new compounds from microorganisms.⁷⁰⁾ We applied the OSMAC method to search for secondary metabolites in Streptomyces sp. 80H647, a strain producing the nucleoside antibiotic ascamycin and found a variety of compounds in different fermentation broths.⁷¹⁾

The producer strain was cultured in six media types containing different ingredients and acetone was added to prepare the broth extracts (Fig. 14). Using the LC/MS analysis results of the ethyl acetate/water and butanol/water solvent layers, we performed principal component analysis of the metabolites. The results of the organic solvent extract of the CDY and the OAT culture broths, and the aqueous layer of different media formed distinct clusters in the principal component analysis plot, suggesting that characteristic secondary metabolites were produced in each culture condition. The compounds found in the specific culture medium were purified using a series of chromatography steps, and three new compounds that differ from ascamycin, i.e., compound X (nocardamin glucuronide), compound Y (N-acetyl-α-hydroxy-β-oxotryptamine), and compound Z (2-methylthio-N⁷-methyl-cis-zeatin), were isolated (Fig. 15).

Fig. 14

Color changes in secondary metabolites induced by different fermentation media. Six kinds of media were used for 72-hours fermentation using starch, glucose, meat extract, dried yeast, soybean medium (C4), corn steep liquor-dried yeast medium (CDY), potato dextrose-tomato juice medium (PV8), oatmeal-peptone medium (OAT), tryptone-yeast extract medium (TY), and glucose-yeast extract medium (GY).

Fig. 15

Chemical structures of nocardamin glucuronide (X), N-acetyl-α-hydroxy-β-oxotryptamine (the stereochemistry marked with an asterisk is a mixture) (Y), and 2-methylthio-N⁷-methyl-cis-zeatin (Z).

The molecular formula of compound X was determined to be C₃₃H₅₆N₆O₁₅ using HR-ESI-MS. The NMR spectrum was similar to that of nocardamin, but the presence of a signal estimated to be derived from a sugar at around 3.5 ppm and an anomeric proton (δ 4.62 ppm, J = 8.0 Hz) suggested that X was a glycoside of nocardamin. Detailed analysis of the 2D-NMR spectra showed that X was a glucuronic acid (GlcA) glycoside of nocardamin, which was confirmed using tandem MS experiments.⁷¹⁾ GlcA was determined to be β-oriented based on the coupling constant of the anomeric proton. The stereochemistry of the sugar was determined to be the D-isomer by derivatizing GlcA obtained by β-glucuronidase and comparing it with a derivative prepared from an authentic sample using LC/MS. The structure of X was determined to be nocardamin β-D-glucuronide. Because nocardamin is known as an iron chelator, X was subjected to an iron-binding assay. X showed a weaker effect than nocardamin suggesting that presence of GlcA affected the activity.

Compound Y has the molecular formula C₁₂H₁₂N₂O₃, and its planar structure was determined to be N-acetyl-α-hydroxy-β-oxotryptamine by 2D-NMR analysis.⁷²⁾ Two peaks of this compound were detected in HPLC analysis using a chiral column, suggesting the presence of stereoisomers. Chiral purification was performed to isolate enantiomers Ya and Yb to determine their structures. By comparing their absolute configurations with the calculated values from the electronic circular dichroism spectra, they were determined to be the R and S forms, respectively. These stereoisomers, Ya and Yb, exist naturally in the fermentation broth and do not undergo isomerization, even after storage at －20°C for several weeks. These results confirmed that N-acetyl-α-hydroxy-β-oxotryptamine exists naturally as a racemate. The biological activity of the racemate was measured, and it showed activity with IC₅₀ values of 27, 28, 34, and 81 μM against HeLa, HL-60, MG-63, and MIA PaCa-2 cells, respectively. The activities of the optical isomers Ya and Yb were evaluated, and both showed similar activities with no differences due to stereochemistry.

Compound Z was determined to have the molecular formula C₁₂H₁₇N₅OS by HR-ESI-MS, and its planar structure was determined to be 2-methylthio-N⁷-methyl-cis-zeatin by detailed analyses of NMR data.⁷³⁾ The geometric isomerism of the side chain double bond was determined to be cis configuration based on the ¹³C NMR chemical shift (21.9 ppm) of Me-15. 2-methylthio-N⁷-methyl-cis-zeatin showed weak activity against HL-60 (growth-inhibitory concentration (GI₅₀) 53 μM) and relatively strong antimalarial activity (GI₅₀ 2.4 μM).⁷³⁾ In addition, because its structure is similar to the plant hormone trans-zeatin, its growth-inhibitory activity against plants was measured; however, no zeatin-like activity was observed.

As mentioned above, the OSMAC method is useful for identifying compounds that cannot be identified through screening using a small number of biological activities as an indicator. When combined with microbial genomic information and the metabolic profiles of small-molecule compounds, it can be used to obtain new secondary metabolites and to understand the significance of microbial secondary metabolism.

5. Phenotypic screenings

Various screening systems have been devised to identify useful bioactive compounds from chemical libraries. This section describes phenotypic screening, which focuses on the phenotypes including the organism’s complex observable characteristics or traits.⁷⁴⁾ In other words, it refers to a method of evaluating drug efficacy by observing the organism’s survival, growth, morphological, or physical form and structure, biochemical and physiological properties, and behavior. Although phenotypic screening is considered an old-fashioned method, it has recently attracted renewed attention.⁷⁴⁾ In this section, fungicide screening based on morphological changes in filamentous fungi are described.

The reduction of yields due to diseases of agricultural crops is a major problem in food production, and more than 80% of diseases are caused by fungi.⁷⁵⁾ Rice blast disease caused by P. oryzae⁷⁶⁾ and powdery mildew caused by Erysiphe necator representative examples. Moreover, many types of pathogens cause damage to Cucurbitaceae, Solanaceae, and strawberry. Botrytis cinerea causes gray mold in many vegetables.⁷⁷⁾ Rice blast disease is a particularly serious problem in Japan, where rice is the staple food. It frequently causes outbreaks during cool summers with low temperatures and heavy rain, causing severe damage.⁷⁸⁾

By screening for anti-rice blast diseases, the Sumiki, Suzuki, and Umezawa groups discovered blasticidin S,⁷⁾ polyoxin⁸⁾ and kasugamycin,⁹⁾ respectively, as antifungal substances from different actinomycetes. Polyoxin inhibits the biosynthesis of chitin,^79)-81) a component of fungal cell walls that does not exist in humans.

5.1. Antifungal screening based on morphological changes.

In filamentous fungi cells where cell wall synthesis is inhibited by polyoxin, cells cannot withstand intracellular pressure and are induced to swell to a spherical shape, a morphology called “swelling.”^82)-84) We focused on the fact that antifungal agents induce unique morphologies that depend on the mode of action of the antifungal compound. The morphological changes induced by compounds in P. oryzae can be used to screen for new antifungal agents and elucidate their mechanisms of action. Therefore, we created a database of morphological changes in P. oryzae induced by antifungal compounds.

Morphological changes induced by known compounds in P. oryzae were observed in a 96-well plate. After adding the test samples to P. oryzae, the culture was incubated at 28°C for 48 hours, and the induced morphological changes were observed. Many of the compounds that showed growth-inhibitory activity induced different morphologies depending on the concentration; however, the morphology in which the mycelia died and turned black was commonly observed at high concentrations. Therefore, we decided to use morphological changes induced near the IC₅₀ values in the catalog.

Approximately 100 types of antifungal compounds with known targets were added to the culture media for P. oryzae, and the morphology was classified into “Short” when growth was inhibited and short hyphae were observed, and “Swelling” when cells swelled into a spherical shape and hyphal growth was inhibited. Complex I and III inhibitors, such as piericidin A and antimycin A, respectively induced thick swelling and short hyphae (“Swelling+Short”), similar to “Short” and unlike normal “Swelling”, were observed. In the case of oligomycin A, an inhibitor of complex V (FoF₁),⁸⁵⁾ the morphology of short hyphae connecting small swellings was observed, and it was named “Short+Beads”(Fig. 16).

Fig. 16

Morphological changes of Pyricularia oryzae induced by antifungal compounds.

Antifungal compounds from microbial metabolites were explored using this morphological change database named fMorphoBase (fungal morphology database). The fermentation broth of the actinomycete YO15-A001 strain was extracted using a mixed solution of MeOH and acetone, and the biological activity of the resulting extract was evaluated. A new active substance, YO-001A, purified from the strain, showed strong growth-inhibitory activity against P. oryzae, and its chemical structure was determined using NMR analysis and other methods (Fig. 17).⁸⁶⁾

Fig. 17

Chemical structure and biological activity of YO-001A. The structural formula and biological activity of YO-001 are similar to those of oligomycin.

The chemical structure of YO-001A is similar to that of oligomycin A, an inhibitor of the mitochondrial complex V (FoF₁-ATPase). When the morphological changes induced by YO15-A001 were compared with those of existing drugs, they were similar to the “Short & Beads” morphology induced by FoF₁-ATPase inhibitors. Because it was assumed that YO-001A has FoF₁-ATPase inhibitory activity similar to that of oligomycin A, the inhibitory activity of YO-001A against FoF₁-ATPase was evaluated using isolated mitochondria. The result revealed that the action of unknown compounds can be inferred from morphological changes in fungi and demonstrated the possibility of discovering new antifungal compounds using morphological changes as an indicator.

5.2. Artificial intelligence-assisted screening system.

Using visual observations, chemical compounds that induced morphological changes in P. oryzae were explored. However, visual observation has drawbacks, such as being qualitative and dependent on the observer’s subjectivity, and it is difficult to obtain quantitative data in a high-throughput manner. We developed a new morphological discrimination system to overcome these drawbacks. The new database named 2G-fMorphoBase (the second generation of fungal MorphoBase) was created using the assistance of artificial intelligence (AI). We used NVIDIA’s DIGITS (a deep learning GPU training system, https://developer.nvidia.com/digits) to build the morphological discrimination system. First, we observed morphological changes in P. oryzae induced by the target-known-compounds and constructed a morphological change database using learning AI from the microscopic images. Next, we screened and observed morphological changes in P. oryzae induced by microbial broth samples. We isolated the active substance from the culture broth and analyzed its mechanism of action. We tried to isolate the sample with low similarity to the morphological change induced by known compounds.

We successfully incorporated an AI system to categorize the P. oryzae morphology mentioned above. After training using several thousand photos for each category, the AI system correctly distinguished morphological changes in P. oryzae induced by antifungal compounds, and the prediction value exceeded 95%.

6. Chemical array screening

With the rise of combinatorial chemistry in the 1990s, it became possible to synthesize a huge number of compounds, and HTS using automated robots became popular.⁸⁷⁾ HTS is typically monitoring the desired biological activity in a small volume of test samples using chromogenic or fluorescent substrates. To increase analysis speed and cost-effectiveness, the sample volume should be smaller. Making the miniaturization of screening samples, the number of wells per multi-well plate increased from 96 to 384 and to 1,536 wells.

Inspired by DNA microarrays, we developed a chemical array in which compounds were immobilized on a chip (glass slide) and established a drug discovery screening system. Comprehensive analysis of the physical interactions (binding strength) between compounds immobilized on glass slides and the target protein has made it possible to screen a wide variety of compounds faster and using smaller amounts than in conventional screening method.

Screening using chemical arrays was pioneered by the Schreiber group at Harvard University.^88)-90) The first chemical array they made was immobilized on a glass substrate by introducing maleimide groups, spotting compounds with thiol groups on the substrate, and forming covalent bonds between the thiol groups of the compounds and the maleimide groups.⁸⁸⁾ The Schreiber group also immobilized compounds on the substrate by activating the substrate with thionyl chloride and reacting compounds with hydroxyl groups,⁸⁹⁾ and immobilizing compounds with acidic functional groups using diazobenzylidene groups.⁹¹⁾ All of these methods used specific functional groups of the compounds to covalently bond with linkers attached to the glass slide. Therefore, these methods require that the compounds possess a certain functional group or a certain functional group must be introduced into the compounds. In addition, when a specific functional group is used to bind to a linker, the area in the vicinity of that functional group cannot interact with the target protein (Fig. 18).

Fig. 18

Various methods for trapping small molecule compounds on a glass slide. Compounds with a functional group in the structure readily attach to the glass slide surface through a selective coupling reaction with a functional group at the end of the linker or the surface of the slide glass. This figure demonstrates three cases for each feature of the compound: thiol group reported by MacBeath et al.,⁸⁸⁾ hydroxy group reported by Hergenrother et al.,⁸⁹⁾ and acid group reported by Barnes-Seeman et al.⁹¹⁾

Therefore, we used carbene species⁹²⁾ generated by irradiating aryldiazirine groups with UV light (365 nm) to immobilize compounds on the linkers. As carbene species are highly reactive and can be inserted into various bonds, including C-H bonds, they are irreversibly bound to or inserted into nearby small molecules in a manner that is independent of the functional groups of small molecules.^93),94) In other words, it is possible in principle to immobilize any organic compound on a glass slide using the same method, without relying on specific functional groups.⁹³⁾ Generally, organic chemists aim for “clean” chemical reactions that produce uniform synthetic products. In contrast, our method, called photo-crosslinking, produces multiple reaction products and thus can be called a “dirty” reaction (Fig. 19).⁹⁵⁾ However, the ability to immobilize a single compound on the glass slide at various reaction sites leads to the retention of binding ability with the target protein, which is a major advantage.

Fig. 19

Functional group independent photo-cross-linking method by UV-irradiation. Various small molecules can be introduced onto glass slides via a photoaffinity reaction, regardless of the differences in their chemical structures. The advantage of this method is that it retains the ability of the compounds to interact with the binding proteins.

Using this method, we developed a chemical array (NPDepo array) carrying approximately 30,000 compounds from NPDepo. We designed a high-performance chemical array that is resistant to organic solvents that suited our purpose and constructed a fusion protein library consisting of red fluorescent protein and target proteins (GLORIA: Gene Library of Osada Laboratory, RIKEN) for large-scale chemical array analysis.^96),97) This chemical array method is useful for finding chemicals that interfere with nonenzymatic proteins, such as receptor proteins and transcription factors, and it is also useful for screening a huge number of compounds against a protein of interest. To date, we have conducted ligand screening of more than 200 proteins in collaboration with domestic and overseas laboratories. As a result, we found inhibitors of various proteins. The following sections introduce the results of inhibitor screening for scytalone dehydratase (SDH1), a melanin biosynthesis enzyme of P. oryzae, and for abscisic acid agonists that enhance the stress resistance of plant, in this section.⁹⁸⁾

6.1. Melanin synthesis inhibitors: agents for controlling rice blast infection.

Rice blast is a disease caused by the fungus Pyricularia oryzae, and controlling this pathogen is an important theme in agriculture.⁹⁹⁾ For P. oryzae to infect rice, it is necessary to synthesize melanin in organelles called appressoria, which are necessary for infection.^100)-102) Therefore, the melanin biosynthesis enzymes of P. oryzae have become targets for controlling rice blast, and various melanin biosynthesis inhibitors (MBIs) have been developed as agrochemicals (Fig. 20). Because MBIs can control disease without showing toxicity to the growth of the fungi, they are expected to be environmentally friendly agrochemicals. Carpropamid (MBI-D), an inhibitor of SDH1, which dehydrates scytalone, an intermediate in melanin biosynthesis, has been put to practical use as a rice blast disease control agent with excellent selective toxicity. However, with the emergence of mutants resistant to MBI-D, the previously developed SDH1 inhibitors were no longer used.^103),104)

Fig. 20

Melanin biosynthetic pathway in plant pathogenic fungi and melanin biosynthesis inhibitors. MBI: melanin biosynthesis dehydratase inhibitor. The targets of MBI were MBI-P (polyketide biosynthesis), MBP-R (THN reductase), and MBP-D (scytalone dehydratase).

We aimed to develop an inhibitor effective against drug-resistant fungi with the SDH1-V75M mutation, in which the 75th amino acid residue, valine, in SDH1 is mutated to methionine, using the chemical array method.^63),98),104) First, we prepared wild-type (SDH1-WT) and MBI-D-resistant (SDH1-V75M) recombinant enzymes and used chemical array technology to search for small-molecule compounds that bind to SDH1-WT and SDH1-V75M from the NPDepo library (approximately 30,000 compounds). Among the compounds found, NPD13731 had the strongest binding ability and inhibited SDH1-WT and SDH1-V75M with IC₅₀ values of 8.4 nM and 9.1 nM, respectively. Thus, NPD13731 binds to both wild-type and drug-resistant enzymes. Incidentally, commercially available MBI-D exhibited strong inhibitory activity against SDH1-WT (IC₅₀ 3.0 nM); however, its inhibitory activity against SDH1-V75M was significantly reduced (IC₅₀ 100 nM).

Although NPD13731 showed strong inhibitory activity at the enzyme level, its inhibitory activity on melanin biosynthesis in pathogenic fungi was weak; therefore, we attempted to synthesize derivatives. In conventional chemical synthesis, 2-(trimethylsilyl)ethoxymethyl (SEM) group is introduced to protect the hydroxyl group, and then the SEM group is removed. Unexpectedly, the derivative with a protecting group (SEM group) remaining at the 5-position hydroxyl group (melabiostin) showed the strongest activity, inhibiting SDH-WT and SDH-V75M at IC₅₀ values of 1.1 nM and 1.4 nM, respectively. Docking simulations predicted the binding between SDH1 and NPD13731 and showed that the 75th amino acid residue, valine, of SDH1 interacts hydrophobically with the benzofuran moiety of NPD13731, which is the cause of the resistant strain (Fig. 21). We presumed that protecting the 5-position hydroxyl group with an SEM group may improve stability, solubility, and uptake into fungal cells.

Fig. 21

Docking simulations of MBI-Ds (carpropamid and NPD13731) and SDH1.

Next, we evaluated the inhibitory activity of melabiostin against MBI-D-resistant strains. Treatment with carpropamid at 30 μM inhibited melanin biosynthesis in the susceptible strain, but not in the resistant strain, even at 100 μM. In contrast, melabiostin inhibited melanin biosynthesis in both strains at a concentration of 30 μM. Furthermore, the effect of melabiostin on the suppression of rice blast infection was analyzed. Carpropamid at 10 μM suppressed infection of the susceptible strain, but not of the resistant strain. In contrast, melabiostin suppressed lesion formation by both strains at a concentration of 10 μM (Fig. 22).

Fig. 22

Effect of melabiostin on the melanin biosynthesis in Pyricularia oryzae. Melabiostin was chemically synthesized based on the structure-activity relationship (Upper panel). Carpropamid (CPD) did not inhibit melanin formation of Pyricularia oryzae KR5-1 strain (drug-resistant), but melabiostin inhibited melanin formation of the resistant strain.

Using our proprietary chemical array technology, we discovered the compound NPD13731, which can also strongly inhibit the V75M mutant enzyme of SDH1. By optimizing its structure, we successfully created the compound melabiostin, which can control the MBI-D-resistant strain. Melabiostin strongly inhibits SDH1, a melanin biosynthetic enzyme, without inhibiting the growth of rice blast fungus and suppresses infection by P. oryzae; therefore, it is expected to be a new rice blast control agent or a lead compound.

6.2. Abscisic acid derivatives that enhance drought tolerance in plants.

As plants cannot move, they have a protective system against abiotic stresses such as drought, high salt, and high temperature. The plant hormone abscisic acid (ABA) plays an important role in abiotic stress responses such as seed dormancy, growth control, and drought in plants.^105)-107) Many ABA receptor proteins, such as PYR/PYL/RCAR, have been identified in plants. The receptor protein controls the downstream signaling cascade by inhibiting protein phosphatase 2C (PP2C) when ABA is not bound. When ABA binds to the receptor protein, PP2C is inactivated, SNF1-related protein kinase 2 is activated, and then the downstream signaling cascade was activated (Fig. 23).¹⁰⁸⁾

Fig. 23

Signal transduction of abscisic acid. Protein phosphatase 2C (PP2C) suppresses the ABA signal transduction when ABA is absent. Once the ABA receptor protein PYR1 binds to ABA, PP2C is recruited to the PYR1 and ABA complex. Then, SNF1-related protein kinase 2 (SnRK2) is activated, and ABA signaling is transferred downstream.

Arabidopsis thaliana has 14 receptor types, classified into three subfamilies (sf I, II, and III) (Fig. 24).¹⁰⁹⁾ However, many aspects of the ABA response to each receptor or subfamily as well as their functional differentiation remain unknown. Although several small molecule agonists/antagonists to subtype-specific ABA receptors have been reported, the variety of compounds is insufficient to discuss the role of the superfamily of receptors.¹¹⁰⁾ Therefore, we have started screening agonists/antagonists for ABA receptors using the chemical array method to identify receptor-specific agonists/antagonists.

Fig. 24

Subfamilies of abscisic acid receptors.

We cloned 14 ABA receptor genes and attempted to express these proteins for chemical array screening. We successfully expressed 11 receptor proteins and prepared highly pure proteins. Initially, we used PYR1 as a target receptor for ABA for 24,275 compounds attached to the chemical array and found a binding compound,¹¹⁰⁾ but it was ineffective in plant experiments due to poor membrane permeability. Hence, we explored more potent and specific binding compounds from the ABA focused library in which different chain lengths were introduced at the 3′ position of ABA. These compounds were examined for their interactions with 11 ABA receptors. When we found hit compounds, we performed a protein phosphatase assay of PP2C using three receptors from different subfamilies.¹¹¹⁾ We revealed that a series of 3′-alkyl ABAs bind to specific receptor subfamilies depending on the alkyl chain length (Fig. 25). We performed phosphatase assays using 11 Arabidopsis ABA receptors, Arabidopsis seed germination analysis, comprehensive gene expression analysis, root elongation analysis, and stomatal conductance measurement for five types of 3′-alkyl ABA with different alkyl chain lengths.¹¹²⁾ We found that activation of sf III and sf II is sufficient to cause an ABA response and that compounds that activate sf III and weakly activate certain sf II receptors induce stomatal closure and expression of ABA-responsive genes but do not inhibit taproot elongation (Fig. 26).¹¹³⁾ These observations suggested that 3′-butyl ABA is a potent compound that protects plants against drought as it induces stomatal closure without affecting taproot length.

Fig. 25

Binding affinity of ABA derivatives to ABA subfamily receptors.

Fig. 26

Drought resistance induced by ABA derivatives. 3′-Butyl ABA induced stomatal closure without shortening of taproots, then showed strong drought resistance to Arabidopsis thaliana.

As described above, chemical array screening is a powerful platform for identifying small-molecule ligands for target receptor proteins.

7. Summary

In the past, compound libraries were owned only by companies, making it difficult for academic researchers to use them. The United States was the first to set up compound libraries for academic researchers, and since then countries around the world have established compound libraries. The University of Tokyo, RIKEN, and other non-profit institutions in Japan distribute compound libraries to support academic drug discovery.

Meanwhile, companies that compete over the number of compound libraries are reconsidering their HTS methods, which rely on numbers, because blind screening of many compounds is costly and has a low hit rate. They improve cost performance by sharing compound libraries that have been kept secret from other companies and using compound informatics to select compounds with a high probability of success before screening.

In HTS, inhibitors of specific targets, such as enzymes, have been screened. However, Swinney and Anthony reported that many new drugs developed between 1999 and 2008 were the result of phenotypic screening,⁷⁴⁾ and phenotypic and high-content screening have been reevaluated recently. Screening based on morphological changes in filamentous fungi has been carried out previously.^82),83) However, we developed a new screening method using AI, as introduced in this review. In addition, if we can search for inhibitors that act specifically on the target molecules of fungi, we can theoretically identify compounds without side effects; therefore, target-based screening is also effective. Chemical array screening can quickly detect the interactions between many compounds and target proteins; therefore, it can be considered as ultra-high-throughput screening. Chemical biology techniques were introduced earlier to the pharmaceutical industry than to the agrochemical industry. The cases introduced in this review are still at the basic research level; therefore, the effects of the compounds found need to be confirmed in field tests. However, it is hoped that the effectiveness of chemical biology research is well understood and used for agriculture.

Acknowledgements

I want to express my sincere thanks to my collaborators at the Antibiotics Laboratory, Chemical Biology Research Group, and Chemical Resource Development Research Unit in RIKEN. Special thanks go to T. Nogawa, J.A.V. Lopez, I. Miyazaki, S. Takahashi, N. Kanoh, T. Motoyama, C.S. Yun, Y. Futamura, and K. Yoshida (RIKEN), who contributed to the studies described in this review.

The research results described in this review article were obtained as a part of projects “Latent Chemical Space” [JP23H04885], and KAKENHI [JP18H03945, JP21H04720] from the Japan Society for the Promotion of Science and Noda Institute for Scientific Research. Part of this work was also supported by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (28011A).

I would like to thank Editage (www.editage.jp) for English language editing.

Notes

Edited by Akira ISOGAI, M.J.A.

Correspondence should be addressed to: H. Osada, Institute of Microbial Chemistry (BIKAKEN), 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan (e-mail: [e-mail]).

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Non-standard abbreviation list

ABA

abscisic acid

AI

artificial intelligence

GlcA

glucuronic acid

HPLC

high performance liquid chromatography

HR-ESI-MS

high resolution electrospray ionization mass spectrometer

HTS

high-throughput screening

ISP

International Streptomyces Project

LC

liquid chromatography

MBI

melanin biosynthesis inhibitor

MorphoBase

morphology database

MS

mass spectrometer

NMR

nuclear magnetic resonance

NPDepo

Natural Products Depository

NPPlot

Natural Product Plot

OSMAC

one-strain-many-compounds

PP2C

protein phosphatase 2C

RM

reveromycin

SEM

2-(trimethylsilyl)ethoxymethyl

SDH

scytalone dehydratase

TA

tenuazonic acid

TAS

tenuazonic acid synthetase

Profile

Hiroyuki Osada was born in Shirakawa-shi, Fukushima Prefecture, Japan, in 1954. He graduated from the Department of Agriculture, the University of Tokyo, and received his Doctor of Agriculture degree in 1983. He joined RIKEN as a research scientist in 1983 and studied the mode of action of the nucleoside antibiotic ascamycin. From 1985 to 1986, he worked at the National Cancer Institute, Bethesda, USA, as a visiting scientist, where he discovered a new cell growth factor, KGF/FGF7.

He was appointed as a head of the Antibiotics Laboratory, RIKEN in 1992 and started chemical biology research using specific inhibitors isolated from microorganisms. After acting as a director of the Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science (RIKEN CSRS), he is currently a unit leader of RIKEN CSRS and a specially appointed director of the Institute of Microbial Chemistry (BIKAKEN).

He has served as a visiting professor at many universities, including Saitama University, Universiti Sains Malaysia (Malaysia), Tokyo Medical and Dental University, Strasbourg University (France), Victoria University of Wellington (New Zealand). He also served as an adjunct scientist of the Korean Research Institute of Bioscience and Biotechnology (KRIBB) and as a director of the Joint Research Center for Systems Chemical Biology of RIKEN-Max Plank Institute. He served as president of the Society of Actinomycetes, Japan (2012-2016), the Japanese Association for Molecular Target Therapy of Cancer (2015-2017) and the Japanese Society for Chemical Biology (2018-2024). He is currently president of the Federation of Microbiological Societies Japan (2021-present). He also played important roles as an editorial board member and as an advisory board member to international journals (Cancer Science, Journal of Antibiotics, Oncology Research, ACS Chemical Biology, Journal of Industry Microbiology and Biotechnology).

He has discovered over 100 kinds of new compounds, including tryprostatin, reveromycin, and epolactaene and revealed the molecular targets of newly isolated bioactive compounds. He has published more than 500 papers and 100 patent applications. His current research interests include the discovery of novel bioactive compounds, biosynthesis of microbial metabolites, and identification of molecular targets of bioactive compounds.

He is a recipient of the Medal with Purple Ribbon and the Sumiki-Umezawa Memorial Award from the Japan Antibiotic Research Association, the Award from the Society for Actinomycetes Japan, the Award from the Minister of Education, Culture, Sports, Science and Technology, the Award from the Bioindustry Association, the Inhoffen Prize in Germany, and the Special Award from the Agricultural Chemical Society of Japan.