The role of azole antifungal agents for systemic antifungal therapy
M-253c A novel antifungal agent discovered from ...
1
A novel antifungal agent discovered from Streptomyces sp. in sea sand Y. MATSUMOTO, K. KOBAYASHI, S.KATO, and M. IWAMI Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan and OP BIO factory,Uruma,Japan Contact Information Iwami Morita, Ph. D. Institute of Scientific and Industrial Research OP BIO factory. 12-2 Suzaki, Uruma, Okinawa 904-2234, Japan Phone +81-9-8982-1331 Fax +81-9-8982-1332 [email protected] Background: Clinically available antifungal drugs are not enough in the increase of fungal infectious diseases. A new type of antifungal agent is needed worldwide. We have found a new small compound active against broad spectrum of fungi from Streptomycin sp. A84 isolated from the sea sand in Cameraman Island, Kagoshima, Japan. The new antifungal agent was named Kakeromycin. Here we show preliminary data of antifungal activity of Kakeromycin. Methods: Kakeromycin was extracted from cultivated broth of Streptomycin sp. with equal volume of Ethyl Acetate. The Ethyl Acetate was then concentrated . The dried sample was dissolved in a small amount of water and was injected in HPLC using ODS column for purification. Macs were determined by a micro-broth dilution method followed by M27-A3 (CLSI). Macs of Kakeromycin were read by IC50 after 24 h for Candida and after 48 h for Cryptococcus and Aspergillums. Results: Kakeromycin had a new structure. Activities of Kakeromycin, micafungin, voriconazole and amphotericin B are displayed in the Table. Kakeromycin demonstrated good activity against Candida spp., Aspergillums spp. and Cryptococcus neoformans. Conclusions: The profile of Kakeromycin activity was different from other antifungal agents. Although the mechanism of antifungal activity of Kakeromycin has not yet been cleared, it seems different from other available antifungal drugs. The structure of Kakeromycin is small and seems easy to synthesize and to modify side chains to prepare derivatives. Kakeromycin would be expected to be a new class of useful antifungal agent. Acknowledgements: This work is supported by grant of Ministry of Economy, Trade and Industry. M-253c ICAAC2013 Fig 1. Picking place of Streptomyces sp. A84, the producer of antifungal agent Abstract Introduction Fig 3. Isolation and purification of Kakeromycin Kakeromycin was purified to 95 % finaly, and its structure was determined from LC-MS and NMR data. Summary Table 1. Antifungal activity of Kakeromycin Producing organism. Streptomyces sp. A84 New species. (Undescribed species) Conclusion Bioactive fraction was concentrated and purified by RP-HPLC. (MeOH) The bioactivities of samples were determined by Saccharomyces cerevisae assay plates . HPLC condition: ODS column, 65 % MeOH (isocratic) . HPLC condition: ODS column, 40 % Acetonitrile (isocratic) . Acknowledgements This work is supported by grant of Ministry of Economy, Trade and Industry. Kakeromycin molecular formula：secret molecular weight ：secret We selected isolated places not frequented by humans for the collection of the microorganisms using boat. The area where the new compound-producing microorganism was collected is shown in the map. We isolated an actinomycetes with antifungal activity after one million microorganisms screening. The actinomycetes was identified by 16SrRNA phylogenetic analysis. As a result of 16SrRNA phylogenetic study, the producing strain showed the highest similarity to Streptomyces nodosus. However, the homology of the 16SrRNA sequences was only 90%. In addition, the morphological characteristics of the spore chain were different from S. nodosus. These lead us to the conclusion that the isolated Streptomyces is a new species. Origin: Sea sand Place: Kakeroma island, Amami islands, Kagoshima Prefecture, Japan The new antifungal agent was named “Kakeromycin”. 【Pre-culture】 100ml SC medium 3 days at 28℃ with shaking in rotary shaker (200rpm). 【Main culture】 Pre-culture was seeded to the fresh SC medium by 5%, and cultured for the following 3 days at 28℃ in the shaker. SC Medium: ○ Soluble Starch 1.0% ○ Casein 0.1% ○ Na2HPO4 0.1% ○ Artificial Sea Water 3.84% Kakeromycin was extracted from10 liter of filtered broth with 5 liter of Ethyl Acetate, concentrated by open ODS column, and was eluted with serial concentrations of MeOH. 【The strain】 Streptomyces sp. A84 Fig 2. Phylogenetic systematics and Cultured plate of the producer A84 Antifungal activity was present in 60% methanol extraction. MICs were determined in the laboratories of BML, Inc. (Kawagoe, Japan) by microbroth dilution method according to approved standard M27-A3 defined by CLSI. Abbreviations: KKRM (Kakeromycin), MCFG (Micafungin), AMPH-B (Amphotericin B), 5-FU (Fulcytosin), FLCZ (Fluconazole), ITCZ (Itraconazole), VRCZ (Voriconazole), MCZ (Miconazole) Fig 4. Morphological changes of C. albicans by Kakeromycin on agar plate Screening of natural products for antifungal agents have been carried out for many years but no new antifungal agent has been discovered in recent years. One possible reason is because no new microorganisms have been found. We decided to pursue the isolation of new microorganisms from marine sources which are not commonly carried out. In addition, we developed a new screening method which did not use any analytical equipment, but instead carried out 1 million microorganisms evaluation manually. As a result, we discovered a compound having a novel core structure with strong antifungal activity. In this report, we present the place of collection, the characteristics of the producing microorganism, the culture method, compound purification and antifungal activity. We have found a new Streptomyses sp. producing a new antifungal agent named kakeromycin using a new isolation method from our unique library of microorganisms. The activity profile of Kakeromycin activity is different from other antifungal agents. Although the mechanism of the activity of Kakeromycin has not yet been fully elucidated, it seems to be different from other known antifungal drugs. The structure of Kakeromycin (undisclosed at the moment) is small and will be easy to synthesize. The side chains can also be modified to prepare derivatives for structure-activity relationship (SAR) studies. Kakeromycin is expected to be a new class of useful antifungal agent. The extract Outside of inhibition zone Edge of inhibition zone Center of inhibition zone 10μ 10μ 10μ 10μ No resistance acquisition by C. albicans to Kakeromycin after single pasage Cells grown in the inhibition zone of Kakeromycin was still sensitive to Kakeromycin as well as control cells. Control cells Cells grown in the inhibition zone MIC: μg/mL KKRM MCFG AMPH-B 5-FU FLCZ ITCZ VRCZ MCZ Candida albicans ATCC24433 0.5 ≦0.015 1 0.25 0.25 ≦0.015 ≦0.015 ≦0.03 ATCC90028 0.125 ≦0.015 0.25 ≦0.125 ≦0.125 ≦0.015 ≦0.015 ≦0.03 ATCC90029 0.125 ≦0.015 0.125 >64 0.25 ≦0.015 ≦0.015 ≦0.03 clinical isolate 1 0.125 ≦0.015 0.25 ≦0.125 ≦0.125 ≦0.015 ≦0.015 ≦0.03 clinical isolate 2 0.125 ≦0.015 0.25 ≦0.125 ≦0.125 ≦0.015 ≦0.015 ≦0.03 clinical isolate 3 0.06 ≦0.015 0.125 ≦0.125 0.25 0.03 ≦0.015 ≦0.03 Candida parapsilosis CC22019 0.015 0.5 0.25 ≦0.125 0.25 ≦0.015 ≦0.015 0.06 ATCC90018 0.5 ≦0.015 0.125 ≦0.125 0.5 ≦0.015 ≦0.015 ≦0.03 clinical isolate 1 0.008 0.5 0.125 ≦0.125 2 0.03 ≦0.015 ≦0.03 clinical isolate 2 0.125 0.25 0.25 ≦0.125 2 0.03 0.06 0.25 clinical isolate 3 0.015 0.25 0.125 ≦0.125 0.5 ≦0.015 ≦0.015 0.06 Candida tropicalis ATCC750 >8 ≦0.015 0.25 ≦0.125 1 0.06 0.03 0.5 clinical isolate 1 >8 ≦0.015 0.125 ≦0.125 1 0.06 0.03 0.25 clinical isolate 2 >8 ≦0.015 0.125 ≦0.125 2 0.25 0.06 1 clinical isolate 3 4 ≦0.015 0.5 ≦0.125 4 0.125 0.125 1 Candida krusei ATCC6258 0.125 0.06 0.5 4 32 1 0.25 1 Candida glabrata ATCC90030 1 0.03 0.5 ≦0.125 8 1 0.25 0.125 clinical isolate 1 0.125 ≦0.015 1 ≦0.125 4 0.5 0.125 0.125 clinical isolate 2 8 ≦0.015 0.25 ≦0.125 4 0.5 0.125 0.125 clinical isolate 3 0.5 ≦0.015 0.5 ≦0.125 4 0.25 0.125 0.125 Candida quilliermondii clinical isolate 1 0.5 1 0.25 1 4 0.25 0.06 0.25 Candida lusitaniae clinical isolate 1 0.5 0.125 1 0.25 0.25 0.06 ≦0.015 0.06 Aspergillus fumigatus clinical isolate 1 2 ≦0.015 1 64 >64 0.125 0.25 0.5 Aspergillus flavus clinical isolate 1 4 ≦0.015 1 4 32 0.125 0.25 2 Aspergillus niger clinical isolate 1 8 ≦0.015 0.5 0.5 >64 0.25 0.125 1 Aspergillus terreus clinical isolate 1 0.25 ≦0.015 0.5 0.25 16 ≦0.015 0.03 0.06 The active compound-producing microorganism was isolated from a place not frequented by humans. Sea water was used in the isolation medium. A new Streptomyces species having strong antifungal activity was found from one million strains screened by a manual evaluation method without using analytical equipment. The antifungal compound named Kakeromycin was purified by HPLC and its structure was determined by LC-MS and NMR. Kakeromycin has a novel core structure (patent- pending). Kakeromycin showed strong antifungal activity against various Candida spp. and Aspergillus spp. MIC levels of the compound against C. albicans were similar to amphotericin B. The shape of C. albicans cells were expanded and elongated by Kakeromycin. Kakeromycin is not completely fungicidal against C. albicans. However, Kakeromycin still cause an inhibition zone showing the sensitivity of C. albicans to the compound.
Transcript of M-253c A novel antifungal agent discovered from ...
A novel antifungal agent discovered from Streptomyces sp. in sea sand Y. MATSUMOTO, K. KOBAYASHI, S.KATO, and M. IWAMI
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan and OP BIO factory,Uruma,Japan
Contact Information
Iwami Morita, Ph. D.
Institute of Scientific and Industrial Research OP BIO
factory.
12-2 Suzaki, Uruma, Okinawa 904-2234, Japan
Phone +81-9-8982-1331
Fax +81-9-8982-1332
Background: Clinically available antifungal drugs are not enough in the increase of fungal infectious diseases. A new type of antifungal agent is needed worldwide. We have found a new small compound active against broad spectrum of fungi from Streptomycin sp. A84 isolated from the sea sand in Cameraman Island, Kagoshima, Japan. The new antifungal agent was named Kakeromycin. Here we show preliminary data of antifungal activity of Kakeromycin. Methods: Kakeromycin was extracted from cultivated broth of Streptomycin sp. with equal volume of Ethyl Acetate. The Ethyl Acetate was then concentrated . The dried sample was dissolved in a small amount of water and was injected in HPLC using ODS column for purification. Macs were determined by a micro-broth dilution method followed by M27-A3 (CLSI). Macs of Kakeromycin were read by IC50 after 24 h for Candida and after 48 h for Cryptococcus and Aspergillums. Results: Kakeromycin had a new structure. Activities of Kakeromycin, micafungin, voriconazole and amphotericin B are displayed in the Table. Kakeromycin demonstrated good activity against Candida spp., Aspergillums spp. and Cryptococcus neoformans. Conclusions: The profile of Kakeromycin activity was different from other antifungal agents. Although the mechanism of antifungal activity of Kakeromycin has not yet been cleared, it seems different from other available antifungal drugs. The structure of Kakeromycin is small and seems easy to synthesize and to modify side chains to prepare derivatives. Kakeromycin would be expected to be a new class of useful antifungal agent. Acknowledgements: This work is supported by grant of Ministry of Economy, Trade and Industry.
M-253c
ICAAC2013
Fig 1. Picking place of Streptomyces sp. A84, the producer of antifungal agent
Abstract
Introduction
Fig 3. Isolation and purification of Kakeromycin
Kakeromycin was purified to 95 % finaly, and its structure was determined from LC-MS and NMR data.
Summary Table 1. Antifungal activity of Kakeromycin
Producing organism. Streptomyces sp. A84 New species. (Undescribed species)
Conclusion
Bioactive fraction was concentrated and purified by RP-HPLC.
(MeOH)
The bioactivities of samples were determined by Saccharomyces cerevisae assay plates .
HPLC condition: ODS column, 65 % MeOH (isocratic) .
HPLC condition: ODS column, 40 % Acetonitrile (isocratic) .
Acknowledgements This work is supported by grant of Ministry of Economy, Trade and Industry.
Kakeromycin molecular formula：secret molecular weight ：secret
We selected isolated places not frequented by humans for the collection of the microorganisms using boat. The area where the new compound-producing microorganism was collected is shown in the map.
We isolated an actinomycetes with antifungal activity after one million microorganisms screening. The actinomycetes was identified by 16SrRNA phylogenetic analysis.
As a result of 16SrRNA phylogenetic study, the producing strain showed the highest similarity to Streptomyces nodosus. However, the homology of the 16SrRNA sequences was only 90%. In addition, the morphological characteristics of the spore chain were different from S. nodosus. These lead us to the conclusion that the isolated Streptomyces is a new species.
Origin: Sea sand Place: Kakeroma island, Amami islands, Kagoshima Prefecture, Japan The new antifungal agent was named “Kakeromycin”.
【Pre-culture】 100ml SC medium 3 days at 28℃ with shaking in rotary shaker (200rpm).
【Main culture】 Pre-culture was seeded to the fresh SC medium by 5%, and cultured for the following 3 days at 28℃ in the shaker.
SC Medium: ○ Soluble Starch 1.0% ○ Casein 0.1% ○ Na2HPO4 0.1% ○ Artificial Sea Water 3.84%
Kakeromycin was extracted from10 liter of filtered broth with 5 liter of Ethyl Acetate, concentrated by open ODS column, and was eluted with serial concentrations of MeOH.
【The strain】 Streptomyces sp. A84
Fig 2. Phylogenetic systematics and Cultured plate of the producer A84
Antifungal activity was present in 60% methanol extraction.
MICs were determined in the laboratories of BML, Inc. (Kawagoe, Japan) by microbroth dilution method according to approved standard M27-A3 defined by CLSI. Abbreviations: KKRM (Kakeromycin), MCFG (Micafungin), AMPH-B (Amphotericin B), 5-FU (Fulcytosin), FLCZ (Fluconazole), ITCZ (Itraconazole), VRCZ (Voriconazole), MCZ (Miconazole)
Fig 4. Morphological changes of C. albicans by Kakeromycin on agar plate
Screening of natural products for antifungal agents have been carried out for many years but no new antifungal agent has been discovered in recent years. One possible reason is because no new microorganisms have been found. We decided to pursue the isolation of new microorganisms from marine sources which are not commonly carried out. In addition, we developed a new screening method which did not use any analytical equipment, but instead carried out 1 million microorganisms evaluation manually. As a result, we discovered a compound having a novel core structure with strong antifungal activity. In this report, we present the place of collection, the characteristics of the producing microorganism, the culture method, compound purification and antifungal activity.
We have found a new Streptomyses sp. producing a new antifungal agent named kakeromycin using a new isolation method from our unique library of microorganisms. The activity profile of Kakeromycin activity is different from other antifungal agents. Although the mechanism of the activity of Kakeromycin has not yet been fully elucidated, it seems to be different from other known antifungal drugs. The structure of Kakeromycin (undisclosed at the moment) is small and will be easy to synthesize. The side chains can also be modified to prepare derivatives for structure-activity relationship (SAR) studies. Kakeromycin is expected to be a new class of useful antifungal agent.
The extract
Outside of inhibition zone Edge of inhibition zone Center of inhibition zone
10µ 10µ
10µ 10µ
No resistance acquisition by C. albicans to Kakeromycin
after single pasage
Cells grown in the inhibition zone of Kakeromycin was still sensitive to Kakeromycin as well as control cells.
Control cells Cells grown in the inhibition zone
MIC: μg/mL KKRM MCFG AMPH-B 5-FU FLCZ ITCZ VRCZ MCZ
Candida albicans ATCC24433 0.5 ≦0.015 1 0.25 0.25 ≦0.015 ≦0.015 ≦0.03 ATCC90028 0.125 ≦0.015 0.25 ≦0.125 ≦0.125 ≦0.015 ≦0.015 ≦0.03
ATCC90029 0.125 ≦0.015 0.125 >64 0.25 ≦0.015 ≦0.015 ≦0.03 clinical isolate 1 0.125 ≦0.015 0.25 ≦0.125 ≦0.125 ≦0.015 ≦0.015 ≦0.03 clinical isolate 2 0.125 ≦0.015 0.25 ≦0.125 ≦0.125 ≦0.015 ≦0.015 ≦0.03 clinical isolate 3 0.06 ≦0.015 0.125 ≦0.125 0.25 0.03 ≦0.015 ≦0.03
Candida parapsilosis CC22019 0.015 0.5 0.25 ≦0.125 0.25 ≦0.015 ≦0.015 0.06 ATCC90018 0.5 ≦0.015 0.125 ≦0.125 0.5 ≦0.015 ≦0.015 ≦0.03
clinical isolate 1 0.008 0.5 0.125 ≦0.125 2 0.03 ≦0.015 ≦0.03 clinical isolate 2 0.125 0.25 0.25 ≦0.125 2 0.03 0.06 0.25 clinical isolate 3 0.015 0.25 0.125 ≦0.125 0.5 ≦0.015 ≦0.015 0.06
Candida tropicalis ATCC750 >8 ≦0.015 0.25 ≦0.125 1 0.06 0.03 0.5 clinical isolate 1 >8 ≦0.015 0.125 ≦0.125 1 0.06 0.03 0.25 clinical isolate 2 >8 ≦0.015 0.125 ≦0.125 2 0.25 0.06 1 clinical isolate 3 4 ≦0.015 0.5 ≦0.125 4 0.125 0.125 1
Candida krusei ATCC6258 0.125 0.06 0.5 4 32 1 0.25 1 Candida glabrata ATCC90030 1 0.03 0.5 ≦0.125 8 1 0.25 0.125
clinical isolate 1 0.125 ≦0.015 1 ≦0.125 4 0.5 0.125 0.125 clinical isolate 2 8 ≦0.015 0.25 ≦0.125 4 0.5 0.125 0.125 clinical isolate 3 0.5 ≦0.015 0.5 ≦0.125 4 0.25 0.125 0.125
Candida quilliermondii clinical isolate 1 0.5 1 0.25 1 4 0.25 0.06 0.25 Candida lusitaniae clinical isolate 1 0.5 0.125 1 0.25 0.25 0.06 ≦0.015 0.06
Aspergillus fumigatus clinical isolate 1 2 ≦0.015 1 64 >64 0.125 0.25 0.5 Aspergillus flavus clinical isolate 1 4 ≦0.015 1 4 32 0.125 0.25 2 Aspergillus niger clinical isolate 1 8 ≦0.015 0.5 0.5 >64 0.25 0.125 1
Aspergillus terreus clinical isolate 1 0.25 ≦0.015 0.5 0.25 16 ≦0.015 0.03 0.06
Ø The active compound-producing microorganism was isolated from a place not frequented by humans.
Ø Sea water was used in the isolation medium. Ø A new Streptomyces species having strong antifungal
activity was found from one million strains screened by a manual evaluation method without using analytical equipment.
Ø The antifungal compound named Kakeromycin was purified by HPLC and its structure was determined by LC-MS and NMR.
Ø Kakeromycin has a novel core structure (patent-pending).
Ø Kakeromycin showed strong antifungal activity against various Candida spp. and Aspergillus spp. MIC levels of the compound against C. albicans were similar to amphotericin B. The shape of C. albicans cells were expanded and elongated by Kakeromycin.
Ø Kakeromycin is not completely fungicidal against C. albicans. However, Kakeromycin still cause an inhibition zone showing the sensitivity of C. albicans to the compound.
