Nornidulin, A New Inhibitor of Plasmodium falciparum Malate: Quinone Oxidoreductase (PfMQO) from Indonesian Aspergillus sp. BioMCC f.T.8501.

This study aimed to obtain a microbial active compound as a novel antimalarial drug from Indonesian isolates. Target-based assays were used to screen for antimalarial activity against the parasite mitochondrial, Plasmodium falciparum malate:quinone oxidoreductase (PfMQO) enzyme. In total, 1600 crude extracts, composed from 800 fungi and 800 actinomycetes extracts, were screened against PfMQO, yielding six active extracts as primary hits. After several stages of stability tests, one extract produced by Aspergillus sp. BioMCC f.T.8501 demonstrated stable PfMQO inhibitory activity. Several purification stages, including OCC, TLC, and HPLC, were performed to obtain bioactive compounds from this active extract. All purification steps were followed by an assay against PfMQO. We identified the active compound as nornidulin based on its LC-MS and UV spectrum data. Nornidulin inhibited PfMQO activity at IC50 of 51 µM and P. falciparum 3D7 proliferation in vitro at IC50 of 44.6 µM, however, it had no effect on the growth of several mammalian cells. In conclusion, we isolated nornidulin from Indonesian Aspergillus sp. BioMCC f.T.8501 as a novel inhibitor of PfMQO, which showed inhibitory activity against the proliferation of P. falciparum 3D7 in vitro.

Exploring natural microbial resources for the discovery of anti-malarial compounds.

Microorganisms in nature are highly diverse biological resources, which can be explored for drug discovery. Some countries including Brazil, Columbia, Indonesia, China, and Mexico, which are blessed with geographical uniqueness with diverse climates and display remarkable megabiodiversity, potentially provide microorganismal resources for such exploitation. In this review, as an example of drug discovery campaigns against tropical parasitic diseases utilizing microorganisms from such a megabiodiversity country, we summarize our past and on-going activities toward discovery of new antimalarials. The program was held in a bilateral collaboration between multiple Indonesian and Japanese research groups. In order to develop a new platform of drug discovery utilizing Indonesian bioresources under an international collaborative scheme, we aimed at: 1) establishment of an Indonesian microbial depository, 2) development of robust enzyme-based and cell-based screening systems, and 3) technology transfer necessary for screening, purification, and identification of antimalarial compounds from microbial culture broths. We collected, characterized, and deposited Indonesian microbes. We morphologically and genetically characterized fungi and actinomycetes strains isolated from 5 different locations representing 3 Indonesian geographical areas, and validated genetic diversity of microbes. Enzyme-based screening was developed against two validated mitochondrial enzymes from Plasmodium falciparum, dihydroorotate dehydrogenase and malate:quinone oxidoreductase, while cell-based proliferation assay was developed using the erythrocytic stage parasite of 3D7 strain. More than 17 thousands microbial culture extracts were subjected to the enzyme- and cell-based screening. Representative anti-malarial compounds discovered in this campaign are discussed, including a few isolated compounds that have been identified for the first time as anti-malarial compounds. Our antimalarial discovery campaign validated the Indonesian microbial library as a powerful resource for drug discovery. We also discuss critical needs for selection criteria for hits at each stage of screening and hit deconvolution such as preliminary extraction test for the initial profiling of the active compounds and dereplication techniques to minimize repetitive discovery of known compounds.

Gentisyl alcohol and homogentisic acid: Plasmodium falciparum dihydroorotate dehydrogenase inhibitors isolated from fungi.

Two Indonesian fungi Aspergillus assiutensis BioMCC-f.T.7495 and Penicillium pedernalense BioMCC-f.T.5350 along with a Japanese fungus Hypomyces pseudocorticiicola FKI-9008 have been found to produce gentisyl alcohol (1), which inhibits Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with an IC50 value of 3.4 µM. Another Indonesian fungus, Penicillium citrinum BioMCC-f.T.6730, produced an analog of 1, homogentisic acid (4), which also inhibits PfDHODH with an IC50 value of 47.6 µM.