Dephospho-Coenzyme A Kinase Is an Exploitable Drug Target against Plasmodium falciparum: Identification of Selective Inhibitors by High-Throughput Screening of a Large Chemical Compound Library.

Malaria is a mosquito-borne fatal infectious disease that affects humans and is caused by Plasmodium parasites, primarily Plasmodium falciparum. Widespread drug resistance compels us to discover novel compounds and alternative drug discovery targets. The coenzyme A (CoA) biosynthesis pathway is essential for the malaria parasite P. falciparum. The last enzyme in CoA biosynthesis, dephospho-CoA kinase (DPCK), is essential to the major life cycle development stages but has not yet been exploited as a drug target in antimalarial drug discovery. We performed a high-throughput screen of a 210,000-compound library using recombinant P. falciparum DPCK (PfDPCK). A high-throughput enzymatic assay using a 1,536-well platform was developed to identify potential PfDPCK inhibitors. PfDPCK inhibitors also inhibited parasite growth in a P. falciparum whole-cell asexual blood-stage assay in both drug-sensitive and drug-resistant strains. Hit compounds were selected based on their potency in cell-free (PfDPCK) and whole-cell (Pf3D7 and PfDd2) assays, selectivity over the human orthologue (HsCOASY) and no cytotoxicity (HepG2). The compounds were ranked using a multiparameter optimization (MPO) scoring model, and the specific binding and the mechanism of inhibition were investigated for the most promising compounds.

First Total Synthesis and Structure-Activity Relationship of Iheyamide A, an Antitrypanosomal Linear Peptide Isolated from a Dapis sp. Marine Cyanobacterium.

Iheyamide A (1) is an antitrypanosomal linear peptide isolated from a Dapis sp. marine cyanobacterium by our group in 2020, and based on structure-activity relationships of its natural analogues, the C-terminal pyrrolinone moiety has been identified as the phamacophore for its antiparasitic activity. Further, we isolated this pyrrolinone moiety by itself as a new natural product from the marine cyanobacterium and named it iheyanone (2). As expected, iheyanone (2) showed antitrypanosomal activity, but its potency was weaker than iheyamide A (1). To clarify more detailed structure-activity relationships, we completed a total synthesis of iheyamide A (1) along with iheyanone (2) and evaluated the antitrypanosomal activities of several synthetic intermediates. As a result, we found that the longer the peptide chain, the stronger the antitrypanosomal activity. As iheyamide A (1) showed selective toxicity against Trypanosoma brucei rhodesiense, these findings can provide design guidelines for antitrypanosomal drugs.

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.

Characterization of Plasmodium falciparum Pantothenate Kinase and Identification of Its Inhibitors From Natural Products.

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum, the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases (PfPanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant PfPanK1 for the first time. PfPanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant PfPanK1 and identified four PfPanK inhibitors from natural compounds.

Isolation, Structure Determination, and Total Synthesis of Hoshinoamide C, an Antiparasitic Lipopeptide from the Marine Cyanobacterium Caldora penicillata.

Hoshinoamide C (1), an antiparasitic lipopeptide, was isolated from the marine cyanobacterium Caldora penicillata. Its planar structure was elucidated by spectral analyses, mainly 2D NMR, and the absolute configurations of the α-amino acid moieties were determined by degradation reactions followed by chiral-phase HPLC analyses. To clarify the absolute configuration of an unusual amino acid moiety, we synthesized two possible diastereomers of hoshinoamide C and determined its absolute configuration based on a comparison of their spectroscopic data with those of the natural compound. Hoshinoamide C (1) did not exhibit any cytotoxicity against HeLa or HL60 cells at 10 µM, but inhibited the growth of the parasites responsible for malaria (IC50 0.96 µM) and African sleeping sickness (IC50 2.9 µM).