Shusaku Mizukami

Our group explores the antimalarial compounds from the Streptomyces culture library. Pilot screening showed significant antimalarial activity in Streptomyces antibioticus HUT6035 (synonym of strain NBRC3117). We here isolated antimalarial compounds through large-scale fermentation and subsequent purification using chromatographies. Strain HUT6035 accumulated actinomycins X ₂ and X _0ß , which were 4-oxo and 4-hydroxy proline derivatives of actinomycin D, respectively. The former showed an antimalaria activity (IC ₅₀ = 0.241 nM) and a cytotoxicity (CC ₅₀ = 6.71 nM), while the latter showed IC ₅₀ = 7.69 nM and CC ₅₀ = 818 nM. Compared with commercially available actinomycin D (IC ₅₀ = 0.469 nM and CC ₅₀ = 6.71 nM), both actinomycins X ₂ and X _0ß have significant selectivity index (SI) values (CC ₅₀ /IC ₅₀ ) with 27.9 and 106. These SI values were two- and seven-fold preferential against that of actinomycin D (SI = 14.3). Thus, actinomycin derivatives are potential antimalarial agents.

Background: Natural products play a key role as potential sources of biologically active substances for the discovery of new drugs. This study aimed to identify secondary metabolites from actinomycete library extracts that are potent against the asexual stages of Plasmodiumfalciparum (P.falciparum). Methods: Secondary metabolites from actinomycete library extracts were isolated from culture supernatants by ethyl acetate extraction. Comprehensive screening was performed to identify novel antimalarial compounds from the actinomycete library extracts (n = 28). The antimalarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) lines of P.falciparum. The cytotoxicity was then evaluated in primary adult mouse brain (AMB) cells. Results: Out of the 28 actinomycete extracts, 17 showed parasite growth inhibition > 50% at a concentration of 50 µg/mL, nine were identified with an IC₅₀ value < 10 µg/mL, and seven suppressed the parasite significantly with an IC₅₀ value < 5 µg/mL. The extracts from Streptomycesaureus strains HUT6003 (Extract ID number: 2), S.antibioticus HUT6035 (8), and Streptomyces sp. strains GK3 (26) and GK7 (27), were found to have the most potent antimalarial activity with IC₅₀ values of 0.39, 0.09, 0.97, and 0.36 µg/mL (against 3D7), and 0.26, 0.22, 0.72, and 0.21 µg/mL (against Dd2), respectively. Among them, Streptomycesantibioticus strain HUT6035 (8) showed the highest antimalarial activity with an IC₅₀ value of 0.09 µg/mL against 3D7 and 0.22 µg/mL against Dd2, and a selective index (SI) of 188 and 73.7, respectively. Conclusion: Secondary metabolites obtained from the actinomycete extracts showed promising antimalarial activity in vitro against 3D7 and Dd2 cell lines of P.falciparum with minimal toxicity. Therefore, secondary metabolites obtained from actinomycete extracts represent an excellent starting point for the development of antimalarial drug leads.

Background Scientists renewed interest in the discovery of drugs from natural products and therefore they continue to play a key role as a potential source of biologically active substances for the discovery of new drugs. This study aimed to identify the secondary metabolite of actinomycete library extracts that are potent against asexual stages of Plasmodium falciparum (P. falciparum). Methods Isolation of secondary metabolite of actinomycete library extracts were conducted using ethyl acetate extraction from the culture supernatant. A comprehensive screening was performed to identify novel antimalarial compounds from secondary metabolites in Actinomycete library extracts (n = 28). The antimalarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) lines of P. falciparum. The cytotoxicity was also evaluated using primary adult mouse brain (AMB) cells. Results Out of 28 secondary metabolites of actinomycete extracts, seventeen of which showed a parasite growth inhibition > 50% at 50 µg/mL, nine hits were identified with the IC₅₀ < 10 µg/mL, seven of which were suppressing the parasite significantly with the IC₅₀ < 5 µg/mL. The extracts from Streptomyces aureus strains HUT6003 (Extract ID number: 2) and antibioticus HUT6035 (8), and Streptomyces sp. strains GK3 (26) and GK7 (27), were found to have the most potent antimalarial activity with the IC₅₀ 0.39, 0.09, 0.97, and 0.36 µg/mL (against 3D7), and 0.26, 0.22, 0.72, and 0.21 µg/mL (against Dd2), respectively. Among them, Streptomyces antibioticus strain HUT6035 (8) showed the highest antimalarial activity with IC₅₀ 0.09 against 3D7 and 0.22 against Dd2 with a selective index (SI) of 188 and 73.7, respectively. Conclusion Secondary metabolite(s) of actinomycete extracts showed promising antimalarial activity against 3D7 and Dd2 lines of P. falciparum in vitro with minimal toxicity. Therefore, a secondary metabolite of actinomycete extracts represents an excellent starting point toward the development of antimalarial drug leads.

Recent studies have suggested that CD8⁺ liver-resident memory T (T_RM) cells are crucial in the protection against liver-stage malaria. We used liver-directed mRNA-containing lipid nanoparticles (mRNA-LNPs) to induce liver T_RM cells in a murine model. Single-dose intravenous injections of ovalbumin mRNA-LNPs effectively induced antigen-specific cytotoxic T lymphocytes in a dose-dependent manner in the liver on day 7. T_RM cells (CD8⁺ CD44^hi CD62L^lo CD69⁺ KLRG1^-) were induced 5 weeks after immunization. To examine the protective efficacy, mice were intramuscularly immunized with two doses of circumsporozoite protein mRNA-LNPs at 3-week intervals and challenged with sporozoites of Plasmodium berghei ANKA. Sterile immunity was observed in some of the mice, and the other mice showed a delay in blood-stage development when compared with the control mice. mRNA-LNPs therefore induce memory CD8⁺ T cells that can protect against sporozoites during liver-stage malaria and may provide a basis for vaccines against the disease.

Background: Current therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo. Methods: As an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging. Results: As a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC₅₀ values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model. Conclusions: For these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.

Lipid nanoparticles (LNPs) are currently in the spotlight as delivery systems for mRNA therapeutics and have been used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. mRNA-LNP formulations have been indicated to require strict control, including maintenance at fairly low temperatures during their transport and storage. Since it is a new pharmaceutical modality, there is a lack of information on the systematic investigation of how storage and handling conditions affect the physicochemical properties of mRNA-LNPs and their protein expression ability. In this study, using the mRNA-LNPs with standard composition, we evaluated the effects of temperature, cryoprotectants, vibration, light exposure, and syringe aspiration from the vials on the physicochemical properties of nanoparticles in relation to their in vitro/in vivo protein expression ability. Among these factors, storage at −80 °C without a cryoprotectant caused a decrease in protein expression, which may be attributed to particle aggregation. Exposure to vibration and light also caused similar changes under certain conditions. Exposure to these factors can occur during laboratory and hospital handling. It is essential to have sufficient knowledge of the stability of mRNA-LNPs in terms of their physical properties and protein expression ability at an early stage to ensure reproducible research and development and medical care.

Three phenylpropanoid-conjugated iridoid glucosides, acetylgaertneric acid (1), acetyldehydrogaertneroside (2), and dehydrogaertneric acid (10), together with nine known related iridoid glucosides (3–9, 11, and 12), two coumaroyl alkaloids, one benzenoid, and three flavonoid glucosides were isolated from leaves of Morinda morindoides (Rubiaceae). Structures of these isolated compounds were determined using spectroscopic analysis. Compounds 1–18 and previously isolated compounds (19–29) were evaluated for anti-trypanosomal activity against Trypanosoma cruzi Tulahuen strain (trypomastigote and amastigote) together with cytotoxicity against host cells, new-born mouse heart cells. Among them, molucidin (21) and prismatomerin (22) exhibited good anti-trypanosomal activity (IC₅₀ of 4.67 and 5.70 µM, respectively), together with cytotoxicity (CC₅₀ of 2.76 and 3.22 μM, respectively). Compounds 1–18 did not show anti-malarial activity against a chloroquine/mefloquine-sensitive strain of Plasmodium falciparum.

Two phenylpropanoid-conjugated iridoids, deglucosyl gaertneroside (1) and morindoidin (2), were isolated from the leaves of Morinda morindoides (Rubiaceae) by activity-guided fractionation using an anti-malarial activity assay. The known related iridoids molucidin (3) and prismatomerin (4), two lignans, abscisic acid, two megastigmanes, and two flavonol glycosides were also identified. The structures of isolated compounds were elucidated using spectroscopic analysis. The isolated compounds were evaluated for anti-malarial activity against the chloroquine/mefloquine-sensitive strains of Plasmodium falciparum together with cytotoxicity against adult mouse brain cells. Potent anti-malarial activity of 3 and 4 (IC₅₀ of 0.96 and 0.80 μM, CC₅₀ of 1.02 and 0.88 μM, and SI of 1.06 and 1.10, respectively) was shown, while new iridoids 1 and 2 and pinoresinol (5) displayed moderate activity (IC₅₀ of 40.9, 20.6, and 24.2 μM) without cytotoxicity (CC₅₀ > 50 μM). These results indicate that 1–5 may be promising lead compounds for anti-malarial drugs. In addition, our results imply the necessity of the quality control of the extract of M. morindoides leaves based on the contents of 1–5 in terms of the safety and efficacy.