Kiyoshi Kita

Background: 5-aminolevulinic acid (5-ALA), a natural amino acid that is marketed alongside sodium ferrous citrate (SFC) as a functional food, blocks severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proliferation in vitro and exerts anti-inflammatory effects. In this phase II open-label, prospective, parallel-group, randomized trial, we aimed to evaluate the safety and efficacy of 5-ALA in patients with mild-to-moderate coronavirus disease 2019. Methods: This trial was conducted in patients receiving 5-ALA/SFC (250/145 mg) orally thrice daily for 7 days, followed by 5-ALA/SFC (150/87 mg) orally thrice daily for 7 days. The primary endpoints were changes in SARS-CoV-2 viral load, clinical symptom scores, and 5-ALA/SFC safety (adverse events [AE] and changes in laboratory values and vital signs). Results: A total of 50 patients were enrolled from 8 institutions in Japan. The change in SARS-CoV-2 viral load from baseline was not significantly different between the 5-ALA/SFC (n = 24) and control (n = 26) groups. The duration to improvement was shorter in the 5-ALA/SFC group than in the control group, although the difference was not significant. The 5-ALA/SFC group exhibited faster improvement rates in “taste abnormality,” “cough,” “lethargy,” and “no appetite” than the control group. Eight AEs were observed in the 5-ALA/SFC group, with 22.7% of patients experiencing gastrointestinal symptoms (decreased appetite, constipation, and vomiting). AEs occurred with 750/435 mg/day in 25.0% of patients in the first phase and with 450/261 mg/day of 5-ALA/SFC in 6.3% of patients in the second phase. Conclusion: 5-ALA/SFC improved some symptoms but did not influence the SARS-CoV-2 viral load or clinical symptom scores over 14 days. The safety of 5-ALA/SFC in this study was acceptable. Further evaluation using a larger sample size or modified method is warranted.

Plasmodium falciparum contains several mitochondrial electron transport chain (ETC) dehydrogenases shuttling electrons from the respective substrates to the ubiquinone pool, from which electrons are consecutively transferred to complex III, complex IV, and finally to the molecular oxygen. The antimalarial drug atovaquone inhibits complex III and validates this parasite’s ETC as an attractive target for chemotherapy. Among the ETC dehydrogenases from P. falciparum, dihydroorotate dehydrogenase, an essential enzyme used in de novo pyrimidine biosynthesis, and complex III are the two enzymes that have been characterized and validated as drug targets in the blood-stage parasite, while complex II has been shown to be essential for parasite survival in the mosquito stage; therefore, these enzymes and complex II are considered candidate drug targets for blocking parasite transmission. In this study, we identified siccanin as the first (to our knowledge) nanomolar inhibitor of the P. falciparum complex II. Moreover, we demonstrated that siccanin also inhibits complex III in the low-micromolar range. Siccanin did not inhibit the corresponding complexes from mammalian mitochondria even at high concentrations. Siccanin inhibited the growth of P. falciparum with IC₅₀ of 8.4 μM. However, the growth inhibition of the P. falciparum blood stage did not correlate with ETC inhibition, as demonstrated by lack of resistance to siccanin in the yDHODH-3D7 (EC₅₀ = 10.26 μM) and Dd2-ELQ300 strains (EC₅₀ = 18.70 μM), suggesting a third mechanism of action that is unrelated to mitochondrial ETC inhibition. Hence, siccanin has at least a dual mechanism of action, being the first potent and selective inhibitor of P. falciparum complexes II and III over mammalian enzymes and so is a potential candidate for the development of a new class of antimalarial drugs.

Malaria parasites cannot multiply in host erythrocytes without cholesterol because they lack complete sterol biosynthesis systems. This suggests parasitized red blood cells (pRBCs) need to capture host sterols, but its mechanism remains unknown. Here we identified a novel high-density lipoprotein (HDL)-delivery pathway operating in blood-stage Plasmodium. In parasitized mouse plasma, exosomes positive for scavenger receptor CD36 and platelet-specific CD41 increased. These CDs were detected in pRBCs and internal parasites. A low molecular antagonist for scavenger receptors, BLT-1, blocked HDL uptake to pRBCs and suppressed Plasmodium growth in vitro. Furthermore, platelet-derived exosomes were internalized in pRBCs. Thus, we presume CD36 is delivered to malaria parasites from platelets by exosomes, which enables parasites to steal HDL for cholesterol supply. Cholesterol needs to cross three membranes (RBC, parasitophorous vacuole and parasite’s plasma membranes) to reach parasite, but our findings can explain the first step of sterol uptake by intracellular parasites.

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC₅₀ of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.

Plasmodium falciparum’s resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is a candidate as an oral antiviral drug for COVID-19.

Background: The Sri Lankan traditional betel quid (TBQ) which had been extensively used in the country before its colonization is claimed to have antiperiodontopathic effects in the Sri Lankan folklore. However, there is no reported scientific evidence to support the claimed antiperiodontopathic effects mediated by this TBQ. The present study was carried out to investigate the protective effect of the Sri Lankan TBQ in the pathogenesis of periodontitis.Methods: We investigate the ethyl acetate extract of the Sri Lankan TBQ for its antibacterial effects against the keystone periodontopathic bacterium,P. gingivalisand also its antioxidant potential, which is important to protect the periodontium from oxidative stress. Further, its safety was analyzed using the cytokinesis block micronucleus assay on human peripheral blood lymphocytes (PBLs).Results: Ethyl acetate extract of this TBQ inhibited the growth ofP. gingivaliswith a minimum bactericidal concentration (MBC) of 125 μg/ml. It was found to be a rich source of polyphenols and displayed considerable DPPH and ABTS radical scavenging activities and a strong ferric reducing antioxidant power. This extract could protect the cultured human gingival fibroblasts from H₂O₂induced oxidative stress. In addition, this TBQ extract was not genotoxic to human PBLs even at a concentration of 2.5 mg/ml. Moreover, it exhibited protective effects against bleomycin induced genotoxicity in PBLs.Conclusion: Ethyl acetate extract of the Sri Lankan TBQ is a source of natural antibacterial compounds againstP. gingivalis. It is also a source of natural antioxidants which can protect human gingival fibroblasts from H₂O₂induced oxidative stress. These properties of the TBQ may have contributed to its claimed antiperiodontopathic effects. Besides, it was found to be relatively non-toxic to human cells. Thus this TBQ extract has a huge potential to be developed as a novel adjunctive therapeutic lead against periodontitis.

In developed countries, it is said that "threats of infectious diseases are already thought as things of the past''. However, as you can see in the case of Ebola hemorrhagic fever that occurred in West Africa, this is a big mistake. Among infectious diseases, only smallpox has been successfully eradicated worldwide. In addition to the three major infectious diseases of HIV/AIDS, tuberculosis, and malaria, there is another group called emerging and reemerging infectious diseases. Recently, neglected tropical diseases (NTDs) have been listed as threats by the WHO, as have drug-resistant bacteria. The spread of these pathogens is increasing due to an increase in global travel. Malaria and more than half of the NTDs are parasitic diseases, such as trypanosomiasis and soil-borne helminthiasis. These are caused by parasites, with eukaryotes similar to their host mammals. In the case of these NTDs, protective immune responses induced by differences between a pathogen and host do not work well, and there is no vaccine against parasites. As for drugs developed to treat these diseases, because the properties of enzymes and target receptors are very similar, and effective drugs simultaneously show efficacy against both the disease and the host, severe side effects often occur. Therefore, the search for targets specifically present in parasites, and screening for drugs that inhibit their physiological functions, is extremely important. Here, as an example of the development of antiparasitic drugs, I will introduce a study on malaria.