Lead optimization of potent Gwt1p inhibitors toward a new antimalarial drug with a novel mechanism of action
Project Completed
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Introduction and Background of the Project


Malaria is a mosquito-borne, life-threating infectious disease caused by Plasmodium protozoa and an estimated 429,000 people die every year, mainly children in African countries. The current standard care for the treatment of malaria typically involves combination therapy with artemisinins. However, there is evidence of emerging resistance to both the artemisinins and the partner drug in some countries such as Cambodia, Thailand, and Vietnam. This highlights the urgent need for new classes of compounds with novel mechanisms-of-action (MoA) to treat resistant strains of malaria parasites and support the malaria eradication strategy.


In this project, we aim to conduct lead optimization on a series of compounds with a well-characterized and novel MoA. Glycosylphosphatidylinositol (GPI) is a common moiety in all eukaryotes which has a role in anchoring many proteins to the cell surface. Gwt1p, one of the essential enzymes in the GPI biosynthesis pathway, was identified by Eisai as a novel target for an antifungal drug. After conducting discovery research, Eisai discovered E1210, an antifungal drug clinical candidate, and found that the GWT1 gene encoding Gwt1p enzyme is highly conserved among eukaryotes, including Plasmodium protozoa, the etiological pathogens for malaria. Eisai has screened an internal compound library targeting fungal Gwt1p and found a hit compound with inhibitory activities on plasmodial Gwt1p. This compound showed anti-Plasmodium activities in vitro and in vivo and was subjected to chemical modification in a GHIT Hit-to-Lead Platform. MMV and Eisai succeeded in improving anti-Plasmodium activity to fulfill the requirement for lead compounds. In this project, we plan to conduct the lead optimization and aim to improve anti-Plasmodium activity, secure a sufficiently long half-life and deliver a compound that meets the MMV Target Candidate Profiles (TCPs). 


Project objective

To deliver a drug candidate that can be progressed to clinical development as a potential new treatment for uncomplicated malaria, chemical modification of current lead compounds will be conducted aiming both improvement of in vitro anti-Plasmodium activities and elongation of in vivo half-life. After the course of chemical modification, several numbers of in vivo effective compounds will be selected as the early candidates. Then these early candidates will be characterized in ADMET, safety and physicochemical studies, and the most promising compound will be selected as the preclinical candidate.

In parallel with the chemical modification, biological characterization will be performed to confirm the target TCPs and expected additional efficacy such as the induction of host immunity. Importantly, construction of anti-P. vivax assay will be performed at first to evaluate the anti-P. vivax activities of Gwt1p-inhibitors. 


Project design

In this project, chemical modification of current lead compounds will be conducted to improve anti-Plasmodium activity and secure a long-half-life. New compounds synthesized at the CRO will be shipped to Eisai’s laboratory located in Tsukuba, Japan. They will be tested according to the defined screening cascade: (1) Primary screening: anti-Plasmodium activity and Gwt1p-inhibitory activity will be tested. Compounds which show good anti-Plasmodium activity will be tested in the next tier. (2) Solubility at neutral pH and stability against human and murine liver microsomes will be tested. (3) Druggable compounds with efficient solubility and acceptable metabolic stability will be evaluated in multiple assays to identify candidates. The most promising compound will be selected as ‘Preclinical candidate’ for the next preclinical stage. Importantly, a 7-day rat dose range finding study will be conducted before the candidate selection.

How can your partnership (project) address global health challenges?

Malaria is still one of the most life-threating diseases and many children still die, especially in sub-Saharan African countries. In these countries, children get malaria time to time again, due to low immunity to malaria. This project is proposed to contribute to the eradication of malaria based on its novel and unique mechanism-of-action (MoA). For the control, elimination and particularly for the eradication of infectious diseases, vector control, use of vaccines to prevent infection and treatment with drug are all equally important. For malaria, each of them is under evaluation. As no vaccines with sufficient prophylactic efficacy are yet available, chemotherapy is also the most effective tool for the treatment and prevention of malaria. Antimalarial drugs have shown memorable improvements in the last century, but this great footprint is at risk of being washed away by the appearance of resistant parasites. As a common standard for new antimalarials toward the eradication of malaria, MMV has defined target product profiles (TPPs) and target compound profiles, i.e. individual characteristics of compounds to be included in such future antimalarials (TCPs). The novel MoA in this project is thought to fulfill several of the target profiles set for the next generation of antimalarial drugs targeting malaria eradication and expected to save many children’s lives in the future.

What sort of innovation are you bringing in your project?

The most promising advantage of this project is a novel MoA with an identified target protein. The MoA is inhibition of GPI-biosynthesis and this is a completely novel concept in the treatment of malaria. Furthermore, the target protein Gwt1p, an acyltransferase essential in GPI-biosynthesis, was discovered in Eisai and no other discovery activities targeting this enzyme are reported. Based on its novel mechanism, Gwt1p-inhibitors are expected to act on Plasmodium strains resistant to existing antimalarials, including artemisinins. The existing data suggests that the series is consistent with TCP1 for malaria treatment, set by MMV, and maintaining efficient drug concentration for 8 days is necessary to be achieved during the lead optimization. As the second advantage, inhibition of GPI-biosynthesis is expected to lead to anti-Plasmodium activities against multiple parasite life stages, because many kinds of stage-specific GPI-anchored proteins are expressed in each life stage of the malaria parasite. For example, in the sexual stages of P. falciparum, a GPI-anchored protein named Pfs48/45 is expressed in male gametocytes and this protein is essential for sexual mating. These expected efficacies would be consistent with other TCP’s, such as transmission blocking (TCP5) or chemoprotection (TCP4), if they are confirmed. As the third advantage, in addition to these direct anti-Plasmodium activities, inhibition of Gwt1p is expected to show additional preferable characteristics. Gwt1p-inhibitors may activate the immunization system of the host by decreasing GPI-anchored antigen proteins which inhibit development of effective immunity. Gwt1p-inhibitors may also decrease the inflammatory response caused by malaria infection. It was reported that the GPI moiety has the role of an endotoxin in the host. Malaria-treatment with a Gwt1p-inhibitor leads to a decrease in the total amount of Plasmodium-GPI released into host’s blood stream and additive efficacy due to the decreased TLR-mediated response can also be expected.

Role and Responsibility of Each Partner

Eisai is responsible for synthesis of new compounds, the primary evaluation of synthesized compounds and preclinical tests. Eisai will contract with Charles River Discovery Research Services UK Ltd., a CRO located in Eisai’s facility in UK for chemistry work. Eisai’s medicinal chemist will lead these contracted chemists by planning new structures and managing chemical synthesis. Contracted chemists will also create new compounds based on their own ideas and experience. All intellectual properties for newly synthesized compounds will be kept by Eisai. Synthesized compounds will be shipped to biologists in Eisai and then will be tested for antimalarial activities, Gwt1p-inhibitory activities and cytotoxicity. Eisai will also test positive compounds for solubility and basic DMPK parameters prior to the characterization in the MMV network. To allow a drug candidate to be identified, Eisai will test and characterize several selected compounds in preclinical studies for ADMET parameters, preliminary dose range finding studies and physicochemical parameters.


MMV is responsible for malaria lifecycle profiling and in vivo evaluation of positive compounds in its research network. MMV will arrange the testing of compounds at its external centers of excellence and will coordinate the study plan and transferring of study reports to the team. MMV is also responsible for the determination of target TCPs based on obtained data, as a leading PDP for antimalarial drug development.


All decisions will be made through scientific discussion between Eisai and MMV. Eisai is a Japanese pharmaceutical company which has varied experiences for drug development including safety and ADMET. MMV is a Product Development Partnership which has expertise for the development of antimalarial drugs designed to support the eradication of malaria agenda. Based on these backgrounds, earnest scientific discussions and proper conclusions will be made from this study. Regarding the responsibilities, Eisai will make decisions based on the scientific discussions with MMV at each milestone.

Others (including references if necessary)



2. Yeung S, Socheat D, Moorthy VS et al. Artemisinin resistance on the Thai-Cambodian border. Lancet 2009; 374: 1418-9.

3. Hawkes M, Conroy AL, Kain KC. Spread of artemisinin resistance in malaria. The New England journal of medicine 2014; 371: 1944-5.

4. Okamoto M, Yoko-o T, Umemura M et al. Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p. The Journal of biological chemistry 2006; 281: 4013-23.

5. Sagane K, Umemura M, Ogawa-Mitsuhashi K et al. Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p. The Journal of biological chemistry 2011; 286: 14649-58.

6. Tsukahara K, Hata K, Nakamoto K et al. Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly. Mol Microbiol 2003; 48: 1029-42.

7. Umemura M, Okamoto M, Nakayama K et al. GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast. The Journal of biological chemistry 2003; 278: 23639-47.

8. Miyazaki M, Horii T, Hata K et al. In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds. Antimicrobial agents and chemotherapy 2011; 55: 4652-8.

9. Burrows et al. New Developments in Anti-Malarial Target Candidate and Product Profiles. Malar J 2017; 16:26    

Final Report

1. Project objective

To deliver a drug candidate that can be progressed to clinical development as a potential new treatment for uncomplicated malaria, chemical modification of current lead compounds was conducted. After the course of chemical modification, compounds with improved activities were assessed in animal models, ADMET, safety and physicochemical studies, and the most promising compound has been selected as the preclinical candidate. In parallel with the chemical modifications, biological characterization was performed to confirm the target TCPs and the anti-P. vivax activities of Gwt1p-inhibitors were also be examined.


2. Project design

Chemical modification of lead compounds was conducted to improve antimalarial activity and secure a long-half-life. New compounds designed and synthesized at the CRO were shipped to Eisai’s laboratory located in Tsukuba, Japan. They were tested according to the defined screening cascade: (1) Primary screening: anti-Plasmodium activity and Gwt1p-inhibitory activity. (2) Solubility at neutral pH and stability against human and murine liver microsomes. (3) Evaluation in MMV’s multiple assays to identify candidates. The most promising compound was selected as for the next preclinical stage. Before the candidate selection, a 7-day rat dose range finding study was conducted.  


3. Results, lessons learned

From 400 compounds synthesized in the Hit-to-Lead Platform project followed by this project, lead compounds were selected and subjected to chemical modifications. About 600 compounds were newly synthesized in two years. All these compounds were evaluated for antimalarial activities in vitro, and many compounds showed improved activities. After the solubility test and the metabolic stability test in human and murine liver microsomes, about 150 compounds were evaluated in the murine malaria model at Eisai. Then two compounds were selected and evaluated in the humanized murine model at MMV. The evaluation of physical chemistry, safety and DMPK were also conducted at Eisai in parallel. Based on the antimalarial activities and DMPK parameters, prediction of the required human doses were performed and safety margins were assessed. Considering the results of all these assessment, one compound was selected as the final candidate and 7-day dose range finding study in rat was performed. This compound was approved as a preclinical candidate in Eisai and it will be discussed in MMV in near future, before the next preclinical studies. Also, the antimalarial activities against each life cycles of parasite and the resistant frequency were evaluated by MMV. Currently Gwt1p-inhibitors are targeting TCP-1, a compound for treatment of uncomplicated malaria, the possibility of TCP-4, a prophylaxis compound, may be discussed.