Current Research Projects
Current research projects beingconducted by members of the program are drug development, drug target identification and expression, X-ray crystallography/molecular modeling and drug design, synthetic and medicinal chemistry, parasite genetic manipulation through transfection technology and drug discovery through screening technologies. We focus mainly on two drug targets in folate metabolism, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). Other enzymes in the folate and related pathways that can be potential new targets are also being exploited in our laboratory. These include thymidylate DHFR in the same protein, serine hydroxymethyltransferase (SHMT), and targets in other metabolic pathways.
Development of Antifolates against Parasitic Diseases
- DHFR from P. falciparum
The laboratory has a long history of research toward understanding of antifolate target of malaria parasites (Yuthavong, Y. et al. 2006). The important breakthrough for our antifolate development has been mainly on the success in obtaining crystal structure of PfDHFR-TS by our group (Yuvaniyama, J. et al. 2003, Chitnumsub, P. et al. 2004), for which the team leader, Prof Yongyuth Yuthavong, was recognized by the Nikkei Asia Award for Science, Technology and Innovation in 2004.
- P. falciparum dihydrofolate reductase (pfDHFR) is a validated target for antifolate antimalarials, essentially due to its role in the dTMP cycle. Resistance to antifolate drugs such as pyrimethamine and cycloguanil has been shown to be caused by mutations that reduce the binding affi nity of the drugs to the target. Series of antifolate resistant mutant of PfDHFR occurred naturally has been engineered from its synthetic gene (Sirawaraporn, W. et al. 1993) and large amounts of wild type and mutant PfDHFRs were produced, enabling the testing of drug affi nities for these enzymes. New antimalarials directed against this target therefore are dev eloped to have high binding affi nity of the mutant enzymes and the wild type PfDHFRs using molecular modeling approaches and structural information (Yuthavong,Y. et al. 2000, Rastelli, G. et al. 2000,Tarnchompoo, B. et al. 2002, Vilaivan, T. et al. 2003, Sirichaiwat, C. et al. 2004,Kamchonwongpaisan, S. et al. 2004).
- DHFR from P. vivax
The enzyme dihydrofolate reductase-thymidylate synthase in another malarial parasite species, Plasmodium vivax, has also been studied. In these studies, P. vivax dihydrofolate reductases (PvDHFRs, wild type and mutantenzymes) were cloned and characterized. The antifolates were shown to inhibit wild type PvDHFR at a low nanomolar range. All the antifolate analogues available in the antifolate collection are currently being tested against these PvDHFRs, and our researchers plan to design more antifolates against PvDHFRs based on enzyme structure and molecular modeling (Leartsakulpanich, U. et al. 2002). Since there is no long term cultivation, bacterial surrogate model has been developed for antifolate testing against PvDHFR (Bunyarataphan, S. et al. 2006). Recent advances in transfection technology for malaria parasite have also made it possible to create plasmodial surrogate models for PfDHFR and PvDHFR in P. falciparum and P. berghei models with supports from TDR transfection Networks technically and from WHO/TDR financially.
- DHFR from Other Parasitic Diseases
DHFR targeting is also a possible strategy for trypanosomiasis treatment. In vitro cultivation of Trypanosoma brucei for antifolate screening has been set up in our laboratory. In addition, TbDHFR-TS gene has also been cloned and expressed in large amount for structure determination and antifolate testing. Apart from T. brucei, we also have succeeded in expressing DHFR-TS from T. cruzi for the same purposes. Structures of these enzymes were obtained.
- SHMT from P. falciparum and P. vivax
SHMT is another enzyme in the triad (DHFR-TS-SHMT) of d-TMP Synthesis Cycle. It is a potential target for antimalarial drugs, as also the corresponding human enzymes for cancer chemotherapy. In collaboration with Mahidol University, and under the leadership of Dr. Ubolsree Leartsakulpanich, a recipient of the 2003 L’Oreal Award for Women in Science, we have characterized the enzymes from both P. falciparum and P. vivax (Maenpuen,S. et al. 2009, Sophitthummakun, K. et al. 2009). A quick assay is being developed for screening of potential inhibitors of these enzymes, which may be drug candidates.
X-Ray Structure Determination and Molecular Modeling for Rational Drug Design
Structure determination of several enzymes from malarial parasites in different metabolic pathways i.e. synthesis of pyrimidines and purines and glycolytic pathways, are being carried out in parallel with the biochemical studies. Our main focus is to understand the interactions of enzyme and substrate or inhibitor to aid the drug development process. For example, crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, complexed with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, have been solved for the first time in our laboratory which reveal unique features for overcoming resistance (Yuvaniyama, J. et al. 2003). Further information obtained from cocrystals of PfDHFR-TS and antifolates has been used in structure activity relationship (SAR) cycles to improve the inhibitor effi cacy specifi c to V1/S. We have successfully obtained lead inhibitors which now are in the animal testing stage.
In parallel with the rational drug design for effective antifolates antimalarials based on structure elucidation using X-ray structure, technologies on molecular modeling and quantitative analysis of structure-activity relationship (QSAR) has also been implemented and used for drug design. Before our success in obtaining X-ray crystal structure of PfDHFR-TS, molecular modeling approach was used in order to gain an insight into enzymatic structures. The homology and alignment of PfDHFR with DHFRs from other species, for which the X-ray structures of which have been established, have made possible the prediction of the active site of PfDHFR. At present, modeling continues to be useful in predicting the modes of interaction between ligands and the enzyme. The effects of the mutation of residues around the active site have been studied by combining modeling and experimental approaches, using cassette mutagenesis to generate mutants harboring one of the 20 amino acids at the particular sites. These studies are mportant
Active site of malarial DHFR
for gaining insights into the mechanisms of the actions of the target enzyme, and the loss of function through nteraction with the inhibitors.
Target-Based and Parasite-Based Antimalarial Development
From our knowledge of the structure of pfDHFR, and how resistance occurred, we could embark on an ambitious project, funded partly by MMV, Wellcome Trust and EU for design, synthesis and optimization of new antifolate drugs based on their interaction with the target and their pharmacological characteristics.
With the success in crystal structure determination, series of antifolates were synthesized based on the crystal structures and molecular models of P. falciparum and P. vivax DHFR-TS, with mutation of dihydrofolate reductase at various sites. Several analogues with Ki in low nanomolar (nM) range for several types of PfDHFR mutants have been identifi ed. Some of these analogues show good in vitro antimalarial activities against strains of P. falciparum harboring these mutant enzymes. These inhibitors also have low toxicity against mammalian cell lines such as Vero, KB and BC cells, with good safety indices for malaria parasites (Tarnchompoo, B. et al. 2002, Vilaivan, T. et al. 2003, Sirichaiwat, C. et al. 2004, Kamchonwongpaisan, S. et al. 2004).
In collaboration with Monash University and London School of Tropical Medicine and Hygiene, a new series of antifolates are being optimized with promising oral effi cacy and lack of host toxicity. A candidate will hopefully soon be selected for further pre-clinical and clinical development, with the assistance of MMV.
In related development, one of our researchers (Dr. Bongkoch Tarnchompoo) has been given grant support in the Grand Challenges Exploration Program of the Bill and Melinda Gates Foundation for design and synthesis of new antifolates which will eliminate or retard development of resistance.
One of our researchers (Dr. Chawanee Thongpanchang) has received Unesco-L’Oreal Fellowship for Young Women in Life Sciences, from which she underwent training from Institute of Organic Chemistry, Zurich, in order to prepare for large-scale synthesis of our drugs and their precursors.
Parasite Genetic Manipulation through Transfection Technology for Drug Screening
A medium-throughput system using a combination of two techniques - parasite lactate dehydrogenase (PfLDH) assay and [3H]-hypoxanthine incorporation assay - has been established for more than 10 years in our laboratory. The techniques have been applied for natural product screening and antifolate development, where compounds from our antifolate library are investigated as a single drug, or in combination with other inhibitors of well-defi ned ntimalarial targets. Other antimalarial screening protocol using fl uorescent dyes has also been established and is being optimized for high throughput format.
Technology enabling the genetic manipulation of malaria parasites has advanced greatly in recent years. Today transfection technology is used as an instrument in assessing numerous aspects of Plasmodium species biology, including gene function and protein targeting. In our laboratory, under the leadership of Dr. Chairat Uthaipibull, a TDR/WHO Career Development grantee, and Dr. Sumalee Kamchonwongpaisan through WHO/TDR Transfection Network supported by WHO/ TDR and HHMI, USA, we use this transfection technology to introduce genes of interest such as fl uorescent proteins (GFP, YFP and RFP) into both human malaria P. falciparum and murine malaria P. berghei in order to create novel antimalarial screening models. The protocol using these transgenic fl uorescent parasites in antimalarial screening has been established. Other work using transfection technology is being explored in our laboratory including switching of dhfr gene between plasmodium species to assess DHFR activity and then use as antimalarial screening models.
Enzyme engineering through site-directed mutagenesis has been an important tool to study PfDHFR-TS structure and function. Based on sequence alignment, several amino acids in the active site of PfDHFR and TS are found conserved in the evolution. The essential role of these amino acids and those identifi ed from antifolate resistant phenotypes in enzyme catalytic activities and drug resistance have been explored (Sirawaraporn, W. et al. 2002,
DSuperimpositiom of Inhibitor-Bound Complexes of
the Quadruple Mutant of PfDHFR-TS
Chusacultanachai, S. et al. 2002, Chanama, M. et al, 2005, Kamchonwongpaisan, S. et al. 2007). In addition, this technique is also proven to be useful in improving solubility of the enzymes for structural study (Japrung, D. et al. 2005).
Directed Evolution, a Potential Tool for Predicting Possible Drug Resistance
Directed evolution techniques and site-directed mutagenesis have been used for the creation of mutants for the identifi cation of their essential role in enzyme catalytic activities and for drug screening in our laboratory. Through the techniques of error-prone PCR, we have been able to make powerful predictions concerning possible resistant mutants that can develop when a particular drug is introduced for malaria treatment. Libraries containing thousands of PfDHFRs variants have been constructed, and selection for drug-resistant PfDHFR enzymes can be performed rapidly and easily by a bacterial complementation assay (Chusacultanachai, S. et al. 2002, Japrung, D. et al. 2007). Recently, application of DNA shuffl ing technique to create a larger pool of mutants from mutant libraries has empowered the understanding of evolution of antifolate resistant in malaria parasites. These techniques, combination of error-prone PCR and DNA shuffl ing, are being applied for evaluating the propensity of our novel compounds to antifolate resistant mutants. These powerful techniques also make it possible to design new antifolate antimalarials prior to the emergence of novel drug-resistant parasites.
Combinatorial chemistry has been applied to the synthesis of antifolate-based antimalarials. Researchers have developed a method for the selection of tight binding inhibitors from the libraries, based on the dissociation constant (Ki) of the enzyme with each inhibitor. Libraries can be tested with enzymes to sort out the best sublibraries for lead identifi cation (Vilaivan, T. et al. 2003). Alternatively, enzymes can also be used to “fish out” the best compounds in the libraries by free enzyme (Kamchonwongpaisan, S. et al. 2005) and immobilized enzyme (Thongpanchang, C. et al. 2007). This technique should facilitate the development of antifolates, and should also be applicable to other inhibitor libraries with well-defi ned protein targets.
Understanding Artemisinin Action
In studies of drug-host-parasite interaction, apparent resistance to artemisinin was found for the parasite infecting thalassemic erythrocytes (Yuthavong, Y. et al. 1989). Malaria parasites reside in genetically abnormal erythrocytes of thalassemic individuals whose globin chain synthesis is reduced. The apparent resistance has been found to be due to increased accumulation of artemisinin in alpha thalassemic erythrocytes (Kamchonwongpaisan, S. et al. 1994), with a preferential binding of the drug with hemoglobin H in HbH-containing erythrocytes (Vattanaviboon, P. et al.1998). At the same time, drug inactivation by the cell components has also been found to be more important than drug binding (Charoenteeraboon, J. et al. 2000). Artemisinin drugs can be inactivated in the cells by the presence of heme on the erythrocyte membrane (Vattanaviboon, P. et al. 2002). Heme and heme containing proteins such as catalase and hemoglobins in red cell cytosol are also responsible for inactivating artemisinin in the cytosol (Ponmee, N. et al. 2007). The fi nding of reducing drug effectiveness of malaria-infecting thalassemic erythrocytes does not invalidate the use of the drug for thalassemic patients, since dihydroartemisinin has been found to promote the elimination of P. falciparum infecting thalassemic erythrocytes to an extent much greater than those infecting normal erythrocytes. Moreover, the antibodies from patient blood can facilitate the rate of phagocytosis. The molecular interaction of artemisininhost-parasite interaction is being explored for better understanding and may lead to the identifi cation of its potential targets, with funding support from Howard Hughes Medical Institute (HHMI), which recognizes Dr. Sumalee Kamchonwongpaisan as its “scholar”, adding international prestige to our group.
Target Research Unit Network (TARUN) of the Thailand Tropical Diseases Research (T2) Programme
In order to strengthen the drug target research community in Thailand, a Target Research Unit/Network (TARUN) has been established by members in this research unit since 2002. TARUN is a sub-programme of the Thailand Tropical Diseases Research (T2) Programme, and is aimed at supporting research on drug/diagnostic/vaccine targets of tropical diseases. It has been set up to promote collaboration in research which requires new knowledge and tools, especially those essential for obtaining relevant data on targets from genomic information and other information from molecular biology, including target identifi cation and validation, target characterization and utilization for drug/diagnostic/vaccine development.
The network focuses on the application of genomics, particularly microarrays and proteomics technologies, and in molecular structure studies of targets in malaria, TB, dengue haemorrhagic fever and other common tropical diseases.The main objectives of the network are to exchange information on target research; to collaborate in research which requires new knowledge and tools, especially those essential for obtaining relevant data on targets from the genomic information available or soon to be available; and to set up the necessary equipment for the collaborative research, especially concerning information systems and tools for target identifi cation and validation, crystal structure determination (Yuvaniyama, J. et al. 2003, Chitnumsub, P. et al. 2004), target characterization and utilization for drug/diagnostic/vaccine development. Microarrays to interrogate transcriptomes of the pathogens described above have been fabricated by the network, and their utility for providing valuable new information demonstrated (Shaw, P.J. et al. 2007). A series of research seminars, workshops and research clinics have been launched for the network members. In addition, international workshops on microarray technology for malaria parasite were arranged with support from TDR/WHO and MR4. The proteomics facility has served the community (Wongtrakoongate, P. et al. 2007) and it was transformed into a proteomics service unit in the year 2005 with more equipment from BIOTEC. This service unit is now conducting research and service for research community under the Genome Institute of BIOTEC, NSTDA.
Our original model for strengthening national research capacity with assistance of the international community now has parallels in some other developing countries. For example, South Africa Malaria Initiative (SAMI) has recently been launched with the specific aim of developing drugs against malaria. With these programmes, indigenous research strength can be built in the developing countries, with benefi t both to the host countries and to the developing world as a whole.