By some estimates, even a simple cell has over 14,000 proteins, each of them acting in concert with several others. The sum of all these proteins and their interactions—known collectively as the proteome—forms the basis of life, regulating the many complex processes in a cell.
Identifying drugs that can bind to and disrupt specific proteins is thus a key challenge of biology. In 2013, groups at Karolinska Institutet, Sweden, and Nanyang Technological University (NTU), Singapore, led by Pär Nordlund who later joined A*STAR’s Institute of Molecular and Cell Biology (IMCB) in 2014, developed a way to study drug–protein interactions based on the temperature at which proteins denature. Called the cellular thermal shift assay (CETSA), the technique was originally used to discover cancer drug targets and has since been adopted by both academia and industry.
Although CETSA allows scientists to identify drug–protein interactions in whole cells, the process can only investigate a few proteins at a time, relying on time-consuming Western blots to quantify the amount of protein present said Radoslaw Sobota, a Principal Investigator at IMCB and a co-corresponding author on the study. Instead, a joint A*STAR and NTU team combined CETSA with mass spectrometry (MS) to increase the throughput to at least 5,000 proteins per experiment, applying their technique to the age-old problem of malaria.
“Despite decades of research, we only have about 20 antimalarial compounds in clinical use, many of which are not very useful anymore due to widespread drug resistance,” said study first author Jerzy Dziekan, a Presidential Post-Doctoral Fellow at NTU.
“We knew that MS-CETSA had serious potential to accelerate ongoing antimalarial drug discovery efforts, therefore the malaria parasite was one of the first organisms we adapted this method for.”
Together with Zbynek Bozdech from NTU, Nordlund and Sobota’s groups first optimized the MS-CETSA protocol for Plasmodium falciparum, the only malaria parasite strain that can be cultivated in the lab. They then validated their method on existing drugs and their known molecular targets, before using it to identify a previously unreported target of the world’s first antimalarial drug, quinine.
“Quinine has remained in clinical use for the treatment of malaria for over 400 years, but despite all this time and decades of research its mechanism of action remained obscure until now. Identifying the malaria parasite’s purine nucleoside phosphorylase as quinine’s target has finally shed some light on how this drug functions, linking its mechanism of action to the inhibition of the parasite’s purine biosynthesis pathway,” Sobota said.
Building on their initial success, the researchers have been awarded an Academic Research Fund Tier 3 grant from the Ministry of Education. The team, led by Bozdech, and A*STAR collaborators Sobota and Laurent Renia of A*STAR’s Singapore Immunology Network (SIgN), plans to use MS-CETSA among other techniques to characterize the parasite’s biology. They will also identify the targets of candidate antimalarial compounds in early to late stages of development.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology (IMCB).