Malaria is a devastating infectious disease killing close to a million people every year and contributing significantly to mortality and morbidity in many developing countries. The lack of economic incentives to develop antimalarial drugs, and the widespread resistance of the malaria parasite to most front-line drugs are two of the main hurdles to fight this disease. In addition, rapid emergence of drug resistance against new therapies such as artemisinin has made the identification of novel therapeutic targets extremely urgent.
Our lab's goal is to identify novel enzyme targets in Plasmodium species and to understand their biological functions. We believe that chemical and biological validation of new targets should be a priority in order to control malaria and fight drug resistance. Our group applies and develops chemical and molecular biology approaches to:
- Identify enzymes that are essential for parasite development.
- Validate them as anti-malarial targets.
- Characterize their biological and molecular functions.
So far, we have focused our efforts on proteases because of their ubiquitous regulatory and effector roles in many biological pathways. Indeed, proteolytic activity has been shown to be essential at most stages of parasite development. Also, proteases are one of the most prominent families of therapeutic targets, and protease inhibitors are currently used for the treatment of cancer, diabetes, hypertension, coagulation disorders and HIV/AIDS.
Because of their central role in many biological pathways, the activity of regulatory enzymes such as proteases or kinases is tightly regulated post-translationally. Therefore, mRNA levels, protein abundance, and protein localization often fail to inform on when and where a regulatory enzyme becomes activated and performs a biological function (Fig. 1A).
Activity-based probes (ABPs) are small molecules that use the catalytic mechanism of the targeted enzyme to covalently modify its active site. Therefore, ABPs can discriminate between the active and inactive forms of an enzyme, thus making them ideal tools to study the biological function of regulatory enzymes. A tag embedded within the structure of the ABP allows for the visualization of enzyme activity in a gel-based format (Fig. 1B). Biotinylated ABPs can be used to pull-down and identify labelled proteins, whilst fluorescent ABPs are excellent tools for live-cell imaging of enzyme activity.
A. Regulation of protease function. In addition to transcriptional and translational regulation, proteases are highly regulated at the protein level. Expressed as zymogens, they are activated in a variety of ways depending on the protease. Mechanisms of activation include environmental changes, protein-protein interactions, allosteric activation by small molecules, and processing by upstream proteases. Endogenous inhibitors and targeted degradation form yet another layer of control.
B. Activity-based probes. ABPs are small-molecule reporters that use the active protease's own chemistry to distinguish it from its inactive forms. Most ABPs consist of three parts: a warhead (an electrophilic moiety that reacts with the active site nucleophile to result in a covalent and irreversible adduct), a spacer and/or recognition element that targets the probe to a specific protease, and a tag (usually a fluorescent dye and/or an affinity handle like biotin). Treatment of a complex proteome, such as crude cell lysates or intact cells, with an ABP results in the labeling of only the active form of the protease whose activity can easily be detected in a gel-based format.
(Adapted from Nat. Struct. Mol. Biol., 2012, 19(1):9-16.)
Because ABPs covalently modify their targets, they can be used in a variety of applications depending on their specificity profile:
- Broad spectrum ABPs take advantage of the conserved catalytic mechanism of an enzyme family to label most of the members of that family (Fig. 2A). Activity-based protein profiling can therefore be used to assign protein function, map changes in enzyme activation under different conditions, or determine the specificity profile of an inhibitor against all members of an enzyme family.
- Specific ABPs are ideal for imaging the activity of a single enzyme at the subcellular, cellular, or animal level (Fig. 2B), thus providing a very valuable tool to study the biological function of a specific enzyme
A. Activity-based protein profiling. Broad-spectrum ABPs enable global profiling of an entire enzyme family. Treatment of a complex proteome (cell lysates, tissue extract, intact parasite, or living animal) with a broad-spectrum ABP results in labeling of all active members of the targeted family. These can then be resolved on a gel and identified by mass spectrometry. Inhibitor treatment prior to probe labeling results in diminished labeling of the inhibited targets, thus providing a specificity profile against all members of an enzyme family. This structure activity relationship information can then be used to design specific inhibitors for the target of interest.
B. ABPs imaging applications. Fluorescently labeled ABPs can be applied to top-down characterization of a target function. Whole-animal non-invasive imaging techniques allow the visualization of target distribution, and extracted tissue can be analyzed ex vivo. Histology shows target distribution on a microscopic level, FACS analysis identifies the types of cells that contain active protease, fluorescent microscopy pinpoints its subcellular localization, and biochemical analysis allows characterization at the protein level. Treatment with a lead compound before labeling provides information on target inhibition.
(Adapted from Nat. Struct. Mol. Biol., 2012, 19(1):9-16, and Chem. & Biol., 2012, 19(5):619-628.)
Our lab couples inhibitor screens with activity-based protein profiling to identify enzymes that are important for parasite development, and to design specific inhibitors and ABPs. We then combine these chemical tools with genetic, biochemical, and proteomic approaches to define the molecular functions of the identified targets. In addition to continuing our work on proteases, we are expanding our approach to the identification of other important regulatory enzymes in _Plasmodium falciparum_.