One of our research goals is to use chemical biology approaches to identify novel enzymatic targets in Plasmodium species and to study their biological functions by combining chemical and genetic tools. We believe that chemical, genetic, and biological validation of new pharmaceutical 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 antimalarial targets.
- Characterize their biological and molecular functions.
So far, we have focused our efforts on hydrolytic enzymes such as proteases and serine hydrolases because of their ubiquitous roles in many biological pathways and because these enzyme families have been successfully targeted by the pharmaceutical industry.
Because of their central role in many biological processes, the activity of regulatory or effector enzymes such as proteases, kinases, or metabolic serine hydrolases is tightly regulated post-translationally. Therefore, mRNA levels, protein abundance, and protein localization often fail to inform on when and where an enzyme becomes active 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 tightly regulated enzymes. A tag embedded within the structure of the probe 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 by MS, whilst fluorescent ABPs are excellent tools for live-cell imaging of enzyme activity.
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.
Figure 1. Activity-based probes report on protease 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.)
Figure 2. Activity-based probes are ideal tools to study protease function.
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.)
Identification of metabolic serine hydrolases as antimalarial targets
In an effort to synergize basic research with antimalarials drug development, the pharmaceutical industry has screened millions of small molecules and identified thousands of new compounds with antimalarial activity. These hits represent a treasure-trove of chemical tools to study parasite biology and identify new targets. However, in order to harness the potential benefits of these compounds it is crucial to understand their mechanism of action.
Our group has been using chemical biology approaches to identify the targets of some bioactive compounds with the goal of validating new antimalarial targets. In particular, we have used broad-spectrum ABPs to simultaneously monitor the activity of dozens of cysteine proteases and serine hydrolases in the malaria parasites. We have used this method to determine whether any small molecule from a collection of 400 anti-parasitic compounds, known as the MMV Malaria Box, was able to inhibit targets belonging to these enzyme families. While none of the compounds was able to robustly inhibit cysteine proteases, three of them consistently targeted unknown serine hydrolases.
We then used a quantitative chemical proteomics approach to identify these targets by measuring whether increasing concentrations of compound decreased the level of ABP labeling of any serine hydrolase in the malaria proteome. Interestingly, two serine hydrolases are inhibited in a dose-dependent manner. We are currently using conditional genetics to determine the biological function of these two enzymes and to confirm that they are the targets responsible for the antimalarial activity of the selected compounds. Importantly, these two serine hydrolases have been recently annotated as essential genes in the malaria parasites based on a recent genetic screen and might therefore be potential antimalarial targets.
Metabolic serine hydrolases play essential biological functions in all living organisms. However, this enzyme family is very poorly annotated in Plasmodium genomes and very little is known about the role of serine hydrolases in parasite development. Our lab has used broad-spectrum ABPs and quantitative chemical proteomics to identify all serine hydrolases in infected red blood cells and to measure their level of activity throughout the asexual replication cycle. This cycle consists of red blood cell invasion, intracellular parasite growth and replication, and escape from the host cell for further RBC invasion. The exponential replication of parasites during this erythrocytic cycle is responsible for all the pathology associated with malaria.
Our chemical proteomic approach has identified 29 different Plasmodium serine hydrolases that are active at different stages of parasite development. Interesting, we observed a substantial number of enzymes that seem to be specifically activated at the time of RBC invasion. We think these enzymes might be important for establishing a metabolic environment within newly infected red blood cells to allow initial parasite development. We have selected a few of these parasite serine hydrolases to determine whether they are essential for parasite development and to study their biological functions.
This is the first comprehensive study on malarial serine hydrolases and opens a new area of research in parasite biology, which we hope will be translated in the development of novel antimalarial therapies. This work has been partially funded by the ERC Marie Curie Career Integration Grant PCIG14-GA-2013-631806: MALARIA TARGETS ID.