Activity-based protein profiling (ABPP) is recognized as a powerful and versatile chemoproteomic technology in drug discovery. Central to ABPP is the use of activity-based probes (ABPs) to report the activity of specific enzymes or reactivity of amino acid types in complex biological systems. Over the last two decades, ABPP has facilitated the identification of new drug targets and discovery of lead compounds in human and infectious disease. Furthermore, as part of a sustained global effort to illuminate the druggable proteome, the repertoire of target classes addressable with ABPs has vastly expanded in recent years. Here, we provide an overview of ABPP and summarise the major technological advances with an emphasis on probe development.
The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as Toxoplasma gondii. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H2O2 to human fibroblasts infected with T. gondii triggers a Ca2+ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H2O2 is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in T. gondii, evidencing a link between extracellular redox and intracellular Ca2+ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.
Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.
SummaryPathogenic infections and the diseases they cause are defined by invasion and colonization of distinct host cell types and tissue niches. In the case of viruses and bacteria, molecular and cellular barcoding has shaped our understanding of within-host pathogen population dynamics, and informed therapeutic intervention strategies. Host brain colonization is a clinically untreatable feature of persistent infection by the eukaryotic pathogen Toxoplasma gondii, and the process remains poorly understood. The host blood-brain barrier is expected to physically restrict parasite colonization of this tissue niche and force the infection through a selection bottleneck, however tools and technologies to test this hypothesis have not been available. Here, we have developed a simple CRISPR-based method to barcode Toxoplasma parasites, and then used complex libraries of barcoded parasites to define how the different phases of an infection shape the pathogen population structure. Unexpectedly, we have discovered that the murine host brain does not restrict parasite colonization, with the population structure predominantly shaped by a bottleneck experienced during the acute phase of infection. These data support an evolutionary strategy to maximize genetic diversity of parasite persister cells within the intermediate host brain for subsequent transmission into the definitive feline host.
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