Functional investigation of nucleophilic cysteines as covalent anti-parasitic targets

Abstract

Small molecules that engage protein-encoded amino acid nucleophiles are resurgent in drug discovery as versatile probes and therapeutic agents. Over recent years, considerable efforts have been devoted to the development of ‘reactivity-based profiling’ (RBP) technologies for the identification of nucleophilic amino acids across proteomes. However, these chemoproteomic approaches cannot discern which sites contribute to protein function and can be prioritised as targets. This has led to researchers to overlook poorly characterised proteins with potential therapeutic value. To address the low throughput of current methods, we have developed a new genetic screen, CRISPR/Cas9-based Oligonucleotide Recombineering (CORe), for systematic prioritisation of reactive amino acids as therapeutic targets. Here, CORe was used to functionally prioritise reactive cysteine residues as prospective covalent antimicrobial targets in the clinically-important apicomplexan parasite, Toxoplasma gondii. First, cysteine reactivity was profiled across the T. gondii proteome using an established RBP methodology. We then applied CORe to investigate the importance of 74 hyperreactive cysteines for the asexual growth of T. gondii. Fitness-conferring reactive cysteines were associated with diverse molecular function and enriched within the T. gondii 80S ribosome. Saturation mutagenesis of fitness-conferring cysteines in ribosome proteins revealed that these reactive sites display heterogeneity in their tolerance to different amino acid substitutions. Intriguingly, many of these cysteines were absent in the human host, leading us to prioritise the apicomplexan translation apparatus as a target for covalent drug discovery. Screening of a cysteine-reactive acrylamide fragment library against the related malaria parasite, Plasmodium falciparum, resulted in the identification of a parasite-specific in vitro translation inhibitor with in-cell activity. These studies introduce cysteine-targeted covalent inhibition of protein biosynthesis as a new therapeutic modality for anti-malarial development. Moreover, this project demonstrates the integration of CORe with RBP into a multidisciplinary approach for promoting new proteins and biological processes for covalent drug discovery.