Whole-cell phenotypic screening is proving to be an effective approach to drug discovery, leading to the identification of numerous lead compounds with antimicrobial properties. However, uncovering the targets and understanding the mode of action remains a challenging and elusive aspect of this approach. In this project, we focused on identifying novel drug candidates against apicomplexan parasites using a comprehensive approach that combines small compound library screening and target deconvolution based on forward genetics with transcriptome sequencing and computational mutation discovery, using Toxoplasma gondii as a relevant model for Apicomplexa. Phenotypic screening included the monitoring of intracellular parasite growth and the cytotoxicity of the drug towards the host cell.

The target deconvolution process employed a forward genetics strategy involving Ethyl Methanesulfonate (EMS) mutagenesis and mRNA sequencing. This approach facilitated the identification of point mutations in genes conferring drug resistance and provided valuable insights into the genetic basis of drug response in apicomplexan parasites. Several novel drug-target pairs were successfully identified through this strategy, identifying new potential therapeutics.

Since certain drug targets are conserved in different apicomplexan parasites, the effectiveness of the identified compounds was systematically assessed across Plasmodium and Cryptosporidium species. This cross-species evaluation reinforces the translational potential of the drug candidates identified, and offers the prospects of a broad-spectrum antiparasitic therapeutics.

The integration of phenotypic screening with forward genetic approaches represents a powerful workflow in the search for effective antiparasitic drugs. The findings from this study significantly contribute to the field of drug discovery, providing a framework for the identification, validation, and assessment of novel drug-target pairs across diverse apicomplexan parasites, addressing the urgent need for innovative solutions against malaria and cryptosporidiosis.