Flagellar pocket receptors as entry point for protein-based drugs against kinetoplastid parasites

Abstract

Aim: Kinetoplastids are flagellated protozoa encompassing multiple parasitic species responsible for severe neglected diseases. Although treatments exist, therapeutic failure and toxic side effects underscore the need for innovative drug development. Recent advances in protein design have accelerated the creation of small protein modules with specific functions, known as miniproteins, with broad pharmacological applications. However, their intrinsic inability to cross biological membranes limits their use against intracellular targets. This work aims to propose and computationally explore a modular delivery strategy that exploits the flagellar pocket (FP) as an entry route to deliver protein-based therapeutics into the parasites.

Methods: Using experimentally determined structures of three FP receptors, we applied a motif-scaffolding pipeline combining RFdiffusion, ProteinMPNN, and AlphaFold2-multimer to design de novo miniprotein modules capable of mimicking the natural cargo recognized by each receptor. Candidate designs were evaluated using a scoring function integrating minimum interaction predicted aligned error (miPAE) and backbone root mean square deviation (RMSD) across five predicted models per design.

Results: The design campaign yielded different outcomes depending on the target. For the transferrin receptor, 67 candidates surpassed the established in silico success thresholds, a pool expected to contain multiple experimentally validated binders. For the invariable surface glycoprotein 65, 17 candidates met the criteria, constituting a tractable experimental panel. Lastly, the haptoglobin-hemoglobin receptor proved a challenging target, with no candidates clearly surpassing both thresholds, likely due to the hydrophilic nature of its binding interfaces and the requirement for direct heme coordination.

Conclusions: This work provides a structural rationale for a novel receptor-mediated intracellular delivery paradigm in kinetoplastid parasites, offering a computational pipeline for generating miniprotein modules ready for experimental validation. We further outline how these delivery modules could be integrated into modular protein-based drugs incorporating protease recognition sequences, cell-penetrating peptides, and subcellular localization signals, laying the conceptual ground for a new therapeutic approach against these neglected diseases.

Keywords

kinetoplastids / flagellar pocket / miniproteins / protein design / protein-based inhibitors / protein binders / structural bioinformatics

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