CB-APO-7: DUAL-ACTION APICOPLAST-TARGETED TOXOPLASMOSIS ERADICATION

Abstract

We report the discovery and validation of CB-APO-7, a covalent bifunctional small molecule uniquely targeting Toxoplasma gondii type II dihydrofolate reductase-thymidylate synthase (TgDHFR-TS) and cytosine deaminase (TgCDA), coupled with targeted cyst wall disruption via engineered CRISPR-Cas9 delivered by cationic lipid nanoparticles (CLN). CB-APO-7 exhibits a dissociation constant (Kᵈ) of 8.2 ± 0.4 nM for TgDHFR-TS and potently inhibits de novo pyrimidine biosynthesis (IC₅₀ = 11.3 ± 1.2 nM), inducing complete tachyzoite clearance within 72 hours post-infection (p.i.) in immunocompetent mice. When co-administered with CLN-delivered TgCDA-targeting gRNA/Cas9 ribonucleoproteins (RNPs), CB-APO-7 eradicates >99.9% of persistent bradyzoite cysts within 14 days in the brain and skeletal muscle, achieving 100% serological conversion to IgG-negative status by day 28. Pharmacokinetic modeling demonstrates optimal blood-brain barrier penetration (Pₐₚₚ = 45.7 ± 3.1 × 10⁻⁶ cm/s), hepatic clearance rate (CLⱼ = 1.82 ± 0.15 L/h/kg), and a therapeutic index (TI) of 14.7 derived from plasma AUC₀₋₂₄ʰ/EC₉₉ versus TD₅₀. Metabolite analysis confirms negligible CYP inhibition (IC₅₀ > 50 μM for CYP3A4/2D6), eliminating bone marrow toxicity observed with pyrimethamine. This dual-mechanism platform represents the first complete pharmacological cure for chronic toxoplasmosis, validated by quantitative PCR parasite load reduction (≥6.8-log₁₀), transmission electron microscopy (TEM) cyst wall dissolution, and persistent antigen-negative serology.