Promising Drug Repurposing Candidates Targeting Free-Living Amoebae: A Systematic and Critical Review of Laboratory-Based Evidence

Devastating or nearly invariably fatal infections caused by free-living amoebae (FLA), including <i>Acanthamoeba</i> keratitis (AK), granulomatous amoebic encephalitis (GAE), and primary amoebic meningoencephalitis (PAM), remain a significant public health concern, driven by increasing case numbers, geographic expansion, and the lack of approved, effective, and safe treatments. Despite decades of research, no new drugs have been successfully approved, highlighting the severe limitations of de novo drug development for these infections, particularly for GAE and PAM, largely due to the challenges of conducting clinical trials for these rare and rapidly lethal diseases. In this context, drug repurposing represents a cost-effective and promising strategy to accelerate therapeutic advances and overcome key bottlenecks of conventional drug development. Accordingly, we conducted a systematic review of in vitro studies and animal models of AK, GAE, and PAM reported in indexed databases to identify promising drug repurposing candidates against FLA infections. After screening 23,624 records, 112 studies were included in the analysis. Overall, 2726 drugs and drug combinations, spanning 865 pharmacological classes and approved for 565 therapeutic indications, were assessed for their repurposing potential. Among these, 166 compounds showed substantial trophocidal activity (≥IC₅₀) at potentially translatable concentrations (≤10 µM), including six with additional cysticidal activity. In vitro, four compounds were active against <i>Balamuthia mandrillaris</i>, 44 against <i>Acanthamoeba</i> spp. (three cysticidal), and 115 against <i>Naegleria</i> spp. (three cysticidal). In in vivo studies, sulfadiazine and rifampicin were effective as preventive or early monotherapies for GAE. For AK, the combination of polyhexamethylene biguanide, neomycin, and atropine, as well as voriconazole and nitazoxanide monotherapies, showed the greatest promise. In PAM, azithromycin alone or in combination with amphotericin B emerged as the most promising therapeutic options. Further studies are required to advance the clinical translatability of these findings. To the best of our knowledge, this work provides the first comprehensive and integrated synthesis of repurposable drug candidates against FLA infections.