De novo designed proteins neutralize lethal snake venom toxins

Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more1,2. Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage3 and inhibition of nicotinic acetylcholine receptors, resulting in life-threatening neurotoxicity4. At present, the only available treatments for snakebites consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs5,6,7. Here we used deep learning methods to de novo design proteins to bind short-chain and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtained protein designs with remarkable thermal stability, high binding affinity and near-atomic-level agreement with the computational models. The designed proteins effectively neutralized all three 3FTx subfamilies in vitro and protected mice from a lethal neurotoxin challenge. Such potent, stable and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our results highlight how computational design could help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for the development of therapies for neglected tropical diseases.

Snakebite envenoming represents a public health threat in many developing regions, notably low-resource settings in sub-Saharan Africa, South Asia, Papua New Guinea and Latin America2. With over two million annual cases, snakebites result in 100,000 fatalities and 300,000 permanent disabilities1. In 2017, the World Health Organization listed snakebite envenoming as a highest-priority neglected tropical disease8. Nonetheless, limited resources have been dedicated to improving the current antivenom treatments2. These therapies rely on plasma-derived polyclonal antibodies from hyperimmunized animals, complemented by medical and surgical care9. Although instrumental in saving lives, antivenom accessibility is hindered by high production costs and inadequate cold-chain infrastructure in remote areas9. Serious adverse effects, including anaphylaxis and pyrogenic reactions, represent additional challenges during antivenom administration2,10,11. Furthermore, these treatments are often ineffective in counteracting neurotoxicity and tissue necrosis owing to suboptimal concentrations of neutralizing antibodies against three-finger toxins (3FTxs)5,6,7. This inefficacy stems from the limited immunogenicity of 3FTxs in antivenom-producing animals, resulting in a failure to elicit a strong antibody response12. Additional issues arise because of the delayed administration of antivenom treatment13. Antibody14,15,16,17,18,19,20,21 and non-antibody-based therapeutics22,23,24,25,26,27,28 have been tested in preclinical studies, but the development of these types of molecules requires either immunization of animals or the development of large libraries that require extensive selection, screening and optimization efforts29.

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