AI-designed proteins revolutionize snakebite treatment

Snakebites cause over 100,000 deaths annually, contributing substantially to the mortality rates in resource-poor regions. Toxins such as three-finger toxins (3FTxs) in snake venom cause significant neurotoxicity and tissue damage. However, traditional snakebite treatments rely on animal-derived anti-venoms, which are not very effective against 3FTxs.

In a recent study published in Nature, a team of scientists explored a novel approach to developing anti-venoms using computationally designed proteins to neutralize 3FTx toxins.

Using advanced deep learning methods, they developed stable, high-affinity protein binders that target specific neurotoxins, offering potential options for cost-effective, accessible antivenom solutions.

Closeup head of king cobra snake.​​​​​​​Study: De novo designed proteins neutralize lethal snake venom toxins. Image Credit: Kurit afshen/Shutterstock.com

Anti-Venom Treatment

Snakebite envenomation is a significant yet neglected tropical disease that disproportionately affects low-resource regions such as sub-Saharan Africa and South Asia.

Current anti-venom therapies rely on antibodies derived from hyperimmunized animals, which are costly and require cold storage, limiting their availability in remote areas.

These treatments also cause adverse immune reactions and often fail to neutralize low-immunogenic toxins such as 3FTxs, a key component of the venom of elapid snakes such as cobras. These toxins disrupt nicotinic acetylcholine receptors, causing neurotoxicity and tissue damage.

Existing efforts to develop antibody-based or alternative therapies are hindered by high production costs and complex development processes.

The limited effectiveness of traditional anti-venom methods highlights the urgent need for innovative therapeutic strategies that are scalable, effective, and tailored for such toxins. Recent advances in computational protein design offer promising avenues to address this need for more effective antivenoms.

About the Study

The present study utilized deep learning and computational tools to design proteins that are capable of neutralizing 3FTxs in snake venom. The researchers employed RFdiffusion, an open-source artificial intelligence (AI) protein design program, to create binders targeting short-chain and long-chain α-neurotoxins, as well as cytotoxins.

For α-neurotoxins, they used a design strategy focused on β-strand pairing interactions to block neurotoxin binding at nicotinic acetylcholine receptors.

This involved computational modeling, sequence optimization, and structural validation using AI tools such as AlphaFold2 and Rosetta metrics.

Synthetic genes for the designed binders were cloned and expressed in Escherichia coli. The researchers then purified the proteins using chromatography and verified them for structural integrity through size exclusion chromatography and other methods.

High-affinity binders were identified through bio-layer interferometry and surface plasmon resonance, while X-ray crystallography provided structural insights into the interactions between the binders and toxins.

For cytotoxins, a consensus sequence derived from 86 snake toxins was used to create binders targeting conserved regions, focusing on loops critical for membrane interaction. These designs were validated through in vitro assays that measured their ability to neutralize venom-induced cytotoxicity in human keratinocyte cultures.

Furthermore, the protein designs were tested for their neutralizing capabilities in human-derived cell lines expressing muscle-type receptors. The protective efficacy of binders was also evaluated in vivo in mice exposed to lethal doses of purified toxins, with survival outcomes tracked for 24 hours.

Major Findings

The results showed that the computationally designed proteins could effectively neutralize 3FTxs in snake venom. These proteins exhibited high thermal stability, strong binding affinity, and structural fidelity to their computational models.

Moreover, the in vitro tests showed that binders for short-chain neurotoxins (SHRT) and long-chain α-cobratoxins (LNG) inhibited toxin binding to nicotinic acetylcholine receptors even at nanomolar concentrations.

The SHRT binder achieved complete neutralization of short-chain toxins, while LNG showed similar efficacy against long-chain α-cobratoxins.

Additionally, the cytotoxin-specific binders (CYTX) were found to protect human keratinocytes from venom-induced cytotoxicity. When preincubated with cobra venoms, CYTX reduced cell death by 70% to 90% and demonstrated broad cross-reactivity with venoms from various cobra species.

Further structural analysis revealed that the CYTX binders targeted the loops in 3FTxs that were essential for membrane disruption.

Furthermore, the in vivo experiments in mice confirmed the efficacy of these binders. Both SHRT and LNG provided 100% protection against their respective toxins when administered at a 1:10 molar ratio of toxin to binder.

While SHRT retained full protective efficacy even when administered 30 minutes after toxin exposure, LNG provided 60% protection under similar conditions. Importantly, the binders caused no adverse effects in control mice, highlighting their safety.

Despite their success, the CYTX binders demonstrated limited effectiveness against venom-induced dermo-necrosis or skin necrosis and tissue death in a murine model. The researchers noted that further optimization was required to enhance cytotoxin neutralization in vivo.

Conclusions

To summarize, the study developed computationally designed proteins that were found capable of neutralizing snake venom toxins, particularly 3FTxs, with high specificity and efficacy.

These proteins exhibited thermal stability, strong binding affinities, and significant protective effects both in vitro and in vivo.

These findings highlighted the potential of de novo protein design to revolutionize anti-venom development and address the limitations of traditional therapies.

Journal reference:
  • ​​​​​​​Torres, V., Valle, B., Mackessy, S. P., Menzies, S. K., Casewell, N. R., Ahmadi, S., Burlet, N. J., Muratspahić, E., Sappington, I., Overath, M. D., RiveradeTorre, E., Ledergerber, J., Laustsen, A. H., Boddum, K., Bera, A. K., Kang, A., Brackenbrough, E., Cardoso, I. A., Crittenden, E. P., & Edge, R. J. (2025). De novo designed proteins neutralize lethal snake venom toxins. Nature. doi:10.1038/s4158602408393xhttps://www.nature.com/articles/s41586-024-08393-x

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