De novo designed proteins neutralize lethal snake venom toxins

Abstract

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 more^1,2. 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 (nAChRs) resulting in life-threatening neurotoxicity⁴. Currently, the only available treatments for snakebite consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs^5,6,7. Here, we use deep learning methods to de novo design proteins to bind short- and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtain protein designs with remarkable thermal stability, high binding affinity, and near-atomic level agreement with the computational models. The designed proteins effectively neutralize all three 3FTx sub-families in vitro and protect 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 computational design methodology should help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for development of therapies to neglected tropical diseases.

Figure 1.. Targets of 3 finger snake toxins (3FTxs).

(a) Structure of 3FTxs (PDB ID: 1QKD). Highly conserved cysteine residues are highlighted in sticks and each of the three fingers indicated (I-III). (b) Representation of a type IA cytotoxin (dark pink) (PDB ID: 5NQ4) interacting with a lipid bilayer. (c) Muscle acetylcholine (Torpedo) receptor (light blue) (PDB ID: 7Z14). Acetylcholine (ACh) binding site is depicted in violet. Left inset: Close-up of the acetylcholine binding protein (AChBP) (teal) (PDB ID: 3WIP) bound to Ach (violet). A set of aromatic residues form a cage around the neurotransmitter. Middle: Close-up of α-cobratoxin (dark purple) blocking access to the ACh binding site in AChBP (teal) (PDB ID: 1YI5). Right: Close-up of ScNtx (dark blue) blocking access to the ACh binding site in the Torpedo receptor (light blue) (PDB ID: 7Z14). (d) Schematic showing α-cobratoxin binder design using RFdiffusion. Starting from a random distribution of residues around the specified β-strands in the target toxin (dark purple), successive RFdiffusion denoising steps progressively remove the noise leading at the end of the trajectory to a folded structure interacting with α-cobratoxin β-strands. Panels (a), (b), and (c) were created with BioRender.com

Figure 2.. Experimental characterization of 3FTx binding proteins.

(a) Design models of protein binders (gray) bound to their 3FTx targets (dark blue: ScNtx, dark purple: α-cobratoxin, dark pink: consensus cytotoxin). (b) SEC traces of purified proteins. (c) SPR binding affinity measurements. Colored solid lines represent fits using the heterogeneous ligand model, with dissociation constant (K_d) values derived from these fits. (d) CD data confirms the presence of αβ-secondary structure in the 3FTx binding proteins and their thermal stability (inset).

Figure 3.. Crystal structures of 3FTx binding proteins closely match design models.

(a) Apo-state crystal structure of SHRT design. Left: Hydrogen bonding between the carbonyl oxygen of Cys41 in ScNtx (dark blue) and the side chain of Tyr45 in the SHRT design model (gray). Middle: Overlay of SHRT design model (gray) with crystal structure (wheat). Right: Backbone hydrogen bonding between the SHRT design model (gray) and ScNtx (dark blue) β strands. (b) Crystal structure of LNG design in complex with α-cobratoxin. Left: Cross-interface hydrogen-bond network involving Arg33 at loop II in α-cobratoxin (light purple) and Glu69, Tyr40, and Tyr49 in LNG crystal structure (wheat). Middle: Overlay of LNG design model (gray) bound to α-cobratoxin (dark purple) with crystal structure of binder (wheat) bound to toxin (light purple). Right: Backbone hydrogen bonding between crystal structure of designed binder (wheat) and α-cobratoxin (light purple) β strands. (c) Crystal structure of CYTX_B10 design in complex with Naja pallida cytotoxin. Left: Cross-interface electrostatic interaction network between loops III and II of Naja pallida cytotoxin (light pink) and binder crystal structure (wheat). Middle: Overlay of CYTX_B10 design model (gray) bound to toxin (dark pink) with crystal structure of binder (wheat) bound to Naja pallida cytotoxin (light pink). Right: Salt bridge between positively charged Lys18 in cytotoxin (light pink) and Asp57 in the binder crystal structure (wheat).

Figure 4.. In vitro and in vivo efficacy of designed proteins against snake venom toxins.

(a) Concentration-response curves comparing SHRT binder and anti-ScNtx V_HH efficacy in preventing nAChR blocking by 1 IC₈₀ of ScNtx. Data represent the toxin’s inhibition of ACh response, normalized to full ACh response, averaged within each group (n=16). (b) Concentration-response curves comparing the efficacy of LNG binder and anti-α-cobratoxin V_HH in preventing nAChR blocking by 1 IC₈₀ of α-cobratoxin. (c) Neutralization of the cytolytic effects of whole venoms from seven different Naja (N.) species and isolated cytotoxin by the CYTX binder. 2 IC₅₀ of the whole venoms or toxin were pre-incubated with CYTX at a 1:5 molar ratio (toxin:binder). Keratinocyte media was used as a positive control (PC). Triton X-100 was used as a negative control (NC). CYTX binder (B) was used as a positive control. (−) denotes 2 IC₅₀ of the whole venoms without binder, and (+) denotes venoms incubated with binder. Experiments were performed in triplicates, and results are expressed as mean ± SD. (d) Mice survival following lethal neurotoxin challenge (n=5). 3 LD₅₀s of ScNtx or α-cobratoxin were preincubated for 30 minutes (−30 min) with the corresponding protein binders at 1:10 ratios and then administered IP into groups of five mice. Toxins administered IP following IP administration of binders at 1:10 or 1:5 molar ratios (toxin:binder) either after 15 (+15 min) or 30 minutes (+30 min) post-toxin injection. Controls included mice receiving toxins alone (C). Specificity was assessed via cross-treatment (CT) experiments, where non-target binders were preincubated with 3 LD₅₀s of ScNtx or α-cobratoxin and administered IP. Signs of toxicity were observed, and deaths were recorded for a period of 24 hours. (d) was created with BioRender.com.