Scorpion envenoming is a severe health problem in many regions causing significant clinical toxic effects and fatalities. In the Middle East/North Africa (MENA) region, Buthidae scorpion stings are responsible for devastating toxic outcomes in human. The only available specific immunotherapeutic treatment is based on IgG fragments of animal origin. To overcome the limitations of classical immunotherapy, we have demonstrated the in vivo efficacy of NbF12-10 bispecific nanobody at preclinical level. Nanobodies were developed against BotI analogues belonging to a distinct structural and antigenic group of scorpion toxins, occurring in the MENA region. From Buthus occitanus tunetanus venom, BotI-like toxin was purified. The 41 N-terminal amino acid residues were sequenced, and the LD₅₀ was estimated at 40 ng/mouse. The BotI-like toxin was used for dromedary immunization. An immune VHH library was constructed, and after screening, two nanobodies were selected with nanomolar and sub-nanomolar affinity and recognizing an overlapping epitope. NbBotI-01 was able to neutralize 50% of the lethal effect of 13 LD₅₀ BotI-like toxins in mice when injected by i.c.v route, whereas NbBotI-17 neutralized 50% of the lethal effect of 7 LD₅₀. Interestingly, NbBotI-01 completely reduced the lethal effect of the 2 LD₅₀ of BotG50 when injected at 1:4 molar ratio excess. More interestingly, an equimolar mixture of NbBotI-01 with NbF12-10 neutralized completely the lethal effect of 7 and 5 LD₅₀ of BotG50 or AahG50, at 1:4 and 1:2 molar ratio, respectively. Hence, NbBotI-01 and NbF12-10 display synergic effects, leading to a novel therapeutic candidate for treating Buthus occitanus scorpion stings in the MENA region.

Scorpion venoms represent complex pharmacological arsenals whose complete molecular inventory remains incompletely defined, particularly for North African buthid species of high medical relevance. Buthus tunetanus is among the most clinically significant scorpion species across the Maghreb. We analysed the toxic fraction BotG50, isolated by Sephadex G-50 gel-filtration, using nanoLC-HRMS/MS coupled with an evidence-tiered, five-stage bioinformatics workflow. The workflow combines Proteome Discoverer database searching, BLAST-guided homology expansion, Casanovo de novo sequencing with variant construction, signal-peptide and propeptide removal, and post-translational modification (PTM) annotation. The analysis revealed 64 components (FDR < 1%), comprising 38 multi-peptide-supported identifications (≥2 peptides) and 26 single-peptide hits, with a median sequence coverage of 33%. The dominant neurotoxic repertoire includes toxins targeting K⁺, Na⁺, and Cl^- channels, α/β toxins, insect- or mammal-specific neurotoxins, and putative depressant toxins, while a minor accessory compartment comprises hyaluronidase, phospholipase A₂, lipolytic peptides, and hemocyanin-like proteins. BLAST analysis revealed strong sequence homologies with peptides from Buthus israelis (32.8%), B. tunetanus (15.6%), and Mesobuthus eupeus (10.9%). Approximately 52% of final identifications (33/64 proteins) required analytical stages beyond classical FASTA database searching. This proteomic atlas provides the most detailed molecular map of BotG50 to date, constituting a critical resource for antivenom epitope design, nanobody target selection, and bioactive peptide discovery.