Novel recombinant protein therapeutics of botulism

描述（申请人提供）：肉毒毒素是最致命的生物制剂之一，具有大规模毁灭能力。 该毒素由受体结合结构域、膜转位结构域和蛋白水解催化结构域组成。 肉毒毒素通过受体结合结构域特异性结合神经肌肉接头处的突触，通过易位结构域穿透神经细胞，通过催化结构域破坏分泌蛋白组装，并导致瘫痪和可能的死亡。 目前没有治疗药物可用于治疗受影响的个体。 鉴于最近炭疽、乳腺癌、关节炎等的肽和重组蛋白治疗的显著成功，拟议项目的目标是开发针对肉毒杆菌中毒的重组蛋白药物。 授予期间的具体目标将是首先分离不仅能特异性抑制蛋白水解而且还能拯救受损神经细胞的肽，其次是开发此类肽的靶向和递送系统。 结合催化结构域的肽最初将通过基于现有抑制剂结构的组合噬菌体展示肽库以及随机搜索来分离。 肉毒杆菌毒素中蛋白酶活性的高通量测定然后将用于鉴定抑制肽。 将对抑制性肽结构进行进一步的修饰和扩展，以提高肽的亲和力和特异性。 将选择从毒性中拯救受损细胞的肽。 然后将合成重组蛋白，其1）具有与肉毒杆菌毒素相同的突触结合特性; 2）含有完整的易位结构域; 3）含有无活性的蛋白水解催化结构域;以及4）与毒素抑制剂、中和蛋白和拯救肽融合。 将在体外和体内检查重组蛋白在中和和抑制肉毒杆菌毒素中的有效性。 预期此类重组蛋白与肉毒杆菌毒素结合至相同的靶神经细胞，以使催化结构域跨膜移位，并最终特异性抑制毒素对突触蛋白的降解并在体内拯救受损细胞。 这些结果可以很容易地应用于同一家族的其他毒素，并有助于疫苗的开发。

DESCRIPTION (provided by applicant): Botulinum toxins are among the most deadly biological agents with the capacity of mass destruction. The tripartite toxin consists of a receptor binding domain, a membrane translocation domain and a proteolytic catalytic domain. Botulinum toxins bind specifically to synapses at the neuromuscular junctions by the receptor binding domain, penetrate the nerve cells by the translocation domain, destroy secretary protein assembly by the catalytic domain, and result in paralysis and possible death. No therapeutic drugs are currently available to treat affected individuals. In view of notable recent successes of peptide and recombinant protein therapeutics of anthrax, breast cancer, arthritis, etc., the objective of the proposed project is to develop recombinant protein drugs for botulism. The Specific Aims for the granting period will be first isolating peptides that will not only specifically inhibit the proteolysis but will also rescue damaged nerve cells and, secondly, to develop a targeting and delivery system for such peptides. Peptides that bind the catalytic domain will be initially isolated through combinatorial phage display peptide libraries based on structures of existing inhibitors as well as random search. A high throughput assay of the protease activity in botulinum toxins will then be used to identify inhibitory peptides. Further modifications and expansion of the inhibitory peptide structure will be made to improve the affinity and the specificity of the peptides. Peptides that rescue damaged cells from the toxicity will be selected. Recombinant proteins will then be synthesized that 1) have identical synaptic binding properties as botulinum toxins; 2) contain the intact translocation domain; 3) contain an inactive proteolytic catalytic domain; and 4) fuse with toxin inhibitors, neutralizing proteins, and rescue peptides. The effectiveness of the recombinant proteins in neutralizing and inhibiting botulinum toxin will be examined in vitro and in vivo. Such recombinant proteins are expected to bind to the same target nerve cells as botulinum toxins, to translocate the catalytic domain across the membranes, and eventually to specifically inhibit the degradation of synaptic proteins by the toxins and rescue damaged cells in vivo. The results could be readily applicable to other toxins of the same family and helpful for vaccine development.