Novel recombinant protein therapeutics of botulism

描述（由申请人提供）：肉毒杆菌毒素是具有大规模杀伤能力的最致命的生物制剂之一。三方毒素由受体结合域、膜易位域和蛋白水解催化域组成。肉毒杆菌毒素通过受体结合域特异性结合神经肌肉连接处的突触，通过易位域穿透神经细胞，通过催化域破坏秘书蛋白组装，导致瘫痪甚至可能死亡。目前没有治疗药物可用于治疗受影响的个体。鉴于最近在炭疽、乳腺癌、关节炎等的肽和重组蛋白治疗方面取得了显著的成功，本项目的目标是开发肉毒杆菌中毒的重组蛋白药物。授权期的具体目标将首先分离出不仅能特异性抑制蛋白水解而且能拯救受损神经细胞的肽，其次，开发出这种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将用于鉴定抑制肽。进一步修饰和扩展抑制肽的结构，以提高肽的亲和力和特异性。从毒性中拯救受损细胞的肽将被选择。重组蛋白将被合成：1)具有与肉毒杆菌毒素相同的突触结合特性；2)包含完整的易位域；3)含有非活性蛋白水解催化结构域；4)与毒素抑制剂、中和蛋白和救援肽融合。重组蛋白中和和抑制肉毒杆菌毒素的有效性将在体外和体内进行研究。这种重组蛋白有望与肉毒杆菌毒素结合到相同的靶神经细胞上，将催化结构域跨膜易位，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。该结果可用于同类毒素的研究，为疫苗的研制提供参考。

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.