NERCE Project 11: COPI interactions mediate toxin entry: a common shared mechanism of translocation, John R. Murphy, Ph.D., Boston Medical Center. It is well known that diphtheria toxin, anthrax toxin, and the bptulinum neurotoxins bind to specific receptors on the surface of their respective target cell and are internalized into an endosomal compartment by receptor-mediated endocytosis. Acidification of the endosomal lumen by the vesicular (v)ATPase results in a dynamic conformational changes of the toxins transmembrane domain resulting in its spontaneous insertion into the plane of the membrane and the formation of a pore or channel. The catalytic domain of each toxin (e.g., fragment A, Lethal Factor and/or Edema Factor, and light chain) is then translocated across the vesicle membrane and released into the cytosol. In the case of both the diphtheria catalytic domain and anthrax Lethal Factor delivery into the cytosol, COPI coatomer complex has been shown to play an early and essential step in the translocation process. In each instance, protein protein interaction between COPI complex and the toxin is mediated through an """"""""entry motif, T1, and at least the beta-COP component of the complex. Bioinformatic analysis of anthrax Edema Factor and the botulinum neurotoxins has shown the presence of either the T1-motif or COPI coatomer binding sites in regions of these toxins that are predicted to emerge from the transmembrane domain channel early in the intoxication process. This grant application proposes to investigate the role of COPI complex in the translocation of Edema Factor and botulinum light chain from the lumen of purified endosomes in vitro. We propose the development of screening assays for inhibitors of T1-motif::COPI coatomer complex interactions in conjuction with the National Screening Laboratories for the Regional Centers of Excellence in Emerging Infectious Diseases and Biodefense. Following initial screening, the activity of small molecule diversity set screening leads in in vitro will be confirmed in whole cell cytotoxicity assays for diphtheria, anthrax, and botulinum toxins. It is anticipated that small molecule inhibitors of the T1 ::COPI interactions would function as broad spectrum inhibitors of diphtheria, anthrax, and botulinum toxin action both in vitro and in vivo.
Research proposed here is extending the observation that the entry of many bacterial protein toxin into the cell follows the same fundamental mechanism. These results strongly suggest that interruption of this entry process will allow for the development of single agents that will have broad application and block the action of multiple toxins. Such inhibitors would be the first of a new class of antitoxin drugs.
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