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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54AI057159-08
Application #
8233433
Study Section
Special Emphasis Panel (ZAI1)
Project Start
2011-03-01
Project End
2014-02-28
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
8
Fiscal Year
2011
Total Cost
$487,954
Indirect Cost
Name
Harvard University
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Nair, Dhanalakshmi R; Chen, Ji; Monteiro, João M et al. (2017) A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism. J Antibiot (Tokyo) 70:1009-1019
Huang, Nai-Jia; Pishesha, Novalia; Mukherjee, Jean et al. (2017) Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin. Nat Commun 8:423
Mertins, Philipp; Przybylski, Dariusz; Yosef, Nir et al. (2017) An Integrative Framework Reveals Signaling-to-Transcription Events in Toll-like Receptor Signaling. Cell Rep 19:2853-2866
de Wispelaere, Mélissanne; Carocci, Margot; Liang, Yanke et al. (2017) Discovery of host-targeted covalent inhibitors of dengue virus. Antiviral Res 139:171-179
Choo, Min-Kyung; Sano, Yasuyo; Kim, Changhoon et al. (2017) TLR sensing of bacterial spore-associated RNA triggers host immune responses with detrimental effects. J Exp Med 214:1297-1311
Zheng, Huiqing; Colvin, Christopher J; Johnson, Benjamin K et al. (2017) Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence. Nat Chem Biol 13:218-225
Coulson, Garry B; Johnson, Benjamin K; Zheng, Huiqing et al. (2017) Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics. Cell Chem Biol 24:993-1004.e4
Vrentas, Catherine E; Moayeri, Mahtab; Keefer, Andrea B et al. (2016) A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection. J Biol Chem 291:21596-21606
Helenius, Iiro Taneli; Nair, Aisha; Bittar, Humberto E Trejo et al. (2016) Focused Screening Identifies Evoxine as a Small Molecule That Counteracts CO2-Induced Immune Suppression. J Biomol Screen 21:363-71
Fink, Avner; Hassan, Musa A; Okan, Nihal A et al. (2016) Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19. MBio 7:e02243

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