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Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A.

Kumaran D, Rawat R, Ludivico ML, Ahmed SA, Swaminathan S
J Biol Chem 283 18883-91 (2008)
Related entries: 3c88, 3c89, 3c8a, 3c8b

Cited: 34 times
EuropePMC logo PMID: 18434312

Abstract

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm.

Articles - 3bwi mentioned but not cited (4)

  1. Substrate binding mode and its implication on drug design for botulinum neurotoxin A. Kumaran D, Rawat R, Ahmed SA, Swaminathan S. PLoS Pathog 4 e1000165 (2008)
  2. Bioinformatic discovery of a toxin family in Chryseobacterium piperi with sequence similarity to botulinum neurotoxins. Mansfield MJ, Wentz TG, Zhang S, Lee EJ, Dong M, Sharma SK, Doxey AC. Sci Rep 9 1634 (2019)
  3. Structural characterization of three novel hydroxamate-based zinc chelating inhibitors of the Clostridium botulinum serotype A neurotoxin light chain metalloprotease reveals a compact binding site resulting from 60/70 loop flexibility. Thompson AA, Jiao GS, Kim S, Thai A, Cregar-Hernandez L, Margosiak SA, Johnson AT, Han GW, O'Malley S, Stevens RC. Biochemistry 50 4019-4028 (2011)
  4. Inhibition of catalytic activities of botulinum neurotoxin light chains of serotypes A, B and E by acetate, sulfate and calcium. Mizanur RM, Gorbet J, Swaminathan S, Ahmed SA. Int J Biochem Mol Biol 3 313-321 (2012)


Reviews citing this publication (6)

  1. Receptor and substrate interactions of clostridial neurotoxins. Brunger AT, Rummel A. Toxicon 54 550-560 (2009)
  2. Clostridial neurotoxins: mechanism of SNARE cleavage and outlook on potential substrate specificity reengineering. Binz T, Sikorra S, Mahrhold S. Toxins (Basel) 2 665-682 (2010)
  3. Botulinum toxin: bioweapon & magic drug. Dhaked RK, Singh MK, Singh P, Gupta P. Indian J Med Res 132 489-503 (2010)
  4. Molecular structures and functional relationships in clostridial neurotoxins. Swaminathan S. FEBS J 278 4467-4485 (2011)
  5. Current strategies for designing antidotes against botulinum neurotoxins. Patel K, Cai S, Singh BR. Expert Opin Drug Discov 9 319-333 (2014)
  6. Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism. Wilson BA, Ho M. Curr Top Med Chem 14 2081-2093 (2014)

Articles citing this publication (24)

  1. Genomic insights into the evolution and ecology of botulinum neurotoxins. Mansfield MJ, Doxey AC. Pathog Dis 76 (2018)
  2. Newly Designed Quinolinol Inhibitors Mitigate the Effects of Botulinum Neurotoxin A in Enzymatic, Cell-Based, and ex Vivo Assays. Bremer PT, Adler M, Phung CH, Singh AK, Janda KD. J Med Chem 60 338-348 (2017)
  3. Synthesis and biological evaluation of botulinum neurotoxin a protease inhibitors. Li B, Pai R, Cardinale SC, Butler MM, Peet NP, Moir DT, Bavari S, Bowlin TL. J Med Chem 53 2264-2276 (2010)
  4. Extreme sensitivity of botulinum neurotoxin domains towards mild agitation. Toth SI, Smith LA, Ahmed SA. J Pharm Sci 98 3302-3311 (2009)
  5. SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E. Agarwal R, Swaminathan S. J Biol Chem 283 25944-25951 (2008)
  6. The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site. Mizanur RM, Frasca V, Swaminathan S, Bavari S, Webb R, Smith LA, Ahmed SA. J Biol Chem 288 24223-24233 (2013)
  7. Basic tetrapeptides as potent intracellular inhibitors of type A botulinum neurotoxin protease activity. Hale M, Oyler G, Swaminathan S, Ahmed SA. J Biol Chem 286 1802-1811 (2011)
  8. Fungal bis-Naphthopyrones as Inhibitors of Botulinum Neurotoxin Serotype A. Cardellina JH, Roxas-Duncan VI, Montgomery V, Eccard V, Campbell Y, Hu X, Khavrutskii I, Tawa GJ, Wallqvist A, Gloer JB, Phatak NL, Höller U, Soman AG, Joshi BK, Hein SM, Wicklow DT, Smith LA. ACS Med Chem Lett 3 387-391 (2012)
  9. Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A. Bremer PT, Pellett S, Carolan JP, Tepp WH, Eubanks LM, Allen KN, Johnson EA, Janda KD. J Am Chem Soc 139 7264-7272 (2017)
  10. Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A. Zuniga JE, Hammill JT, Drory O, Nuss JE, Burnett JC, Gussio R, Wipf P, Bavari S, Brunger AT. PLoS One 5 e11378 (2010)
  11. Light chain separated from the rest of the type a botulinum neurotoxin molecule is the most catalytically active form. Gul N, Smith LA, Ahmed SA. PLoS One 5 e12872 (2010)
  12. Identification of small molecule inhibitors of botulinum neurotoxin serotype E via footprint similarity. Zhou Y, McGillick BE, Teng YG, Haranahalli K, Ojima I, Swaminathan S, Rizzo RC. Bioorg Med Chem 24 4875-4889 (2016)
  13. Cleavage of SNAP25 and its shorter versions by the protease domain of serotype A botulinum neurotoxin. Mizanur RM, Stafford RG, Ahmed SA. PLoS One 9 e95188 (2014)
  14. Computer-aided identification, synthesis, and biological evaluation of novel inhibitors for botulinum neurotoxin serotype A. Teng YH, Berger WT, Nesbitt NM, Kumar K, Balius TE, Rizzo RC, Tonge PJ, Ojima I, Swaminathan S. Bioorg Med Chem 23 5489-5495 (2015)
  15. Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening. Hu X, Legler PM, Southall N, Maloney DJ, Simeonov A, Jadhav A. J Comput Aided Mol Des 28 765-778 (2014)
  16. Tyrosine phosphorylation of botulinum neurotoxin protease domains. Toth S, Brueggmann EE, Oyler GA, Smith LA, Hines HB, Ahmed SA. Front Pharmacol 3 102 (2012)
  17. Inhibition in vivo of the activity of botulinum neurotoxin A by small molecules selected by virtual screening. Eichhorn T, Dolimbek BZ, Deeg K, Efferth T, Efferth T, Atassi MZ. Toxicon 60 1180-1190 (2012)
  18. Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA. Lam KH, Sikorra S, Weisemann J, Maatsch H, Perry K, Rummel A, Binz T, Jin R. Pathog Dis 76 (2018)
  19. Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A. Ho M, Goh CH, Brothers MC, Wang S, Young RL, Ou Y, Lui JN, Kalafatis M, Lan X, Wolf AE, Rienstra CM, Wilson BA. Protein Sci 21 318-326 (2012)
  20. Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice. Singh P, Singh MK, Chaudhary D, Chauhan V, Bharadwaj P, Pandey A, Upadhyay N, Dhaked RK. PLoS One 7 e47110 (2012)
  21. Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method. Kitamura K, Komatsu M, Biyani M, Futakami M, Kawakubo T, Yamamoto K, Nishigaki K. J Pept Sci 18 711-719 (2012)
  22. Structure-based substrate screening for an enzyme. Xu T, Zhang L, Wang X, Wei D, Li T. BMC Bioinformatics 10 257 (2009)
  23. Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication. Guo J, Wang J, Gao S, Ji B, Waichi Chan E, Chen S. Sci Rep 5 16981 (2015)
  24. Utilizing Ayurvedic literature for the identification of novel phytochemical inhibitors of botulinum neurotoxin A. Yalamanchili C, Manda VK, Chittiboyina AG, Guernieri RL, Harrell WA, Webb RP, Smith LA, Khan IA. J Ethnopharmacol 197 211-217 (2017)


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