Identification of novel small molecules that bind to two different sites on the surface of tetanus toxin C fragment

Monique Cosman, Felice C Lightstone, Viswanathan V Krishnan, Loreen Zeller, Maria C. Prieto, Diana C. Roe, Rod Balhorn

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A combination of computational methods, electrospray ionization mass spectroscopy (ESI-MS), and NMR spectroscopy has been used to identify novel small molecules that bind to two adjacent sites on the surface of the C fragment of tetanus toxin (TetC). One of these sites, Site-1, binds gangliosides present on the surface of motor neurons, while Site-2 is a highly conserved deep cleft in the structures of the tetanus (TeNT) and botulinum (BoNT) neurotoxins. ESI-MS was used to experimentally determine which of the top 11 computationally predicted Site-2 candidates bind to TetC. Each of the six molecules that tested positive was further screened, individually and as mixtures, for binding to TetC in aqueous solutions by NMR. A trNOESY competition assay was developed that used doxorubicin as a marker for Site-1 to provide insight into whether the predicted Site-2 ligands bound to a different site. Of the six predicted Site-2 ligands tested, only four were observed to bind. Naphthofluorescein-di-β-galactopyranoside was insoluble under conditions compatible with TetC. Sarcosine-Arg-Gly-Asp-Ser-Pro did not appear to bind, but its binding affinity may have been outside the range detectable by the trNOESY experiment. Of the remaining four, three [3-(N-maleimidopropionyl)biocytin, lavendustin A, and Try-Glu-Try] bind in the same site, presumably the predicted Site-2. The fourth ligand, Ser-Gln-Asn-Tyr-Pro-Ile-Val, binds in a third site that differs from Site-1 or predicted Site-2. The results provide a rational, cost- and time-effective strategy for the selection of an optimal set of Site-1 binders and predicted Site-2 binders for use in synthesizing novel bidendate antidotes or detection reagents for clostridial neurotoxins, such as TeNT and BoNT.

Original languageEnglish (US)
Pages (from-to)1218-1228
Number of pages11
JournalChemical Research in Toxicology
Volume15
Issue number10
DOIs
StatePublished - Oct 1 2002
Externally publishedYes

Fingerprint

Electrospray ionization
gag protein (129-135)
Ligands
Molecules
Binders
Mass Spectrometry
Spectroscopy
Sarcosine
Tetanus Toxin
Antidotes
Gangliosides
Neurotoxins
Tetanus
Motor Neurons
Computational methods
Galactose
Doxorubicin
Nuclear magnetic resonance spectroscopy
Neurons
Assays

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Identification of novel small molecules that bind to two different sites on the surface of tetanus toxin C fragment. / Cosman, Monique; Lightstone, Felice C; Krishnan, Viswanathan V; Zeller, Loreen; Prieto, Maria C.; Roe, Diana C.; Balhorn, Rod.

In: Chemical Research in Toxicology, Vol. 15, No. 10, 01.10.2002, p. 1218-1228.

Research output: Contribution to journalArticle

Cosman, Monique ; Lightstone, Felice C ; Krishnan, Viswanathan V ; Zeller, Loreen ; Prieto, Maria C. ; Roe, Diana C. ; Balhorn, Rod. / Identification of novel small molecules that bind to two different sites on the surface of tetanus toxin C fragment. In: Chemical Research in Toxicology. 2002 ; Vol. 15, No. 10. pp. 1218-1228.
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