Identification of Novel Small Molecules That Bind to Two Different Sites on the Surface of Tetanus Toxin C Fragment

• Published in: Chemical research in toxicology 2002-10, Vol.15 (10), p.1218-1228
• Main Authors: Cosman, Monique, Lightstone, Felice C, Krishnan, V. V, Zeller, Loreen, Prieto, Maria C, Roe, Diana C, Balhorn, Rod
• Format: Article
• Language: English
• Subjects: Antidotes; Binding Sites; Clostridium - pathogenicity; Clostridium tetani; Drug Design; Humans; Ligands; Magnetic Resonance Spectroscopy; Molecular Structure; Motor Neurons; Peptide Fragments - analysis; Peptide Fragments - chemistry; Spectrometry, Mass, Electrospray Ionization; tetanus toxin; Tetanus Toxin - analysis; Tetanus Toxin - chemistry
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx025570m

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.