title: Identification of novel small molecules that bind to two different sites on the surface of tetanus toxin C fragment.
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-beta-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.
authors.affiliation: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551-0808, USA. cosman1@llnl.gov
authors.forename: Monique
authors.initials: M
authors.lastname: Cosman
authors.forename: Felice C
authors.initials: FC
authors.lastname: Lightstone
authors.forename: V V
authors.initials: VV
authors.lastname: Krishnan
authors.forename: Loreen
authors.initials: L
authors.lastname: Zeller
authors.forename: Maria C
authors.initials: MC
authors.lastname: Prieto
authors.forename: Diana C
authors.initials: DC
authors.lastname: Roe
authors.forename: Rod
authors.initials: R
authors.lastname: Balhorn
mesh_terms: D000931:Antidotes; D001665:Binding Sites; D003013:Clostridium / Q000472:pathogenicity; D015195:Drug Design; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009046:Motor Neurons; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013744:Tetanus Toxin / Q000032:analysis / Q000737:chemistry*
chemical_list: D000931:Antidotes; D008024:Ligands; D010446:Peptide Fragments; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C