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.

Selective High-Affinity Ligand Antibody Mimics for Cancer Diagnosis and Therapy: Initial Application to Lymphoma/Leukemia

“…Whereas it is recognized that the force fields and energetics used by the present docking programs are less than ideal, the results we report here for HLA-DR10 and our previous studies with the C fragment of tetanus neurotoxin (24,44) show that these methods can provide sufficient discrimination to effectively screen very large libraries of small molecules and identify a small set of compounds for experimental testing. In each of these studies, <40 compounds were screened for binding to the target proteins and 25% to 56% of the compounds tested were experimentally determined to bind to the protein.…”

Mass spectrometry and non-covalent protein-ligand complexes: Confirmation of binding sites and changes in tertiary structure

“…Solution based NMR experiments used to detect doxorubicin binding to TetC, a 1:1 protein:ligand molar ratio was not sufficient to detect ligand binding and optimal molar ratios were 1:16 to 1:22 [30]. In ligand competition experiments using NMR, another ligand predicted to bind to the same site as doxorubicin, 3'-sialyllactose, displaced doxorubicin from TetC suggesting a specific interaction.…”

“…This requires an understanding of protein structure and protein-ligand binding sites. Doxorubicin has been computationally predicted and experimentally shown to bind TetC [29,30]. Herein, we implement time resolved limited proteolysis of the non-covalently bound TetC-doxorubicin complex, using MALDI TOF MS analysis, to determine doxorubicin binding site(s).…”

DHS Summer Student Project Report

“…Among these molecules are the anticancer agent doxorubicin (Dox) and the tripeptides WEY and YEW ( Figure 1; Cosman et al, 2002). The crystal structure of botulinum toxin and Dox (PDB code: 1I1E) demonstrates that Dox binds in a surface groove of in C-terminal fragment that is conserved in both botulinum and tetanus toxins.…”